atlas journal of biology 1 (3): 47-51, 2011 doi: 10.5147/ajb.2011.0047 a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) punica granatum: a new host of bipolaris spicifera in morocco oumama kadri*, rachid benkirane, amina ouazzani touhami, and allal douira* laboratoire de botanique et de protection des plantes, université ibn tofail, faculté des sciences, kénitra, morocco received: february 12, 2011 / accepted: may 8, 2011 __________________________________________________ *corresponding authors: douiraallal@hotmail.com & ok_ouma@ hotmail.com. introduction pomegranate (punica granatum l.) is a fruitful ornamental tree in the tropical and subtropical areas of the world, including the mediterranean basin and asia. several varieties are generally associated with this species. punica granatum l. ‘nana’ is a dwarf variety more valuable for its numerous small flowers and long flowering period (jianzhu et al., 2003). in march 2009, the survey in the public and the university of sciences gardens of kenitra city (morocco) showed that 60% of leaves of 100 plants of punica granatum l. ’nana’ presented foliar lesions affecting the aesthetic appearance of this ornamental plant. at the time of attacking, temperatures varied between 10 and 25°c (average of 17.5°c) and the rainfall for the month was 19 mm. the necrotic lesions developed on the periphery and in the middle of leaves were sometimes numerous, often circular with dark brown color and measuring 1 to 4 mm in diameter. later, the midst of these lesions becomes more clear and surrounded by a border of dark purple (fig. 1). this was the first time that these symptoms were observed on punica granatum l. ’nana’. 47 short communication abstract during march 2009 in the public gardens and the university of sciences of kenitra city (morocco), 60% of leaves of 100 plants of pomegranate (punica granatum l. ’nana’) presented foliar lesions that affecting the aesthetic appearance of this ornamental plant. bipolaris spicifera was isolated from ten necrotic lesions of 25 leaves. the artificial inoculation of the healthy leaves of three plants of punica granatum l. ’nana’ by conidial suspension of the pathogen induced the same lesions to that observed in nature. the diseased foliar surface and the total number of diseased leaves 30 days after inoculation with b. spicifera conidial suspension were 37.5% and 78. conidia production of b. spicifera on inoculated leaves was 0.81 x 105 spore cm-2 and the fungus was re-isolated from lesions on inoculated plants. this is the first report of b. spicifera on pomegranate in morocco. keywords: punica granatum l.’nana’, bipolaris spicifera, lesions, inoculation. 48 material and methods twenty five leaves showing necrotic lesions were collected from 5 plants of punica granatum l. ‘nana’. leaves with lesions were cut in small fragments, washed with water, disinfected in alcohol and placed in 90 mm petri dishes on three filter paper discs moistened with sterile distilled water with 5 lesions per petri dish. the lesions were incubated at 22°c under continuous fluorescent lighting. lesions were examined using an optical microscope after 4 days and conidia were taken with a capillary tube and placed on psa media (potato sucrose agar: 200 g potato, 20 g sucrose, 15 g agar-agar, and 1000 ml distilled water) and incubated at 28°c to determine the pathogen. three plants of punica granatum l. ’nana’ (plant i: 252 total healthy leaves; plant ii: 327 total healthy leaves; plant iii: 349 total healthy leaves) were maintained in a greenhouse at 17 25°c (april 2009). these plants were inoculated by spraying with 60 ml of a conidial suspension containing 105 conidia ml-1 of fungus. the conidia concentration was adjusted with distilled water, containing 0.02% of tween 20 and 1% gelatin. the control plants were inoculated with distilled water containing tween 20 and gelatin. the inoculated plants were kept covered for 48h with black bags and then moved to a greenhouse. the test was repeated twice and the same fungus was consistently re-isolated from these lesions. the notation of results was made seven days after inoculation and during one month. the diseased foliar surface (%) was scored according to the scale of notteghem et al. (1980). the conidia production (conidia cm-2) of fungus on the infected pomegranate leaves was estimated according to the technique of hill and nelson (1983). one month after inoculation, the leaves with lesions were harvested from the inoculated plants, cut into pieces of 1cm2 and placed in 90 mm petri dishes on three filter paper discs moistened with sterile distilled water. the dishes were incubated from 48 to 72 hours at 22°c una tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 1. foliar symptoms of bipolaris spicifera infection on pomegranate plants (punica graminum l. ‘nana’) in nature. (a): dark brown circular lesions; (b) lesion surrounded by a border of dark purple color; (c): lesions are sometimes numerous. a b c 49 der continuous fluorescent lighting. then, each fragment was placed in a test tube containing 1 ml of sterile distilled water and agitated by a vortex mixer for 2 min. the conidia of the pathogen were counted using a malassez slide under an optical microscope at magnification ×100 with three counting of each sample. results and discussion on psa, the monospore culture of the fungus isolated was black olive, dark inside, becoming clearer toward the periphery. the conidia were brown, 3 septates, elliptical often cylindrical (16.75±4) μm long, (8.25±2) μm wide, rounded at the ends, each with an evident hilum (figure 2). the conidiophores were erected, unbranched, partitioned, 4-8 μm wide and geniculated. based on the morphological description (ellis, 1971), the fungus was identified as bipolaris spicifera (bainier) subram (1971). symptoms on leaves of punica granatum l. ‘nana’ appeared seven days after inoculation by b. spicifera and the necrotic lesions were identical to that observed in nature (figure 3). the tested plants of p. granatum l. ‘nana’, all showed sensitivity relative to b. spicifera. between the 7th and 30th day after inoculation, the amount of disease increased with time. the diseased foliar surface of inoculated plants was 0.5% on the 7th day and increased to 37.5% on the 30th day pot inoculation (dpi). the total number of leaves with symptoms increased from 10 on the 7th day to 78 on the 30th. bipolaris spicifera was also able to produce conidia on the lesions of p. granatum l. ‘nana‘plants with an average of 0.81×105 spore cm-2. bipolaris spicifera isolated from 77 host plants, from air and soil (ellis, 1971). in morocco, this pathogen was isolated for the first time from foliar lesions of oryza sativa (ennaffah et al., 1997), foliar lesions of hibiscus rosa-sinensis (meddah et al., 2007) and from watermelon (el mhadri et al., 2009). this fungus was also isolated from chloris verticillata, eragrostis cilianensis and paspalum leave (roane and roane, 1996, 1997); cynodon a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 2. colonies (a), conidiophore (b) and conidia (c) of bipolaris spicifera isolated from punica granatum l. ‘nana’ and cultivated on psa medium. a b c 50 dactylon and deschampsia flexuosa (roane, 2004, 2009); avena sativa, hordeum vulgare, secale cerea, and triticum aestivum (cook and dubé, 1989); oryza sativa, phaseolus vulgaris, sorghum bicolor, and from zea mays (mendes et al., 1998). moreover, bipolaris spicifera was isolated from setaria glauca and sorghum halepense (pratt, 2006); arachis hypogaea (pourabdollah and ershad, 1997); commelina benghalensis (caretta et al., 1999); pistacia vera (chen et al., 2002); quercus xalapensis (mena-portales et al., 1995) and from the soil (liu and zhang, 2004). to our knowledge, this is the first report of necrotic lesions caused by b. spicifera on p. granatum l. ‘nana’ in morocco. the pathogen was a real problem for the aesthetic quality of this ornamental plant. references caretta g, piontelli e, picco am, del frate g (1999) some filamentous fungi on grassland vegetation from kenya. mycopathologia 145: 155-169. chen wq, ntahimpera n, morgan dp, michailides tj (2002) mycoflora of pistacia vera in the central valley, california. mycotaxon 83: 147-158. cook rp, dubé aj (1989) host-pathogen index of plant diseases in south australia. south australian department of agriculture 142. el mhadri m, benkirane r, ouazzani touhami a, douira a (2009) citrullus lanatus, a new host of bipolaris spicifera in morocco. phytopathol mediterr 48: 291-293. ellis mb (1971) dematiaceous hyphomycetes, commonwealth mycological institute, kew, surrey, england, 608 pp. ennaffah b, bouslim f, benkirane r, ouazzani touhami a, douira a a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a b b b fig. 3. lesions developed on pomegranate leaves after artificial inoculation by bipolaris spicifera. (a): control plant; (b): inoculated plant. a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) (1997) helminthosporium spiciferum, foliar parasite of rice in morocco. agronomie 17: 299-300. hill jp, nelson rr (1983) genetic control of two parasiticfitness attribuates of helminthosporium maydis race t. phytopathology 73: 455-457. jianzhu s, chunli c, xiuxin d (2003) in vitro induction of tetraploid in pomegranate (punica granatum).national key laboratory of crop genetic improvement. plant cell tissue and organ culture 75: 241–246. liu hm, zhang ty (2004) a preliminary report of soil dematiaceous hyphomycetes from the yellow river delta i. mycosystema 23: 338344. meddah n, ouazzani touhami a, douira a (2007) l’hibiscus rosa-sinensis, un nouvel hôte pour le cochliobolus spicifer et le setosphaeria rostrata. phytoprotection 88 : 57-60. mena-portales j, heredia-abarca g, mercado-sierra a (1995) species of bipolaris and curvularia on leaves of quercus and liquidambar from the state of veracruz, mexico. revista mex micol 11: 109-121. mendes mas, da silva, vl, dianese jc (1998) fungos em plants no brasil. embrapa spi/embrapa-cenargen, brasilia. 555 pp. notteghem jl, andriatompo gm, chatel m, dechanet r (1980) techniques utilisées pour la sélection de variétés de riz possédant la résistance horizontale à la pyriculariose. ann phytopathol 12: 199266. pratt rg (2006) johnsongrass, yellow foxtail, and broadleaf signalgrass as new hosts for six species of bipolaris, curvularia, and exserohilum pathogenic to bermudagrass. plant disease 90: 528. pourabdollah s, ershad d (1997) an investigation on mycoflora of peanut seeds in iran. iran j plant pathol 33: 64-68. roane cw, and roane mk (1996) graminicolous fungi of virginia: fungi associated with genera aegilops to digitaria. virginia j science 47: 197-224. roane cw (2009) graminicolous fungi of virginia: fungi in collections 2004-2007. virginia j science 60: 13-50. roane cw (2004) graminicolous fungi of virginia: fungi in collections 1995 2003. virginia j science 55: 139-157. roane cw, roane mk (1997) graminicolous fungi of virginia: fungi associated with genera echinochloa to zizania. virginia j science 48: 11-46. 51 atlas journal of biology 1 (3): 66-69, 2011 doi: 10.5147/ajb.2011.0060 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) the endemic plant limonium mucronatum l. (fil.) chaz., a new host for uromyces limonii (dc.) lev., 1849 in morocco mohammed khouader, rachid benkirane, amina ouazzani touhami, el houcine boussalwa, and allal douira* laboratory of botany, biotechnology and plants protection, university ibn tofail, faculty of sciences, p. o. box 33, 14000 kénitra, morocco received: april 5, 2011 / accepted: june 25, 2011 __________________________________________________ * corresponding author: douiraallal@hotmail.com introduction the genus uromyces, rust cosmopolitan, has 600 species widespread in all regions of the world and on different host plants (lindquist, 1951; savulescu, 1953). in morocco, uromyces is represented by 91 species (khouader et al., 2008), whose uromyces limonii (dc.) lev., 1849. in morocco, u. limonii was encountered on statice sinuata, between essaouira and tamanar (oued dbib), at mohammadia (oued mellah) and settat (guyot and malençon, 1963) and l. beaumieranum, l. bonduelii, l. mouretii, l. sinuatum, and l. thouin (locality not specified) (rieuf, 1970). this species has never been reported before on limonium mucronatum l. (phil.) chaz. endemic of morocco (fennane et al., 1999). u. limonii is a heteroxenous parasite whose cycle of development takes place on two hosts “statice gmelini and s. limonium” (savulescu, 1953). eciospores are observed on s. gmelini while urediospores and teliospores are found in combination on s. limonium (savulescu, 1953). 66 short communication abstract uromyces limonii (dc.) lev., 1849 was observed for the first time in morocco to a moroccan endemic limonium mucronatum l. (phil.) chaz. harvested in coastal rocky cliffs (near sale). the host species and the pathogen have been identified based on phenotypic traits. symptoms rated on the host plant were described in this study. keywords: morocco, rust, limonium mucronatum, uromyces limonii. 67 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) limonium mucronatum is a plant that lives at the base of cliffs or on small ledges and rocky outcrops in the levels which are greatly influenced by ocean spray and set over the dunes (gehu and biondi, 1998; fennane et al., 1999). it was observed irregularly around sidi ifni / mirleft at the base of the cliffs north of safi (gehu and biondi, 1998). limonium mucronatum hemicryptophyte species that is found in the arid, semi-arid mild, subhumid, the saharan morocco, anti atlas, north atlantic morocco and atlantic morocco through (fennane et al., 1999). it is adapted to saline soils and wetlands (simone, 2000). materials and methods surveys in the coastal cliffs between rabat and kenitra allowed us to harvest between may and june 2010, a dozen diseased plants of l. mucronatum. the host plant was determined on the basis of phenotypic characters (fennane et al., 1999). leaf symptoms are in the form of brown, black or orange pustules. the identification of the causative agents responsible for different symptoms, was conducted by consulting some determination keys, including those of savulescu (1953), hiratsuka (1973), and the descriptions of saccardo (1888), lindquist (1951), cummins and hiratzuka (1996), and garcia-hernandez et al. (2008). the description of symptoms was performed using a hand lens or dissecting microscope to better visualize the pustules observed on both sides of the leaves of l. mucronatum. to study the fungus, scraping was performed at pustules developed on leaves of l. mucronatum. preparations were made to observe, through an optical microscope (x 400), urediospores and mostly teliospores because it is the latter aspect that determines the kind of fungus. mounting medium is tap water and sometimes is added to the preparation a drop of bleach to the elucidation of the spore wall. measurements of urediospores, teliospores (at least 50 spores), and pedicel were made by the means of ocular micrometer. results and discussion l. mucronatum grows among the stones on a rocky cliff to the nations beach near bouknadel (near sale) (fig.1a). the substrate is influenced by ocean spray. phenotypic characteristics of the genus limonium radical leaves in rosettes, persistent and non-flowering stem leaves scattered in tiny scales or in small axillary fascicles. inflorescence spikelet base containing one or more flowers each spikelet is bracted by 3 bracts, an external and an average internal sessile spikelets often unilateral and usually arranged in panicles or corymbs. flowers often bracted. calyx usually tubular or funnel-shaped, 5 or 10 ribs scarious, brightly colored in some species. petals free or more or less united at the base. stamens inserted at base of petals. styles glabrous, free or fused at base. fruit dehiscent or indehiscent (fennane et al., 1999). phenotypic characteristics of limonium mucronatum palm three garlic wing well developed, wavy curly. bract internal spikelets 6-7 mm long. mucronate leaves, the basal rather abruptly attenuated into petiole. panicle corymbose, with unilateral spikes. bract external and internal spikelets broadly hyaline on margins, reddish on the back. the corolla is purple (fennane et al., 1999). description of symptoms and characteristics of uromyces limonii forms of reproduction observed are stages ii and iii disease. pustules developed are brown, black or orange, arranged on both sides of the leaves of l. mucronatum (fig. 1b and fig. 2a). two stages of the disease were observed. the telia are observed in powder, combined black and brown. teliospores (25 to 30 μm in length and 23.30 to 26.65 μm in wide) are unicellular, oblong, rarely ovate or ellipsoid, stalked, colored yellow-orange to brown. the wall is light brown, smooth and thick (thicker a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 1a. limonium mucronatum l. (fil.) chaz. located at the base of a cliff. plant on the ground. fig. 1b. limonium mucronatum l. (fil.) chaz. located at the base of a cliff. plant after harvest. at the top). the pedicel (53.30 microns) is hyaline, thick and long (2 times the length of teliospores) (fig. 2b). the uredinia are pulverulent scored, oblong and orange. urediospores (30.35 μm in diameter) are yellow-orange, warty, ellipsoidal or globular (2c). aecial phase is absent. the description of these forms of reproduction is that of u. limonii (savulescu, 1953; malençon and guyot, 1963; garcia-hernandez et al., 2008). in morocco, guyot and malençon (1963) reported the complete absence of aecial phase of u. limonii. urediospores and teliospores can be encountered on statice sinuatum. according to rieuf (1970), u. limonii attack five species of the genus limonium “l. beaumieranum, l. bonduelii, l. mouretii, l. sinuatum, and l. thouin”. statice mucronata (=limonium mucronatum), encountered in the bush of essaouira, the cliffs of cape cantin between el jadida and safi and botanical garden cherifian the scientific institute in rabat, may be attacked by uromycesstatice mucronatae (malençon, 1936; guyot and malençon, 1963). this species differs from u. limonii by the following characters: • remarkable predominance of aecial stage, • uredo stage is very small, • early onset of teleuto stage is in contact with aecial groups, • longer pedicel (can reach up to 87μm in length). on the world scale, uromyces limonii was found on several host plants: armeria sp. and limonium spp. in chile (lindquist, 1951; garcia-hernandez et al., 2008), statice gmelini and statice limonium to romania (savulescu, 1953 ), statice japonica in japan (hiratsuka, 1973), limonium vulgare and limonium sp. in the british isles (ellis, 1985; ellis, 1997), l. vulgare to netherlands (zadoks, 1988, 1992, 2005), l. vulgare and statice limonium in belgium (mathieu, 1855; vanderweyen and fraiture, 2008). between may and early june 2010, we met for the first time u. limonii on l. mucronatum. aecial phase was not observed, but the urediospores and teliospores were found in association on leaves of this new host species. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) references cummins g and y hiratsuka (1996) illustrated genera of rust fungi. edition. the american phytopathological society. st. paul, minnesota, usa, pp. 152. ellis mb and jp ellis (1985) microfungi on land plants. an identification guide. macmillan publ. co., new york, pp. 818. ellis mb and jp ellis (1997) microfungi on land plants an identification handbook. the richmond publishing co ltd., uk, pp. 868. fennane m, m ibn tattou, j mathez, a ouyahya, and j el oualidi (1999) flore pratique du maroc (manuel de détermination des plantes vasculaires), inst. scient.univ. mohammed v, rabat, vol. 1, pp. 558. garcia-hernandez d, m rojas-jara, g sepulveda, and f chavera (2008) presencia de uromyces limonii (dc) lév. (roya del limonium) primer registro para el valle de lluta, région de arica y parinacota, chili. vol. 26: 73–75. géhu jm and e biondi (1998) nature et limites de quelque végétations littorales de type macronésien sur les côtes sud occidentales du maroc. acta bot barc 45: 439-453. guyot al and g malencon (1963) uredinees du maroc ii. trav inst sci cherifien 28:1-161. hiratsuka n (1973) revision of taxonomy of the genus uromyces in the japanese archipelago. report of the tottori mycological institute 10: 1–98. khouader m, r benkirane, a ouazzani touhami, e boussalwa, and a douira (2008) biodiversité au service du développement durable. vème journées nationales de biodiversité, 18-20 décembre, fès, maroc. lindquist jc (1951) urédinales de la provincia de mendoza. rev. fac. agr. univ. la plata 28: 65-144. malençon g (1936) notulae mcologiacae. maroccanae. i. uredinales. puccinia. rev. de mycologie i: 43-74. mathieu c (1855) supplément de la flore générale de belgique. additions, observations, correction bruxelles, gand, leipzig, c. muquardt 1855. rieuf p (1970) parasites et saprophytes des plantes au maroc. les cahiers de recherche agronomique 28: 179-357. saccardo pa (1888) sylloge fungorum omnium hucusque cognitorum. uromyces limonii (dc.) lév. vol. vii: 532-533. savulescu a (1953) monografia uredinalelor din republica populara romania. tome ii. editura academiei republicii populare romane 68 figure 2. (a) symptoms of uromyces limonii (dc.) lév. on limonium mucronatum l.(fil.) chaz.; (b) teliospores x400; (c) uredospores x400. pp. 1166. simone c (2000) le geosystème dunaire anthropisé d’essaouira-est (maroc atlantique) : dynamique et paléoenvironnement. thèse de doctorat. université aix-marseille i – université de provence, france, pp. 204. vanderweyen a and fraiture (2008) catalogue des urédinales de belgique (2ème partie). lejeunia, série n°185, p. 31. zadoks jc (1988) a salty act. notes on the dispersal of uromyces limonii. zeitschrift pflanzenkrankheiten pflanzenschutz 95: 384-391. zadoks jc (1992) studies on the haploid stage of the sea lavender rust, uromyces limonii, on limonium vulgare. journal plant disease and protection 99: 168-173. zadoks jc (2005) sea lavender, rust and mildew – a perennial pathosystem in the netherlands. wageningen academic publishers. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 69 comparative study of the effects of salinity on plant growth, nodulation, and legheamoglobin content in kabuli and desi cultivars of cicer arietinum (l.) varsha mudgal1*, nidhi madaan1, anurag mudgal2, alka singh3, p. kumar3 1department of biotechnology; 2department of mechanical engineering college of engineering and technology, iftm campus, moradabad, up, india; 3department of botany, hindu college, moradabad, up, india kbm journal of biology (2010) 1: 1-4. doi: 10.5147/kbmjb.2010.0001 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) abstract this study was conducted to compare plant growth, nodulation and legheamoglobin content in kabuli and desi cultivars of chickpea (cicer arietinum (l.)) under saline conditions. all the varieties expressed greater adverse effects on the plant height, dry weight of shoot and root at 30 and 60 das whereas the deleterious effects of the salinity minimized at 90 das. it is evident that variety bg-256 registered maximum inhibition at all the three growth stages (30, 60 and 90 das) as compared to varieties pusa-939 and pusa-1053. relative growth rate (rgr) is high in pusa-1053 (kabuli tolerant), stands midway in pusa-939 (kabuli moderate tolerant) and is low in bg-256 (desi sensitive). maximum nodulation was reported in variety pusa-1053 at all the growth stages and all salinity levels (4– 16 ec) while minimum nodulation occurred in variety bg-256. the data clearly indicate that legheamoglobin content of the nodules increased at 60 das and declined thereafter. kabuli tolerant variety (pusa-1053) showed highest and desi sensitive (bg256) showed least legheamoglobin content at all the salinity levels from 4-16 ec and at all the growth stages from 30-90 das. the dry weigh of shoot and number of nodules were affected more adversely than nodule dry weight and legheamoglobin content. it indicates that kabuli cultivars are more tolerant to saline stress and show less deleterious effects on nodules and nitrogen fixation efficiency. key words: cultivar, salinity, ec (electrical conductivity), leghaemoglobin, das (days after sowing), nodulation. received: december 19, 2009 / accepted: february 27, 2010 introduction salinity produces harmful effects on plant growth and yield. permanent and complementary solution to minimize the deleterious effects of the salinity is to select and grow cultivars that can provide economic yield under saline conditions. among legume crops, chickpea ranks first in terms of area and production. it provides a high quality protein to the people of developing countries. it is usually grown mixed with wheat, barley, linseed and mustard. selected cultivars of chickpea are desi type with small and brown seed (nearly 90% area) and kabuli type with bold and cream-colored seed (growing in 10% area). desi chickpeas have markedly higher fiber content and lack pigmentation than kabulis and hence a very low glycemic index which may make them suitable for people with high blood sugar problems. kabuli genotype show higher tolerance to salinity (rao et al., 1995; soussci et al., 1998). the present study compares kabuli and desi genotypes of chickpea in terms of growth, physiological and biochemical markers of salt tolerance. it will be helpful to select of chickpea varieties with improved salt tolerance for sustaining food production. materials and methods to assess the effect of saline water irrigation on chickpea, a field experiment was conducted in year 2007-2008. plots of 1x1 m2 area were prepared and separated from each other by polythene sheets of 0.2 mm thickness, up to depth of 30 cm to stop leaching of salts between two adjacent plots. plant to plant distance was 15 cm and plots were separated by a distance of 45 cm. the experiment was laid out in randomized bloke design with six replicates. tubewell water of 1 dsm-1 ec was used to irrigate control sets. saline solutions of 4, 8, 12, and16 ec were prepared by mixing the salts nacl, na2so4, nahco3 and cacl2. three selected varieties pusa-1053 (tolerant kabuli), pusa-939 (moderately sensitive kabuli) and bg-256 (sensitive desi) were grown in the experimental plots. study the effect of __________________________________________________ * corresponding author: varsha.mudgal@gmail.com 1 saline water irrigation on plant growth, nodulation, and legheamoglobin content at different growth stages. saline water of 4, 8, 12 and 16 dsm-1 ec used to irrigate the plants at 20, 50 and 70 days after sowing (das). first sample of plants were collected at 30 das. plant height, root and shoot length were recorded at 30 das. root, shoot and leaves were dried in hot oven at 60°c for 48 hours and then dry weight of each were recorded. same exercise was repeated at 60 and 90 das. relative growth rate (rgr) calculated as: rgr = (dm2 dm1) (t2 t1) -1 (g-g x d-1) where dm1 = initial total (shoot + root) dry mass, dm2 = final total dry mass, and (t2 t1) = time difference between two samplings (30 d). at each stage number of nodules were counted and separated from the root. detached nodules were then dried in hot oven at 60°c for 48 hours and dry weight was recorded. afterwards this dried material was used for estimation of legheamoglobin fig. 1. effect of salinity on plant height (cm) of three chickpea cultivars at 30, 60 and 90 das. table 1. effect of saline water irrigation on relative growth rate (rgr) in three cultivars of cicer arietinum l with the help of dry weight of root (dwr) and dry weight of shoot (dws) after 30 and 60 days after sowing (das). fig. 2. effect of salinity on number of nodules/plant in three chickpea cultivars at 30, 60 and 90 das. fig. 3. effect of salinity on dry weight of nodules/plant in three chickpea cultivars at 30, 60 and 90 das (data are expressed in mg/plant). kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 2 variety ec of water dsm1 dwr (mg) dws (mg) dm1 dm2 dm2-dm1 rgr 30das 60das 30das 60das pusa-1053 0 63.1 (±4.33) 75 (±5.22) 430 (±4.25) 535 (±4.33) 493.1 610 116.9 3507 4 60 (±3.32) 65.6 (±3.43) 300 (±3.27) 350 (±2.45) 360 415 55.6 1168 8 55 (±4.33) 58 (±2.25) 275 (±3.25) 320 (±4.53) 330 378 48 1440 12 40 (±4.21) 50 (±2.45) 210 (±4.33) 240 (±3.54) 250 290 40 1200 16 35 (±3.35) 45 (±3.25) 195 (±4.53) 220 (±3.35) 230 265 45 1050 pusa-939 0 52 (±3.26) 72 (±3.54) 300 (±4.37) 400 (±3.28) 372 472 100 3000 4 50 (±4.25) 60 (±3.53) 260 (±4.33) 310 (±3.27) 316 370 54 1620 8 35 (±3.35) 40 (±3.43) 240 (±3.26) 280 (±4.21) 275 320 45 1350 12 27 (±5.55) 30 (±3.44) 180 (±3.28) 215 (±3.27) 207 245 38 1190 16 27 (±3.27) 35 (±3.28) 130 (±3.26) 160 (±5.55) 157 195 32 960 bg-256 0 50 (±3.33) 68 (±3.13) 72 (±3.54) 320 (±3.38) 308 388 80 2400 4 36 (±3.44) 40 (±3.38) 60 (±3.53) 278 (±3.26) 278 318 40 1200 8 28 (±3.67) 45.6(±4.25) 180 (±2.25) 280 (±3.25) 208 325.6 35 1050 12 20.2 (±3.74) 38.7 (±3.26) 150 (±3.38) 250 (±3.53) 170.2 288.7 30 900 16 18.3 (±3.77) 25.1 (±4.47) 100 (±3.33) 220 (±4.25) 118.3 245.1 20 600 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) content of the nodules. for the sake of convenience and accuracy all the above observations were taken in six replicates and each replica carried ten plants. for the estimation of legheamoglobin nodules were picked up at 30, 60 and 90 das. nodules were carefully picked up from the roots and thoroughly washed with prechilled double distilled water. after washing nodules, blotted on the filter paper, weighed and finally crushed in prechilled sterilized mortar-pestle containing 50 mm hci, 5mm mgci2, 20 mm kci, 5 mmmercapto ethanol. the slurry was centrifuged at 40c at 8,000 xg for 15 minutes. the pellets were discarded and supernatant (sn) was made to a known volume i.e. 4ml/ gm fresh weight of nodules. in this supernatant, legheamoglobin content was estimated by using hemochromogen method with a slight modification. legheamoglobin contents are expressed as mg heamin/ gm fresh weight of nodules. results and discussion salinity retards the plant growth from germination to maturity. this retardation is due to an increase in the osmotic pressure of the root medium and/or by the specific ion effects or the combination of both. the effect of salinity on plant height is shown in (figure 1). effect of salinity on relative growth rate summarized with the help of dry weight of shoot and dry weight of root, there is a significant reduction in the overall growth of plants irrespective of the type of cultivar. this was evident from the decline in relative growth rate with increasing stress (table 1). the smallest reduction was seen in pusa-1053 (kabuli) and the largest in bg-256 (desi). the decline in root dry mass is probably due to the reduction of nutrients transport from soil to the growing shoots. therefore, a decrease in shoot dry matter accompanied by a decline in root dry matter is expected. we compared the response of chickpea varieties to salinity and we found that there was about 50% reduction in shoot growth at 16 ec as compared to control. root and shoot mass reduction in response to salinity has been reported earlier for desi chickpea (elsheikh table 2. effect of salinity on leghaemoglobin content of nodules in cicer arietinum l. at 30, 60 and 90 das (data are expressed in mg/gm fresh wt. of nodules). and wood, 1990). similar reduction in crop growth has earlier been reported in barley, mung bean, chick pea and soybean (garg, 2004; mudgal, 2004; rao et al., 2002; zurayk et al., 1998). in kabuli tolerant variety (pusa-1053) shoot appear to be more sensitive to salinity than roots even at low salinity levels but in sensitive variety pusa-939 and bg-256 roots are more sensitive. cultivar bg-256 was adversely affected at all stages as compared to pusa-1053 and pusa-939. the effect of salinity on the number and dry weight of nodules/plant at three durations (30, 60, 90 das) at 0, 4, 8, 12 and 16 dsm -1 salinity levels are shown in (figure 2) and (figure 3), respectively. salinity delayed the onset of nitrogen fixation and prevented nitrogen fixation from resulting in significant growth but did not eliminate nodulations or the supply of nitrogen from nodule to shoot. subbaroa et al. (1990) also reported that of the 4 strains effective under control only 2 were effective under salinity. in variety pusa-1053 at 60 das had 60 nodule/ plant in control sets and the number decreased to 22 nodules/ plant at 16 dsm-1. the weight of nodules/plant was significantly decreased by salinity 30 days after sowing pusa-1053 shows (68%), pusa-939 (70%) and bg-256 (73%) reduction in weight of nodules/plant at highest salinity level as compared to control. the failure of nodule formation at high salinity might be attributed to shrinkage of root hairs; consequently the reduction in plant growth under conditions of high salinity could only be partly accounted by the reduction or failure in nodulation (tu, 1981; mudgal et al., 2009). zahran (1999) also showed that nacl affected infection and nodulation. dry weight of nodules is affected less adversely than its number. effect of salinity on leghaemoglobin content of nodules in three varieties of chickpea at 30, 60 and 90 das is given in (table 2). it is evident from the data that leghaemoglobin content of the nodules decreased when the levels of salinity increase, however varietal differences were evident. in all varieties maximum legheamoglobin content was observed at 60 dag, which decreased thereafter due to aging and irreversible oxidation of legheamoglobin, also observed by siddiqui et al. (1985) in kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 3 variety ec of water (dsm-1) days after sowing 30 das 60 das 90 das pusa-1053 control 4 8 12 16 57 (±3.27) 47 (±2.25) 38 (±4.25) 20 (±3.26) 11 (±4.25) 93 (±3.53) 76 (±4.25) 64 (±4.21) 58 (±3.13) 47 (±3.74) 58 (±4.25) 45 (±3.44) 43 (±3.35) 35 (±3.43) 24 (±3.13) pusa-939 control 4 8 12 16 54 (±3.25) 43 (±3.54) 34 (±3.45) 16 (±4.54) 8 (±5.13) 91 (±4.21) 74 (±4.67) 60 (±4.56) 55 (±3.53) 45 (±3.52) 72 (±3.53) 40 (±2.53) 35 (±3.54) 27 (±4.21) 20 (±3.58) bg-256 control 4 8 12 16 50 (±4.53) 48 (±3.53) 30 (±4.24) 14 (±3.21) 4 (±4.34) 88 (±2.21) 70 (±2.26) 54 (±2.21) 47 (±3.27) 38 (±4.21) 62 (±2.25) 30 (±2.36) 25 (±4.33) 21 (±3.46) 11 (±2.27) pea nodules. in the early intracellular infection stage characteristic nitrogenase and legheamoglobin are absent or inactive. in the mature stage nitrogen fixation activity and legheamoglobin concentration become high. sheokand et al. (1995) observed that nacl treatment of the plants accelerated nodule greening, accompanied by a concomitant decrease in leghemoglobin (lb) content of the nodules. with 50 mm nacl, increase in duration of the treatment from 7 to 14 days, the lb content recovered by 15% (rising from 41% to 56% of the control). however, under nacl 100 mm treatment lb content declined further dropping from 27% to 18% of the control 14 days after treatment (dat). babber et.al.( 2000) reported that leghemoglobin content of control plants decreased by 50 % at day 85 indicating senescence of nodules. this senescence was further accelerated by salt treatment after which the leghemoglobin content fell to negligible levels. the structural changes associated with salt stress were mainly reduction in size of the nodules, decreased meristematic zone, reduced number and degradation of symbiosomes, reduced intercellalar spaces and deposition of electron dense material in the intercellular spaces in the cortex of nodules.intercellalar spaces and deposition of electron dense material in the intercellular spaces in the cortex of nodules. references babber s, s sheokand, and s malik (2000) nodule structure and functioning in chickpea (cicer arietinum) as affected by salt stress. biologia plantarum. 43(2): 269-273. elsheikh eae, and m wood (1990) effect of salinity on growth, nodulation and nitrogen yield of chickpea (cicer arietinum l.). j exp bot 41: 1263-1269. garg n, r singla (2004) growth, photosynthesis, nodule nitrogen and carbon fixation in the chickpea cultivars under salt stress. braz j plant physiol 16(3): 137–146. mudgal v (2004) physiological studies on growth and nitrogen metabolism in cicer arietinum l. under saline conditions. phd dissertation, rohilkhand university, india. mudgal v, n madaan, a mudgal, and s mishra (2009) changes in growth and metabolic profile of chickpea under salt stress. j appl biosci 23: 1436–1446. rao dln, and pc sharma (1995) alleviation of salinity stress in chickpea by rhizobium inoculation or nitrate supply. biologia plantarum 37(3): 405-410. rao dl, n giller, ke yeo, and tj flowers (2002) the effects of salinity and sodicity upon nodulation and nitrogen fixation in chickpea (cicer arietinum) annals of botany 89: 563-570. sheokand s, s dhandi, and k swaraj (1995) studies on nodule functioning and hydrogen peroxide scavenging enzymes under salt stress in chickpea nodules. plant physiology and biochemistry 33: 561-566. siddiqui s, and s kumar (1985) indian j plant physiol 28(2): 151-156. soussci mq, a ocana, and c lluch (1998) effects of salt stress on growth, photosynthesis and nitrogen fixation in chick-pea (cicer arietinum (l.)). j exp bot 49(325): 1329-1337. subbaroa gv, c johansen, mk jona, r kumar, and jv d.k. (1991) comparative salinity responses among pigeonpea genotypes and their wild relatives. crop sci 31: 415-418. tu jc (1981) effect of salinity on rhizobium-root hair interaction, nodulation and growth of soybean. can j plant sci 61: 231-239. zahran hh (1999) rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. microbiology and molecular biology reviews 63(4): 968-989. zurayk r, m adian, r baalbaki, and mc saxena (1998) interactive effects of salinity and biological nitrogen fixation on chickpea (cicer arietinum (l.)) growth. j agron crop sci 180 (4): 249-258. kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 4 atlas journal of biology 2 (3): 171–174, 2013 doi: 10.5147/ajb.2013.0131 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) first report of olive anthracnose, caused by colletotrichum gloeosporioides, in morocco el hassan achbani1*, abdellatif benbouazza1, and allal douira2 1 centre régional de la recherche agronomique, unité de recherche en protection des plantes, laboratoire de phytobactériologie et de lutte biologique, km 11, route hadj kaddour, 50100, meknès, morocco ; 2 faculté des sciences, université ibn tofaïl, laboratoire botanique, biotechnologie et protection des plantes, kénitra, morocco. received: june 30, 2013 / accepted: november 17, 2013 __________________________________________________ * corresponding author: achbani105@gmail.com 171 abstract ripe and overripe olive fruits (picholine marocaine) showing circular spots of 5 to 20 mm in diameter, slightly depressed and reddish-brown in color, were collected from two orchards located in the regions of ouazzane in morocco. colletotrichum gloeosporioides was isolated from symptomatic fruits and koch’s postulate was fulfilled. according to the literature, this is the first report of colletotrichum gloeosporioides causing anthracnose in olives in morocco. key words: olea europea, picholine marocaine, colletotrichum gloeosporioides, morocco. introduction olive (olea europaea l.) is subjected to be attacked with a variety of fungal pathogens, which affect its health, yield and its oil quality (sanei et al., 2011). anthracnose caused by colletotrichum acutatum j.h. simmonds and colletotrichum gloeosporioides (penz.) penz. & sacc. (syn. gloeosporium olivarum alm.), widespread disease of olives in most olive-growing regions in the world, causing pre-and post-harvest problems (sergeeva et al., 2008). the disease was first reported in portugal. subsequently, it was reported in the mediterranean countries such as italy (ciccarone, 1950; agosteo et al., 2002; moral et al., 2008), spain (martín et al., 2002), and serbia and montenegro (latinovic and vucinic, 2002), and tunisia recently (rhouma et al., 2010). the disease has also been reported in other countries such as japan, uruguay, argentina, brazil, south africa, california, china, india, australia and new zealand (margarita et al., 1986; bompeix et al., 1988; mugnai et al., 1993; latinovic and vucinic, 2002; sergeeva et al., 2008; duarte et al., 2010;). anthracnose symptom was, in most cases found on immature or mature olive fruits (between 50 and 80%)), while in some cases the leaves were also affected in two orchards (10 to 15%) located in the region of ouazzane (north-east of morocco). short communication this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 172 the aim of the present work was to investigate the etiology of the anthracnose observed in december 2012 on fruit olives. materials and methods samples of infected olive fruits were collected from two olive orchards in ouazzane region (north-east of morocco), placed in a cooler and taken to the laboratory. (refrigerated at 4 °c). these olives showed a soft circular rot on their surface consisting of slightly depressed reddish brown spots. such spots expanded to up to 20 mm in diameter and coalesced to form the characteristic circular sunken lesions (fig. 1). at the laboratory, portions (5 mm2) of the infected fruits, were removed at the point of progression of disease symptom; cut into small pieces and then soaked into in 10% sodium hypochlorite (naocl) for 3 min, rinsed three times with sterile water, dropped after on sterile paper towels before plating them onto potato dextrose agar (pda) and incubated at 25°c for 10 days. isolated colonies were, sub-cultured into fresh plates until pure cultures were obtained. pure cultures obtained were identified by visual examinations and viewing under stereo microscope. they were then described and classified based on conidia and colony morphology as described by barnett et hunter barry (1999). pathogenicity tests preparation of spore suspension in order to verify the pathogenicity of the isolated pathogen, ten olives (cv. picholine marocaine) were surface-sterilized as above and the disinfected fruits were, then rinsed in three changes of sterile distilled water and air before inoculation. suspension of conidia was prepared by suspending mycelia scraped from 10 days old cultures of pathogens fungi in pda. the resulting suspension was filtered through 2-layer cheesecloth. the concentration of spore suspension was adjusted to 106 conidia.milliliter-1 using haemacytometer. inoculations the fruits were each, pierced with sterilized needle in one place; each fruit was injected with 25 µl of spore suspension of pathogen, then sealed in moist plastic bags, and incubated for 5 days in a moist chamber. control fruits were, inoculated with sterile distilled water. typical anthracnose symptoms were evaluated after 5 days (100%). re-isolation of isolated fungal pathogens the causative organism in the diseased parts was re-isolated on potato dextrose agar as described in isolation of pathogen. the characters of the re-isolated pathogens were compared with their original isolates. results and discussion obtained isolates on pda from the affected fruit of olive trees, consistently produced one type of colonies. the cultures contained dense, white mycelium with a few orange conidial masses near the inoculum point (fig. 2), simple conidiophores with an hyaline ovoid conidia, conidia size is 9-16.65 µm in lengh × 3,335 µm in width (fig. 3). the waxy acervuli (fig. 4), a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. olive fruits showing brown spot symptoms of anthracnose. figure 2. macroscopic aspect of colletotrichum gloeosporioides after ten days on pda medium. figure 3. colletotrichum gloeosporioides spores colored with cotton blue × 400. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 173 typically with setae, and simple, short, erect conidiophores (fig. 5). according to these characters and to barnett key, this species could be colletotrichum gloeosporioides. the colonization process of olive fruits by colletotrichum such as spores adhesion and germination on cuticle to form an appressoria (gomes et al., 2012). c. gloeosporioides usually attacks ripe or overripe fruits, and only rarely the leaves, peduncles and shoots. on the fruits, the disease causes soft circular rotted spots, on which slimy orange-colored masses of spores are produced under high humidity. the pathogenicity of c. gloeosporioides was confirmed by artificial inoculation using a spore suspension (106 conidia.milliliter-1). symptoms began to appear after 5 days of inoculation (fig. 6). c. gloeosporioides was re-isolated from these lesions fulfilling koch’s postulate. no symptoms were produced in olive fruit sprayed with sterile distilled water. although c. gloeosporioides was reported on many hosts in different countries of the world (farr et rossman, 2011), to our knowledge, this is the first report of c. gloeosporioides affecting particularly the olive fruit in morocco. anthracnose caused by c. gloeosporioides is one of the most important fungal foliar diseases affecting olive trees and the major disease of olive fruits. fruit rot and mummification are the most important disease symptoms which result in high acidity and a reduced organoleptic quality of olive oil. besides c. gloeosporioides, c. acutatum also sporadically causes olive anthracnose. the co-occurrence of these two fungi was reported in spain on a very limited scale (martín and garcía-figueres, 1999). however, talhinhas et al. (2005) stated that these two species were not seen together on the same plant. with molecular and phenotypic assays, these authors reported that the occurrence of c. acutatum in portuguese olive orchards was higher (>97%) than c. gloeosporioides (<3%). colletotrichum acutatum produced orange to pink coloured colonies with whitish aerial mycelium on potato dextrose agar (pda). colletotrichum gloeosporioides produced grey colonies with whitish aerial mycelium on pda (sergeeva et al., 2008). also, conidia of c. gloeosporioides isolates are cylindrical with obtuse ends and measured 13 to 24 µm in length, whereas conidia of the c. acutatum isolates are elliptical-fusiform, tapered and acute at both ends, and measured 13 to 20 µm in length (gunnell et al., 1992). in olive-growing areas where anthracnose was endemic, disease control was primarily based on early harvesting in order to escape secondary infections of the very susceptible mature and overripe drupes. direct control measures involving regular fungicide sprays were used both to prevent defoliation and to avoid yield losses. aerial spraying of bordeaux mixture or copper oxychloride had been successfully attempted; two or three a b figure 4. colletotrichum gloeosporioides acervuli (a) with setae (b) colored with cotton blue × 400. a b figure 5. colletotrichum gloeosporioides conidia (a) with a short erect conidiophore (b) colored with cotton blue × 400. figure 6. typical anthracnose symptoms were observed after 5 days on the olive fruits after their inoculation with c. gloeosporioides (b) and no symptoms in olive fruits sprayed with sterile distilled water (a). preventive treatments from late september to the end of december proved effective against fruit anthracnose (martelli and piglionica, 1961; graniti et al., 1993; pennisi et al., 1993). references agosteo ge, g magnano di san lio, so cacciola, and s frisullo (2002) characterisation of the causal agent of olive anthracnose in southern italy. acta horticulturae 586: 713-716. barnett hl, and hunter barry b (1999) illustrated genera of imperfect fungi (4th edition). the american phytopathological society st. paul, minnesota, 218pp. bompeix g, evr julio, and dh phillips (1988) glomerella cingulata (stoneman) spaulding et von schrenk. in: smith im, j dunez, ra lelliott, dh phillips, archer s.a.(eds) european handbook of plant diseases. blackwell scientific publications, oxford, uk, pp. 325-327 ciccarone a (1950) considerazioni biologiche e sistematiche sull’agente della “lebbra” delle olive, recentemente osservata nel leccese. bollettino della regia stazione di patologia vegetale di roma, s. iii 5: 143-165. de almeida mjv (1899) la gaffa des olives en portugal. bulletin de la société mycologique de france 15: 90-94. duarte hss, pgc cabral, ol pereira, l zambolim, ed gonçalves, j vieira neto, em zambolim, and v sergeeva (2010) first report of anthracnose and fruit mummification of olive fruit (olea europaea) caused by colletotrichum acutatum in brazil. new disease reports 20:37. farr df, ay rossman (2011) fungal databases. systematic mycology and microbiology laboratory, ars, usda. retrieved march 15, 2011 from http://nt.ars-grin.gov/fungaldatabases/. gomes s, bacelar e, martins-lopes p, carvalho t, and guedes-pinto h (2012). infection process of olive fruits by colletotrichum acutatum and the protective role of the cuticle and epidermis. journal of agricultural science, 4 (1): 101-110. gunnell ps, and gubler, wd (1992) taxonomy and morphology of colletotrichum species pathogenic to strawberry. mycologia, 84:157-165. graniti a, s frisullo, am pennisi, and g magnano di san lio (1993) infections of glomerella cingulata on olive in italy. bulletin oepp/ eppo bulletin 23: 457-465. latinovic j, and z vucinic (2002) cultural characteristics, pathogenicity, and host range of colletotrichum gloeosporioides isolated from olive plants in montenegro. acta horticulturae 586: 753-755. margarita l, a porta-puglia, and a quacquarelli (1986) colletotrichum acutatum, nuovo patogeno dell’olivo in cina e confronto con l’agente della “lebbra” dell’olivo. annali dell’istituto sperimentale per la patologia vegetale 11: 125-133. martelli gp, v piglionica (1961) tre anni di lotta contro la lebbra delle olive in puglia. phytopathologia mediterranea 3: 101-112. martín m, f garcía-figueres and a trapero (2002) iniciadores específicos para detectar las especies de colletotrichum causantes de la antracnosis de los olivos. boletin de sanidad vegetal, plagas 28: 43–50. martín mp, and f garcía-figueres (1999) colletotrichum acutatum and c. gloeosporioides cause anthracnose on olives. european journal of plant pathology 105: 733-741. moral j, k bouhmidi, and a trapero (2008) influence of fruit maturity, cultivar susceptibility, and inoculation method on infection of olive fruit by colletotrichum acutatum. plant disease 92: 1421-1426. mugnai l, g surico, and a ragazzi (1993) glomerella cingulata on olive in india: morphological and pathological notes. bulletin oepp/ eppo bulletin 23: 449-455. pennisi am, ge agosteo, and s grasso (1993) chemical control of the olive rot caused by glomerella cingulata. bulletin oepp/eppo bulletin 23: 467-472. rhouma a, ma triki, and m msallem (2010) first report of olive anthracnose caused by colletotrichum gloeosporioides in tunisia. phytopathologia mediterranea 49: 95-98. ritchie bj and pf cannon (2003) colletotrichum in: plant health diagnostics, introduction to fungi. cabi bioscience 2003, cab international, wallingford, uk, 123 pp. sanei sj, se razavi, and k ghanbarnia (2011). fungi on plants and plant products in iran. peik-e-reihan publication, gorgan, 680 pp. in press. sergeeva v, ng nair, and r spooner-hart (2008). evidence of early flower infection in olives (olea europaea) by colletotrichum acutatum and c. gloeosporioides causing anthracnose disease. australasian plant disease notes 3: 81-82. sergeeva v., naira n. g., and spooner-hart r., 2008. first report of colletotrichum acutatum and c. gloeosporioides causing leaf spots of olives (olea europaea) in australia. australasian plant disease notes, 3 : 143–144. talhinhas p, s sreenivasaprasad, j neves-martin, and h oliveira (2005) molecular and phenotypic analyses reveal the association of diverse colletotrichum acutatum groups and a low level of c. gloeosporioides with olive anthracnose. applied and environmental microbiology 71: 2987-2998. 174 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) kbm journal of biology (2010) 1 (2): 30-33 doi: 10.5147/kbmjb.2010.0008 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) archaeal diversity in a municipal wastewater sludge daniel williams1 and james w. brown2* 1 department of biology, north carolina central university, durham, nc 27707, usa; 2* department of microbiology, north carolina state university, raleigh, nc 27695, usa received: september 21, 2010 / accepted: november 11, 2010 abstract the diversity of archaea in a municipal wastewater sludge sample was investigated by amplification of ribosomal rna genes from sludge dna using archaeal-specific primers. surprisingly, a large fraction (32%) of these sequences were from halobacteriales, not previously seen in surveys of wastewater sludge. other abundant sequences were from members of uncultivated ‘environmental’ archaeal groups that are commonly detected in sludge and sediment environments. only a few distant relatives of methanosarcina (which are commonly thought of as the predominant methanogenic species in sludge environments) were detected. key words: archaea, methanogen, halophile, euryarchaea, crenarchaea. introduction anaerobic digestion of wastewater sludge involves the conversion of organic carbon compounds into acetate, formate, hydrogen, and co2 by a consortium of microorganisms (reviewed in ferry, 1993). methanogenic archaea utilize these substrates to produce methane. classically, the methanogens attributed to this process are methanosarcina barkeri and relatives, which can use a relatively wide range of substrates for methanogenesis (reviewed in whitman et al., 1991). surveys of archaeal populations in wastewater sludges have generally found primarily methanomicrobia, including both methanosarcinales and methanomicrobiales, as well as ‘environmental’ euryarchaea and crenarchaea of unknown phenotypes. as part of an early __________________________________________________ * corresponding author: james_brown@ncsu.edu comparative analysis of ribonuclease p (rnase p) rna structure in archaea, we used dna isolated from digested anaerobicsludge from a municipal wastewater cesspool (euphemistically known as ‘lagoon’s) as a source of environmental rnase p rna gene sequences (harris et al., 2001). we were surprised that no sequences related to those of methanosarcina were obtained, knowing that the primers used in the polymerase chain reactions were capable of amplifying the rnase p rna sequence from at least m. barkeri. we therefore investigated the phylogenetic diversity of archaea in this environment by analysis of ribosomal rna sequences amplified from this dna using archaeal-specific polymerase chain reaction (pcr) primers. materials and methods dna from a sample of anaerobically-digested sludge from the cary south municipal treatment plant was isolated previously (harris et al., 2001). small subunit ribosomal rna (ssu-rrna) genes were amplified from this dna using primers 8fapl (ggctgcagtctagatccggttgatcctgccgg) and 1492rpl (ggctcgagcggccgcccgggttaccttgttacgactt) (edwards et al., 1989; stackebrandt & liesack, 1993). pcr reactions were performed in buffer containing 50 mm kcl, 10 mm tris(hydroxymethyl)aminomethane hydrochloride (tris-cl) (ph 8.3), 1.5 mm each dgtp, dctp, datp, and dttp, 0.05% nonident p40, 5% acetamide, and 2mg/ml of each primer. reactions were incubated for an initial 2 min at 94 0c and amplified for 30 cycles at 92 0c for 1.5 min, 55 0c for 1.5 min, 72 0c for 0.5 min followed by a final extension step at 72 0c for 7 min. 30 short communication kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 31 amplification products were digested with noti and psti and separated by agarose gel electrophoresis (3% nusieve gtg; fmc, rockland, me). gel slices containing ca. 1.5 kbp products were excised, melted at 65 0c, liquified with agarace (promega, madison, wi), and used directly in ligation reactions containing noti and psti-digested pbluescript ks+ dna (stratagene, now agilent tech., santa clara, ca). ligation reactions were used to transform competent e. coli strain dh5 . fifty-two clones containing ca. 1.5 kbp insertions were initially assessed from sequence data obtained from either end of each clone. the 35 non-chimeric (identified using check_chimera {maidak et al., 2000}), full-length archaeal ssu-rrna clones fell into 13 groups of 99% sequence identity. the complete sequence of a single representative from each of these 13 groups was determined (accession numbers af424763 af424775). each sequence was used to search the national center for biotechnology information (ncbi) database for the most similar sequence (and in cases where this was an uncultivated ‘environmental’ sequence, the most similar sequence from a named, cultivated specie); these sequences were added to an alignment fig. 1. phylogenetic analysis of wastewater sludge ssu-rrna sequences. this tree was constructed by the neighbor-joining method using phylip (felsenstein, 1989). the alignment contained a representative sampling of sequences from the rdp (cole et al., 2009) and the sequence most similar to each sludge sequence available in the ncbi/genbank database (see text). the escherichia coli and thermotoga maritima sequences served as outgroup. the percentage of internal branches present in trees from 1000 bootstrap sampling of the alignment are shown above each branch; values below 50% and those not present in the consensus of the bootstrapped trees are not shown. the sludge sequences fell into the same phylogenetic groups in both parsimony and maximum-likelihood analyses (data not shown). α ≥ kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 32 of sequences representing the major phylogenetic groups of archaea extracted from the ribosomal database project (cole et al., 2009). phylogenetic analysis of this alignment was performed using phylip (http://evolution.genetics.washington.edu/ phylip.html; felsenstein, 1989) (see fig. 1). results and discussion the archaeal wastewater sludge ssu-rrna sequences fell into six phylogenetic clusters, five of which are euryarchaeal, the remainder (containing only 2 sequences) crenarchaeal (see figure). no bacterial, eukaryotic or organellar sequences were obtained. the largest group of sequences (46-1, 39-2, 44a-1, and 691, 13 sequences representing 38% of the total) was related to a large group of ‘environmental’ euryarchaea known as the ‘arc1’ group, from which no cultivated species have been identified, and therefore no specific phenotypic data are available (chouari et al., 2005). however, arc1-group sequences are commonly found in surveys of wastewater sludge, sediment, and other methanogenic environments, and the phylogenetic placement of this group suggests that they are methanogenic, most likely utilizing hydrogen and one-carbon compounds (riviere et al., 2009). furthermore, members of this group have been shown to grow in the presence of formate or co2 and hydrogen, although the production of methane specifically by these organisms has not been demonstrated (chouari et al., 2005). surprisingly, the next largest cluster of sequences, comprising 32% of the total (eleven 41-1 sequences and a single 38b-1 sequence), was related to alkaliphilic members of the halobacteriales (which, despite the name are archaea rather than bacteria). sequence 41-1 was the most frequent single sequence to occur in the data set. halobacteriales have not previously been reported in wastewater sludge. consistent with the identification of these sequences is the observation that aerobic enrichment cultures in media designed for halobacteria (american type culture collection media 974 and 1590, and luria broth with 4m nacl prepared using mineral water) grow quickly when inoculated with small amounts of this wastewater sludge. the presence of these organisms in wastewater sludge implies the presence of alkaline hypersaline microenvironments. in this anaerobic environment, it is expected that these organisms are growing photoheterotrophically, as do the purple non-sulfur alphaproteobacteria that are well-known inhabitants of wastewater lagoons (see, for example, do et al., 2003). sequence 33-1 (4 sequences, 12% of the total) is related to another environmental euryarchaeal group, exemplified by sequence sbar16, from the santa barbara channel (delong, 1992) and distantly related to the genus thermoplasma. the phenotypes of this group of organisms are unknown, but they presumable are not methanogenic. no sequences closely related to the genus methanosarcina were obtained. however, four sequences, in two groups (with each of the four sequences occurring only once in the data set), were members of the class methanomicrobia. clones 61-2 and 120a-4 were members of the methanosarcinales, but were closely related to the genera methanothrix and methanosaeta rather than to methansarcina. clones 19-1 and 57-1 were members of the methanomicrobiales, and most closely related to environmental clone wchd3-07 and the genera methanocullex and methanospirillum. indeed, the most obvious green autoflourescent (from methanogenic cofactors f420 (dimarco et al., 1990)) organisms seen in these sludge samples, and in mesophilic methanogen enrichment cultures from these samples grown using either co2/h2 or methanol, are morphologically similar to methanospirillum. these sequences presumably represent the methanogens in the wastewater environment utilizing hydrogenindependent substrates, e.g. methanol and acetate. sequences 72-1 and 52-2 (2 sequences; 6% of the total) are related to a little-known group of environmental crenarchaeal sequences exemplified by pjp89 from obsidian pool, yellowstone national park (barnes et al., 1994), and most closely related to the presumably mesophilic pgrfc26, from a freshwater lake sediment (hershberger et al., 1996). the phenotypes of these organisms is not known, but environmental crenarchaea sequences are commonly found in surveys of wastewater environments, soils and sediments, and these organisms have been shown to be physically associated with methanogenic euryarchaea in sludge environments (collins et al., 2005). sequences 72-1 and 52-2 are not related to known ammonia-oxidizing crenarchaea. this analysis represents a qualitative view of archaea a very complex and dynamic environment. nevertheless, the phylogenetic groups seen in this study, and even their relative proportions, are consistent with those seen in a number of molecular phylogenetic analyses of sludge environments (for example, riviere et al., 2009) except for the detection of halobacteria. the relative abundance of the halobacterial sequences obtained could well reflect bias in the pcr reactions, a well-established issue with any pcr-based survey (reviewed in von wintzingerode, et al., 1997), but the presence of halobacteria in the original sludge sample, and others taken at the same site over the course of 3 years, was confirmed by their growth in enrichment cultures. what role they play in this ecosystem, and whether their presence is an idiosyncrasy of this site or has been overlooked in other wastewater sites, are not known. acknowledgments we thank mary ellen woods, beverly vucson, dr. shermalyn greene, and dr. elizabeth haas for assistance with this work. this work was supported by the north carolina waste management program, the north carolina agriculture foundation, and nih grant gm52894 to jwb. references barnes sm, re fundyga, mw jeffries, and nr pace (1994) remarkable archaeal diversity detected in a yellowstone national park hot spring environment. proc. natl. acad. sci. usa 91(5):1609-1613. chouari r, d le paslier, p daegelen, p ginestet, j weissenbach, and a. sghir (2005) novel predominant archaeal and bacterial groups revealed by molecular analysis of an anaerobic sludge digester. environ. microbiol. 7(8):1104-1115. kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 33 cole jr, q wang, e cardenas, j fish, b chai, rj farris, as kulam-syedmohideen, dm mcgarrell, t marsh, gm garrity, and jm tiedje (2009) the ribosomal database project: improved alignments and new tools for rrna analysis. nucleic acids res. 37(database issue): d141-d145. collins g, l o’connor, t mahony, a gieseke, d de beer, and v o’flaherty (2005) distribution, localization, and phylogeny of abundant populations of crenarchaeota in anaerobic granular sludge. applied environ. microbiol. 71(11):7523-7527. delong ef (1992) archaea in coastal marine environments. proc. natl. acad. sci. usa 89(12):5685-5689. dimarco aa, ta bobik, and rs wolfe (1990) unusual coenzymes of methanogens. ann. rev. biochemistry 59:355-394. do ys, tm schmidt, ja zahn, es boyd, a mora, and aa dispirito (2003) role of rhodobacter sp. strain ps9, a purple non-sulfur photosynthetic bacterium isolated from an anaerobic swine waste lagoon, in odor remediation. appl. environ. microbiol. 69(3):1710-1720. edwards u, t rogall, h blöcker, m emde, and ec böttger (1989) isolation and direct complete nucleotide determination of entire genes: characterization of a gene coding for 16s ribosomal rna. nucleic acids res. 17(19):7843–7853. felsenstein j (1989) phylip phylogeny inference package (version 3.2). cladistics 5:164-166. ferry jg (1993) methanogenesis. chapman & hall. harris jk, es haas, d williams, dn frank, and jw brown (2001) new insight into rnase p rna structure from comparative analysis of the archaeal rna. rna 7(2):220-232. hershberger kl, sm barnes, al reysenbach, sc dawson, and nr pace (1996) wide diversity of crenarchaea. nature 384(6608):420 maidak bl, jr cole, ct parker jr, gm garrity, n larsen, b li, tg lilburn, mj mccaughey, gj olsen, r overbeek, s pramanik, tm schmidt, jm tiedje, and cr woese (2000) the rdp (ribosomal database project) continues. nucleic acids res. 28(1):173-174. riviere d, v desvignes, e pelletier, s chaussonnerie, s guermazi, j weissenbach, t li, p camacho, and a sghir (2009) toward the definition of a core of microorganisms involved in anaerobic digestion of sludge. isme j. 3:700-714. stackebrandt e, and w liesack (1993) nucleic acids and classification. in: goodfellow m, ag o’donnell (ed) handbook of new bacterial systematics. academic press. pp. 152-189. von wintzingerode f, ub göbel, and e stackebrandt (1997) determination of microbial diversity in environmental samples: pitfalls of pcr-based rrna analysis. fems microbiol. rev. 21(3):213-229. whitman wb, tl bowen, and dr boone (1991) the methanogenic bacteria. in: a. barlows, hg truper, m dworkin, w harder, kh schleifer (ed) the prokaryotes. springer-verlag. pp 717-768. atlas journal of biology 2 (2): 130–135, 2013 doi: 10.5147/ajb.2013.0085 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) phytophthora palmivora: a new pathogen of olive trees in morocco mohamed chliyeh1, amina ouazzani touhami1, abdelkarim filali-maltouf2, cherkaoui el modafar3, abdelmajid moukhli4, ahmed oukabli5, rachid benkirane1, and allal douira1* 1 laboratoire de botanique et de protection des plantes, ufr de mycologie, département de biologie, faculté des sciences bp. 133, université ibn tofail, kénitra, maroc; 2 laboratoire de microbiologie et biologie moléculaire, faculté des sciences, université mohammed v agdal, av ibn batouta, bp 1014 rabat, maroc; 3 laboratoire de biotechnologie, valorisation et protection des agroressources, faculté des sciences et techniques guéliz, b.p. 618, 40 000 marrakech, maroc; 4 ur, amélioration génétique des plantes, institut national de la recherche agronomique f40 000 marrakech, maroc; 5 institut national de la recherche agronomique, amélioration des plantes et conservation des ressources phytogénétiques crra, bp 578, meknès, maroc. received: january 17, 2013 / accepted: march 11, 2013 __________________________________________________ * corresponding author: douiraallal@hotmail.com 130 abstract in spring of 2012, olive-trees with crown dieback, root rot and defoliation were observed in two years old olive tree in commercial plantations of tree nurseries in sidi taibi and in twenty to fifty years old field trees in souk el arbaa olive crops in northwest of morocco (gharb area). the objective of this study was to isolate the responsible pathogen of the observed symptoms to the olive trees, to demonstrate its pathogenicity and fulfill the koch´s postulate. phytophthora palmivora was consistently isolated from roots (56%) and stems (73.6%) of the young olive trees and 85% from stems of field trees. koch’s postulate was completed using two isolates of phytophthora palmivora on 2-year old plants of dahbia and haouzia varieties grafted onto wild olive-trees. the affected branches percentages (pab%) of the inoculated olive plants with the isolate 1 were higher (81.8% for dahbia and 68% for haouzia) than those what were inoculated with the isolate 2 (43% for dahbia and 32% for haouzia). the reisolation percentages (pr%) of isolate 1 (84%) and isolate 2 (76%) in the roots of dahbia variety were higher than isolate 1 (48%) and isolate 2 (55%) in roots of haouzia variety. the reisolation percentage of isolate 1 in the stem of dahbia (64%) was higher than that in the stem of haouzia (41.33%). no significant difference was observed between the reisolation percentages of isolate 2 in stem of dahbia olive plants (38%) and in stem of haouzia olive plants (33%). introduction olive-tree (olea europaea l.) is one of the most ancient domesticated fruit trees and the most extensively cultivated fruit crop in the world (fabbri, 2009). in morocco, the olive plantation is estimated at about 650 000 ha accounting for 50% of the national surface dedicated to arboreal crops (ministry of agricultural and marine fischeries, 2009). this cultivar is susceptible to major fungal diseases particularly to leaf-spot disease caused by spilocaea oleagina, the most widespread fungal disease of olive tree in the world (anton & laborda, 1989), verticillium dahliae responsible to defoliation and wilting of olive trees and death of young trees (vossen et al., 2008), and fusarium solani that provokes the root rots to the olive trees (perez et al., 2011). in 2012, a survey of nurseries in sidi taibi and in olive trees field in souk el arbaa, allowed us the pathogenicity of p. palmivora was demonstrated in the olive plants and this was the first report of this pathogen in moroccan olive trees. key words: olive tree crown dieback, root rot, defoliation, phytophthora palmivora, koch’s postulate, pathogenicity. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 131 to remark the presence of leaf chlorosis, defoliation, wilting and twig dieback in some olive plants (figure 1). the objective of this study was to isolate the responsible pathogen of the observed symptoms to the olive trees, to demonstrate its pathogenicity and to fulfill the koch´s postulate. material and methods pathogen isolation one hundred stem segments and one hundred root segments of olive trees were taken from commercial plantations of tree nurseries in sidi taibi and one hundred stem segments from the olive crops in souk el arbaa, washed with water, disinfected with alcohol for five minutes, put on sterile distilled water and then dried with sterile filter paper. then they were put on psa agar plates (potato sucrose agar: 200 g potato, 15 g sucrose, 20 g agar-agar, and 1,000 ml distilled water) and incubated on darkness at 28oc. the developing colonies were then observed for species determination. isolation percentage (pi %) was obtained by applying the following formula: pi = nsx/ nt × 100 nsx: number of segments containing the fungal species x. nt: total number of used segments. nine mm diameter agar plugs from these plates were placed in a 5 cm diameter petri dishes and soil extract was added to just cover the plugs. soil extract was prepared by mixing 10 g of soil and 1 litre of distilled water. after 24 h at 20oc, the mixture was filtered and petri dishes were incubated in darkness at 15oc. after each 24 h incubation, the soil extract was removed and replaced with fresh extract previously stored at 4 oc. sporangial production and development, occurring between 12 and 120 h, was assessed by direct observation using an optical microscope. mature sporangia were removed and placed on glass microscope slides and stained with blue cotton. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) inoculum production zoospores of p. palmivora were produced by growing cultures on oatmeal agar at 28oc in the dark for 14-21 days. the mycelium was transferred to a sterile petri dish, covered with sterile distilled water (sdw) and incubated overnight at 28°c, under lights. the mycelia plates were chilled for 5 min at -20oc to induce zoospores release. the concentration of the inoculum was adjusted at 105 zoospores/ml by sdw. inoculation test the koch’s postulate was verified by inoculating twelve olive plants of haouzia and dahbia varieties (six plants per variety) with two isolates of phytophthora palmivora (isolate 1: isolated from the nursery olive trees and isolate 2 : isolated from the souk el arbaa olive trees field). three plants from each variety were none inoculated and used as a control. plants were inoculated according to the method described by olbricht et al. (2006). the roots of investigated plants were washed under running water to discard soil remnants, trimmed to 2/3 of their length, and subsequently dipped in the prepared inoculum during 6 hours. the olive trees plants were potted into universal soil substrate, watered with the remaining fungal suspension (about 20 ml per plant), and cultivated in a greenhouse. for control plants, sterile water was used instead of fungal suspension. pathogen reisolation at the end of each test, twenty five roots and stems segments were taken from inoculated plants and controls, washed with water, disinfected with alcohol for five minutes, put on sdw and then dried with sterile filter paper. after, they were plated on psa and incubated on obscurity at 28oc the symptoms were evaluated after five weeks of inoculation, the number of new formed branches (nnb) and the number of affected branches (nab) were estimated on the inoculated olive plants and the percentages of the affected branches (pab%) were calculated using the formula : a b figure 1. leaf chlorosis, defoliation, wilting and twig in some olive trees collected from sidi taibi nurseries (a) and souk el arbaa fields in the gharb area (b). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 132 pab(%) = nab/ nnb × 100 reisolation percentage (pr %) was obtained by applying the following formula: pr = nspp/ nt × 100 nspp: number of segments containing phytophthora palmivora. nt: total number of segments used in the reisolation. analysis of the variance and of the mean comparisons using the lsd test (p = 5%) were performed using the software statistica program. statistical analyses focused on the results of five plants, for the affected branches and five repetitions for the reisolation percentage of the inoculated and non inoculated plants. results and discussion fungi morphological characterization showed that all isolates produced papillate sporangia on the soil extract medium (figure 2a), which were ellipsoid to ovoid with a length of 18.31 µm and a larger of 13 µm. some isolates produced subglobose, non-papillate sporangia (figure 2b) and abundant chlamydospores (figure 2c), sexual forms were absents (heterothallic species). the pathogen causing disease on the olive trees was identified as phytophthora palmivora on the basis of morphological and cultural characters especially on sexual and asexual reproduction forms (ho et al., 1992. cacciola et al., 2000. lucero et al., 2006. gallegly and hong, 2008). on the psa plates, some colonies appeared four days later on the segments of stems and roots of the diseased olive trees (figure 3). the microscopic observations leaded us to identify simultaneously the isolated species as phytophthora palmivora (pi=73.6%) and alternaria alternata (pi=26.4%) in the stem fragments of the young olive trees from the nurseries. in the field olive-trees, the isolated species from the stem fragments were p. palmivora (85%) and a. alternata (15%). the root segments of the young olive trees showed the presence of p. palmivora and fusarium solani with 56 and 44% frequencies respectively. after five weeks of inoculation, all the plants showed defoliation (figure 4-a2, a3, b2 and b3), wilting, degradation and reduction of root system (figure 4c2, c3, d2 and d4) necrosis and dropping of the new branches and vascular browning primarily of the xylem tissue (figure 4). table 1 showed that dahbia olive trees inoculated with isolate 1 formed on average of 12.8 branches (figure 5a) significantly fewer at p = 5% than those inoculated with isolate 2 (21.6). haouzia olive plants inoculated with isolate 1 and isolate 2 formed the same number of new branches (17). the obtained results were statistically significant at the 5% level for the isolate 2 of phytophthora palmivora, this isolate affected more new branches in haouzia (5.6) and dahbia (9) varieties than isolate 1 in the same varieties (11.8 and 10.8, respectively) (figure 5b). the affected branches percentages of the inoculated olive plants with the isolate 1 were significantly higher (81.8% for dahbia and 68 % for haouzia) than those of plants varieties inoculated with isolate 2 (43% for dahbia and 32% for haouzia) (table 1). a b c d figure 2. morphological characterization of phytophthora palmivora. papillate sporangia (a), non semi-papillate sporangia (b), hyphal swellings (c), terminal chlamydospore (d) on cotton blue (×400). 133 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a b c d figure 3. phytophthora palmivora (a , c), alternaria alternata (b) isolated from the olives stem segments on psa agar and phytophthora palmivora in the age of 12 days on the psa agar plate (d). figure 4. symptoms on different organs of olive trees after inoculation with two isolates of phytophthora palmivora: olive trees of dahbia variety(a), non inoculated plant (a1), inoculated plant with isolate 1 (a2), inoculated plant with isolate 2 (a3), olive trees of haouzia variety (b), non inoculated plant (b1), inoculated plant with isolate 1 (b2), inoculated plant with isolate 2 (b3), olive trees root system of dahbia variety (c), non inoculated plant (c1), inoculated plant with isolate 1 (c2), inoculated plant with isolate 2 (c3), olive trees root system of haouzia variety (d), non inoculated plant (d1), inoculated plant with isolate 1 (d2), inoculated plant with isolate 2 (d3), roots degradation due to the inoculation with the isolates of phytophthora palmivora (ellipse). table 1. percentage of the affected branches relative to the new formed branches on the inoculated olive trees of dahbia and haouzia varieties with phytophthora palmivora after five weeks of inoculation. a b c d a1 a2 a3 c1 c2 c3 d1 d2 d3 b1 b2 b3 ellipse ellipse variety phytophthora palmivora new formed branches affected branches percentage of the affected branches (%) dahbia isolate 1 12.8 c 10.8 ab 81.8 isolate 2 21.6 9 b 43 c haouzia isolate 1 17 c 11.8 68 b isolate 2 17 c 5.6 c 32 d the results of the same column followed by different letters differ significantly at 5%. 134 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) the two isolates of p. palmivora were reisolated from the root and stem with a. alternata (figure 6). as shown in table 2, the reisolation percentage of isolate 1 (84%) was significantly higher at 5% than that of isolate 2 (76%) in the root of dahbia trees. the reisolation percentage of isolate 1 (48%) was lower than that of isolate 2 (55%) in the root of haouzia trees. in the stems of dahbia, isolate 1 was higherly reisolated (64%) than isolate 2 (38%). the reisolation percentage of isolate 1 (41.33%) was higher than isolate 2 (33%) in the stem of haouzia. the koch’s postulate was verified by inoculating olive trees of haouzia and dahbia varieties with two isolates of phytophthora palmivora isolated from the nursery olive tree and the olive trees field. sanchez hernandez et al. (1998) described the effect of phytophthora palmivora in southern spain on young olive trees as wilt or dieback and death, and they announced that its pathogenicity depends on soil water content. cacciola et al. (2000) showed decline symptoms in new plantations of 1 to 2 years old plants in catanzaro province (calabria) and considered phytophthora palmivora as a pathogen of olive tree in italy that provoked leaf chlorosis, defoliation, wilting, twig dieback and eventual plant collapse associating the symptoms with the root rot. olive trees infected by p. palmivora in argentina were indicated by lucero et al (2006). similarly to this current report, all table 2. reisolation percentage of phytophthora palmivora from roots and stems of the inoculated olive trees of haouzia and dahbia varieties after five weeks of inoculation. figure 5. inoculated olive plant with phytophthora palmivora. affected branch (a), new formed branch (b). a b figure 6. reisolation of phytophthora palmivora from the inoculated olive tree on psa agar. phytophthora palmivora reisolated from root segments (a), the control root segments (b), phytophthora palmivora reisolated from stem segments (c), the control stem segments (d). a b c d the results of the same column followed by different letters differ significantly at 5%. variety phytophthora palmivora reisolation percentage (%) root stem dahbia isolate 1 84 a 64 a isolate 2 76 b 38 bc haouzia isolate 1 48 c 41,33 b isolate 2 55 d 33 c isolates produced papillate sporangia . olive plants could have been contaminated by p. palmivora from other host species as suggested by these authors. p. palmivora infects more than 200 species of ornamental, shade and hedge plants, mostly from tropical areas. in argentina, for instance, p. palmivora was first recorded in citrus spp in 1937. the pathogen may have been introduced through rooted olive plants of mediterranean varieties currently used in argentina (lucero et al., 2006). as regards the ability of phytophthora palmivora to reduce and to degrade the olive tree roots, we could find the same symptoms that lucero announced in 2006. lo giudice et al. (2010) outlined that leaf chlorosis, defoliation, rot of fine roots, twig dieback and wilt were observed in 4-year-old olive trees cv. tonda iblea in drip-irrigated orchard in eastern sicily. trees declined slowly or collapsed suddenly with withered leaves still attached. incidence of affected trees was 10 %. a fungus identified as v. dahliae kleb. was isolated from the xylem of main roots and basal stem. an oomycete was isolated and identified from roots and basal trunk bark. both pathogens were recovered from symptomatic trees with means frequencies of positive isolations per tree of 80 and 30% for v. dahliae and p. palmivora, respectively. the pathogenicity of p. palmivora was demonstrated in the olive plants. this was the first report of this pathogen in moroccan olive trees. in the long-term, it may constitute a real danger to this culture in the nurseries as in the fields. so, due to the severe symptoms and the increasing incidence recorded; p. palmivora should be considered a potential threat to olive cultivation in morocco. acknowledgments this study was conducted under the project ‘rhizolive: selection and use of soil rhizospheric microorganisms to optimize the arbuscular mycorrhization of olive tree in morocco’s soils’ funded by the hassan ii academy of sciences and technology. we thank the academy for its financial support. references anton fa and e laborda (1989) estudio de la susceptibilidad/resistancia de variedades del olivo (olea europaea l.) al patogeno cycloconium oleaginum (cast.) (spilocaea oleaginae hugh.). bol. san. veg. plagas, 4: 385–403. cacciola so, agosteo ge, and a pane (2000) first report of phytophthora palmivora as a pathogen of olive in italy. plant disease 84 (10): 1153. fabbri a, lambaradi m, and yo tokatli (2009) olive breeding in breeding plantation tree crops : tropical species. spinger new york pp. 423-465. ho hh (1992) keys to the species of phytophthora in taiwan. plant pathol. bull. 1: 104-109. lo giudice v, raudino f, magnano di san lio r, cacciola so, faedda r, and a pane (2010) first report of a decline and wilt of young olive trees caused by simultaneous infections of verticillium dahliae and phytophthora palmivora in sicily. plant disease 94 (11): 1372. lucero g, vettraino am, pizzuolo p, di stefano c, and a vannini (2006) first report of phytophthora palmivora on olive trees in argentina. new disease reports 14: 32. ministry of agriculture and marine fischeries (2009) plan vert du maroc: de la stratégie à l’action du maroc. gallegly me and cx hong (2008) phytophthora: identifying species by morphology and dna fingerprints. american phytopathology society press 158pp. olbricht k, ulrich d, and b dathe (2006) cross-breeding with accessions of fragaria chiloensis resulting in selections with outstanding disease resistance and fruit quality characteristics. acta hortic. 708: 507–509. perez ab, farinon om, and mf berretta (2011) first report of fusarium solani causing root rot of olive in southeastern argentina. plant disease 95 (11): 1476. sanchez hernandez me, ruiz davila a, perez de algaba a, blanco lopez ma, and a trapero casas (1998) occurrence and etiology of death of young olive trees in southern spain. european journal of plant pathology 104 (4): 347-357. vossen p, gubler d, and ma blanco (2008) verticillium wilt of olive. newsletter of olive oil production and evaluation. univ. of california cooperative extension. vol. 3 (4): 1-4. 135 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) status of bay of bengal: an ecological point of view pai ik department of zoology, goa university, goa-403206, india kbm journal of biology (2010) 1 (1): 5-9 doi: 10.5147/kbmjb.2010.0002 abstract the marine resources are a major source of food, a reservoir of minerals, major suppliers of oxygen, regulator of climate. it is also an ultimate dumping ground for the mounting burden of human waste material. due to unchecked anthropogenic activity in recent years, the physicochemical and biological status of the marine environments have changed significantly. this has resulted in the changing environmental scenario of the world. the bay of bengal, is one of the largest bay in the world known to receive large flow of sediments from several rivers and other water bodies from india, bhutan, bangladesh, myanmar, indonesia etc., many of these rivers bring along with them, a large quantity of effluents from cities / towns located on either side of these rivers, thus making the bay nutrient rich. this bay also plays a major role in determining the climatic conditions of india and other south east asian countries. thus its ecology is of paramount interest. further, the bay is also known for its oligotrophic nature as well low productivity, thus resulting in high diversity of flora and fauna. the ecological status of the bay has a direct bearing on the lifescape of the bay, and therefore, a study was undertaken to understand abiotic and biotic factors with special reference to zooplankton. based on the observations, the study recommends certain appropriate measures to be taken to conserve the ecology of one of the largest bay in the world. key words: ecology, bay of bengal, physico-chemical parameters, zooplankton introduction approximately 71% of the surface of the planet earth is covered with marine waters, with average depth 3.8 km, volume about 1370 x 106 km3 (prasad, 2000). only recently, it has been recognized as a promising and a major source of food, a reservoir of minerals, a major supplier of oxygen, and a regulator of climate. it is also used as ultimate dumping ground for the mounting burden of human waste material. it is known that, 32 out of 33 animal phyla exist in sea. it is also said, 173 animal classes live in sea, 35 in freshwater and 33 on land (nicoll, 1971). the possible reasons for the same are listed by may (1994). grassle et al. (1991) have reported that, 13 out of 28 phyla found in marine environment are endemic to marine environment and only one of 11 phyla is endemic to terrestrial ecosystem. this makes marine ecosystem as single largest repository of living organisms. it is also the medium, in which various chemical reactions take place, both inside and outside living organisms (nybakken, 1997). physico-chemical parameters the marine water consist of an average of 35g/1000 ml of dissolved compounds collectively called as salts or practical salinity units (psu) which include ci(55.04%), na+ (30.61%), so4 2 (7.68%), mg2+ (3.69%), ca2+ (1.16%), k+ (1.10%) as major constituents and hco3 -(0.41%), br (0.19%), h3b03(0.07%) and st2+(0.04%) apart from 0.01% of dissolved substances of several inorganic salts needed for living of the organisms in sea. martin (1994), has reported that, some of the organisms like diatoms and radiolarians show their existence in a place where there is availability of silicon dioxide, which is required for construction of their skeleton. thus, acts as bio-indicator. while, the other elements such as fe, mn, ca, cu, though may exist in trace amounts, but can very well act as limiting factor for sustenance of life (martin, 1994). among gases, 02 and c02 dissolved in sea water, has metabolic importance. their solubility depends on temperature of the water. arctic sea, temperate northern pacific, which show summer bloom and temperate north atlantic sea, which exhibit spring kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) received december 17, 2009 / accepted march 2, 2010 __________________________________________________ * corresponding author: ikpai@unigoa.ac.in 5 bloom (parsons et ai., 1984); but, tropical seas show thermal stratification. thus, their productivity and biomass in general and zooplankton in particular is some what constant. further, there is a new widespread recognition that, chemical monitoring is not enough and that, pollution is essentially biological phenomenon (wright et al., 1994) and the need for biological methods has been accepted (newman et al., 1992; rosenberg and resh, 1993). at community level too, use of biological approach is already well established and accepted (cairns and pratt, 1993). further, the advantage of using these bio-indicators has been listed and discussed by rosenberg and resh (1993). mcaliister et al. (1994), while analyzing global distribution of coral reef fishes, have reported that in indian subcontinent, laccadive maldives-chagos and sri lankan regions have high animal diversity but, sampling is weak in western sumatra i.e., in bay of bengal in general. keeping in view of the above, to fill the lacunae in our knowledge on the ecology of bay of bengal, an attempt has been made to evaluate the same. materials and methods department of ocean development (dod) (government of india) and national institute of oceanography regularly organize cruises, to various part of the country. cruise no. sk-118 on ocean research vessel, sagar kanya, had a pre-determined 13 stations, at bay of bengal. at each station, conductivity, temperature-depth (ctd) profile system, with rosette samplers was lowered to 30m depth for recording the relevant data. water samples were collected from that depth, by triggering the sample bottles from control panel on deck. bathythermograph and thermo-salinograph were also run, at all the stations to record the parameters. the water thus collected, was analyzed for various physico-chemical parameters, such as, temperature, ph, oxygen contents, salinity, chlorides, sodium, sulphates, magnesium, calcium, potassium, bicarbonates, bromide, boric acid, strontium etc., by following standard analyses methods (apha, 1992). results obtained for three samples each, at every station were pooled. simultaneously, at every station, sampling for zooplankton was also done by both vertical (30m to surface) and horizontal hauls (on water surface) by using bongo net (dia. 0.6m, length 2.5m, mesh width 300um). a pre-calibrated flow meter (t. s. flow meter no. 4512), was also attached to the net mouth, to calculate the actual quantity of water filtered during the operation. thus collected samples were brought to the deck and later isolated and separated in the laboratory on board of the vessel. later, the samples were preserved in 4% formalin and were brought to land laboratory for taxonomic identification and classification by following available literature (kasturirangan, 1963; mon, 1964; daniel, 1985; zheng, 1989; santanam and srinivasan, 1994). results the samples from all the 13 stations (tables1 and 3) were collected by the following regular procedures: table-2a, 2b, 3a and 3b provide information on physico-chemical parameters of bay of bengal. the data obtained, does not differ much with results obtained elsewhere, by working at other marine environment (nybakken, 1997). table 4 exhibits the presence of 433 number species collected in the surveyed area. the various data obtained on abiotic and biotic factors (tables 2a, 2b, 3a, 3b, and 4) were compared with that of available earlier reports (anonymous; 1981; madhupratap, 1981; vijayalaxmi, 1981) and the possibility of making use of modern tools such as 6 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g)statio n no. day/ night latitude longitude biomass ml/100m3 organisms/ 100m3 01 d 20024’00”n 88039’00”e 0.9 1625 02 n 18000’00”n 90000’00”e 1.6 7468 03 d 17000’00”n 90000’00”e 0.5 4053 04 n 16000’00”n 90000’00”e 1.4 4676 05 d 15000’00”n 90000’00”e 1.5 5016 06 d 14000’00”n 90000’00”e 1.0 2445 07 n 13000’00”n 90000’00”e 1.5 6993 08 d 12000’00”n 90000’00”e 0.7 2638 09 n 11000’00”n 90000’00”e 1.1 8617 10 d 10000’00”n 90000’00”e 0.8 3880 11 d 09000’ 00”n 90000’00”e 0.6 2744 12 d 08000’00”n 90000’00”e 0.5 3589 13 d 07000’00”n 90000’00”e 1.1 8165 table 1. sampling sites at bay of bengal (vertical sampling) (30m-0m) stn no. ph temp (oc) salinit y %o alkalinity (ppt) hardness (ppt) chloride (ppt) calcium (ppt) magnesium (ppt) 1 7.0 18.35 34.86 5 8 29 2 6 2 7.0 18.60 34.86 4 5 18 3 2 3 7.0 19.37 34.80 3 11 18 3 11 4 7.0 17.58 34.90 3 8 18 4 4 5 7.0 16.38 34.90 3 7 25 4 3 6 7.0 16.52 34.86 3 8 25 5 3 7 7.0 19.33 34.83 4 8 17 4 4 8 7.0 18.19 34,85 3 6 18 4 2 9 7.0 18.44 34.94 3 6 21 3 3 10 7.0 17.38 34.96 4 6 21 3 3 11 7.0 17.17 34.07 2 5 11 2 3 12 7.0 18.64 35.96 5 5 22 2 3 13 7.0 19.00 34.93 4 5 27 2 3 table 2a. physico-chemical parameters at bay of bengal (vertical sampling) (30m-0m) anpp (anal net primary productivity), avris (air borne visible infra-red imaging spectrometer), bioclim (biological climate analyses and prediction system), erin (environmental resources information net work, gems (global environmental monitoring system), grid (global resource information database), hrv / mla (high resolution visible multispectral linear array), mscp (multiple species conservation plan), mss (multiple spectral scanner), rap (rapid assessment procedures) etc., to have a constant monitoring of the ecology of this bay. 7 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) table 2b. physico-chemical parameters at bay of bengal (vertical sampling) (30m-0m) nt: not traceable stn. no. sulphate (ppt) d.o. (mg/l) phosphate (umol/l) nitrate (umol/l) silicate (umol/l) 1 18 1.60 1.90 0.45 26.42 2 18 1.34 1.80 0.14 3012 3 17 1.57 2.10 0.16 3042 4 17 1.20 1.67 nt 23.26 5 16 1.32 1.54 nt 23.24 6 17 2.30 1.69 0.19 30.19 7 17 1.54 1.72 1.07 30.16 8 17 1.69 1.82 0.04 29.24 9 16 1.72 1.90 0.04 25.22 10 16 1.82 2.14 nt 30.18 11 18 1.90 1.10 0.11 30.24 12 18 2.14 1.15 nt 27.03 13 18 1.10 1.75 0.07 28.67 stat. no. day/ night latitude longitude biomass ml/100m3 organisms/ 100m3 01 d 20024’00”n 88039’00”e 0.9 590 02 n 18000’00”n 90000’00”e 0.4 1622 03 d 17000’00”n 90000’00”e 0.5 288 04 n 16000’00”n 90000’00”e 1.5 2871 05 d 15000’00”n 90000’00”e 0.5 641 06 d 14000’00”n 90000’00”e 1.3 1374 07 n 13000’00”n 90000’00”e 2.0 5083 08 d 12000’00”n 90000’00”e 0.3 1256 09 n 11000’00”n 90000’00”e 0.9 4144 10 d 10000’00”n 90000’00”e 1.3 4033 11 d 09000’00”n 90000’00”e 2.0 7182 12 d 08000’00”n 90000’00”e 0.5 1581 13 d 07000’00”n 90000’00”e 0.5 2255 table 3a. sampling sites at bay of bengal (horizontal sampling) discussion it is well known that, quality of an ecosystem can be assessed by analyzing its abiotic and biotic components. in a marine environment, it is not only oxygen, salinity and chlorides are important yard sticks as major components, but even minor components such as calcium, strontium, potassium, bicarbonate, bromide also would hold a key to the success of flora and fauna of the area as limiting factors (nybakken, 1997). in the present studies, the results (table.2a, 2b, 3a and 3b) indicates, physicochemical parameters analyzed are all on par with other ideal, unpolluted marine ecosystems. the values are in agreement with earlier findings. this indicates that, the bay of bengal has neither significantly changed nor polluted bio-indicators, at lower levels of organization, correlates more directly with environmental levels of known stress than, those at the higher level. many organisms have been used as bio-indicators. metallothioneins (langston and zhou, 1986) provides cellular indices (moore et al., 1982; moore, 1991) or at the individual level (widdows et al., 1980). using of gastropods, barnacles in general and mytilus edulis in particular, as bio-indicator, is in vogue, since 1939 (more and kitching, 1939; southward and crisp 1954; 1956), dogwelks nucella lapillus and nassarius obselata are helpful in analyzing tributyline induced pollution (hawkins et al., 1994), mussel egg has also been identified as an indicator of mutagen (dixon and pascoe, 1994). patella vulgata, patella dispersa, monodonta lineata, littorina spp. etc., are some of the well known bio-indicators of oil spill and red tides (southward and southward, 1978). southward (1984) indicated, the role of sagitta setosa and sagitta. elegans as bio-indicators for phosphates. as can be seen from table 4, a rich fauna of zooplankton in general and bioindicator species such as chaetognaths and other molluscs are present in abundance in bay of bangal. it can be judged that, biotic factors also functioning perfectly well in this bay. it is quite understandable that, when zooplankton are present in abundance, there must be sufficient phytoplankton to feed on. fuether, there must also be sufficient secondary consumers, like fish and other higher organisms, which feeds on these zooplankton. thus, completing a marine food chain systematically. this shows that, at bay of bengal, the ecosystem is a mature, complete, self regulating and self sustaining one. although the fact remains that, a large number of rivers from table 3b. pysico-chemical parameters of bay of bengal (horizontal sampling) stn no. ph temp (oc) salinity %o alkalinity (ppt) hardness (ppt) chloride (ppt) calcium (ppt) magnesium (ppt) 1 7.5 25.7 30.50 7 13 26 3 1 2 7.0 26.4 31.89 6 7 25 1 6 3 7.0 27.1 32.19 5 7 21 4 3 4 6.5 27.0 32.57 4 7 20 3 4 5 7.0 27.2 32.88 2 8 31 5 3 6 7.5 27.4 33.23 6 9 36 3 6 7 7.0 27.9 33.65 4 9 22 7 2 8 7.0 27.7 33,77 7 9 22 6 3 9 7.0 27.9 33.71 8 12 35 7 5 10 7.0 27.9 34.35 6 10 27 5 5 11 7.0 27.9 34.40 4 4 17 2 2 12 7.5 27.9 34.45 4 12 35 6 6 13 7.0 27.9 34.29 3 9 35 9 6 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 8 stn. no. sulphate (ppt) d.o. (mg/l) phosphate (umol/l) nitrate (umol/l) silicate (umol/l) 1 16 4.50 0.07 0.072 0.84 2 17 4.70 0.07 0.021 0.09 3 16 4.20 0.08 0.071 0.74 4 16 5.60 0.06 nt 2.66 5 17 5.20 0.05 nt 1.87 6 18 5.70 0.06 nt 1.92 7 18 4.20 0.06 0.071 1.42 8 18 4.90 0.08 nt 2.32 9 18 5.00 0.07 nt 3.24 10 17 4.80 0.05 nt 0.74 11 17 5.10 0.09 0.046 0.56 12 17 4.70 0.04 nt 1.04 13 18 4.30 0.06 0.060 0.78 table 3c. pysico-chemical parameters of bay of bengal (horizontal sampling) adjoining lands bring in large run-offs along with pollutants to bay of bengal, the reasons for not recording noticeable pollution in this area may be due to its large size. it may also be due to, degradation of most of the pollutant, before they reach this area. it can also be suspected that, most of the heavy pollutants sink to the bottom of the sea, from where they can not disperse further, due to almost stagnant conditions of water. one more plausible reason is that, the countries surrounding the bay of bengal, have recently been industrialized, and the quantity of effluents released is not so much, so that, it could pollute the bay to a significant level so far. but, one has to be careful to see that this water body does not get polluted. however, as the bay of bengal environment is prone to pollution, at the rate at which, the coastal areas are becoming industrialized, one has to have a close, regular and careful monitable 4. number of zooplancton species observed in bay of bengal toring of the ecology of bay of bengal. it could be done by using modern techniques such as anpp, aviris, bioclim, erin, gems, grid, hrvimla, mscp, mss, rap etc., apart from regular survey, sampling for biological organism as well for physicochemical parameters to see that, this pristine environment remains unpolluted for years to come. references anonymous (1981) andaman sea. ind j mar sci 10 (7): 209-210. apha (1992) standard method for examination of water and waste water 18th ed. american public health association, awwa, wpcp, washington, dc, usa. cairns j and jr pratt (1993) a history of biological monitoring using benthic macro-invertebrates. in freshwater bio-monitoring and benthic macro-invertebrates. (eds.) rosenberg dm and vh rash, chapman and hall, new york. pp. 10-27. daniel r (1985) fauna of india. coelenterata; hydrozoa, siphonophora, z. s. l. calcutta. dixon dr and pl pascoe (1994) mussel eggs as indicators of mutagen exposure in coastal and estuarine marine environment. in: water quality and stress indicators in marine and fresh water systems: linking levels of organization (eds.) sutchife dw, freshwater biological association, uk. pp. 124-137. grassle jf, p lessrre, ad mcintyre, and gc ray (1991) marine biodiversity and ecosystem function. biol internat special issue 23: 1-19. hawkins sj, sv proud, sk spence, and aj southward (1994) from the individuals to the community and beyond; water analysis, stress indicators and key species in coastal ecosystem. in: water quality and stress indicators in marine and fresh water systems: linking levels of organization (eds.) sutchife dw, freshwater biological association, uk. pp. 35-62. kasturirangan lr (1963) a key for the identification of the more common planktonic copepods of indian coastal waters. csir, new delhi, india. langston wj and m zhou (1986) evaluation of significance of metal binding proteins in gastropods littorina littorea. mar biol 9z:505515. madhupratap m (1981) thermocline and zooplankton distribution. ind j mar sci 10(7): 262-265. martin j (1994) testing the iron hypothesis in ecosystem of the equatorial pacific. nature 371:123-129. may rm (1994) biological diversity, differences between land and sea. phil trans roy soc lond. b 343: 105-111. mcallister de, fw schueler, cm roberts, and jp hawkins (1994) mapping and gis analysis of the global distribution of coral reef fishes on an equal area grid. in mapping the diversity of nature (eds.) ri miller, chapman and hall pubi. london. pp. 155-175. mon t (1964) the pelagic copepods from neighboring waters of japan, tokyo, the soyo co., pj newman, ma piavaux, and ra sweeting (eds.) river water quality-ecological assessment and control cec, brussels: pp 751. moore mn (1991) lysosomal changes in the response of molluscan hepato-pancreatic cells to extra-cellular signals. histochemical j 23: 495-500. moore mn and ja kitching (1939) the biology of chthamalus stella (poli). j. mar. biol. assn. uk, 23:521-541. moore mn, rk pipe, and sv farrar (1982) lysosomal and microsomal responses to environmental factors in littorina littorea from sullom vol. mar poll bull 13: 340-345. nicol d (1971) species, class and phylum diversity of animals. q. ji. fla acad sci 34: 191-194. nybakken jw (1997) marine biology; an ecological approach. iv ed. addison wesley longman inc. california. parsons tr, m takahashi, and b hargrove (1984) biological oceanotaxonomic group number of species observed and collected protozoa foraminifera 14 radiolarian 06 tintinnida 02 coelenterata hydrozoa 15 schyphozoa 1 ctenophora 14 nemertinia enopla 06 annelida errentia 18 chaetognatha 23 arthropoda cladocera 06 ostracoda 19 calanoida 124 cyclopoida 27 herpecticoida 11 monstrilloida 13 mysidae 17 hyperiidea 12 gemmaridea 17 euphausiacea 24 decapoda 18 mollusca heteropoda 04 pteropoda 13 chordata prochordata 11 appendicularia 13 thallacea 05 total 433 species kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 9 graphic processes. 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pollution gradient in narragansett bay. mar env res 4: 181-194. wright jf, mt furse, and pd armitage (1994) use of macro invertebrate communities to detect environmental stress in running water. in water quality and stress indicators in marine and fresh water systems: linking levels of organization (ed; d. w sutcliffe, freshwater biological association, uk. pp. 15-34. zheng z (1989) marine planktology, china ocean press, beijing. atlas journal of biology 2 (1): 94–99, 2012 doi: 10.5147/ajb.2012.0064 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) modeling absolute and allometric growth in houbara bustard (chlamydotis undulata undulata) in captivity amal korrida1,2,*, samuel n. nahashon3, amal amin-alami2, sami jadallah1, and samuel e. aggrey4 1 hrh prince sultan bin abdul aziz al saud international foundation for conservation and development of wildlife, genetics department, agadir 80350, morocco; 2 laboratoire des substances naturelles, faculté des sciences d’agadir, équipe d’océanographie biologique, université ibn zohr, agadir, maroc ; 3 department of agriculture, university of tennessee, nashville, tn, usa; 4 department of poultry science, university of georgia, athens, ga 30602-2772, usa. received: april 11, 2012 / accepted: may 6, 2012 __________________________________________________ * corresponding author: amalkorrida11@aol.com 94 abstract absolute and allometric growth of houbara bustard (chlamydotis undulata undulata) were studied. using body weight measurements from hatch till 120 days of age, the gompertz-laird growth model was used to describe the growth pattern of the houbara. whereas the model underpredicted the hatching weight (34 vs 40 g), the predicted asymptotic body weight was in concordance with the data (1193 g). the same growth model was also used to describe the growth pattern of beak and shank length, chest girth and wing span. the beak reached asymptotic length faster than the shank, chest girth and wing span. we used the log transformed relationship of huxley to determine the allometric relationship of beak length, shank length, chest girth and wing span relative to body weight from hatch to 120 days. the allometric growth analysis indicated that, beak length, shank length, chest girth and wing span all followed a hypometric allometry (b<1) relative to body weight growth. however, the wing span has the highest allometric growth compared to the other measurements indicating the evolutionary importance of developing wings for quick flights from predators. keywords: houbara bustard, gompertz model, allometry, growth, captive breeding. introduction the houbara bustard (chlamydotis undulata) is a medium sized bustard of the otididae family which breeds in deserts and arid sandy areas with relatively large range of habitats. the houbara bustard species can be found in the canary islands (chlamydotis undulata fuertaventurae) and north africa (chlamydotis undulata undulata) from morocco to egypt, whereas the macqueen’s species (chlamydotis macqueenii) occurs in the middle east, and asia (iran, india, pakistan, kazakhstan and china). the two species are the prized quarry for arab falconers, and widespread hunting and loss of habitat have greatly reduced their numbers. the international union for conservation of nature (iucn, 2011) classifies the houbara species as vulnerable. the houbara is also prohibited from international trade (cites, 2012). since the early 1990s, several conversation centers have been established in northern africa and the middle east to propagate the houbara and macqueen’s in captivity and establish proper conservation practices in order to preserve the houbara. since then there have been studies on the biology (saint jalme et al., 1994; tieleman et al., 2002), captive breeding (gélinaud et al., 1997; hémon et al., 2000) diseases (ostrowski et al., 1995; bailey et al., 2008), ecology (van heezik and ostrowski, 2001), behavior (gaucher et al., 1996, van heezik and seddon, 1998), genetics (chbel et al., 2002; idaghdour et al., 2004; lesobre et al., 2010) and phylogeny (granjon a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 95 et al., 1994; broders et al., 2003) of the houbara. however, data on the entire growth characteristics of the houbara is very scant. this is because most of the houbara bred in captivity are released into the wild at the middle or end of the breeding season. to date, only few studies exist on the juvenile growth characteristics of the houbara and macqueen’s (lawrence et al., 1999; van heezik and seddon, 2001; stiévenart, 2002). prediction of growth patterns has been studied extensively in other avian species, especially domestic fowl (grossman and bohren, 1982; barbato, 1991; aggrey, 2002; aggrey, 2004). growth comprises of hyperplasia, hypertrophy, cell division and apoptosis, however, these processes can also be affected by environmental fluctuations, dietary status, diseases and other by random events causing growth to fluctuate thereby making the study at a single point unattractive. also, the pattern of growth reflects the architecture of body composition (aggrey, 2002). in a species, such as the houbara, the body weight at sexual maturity may become important because of the difference in growth patterns. mathematical equations have been used to model growth characteristics (e.g., gompertz, 1925; richards, 1959). growth characteristics derived from such mathematical equations smoothens body weigh fluctuations and uses previous and future weights to predict age-weight points (denise and brinks, 1985). the objective of this study was to use the gompertz model to determine multiple growth phases, and asymptotic growth of the houbara, and also determine the allometric growth of the beak length, shank length, chest girth, and wing span relative to body weight. materials and methods flock and experimental design data were collected on a total of 178 birds that were hatched from a captive breeding program at hrh prince sultan bin abdul aziz al saud international foundation for conservation and development of wildlife (ifcdw). the houbara bustards hatched and were raised from eggs laid by a captive-bred flock vaccinated against the avian poxvirus using an attenuated live vaccine and against the newcastle disease virus (ndv) using an inactivated vaccine. the houbara chicks were raised artificially and handfed every 2 hours for 14 days with a diet consisting of wet poultry meat extruded pellets (~25% crude protein; 1.5% calcium, 1% phosphorus, 20,000iu vitamin a, 2,000iu vitamin d3, and 200iu vitamin e), supplemented with meal worms (tenebrio molitor), crickets (grillus bimaculatus), carrots, minced meat and fresh alfafa. feed crumbs were always available to stimulate pecking at food. the chicks were raised indoors in small boxes in groups of 3 or 4 at 25 °c. after 7 days, they are moved into an outdoor facility with very small pebble gravels as substrate and hereafter, transitioned into feeding independently and stimulate locomotion. the chicks were monitored at feeding times and abnormalities were reported to veterinarians who attended to them immediately. body weight, beak length, shank length, chest girth (circumference at the largest portion of the breast) and wing span were measured on day of hatch, days 14, 30, 60, 90 and 120. wing span was measured by placing the bird on its feet and the wings held at the wrist joints, and the distance was measured between the tips of the longest primary feathers on each wing. absolute growth model in order to estimate the expected body weight, beak length, shank length, chest girth and wing span at a specific age, the laird form of the gompertz equation (laird et al., 1965) was fitted to the data. the following gompertz equation was used to describe the data: where, wt is the measurement, e.g. body weight of bird at age t, w0 is the initial (hatch) measurement, l is the instantaneous growth rate (per day), k is the rate of exponential decay of the initial specific growth rate, l, which measures the rate of decline in the growth rate. the asymptotic measurement was derived as: growth curve parameters were estimated using proc nlin (marquart algorithm) (sas inst. inc., (2002). scaling relationships we tested the hypotheses on the scaling of beak length, shank length, wing span, and chest girth on body weight using the power function y = axb (huxley, 1932) in its log transformed form: ln(y) = ln(a) + b ln(x) in isometric relationships, the expected slope (b) is 1.0 as both y and x are of linear dimension. we analyzed all linear relations using reduced major axis (rma) regression because it accounts for the error in the ‘independent’ variable (sokal and rohlf, 1995). the rma regression coefficients were computed using rma for java v. 1.21 (bohonak and van der linde, 2008). results and discussion the means and standard deviations for body weight, shank length, wing span and chest girth are presented in table 1. like most longitudinal data, the standard deviations increased with age. the means and standard errors (se) of growth and body dimensions predicted with the gompertz model are presented in table 2. except for beak length, the gompertz model underpredicted the hatching measurements. the hatching weight predicted by the gompertz model (34.5g) was lower than the measured weight (40.3g). the underprediction of initial measurement is a common feature of most growth models, and as a result, grossman and bohren (1982) suggested constraining the initial measurement. however, pasternak and shalev (1994) recommended weighting the initial measurement by the inverse a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ( )1 0 ktl e k tw w e −− = 0 l k aw w e= aw a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 96 of the variance. the fits of the absolute growth measurements are presented in figures 1, 2 and 3. it is expected that the lower the initial growth rate, the earlier the asymptotic measurement is reached (aggrey, 2002). from the estimates in table 2, body weight and wing span had the highest instantaneous rates compared to beak and shank lengths, and chest girth. beak length reached its asymptote earlier than shank length (figure 2). in the wild, the houbara bustard mainly feeds on seeds, invertebrates and small vertebrates, and underdevelopes beak to limit its ability to feed. thus, the beak growing to maturity earlier than other measurements signifies its importance in early feeding ability and survival. the shank grew at a rate of 0.07 cm per day until the age of maximum growth, and hereafter, grew at 0.04 cm per day until maturity. improper development of the table 1. means (±sd) for body weight (bw), beak length (bl), shank length (sl), wing span (ws), and chest girth (cg) at different ages in houbara bustard (n=178). age (days) bw (g) bl (cm) (sl) cm ws (cm) cg (cm) 0 40.33± 4.85 1.46±0.21 1.95±0.23 10.82±0.72 10.11±0.94 7 65.22± 10.17 2.03±0.21 2.77±0.29 16.93±2.06 12.13±1.09 14 146.79± 22.93 2.60±0.25 3.67±0.35 30.02±3.84 14.82±1.27 30 408.89± 59.52 3.36±0.30 5.84±0.48 69.90±9.69 21.56±2.26 60 852.35±128.29 3.94±0.38 8.32±0.55 106.07±6.70 30.36±2.89 90 1026.21±176.71 4.03±0.33 8.97±0.60 115.35±5.23 31.66±2.71 120 1194.06±213.51 4.17±0.41 8.85±0.54 116.09±6.24 32.46±3.24 table 2. estimated coefficients (se) and confident intervals (ci) for gompertz growth, beak length, shank length, wing span, and chest girth parameters in houbara bustard. intercept 95% ci slope 95% ci r2 allometry beak length -0.66±0.02 -0.69 -0.62 0.31±0.00 0.30 – 0.31 0.8763 negative shank length -0.94±0.02 -0.98 -0.92 0.45±0.00 0.45 – 0.46 0.9644 negative wing span -0.26±0.02 -0.31 -0.22 0.73±0.01 0.72 – 0.74 0.9719 negative chest girth 0.99±0.01 0.96 1.01 0.35±0.00 0.35 – 0.36 0.9571 negative table 3. parameter from reduced maximum axis (rma) regression estimates (±se) and confidence intervals (ci) of allometric growth of some morphometric characters, as a function of body weight in houbara bustard. parameter coefficient 95% ci body weight hatching weight (w0), g 34.5326±2.96 28.7329 – 40.3323 initial growth rate (lb), g/d 0.1374±0.01 0.1242 – 0.1506 rate of decay (kb), g/d 0.0388±0.00 0.0366 – 0.0410 asymptotic weight1 (wa), g 1192.9300 beak length hatching length (b0), cm 1.4569±0.02 1.4219 – 1.4920 initial growth rate (lb), cm/d 0.0598±0.00 0.0562 – 0.0634 rate of decay (kb), cm/d 0.0588±0.00 0.0561 – 0.0616 asymptotic length1 (ba), cm 4.0248 shank length hatching length (s0), cm 1.8621±0.02 1.8167 – 1.9076 initial growth rate (ls), cm/d 0.0666±0.00 0.0641 – 0.0692 rate of decay (ks), cm/d 0.0418±0.00 0.0406 – 0.0430 asymptotic length1 (sa), cm 9.1546 wing span hatching weight (ws0), cm 7.3616±0.24 6.8925 – 7.8307 initial growth rate (lws), cm/d 0.1509±0.00 0.1441 – 0.1577 rate of decay (kws), cm/d 0.0545±0.00 0.0531 – 0.0558 asymptotic length1 (wsa), cm 117.5562 chest girth hatching girth (g0), cm 9.5129±0.11 9.2951 – 9.7307 initial growth rate (lg), cm/d 0.0430±0.00 0.0407 – 0.0453 rate of decay (kg), cm/d 0.0341±0.00 0.0326 – 0.05772 asymptotic diameter1 (ga), g 33.5572 1derived parameters. skeletal system can be significant in the health of the houbara. according to naldo et al., (1998), rotational and angular limb development, rolled toes and fractures are common limb abnormalities that affect bustards. growth analysis of the metatarsus, tibiotarsus and humerus in houbara (naldo et al., 2000) suggests the growth pattern of the shank in this study was within expectation of the general skeletal development of houbaras. the initial growth rate of the chest girth lags behind wing span and overall growth. wing span has the highest initial growth rate compared to the overall growth. in the wild, the houbara makes high thrust initial flight from predators but these flights are of shorter durations. perhaps the initial growth rate allows them to develop wing span necessary for such high thrust flights, but underdeveloped keel could be responsible for their short duration. similar to most avian species (barbato, 1991; aggrey, 2002), the correlation between the l and k growth parameters are high (r = 0.92-0.96). the literature is scant on growth characteristics and modeling in the houbara bustard, however, graphical representation of growth by naldo et al., (2000) suggests that the growth data from the current study was in concordance with expectation. even though the houbara exhibit sexual dimorphism, the birds used in this study were not sexed. future studies should include gender and data should be conducted for a longer duration to encompass both summer and winter in order to understand how the houbara regulate its body weight through different seasons. allometry allows for the study of the proportional change in the dimensions of one character relative to another. in essence, it is the scaling relationship between characters. the log transformed relationship of huxley (1932) often reveals linear allometry with a slope of b (figure 4), and such allometries are classified according to the value of b. characters scale hypometrically when b<1, isometrically, when b=1, and hypermetrically when b>1. the scaling exponent of the allometric analysis indicated that the beak, girth, shank, and wing span all exhibited negative allometric growth in relation to body weight (table 3, figure 4). however, of all the parts measured, the wing grew fastest compared to the beak in relation to body weight. the disproportionate growth of different parts of the body relative to body weight is an indication of the relative importance of the 97 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. growth curve of the houbara bustard predicted by the gompertz model. figure 2. beak and shank length curves of the houbara bustard predicted by the gompertz model. figure 3. wing span and girth circumference curves of the houbara bustard predicted by the gompertz model. figure 4. allometric growth functions of beak length, shank length, wing span and chest girth versus body weight. 98 different parts to the survival of the houbara. in early development, the beak and shank are relatively important to foraging and locomotion as the parents shelter the chicks against predators. however, the wing span has the highest allometric growth compared to the other measurements indicating the evolutional importance of developing wings for quick flights from predators. captive breeding of houbara bustard is arduous, and survival of the hatchling is crucial. hand-feeding of chicks may introduce variation in chick body weight especially when there are multiple gamekeepers undertaking the feeding. nevertheless, this study presents the first unique insight into the absolute growth and allometric growth characteristics of the houbara. the average hatching weight was about 40 g, and by 4 months, the average body weight was approximately 1200 g. the beak reaches mature length faster than shank, chest girth and wings, however, the wing span even though has a negative allometry to body weight, still grows faster than the beak, shank and chest girth illustrating the evolutionary importance of developing an appropriate sized wings for quick but short flights from potential predators. acknowledgements the authors dedicate the present study to the late crown prince, his royal highness prince sultan bin abdul aziz al saud, sponsor and patron of the foundation for his dedications, leadership and generosity to the environment and protection of wildlife. we would like also to extend our gratitude to sheikh mohamed bin khalid bin hethlain for his supports and encouragements. this work was made possible also by the support of colleagues and technical staff from ifcdw in agadir and ders in errachidia centers, especially a, s and s. bouaich, m and a. agouram, h. idaghdour, a. bayla, j. amghar, s. boucham, a. zagmouz, a. el abbassi, l and h. gaboula, y. redouane, d. moujani, y. debiani, m. lahrar and a. louzani. we thank also the anonymous referees for reviewing and evaluating this manuscript. this work is a part of the doctoral thesis of amal korrida on houbara bustard genetics. references aggrey se (2002) comparison of three nonlinear and spline regression models for describing chicken growth curves. poultry science 81: 1782-1788. aggrey se (2004) modelling the effect of nutritional status on preasymptotic and relative growth rates in 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(poeciliidae: poecilia reticulata) andre n. porter and jack s. frankel* department of biology, howard university, washington, dc 20059, usa. received: july 16, 2014, 2014 / accepted: august 16, 2014 __________________________________________________ * corresponding author: jfrankel@howard.edu 212 abstract heterogeneity within a population enhances its long-term survival. a fundamental method of maintaining population heterogeneity is the retention of rare or uncommon phenotypes by selective mating strategies. employing two color morphs, red tuxedo and red, of the guppy (poecilia reticulata, poeciliidae), this study was designed to investigate whether p. reticulata females would preferentially seek out heterogeneous groupings of males exhibiting two color morphs. adult female guppies were exposed simultaneously to two groups of males (n=10); one comprised of only the tuxedo color morph (n=5) and the other of both color morphs (n=5). for the latter group, the ratio of males exhibiting the two colorations was changed incrementally over multiple trials, beginning and ending with homogeneous groupings (5:0, 4:1, 3:2, 2:3, 1:4, 0:5). experiments were conducted employing a 76 l aquarium partitioned at both ends to house the male groups. individual females were placed into an acclimation, holding column located in the center compartment of the experimental aquarium. subsequent to a five minute acclimation period in the holding column, each female was released. courting behaviors and population affinities (i.e. female location relative to the male compartments) were recorded every five seconds over a five minute observation period. females did not show a preference for male groupings exhibiting either the 4:1 or 1:4 color ratios. however, females clearly showed affinity for the male groupings of 3:2 and 2:3 (p <0.05). this investigation on female mate choice in p. reticulata clearly reveals that a mating strategy is occurring in this species and that it is frequency-dependent. key words: poecilia reticulata, guppy, mate choice, frequencydependent selection, color morphs. introduction genetic variation and phenotypic diversity are central concepts in evolutionary biology and closely linked to the complexity of organisms, ecosystem recovery, and the ability of species to respond to environmental changes (bazin et al., 2006). mate choice is known to be a powerful evolutionary mechanism influencing the stability and maintenance of polymorphisms within a species (pryke and griffith, 2007). in general, females of a species are more selective than males in discriminating among prospective mates, as they evaluate genetic quality and resource-holding potential based on secondary sexual characteristics, such as body size, coloration, ornamentation, and aggressiveness (basolo, 1990; clotfelter et al., 2006; bierbach et al., 2013). elucidating mate choice strategies can assist in ascertaining and accessing possible cause and effect relationships in mating preference. within non-random mating systems, selective mechanisms must be in place to maintain population heterogeneity. the retention of rare or uncommon phenotypes is a fundamental method of maintaining such heterogeneity. thus, in species where mating is non-random, females are often found to be particularly selective when the parental male investment is low (baldauf et al., 2009). here, females would be expected to select and court with males that exhibit traits present in low phenotypic frequency (i.e. uncommon phenotypes), thereby ensuring that those phenotypes remain within the population. utilizing such a frequency-dependent selection mating strategy, populations would ultimately exhibit an increase in the frequency of those rarer phenotypes, thereby insuring their genetic diversity (gross, 1991; pilastro et al., 1997; punzalan et al., 2005). coloration has been shown to affect both species recognition and mate choice in poeciliid fishes (endler, 1983). in fact, the maintenance and benefits of color variation in numerous species of poeciliids by selective mating strategies has been investigated in both natural and artificial populations (borowsky and this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 213 kallman, 1976; houde and endler, 1990; baer et al., 1994; royle et al., 2008; culumber and rosenthal et al., 2013). additional studies have illustrated the importance of coloration in mate selection by poeciliid females, where they exhibit a natural affinity towards certain colorations when shown a population of males containing phenotypically variable color morphs (houde, 1997; kingston et al., 2003). the study presented here was designed to investigate the validity of frequency-dependent selection based on a body color polymorphism in the freshwater guppy poecilia reticulata (poeciliidae). the poeciliidae include over 40 species from north, south and central america, which occupy a wide range of habitats within these regions and exhibit both morphological and behavioral differentiation within and between species (breden et al., 1999). as a group, they are relatively small fishes, ranging from 1.5 – 6.0 cm in length, with highly developed sexual dimorphisms. the guppy, p. reticulata, one of the more common poeciliids, is distributed world-wide. this species ranges in size from 1.5 to 3.5 cm for adult males and from 3.0 to 6.0 cm for adult females. while native to antigua, barbados, jamaica, trinidad, tobago, brazil, venezuela, and the u.s. virgin islands, populations of p. reticulata have been introduced to many other habitats and have readily occupied bodies of freshwater made accessible to them. the guppy is extremely popular in the aquarium trade, due to ease of upkeep and the colorfully ornamented caudal and dorsal fin morphs found in males through artificial selection. this species is not only sexual dimorphic with regard to both coloration and color intensity, but exhibits a wide array of shape and fin morphology polymorphisms as well. as with other poeciliids, p. reticulata serves as an excellent model organism to employ in a study on frequency-dependent mate selection, since it exhibits both distinct and uniform color morphs. utilizing red and red tuxedo color morphs, this study investigated whether p. reticulata females would preferentially seek out heterogeneous groupings of males as opposed to homogeneous ones. it is hypothesized that phenotypic heterogeneity amongst potential male mates is more attractive to females than a homogeneous grouping, as determined by observing the courting behaviors of females when confronted with these diverse male groupings. materials and methods to investigate female preference for male groups based on the phenotypic frequencies of two color morphs, a series of experimental trials were conducted employing similar constructs as those described for examining female color preference in the pygmy swordtail, xiphophorus pygmaeus (baer et al., 1994). in the work presented here, two phenotypes were chosen; a red tuxedo color morph exhibiting a red trunk with a dark blue caudal peduncle, red dorsal and caudal fins with dark blue margins, and a red color morph which displays a red trunk with red dorsal and caudal fins. these phenotypes were selected for their uniformity in overall coloration and color pattern from individual to individual within these two color morphs. adult guppies were obtained from g & g aquatics inc., lorton, va., usa. male and female guppies were housed in 76 l capacity, high configuration (61cm x 32cm x 42cm) holding aquaria during the course of this investigation. females were chosen at random from an all-female stock population (n = 120) maintained in a dedicated aquarium. males of each color morph were selected from initial stock populations (n = 80) for uniformity in total body length, dorsal and caudal fin lengths, and coloration. males selected from the stock populations were maintained with individuals of like phenotypic coloration, i.e. red males with red males, tuxedo males with tuxedo males. water temperature was maintained at 23 ± 2 °c by the laboratory hvac system. fish were fed tetramin flakes twice daily. water quality (ammonia, nitrite and nitrate) was assessed weekly. the ph level in all aquaria was maintained between 6.8 and 7.2. experimental trials were conducted in a 76 l capacity, long configuration (74cm x 20cm x 30cm) aquarium (fig. 1). groups consisting of five males (all-tuxedo, all-red, or tuxedo/red combinations) were held on either side of the experimental aquarium in 11cm x 20cm x 30cm male compartments, designated as sections a and b. male compartments were constructed with solid plexiglas sheets secured to the aquarium with a continuous strip of aquarium silicon to physically separate males from females during the trials. lighting consisted of fluorescent lights directly above the experimental aquarium. additionally, three sides of the experimental aquarium were covered with white paper, leaving only the front and top uncovered to allow for observation of female location and courting behavior while eliminating distractions from the sides and back. to begin each trial, two groups of five males were collected and placed in each of the male compartments. initially, one section was selected for placement of all-tuxedo males and the other for all-tuxedo males, combinations of tuxedo and red males, or all-red males. male groups were then subsequently designated as a or b; correlating with the corresponding partitioned side as seen in figure 1. one female was then randomly chosen and placed in a plexiglas acclimation column (11cm x 11cm x 50cm) located in the center of the experimental aquarium, designated as “no preference” section d. once a female was placed in the acclimation column, she was given a five minute acclimation period. when the acclimation period expired, the column was removed, releasing a female within section d and allowing her five minutes to move freely within that section and into areas a and b which are juxtaposed to the male compartments. the relative position of each female was designated by her location in a, b, or d and was recorded at five second intervals throughout the duration of each five minute trial period. time intervals spent in section d was interpreted as “no preference,” while time intervals spent in preference areas a or b as “preferentially courting” males in sections a or b. after each trial, females were captured and placed in a separate holding aquarium to prevent reuse for a given trial set. to avoid side bias, all trial sets were repeated with male groupings switched from section a to b. eleven trials were conducted per paired male groupings. adult female guppies were exposed simultaneously to two groups of males (n = 10); one comprised of only the tuxedo color morph (n = 5) and the other of the two color morphs (n = 5). for the latter group, the ratio of males exhibiting the two a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 214 colorations was changed incrementally over multiple trials, beginning and ending with homogeneous groupings (5:0, 4:1, 3:2, 2:3, 1:4, 0:5). for this study, two complete trial sets were undertaken. for each individual trial (e.g. 1, 2a, 2b, 3a, 3b, etc.), 11 observations were recorded for 11 different females. designations “a” and “b” indicate replicate trial observations. a total of 60 observations were recorded (one every five seconds for five minutes) per trial female, giving a total of 660 observations for each group of 11 females/trial. at the completion of all trial sets, a total of 12,540 data points were recorded. the data was then subjected to chi-square analysis for the probability of females deviating from the expected time interval allocation of 1:1 for movement into preference areas a or b due to chance alone. all trials were conducted under an approved howard university iacuc protocol (iacuc-gsas-11-03). results and discussion table 1 presents the total five-second time allotments for 11 female guppies/trial occupying preference areas a and b when exposed to (i) all-tuxedo groupings in both male compartments (trial 1), (ii) all-tuxedo groupings opposed to tuxedo/ red combinations (trials 2a – 9b), and (iii) all-tuxedo groupings opposed to all-red groupings (trials 10a and 10b). when confronted with paired homogeneous tuxedo groupings (trial 1), females clearly showed no preference towards males in either compartment (p = 0.491). further, females did not favor either all-tuxedo or all-red male groupings (trials 10a and 10b), nor did they show an affinity towards 4 tuxedo: 1 red or 1 tuxedo: 4 red combinations (trials 2a, 2b, 3a, 3b, 8a, 8b, 9a and 9b). in fact, females preferentially exhibited courting behaviors directed toward the homogeneous, all-tuxedo groupings when the 1 tuxedo: 4 red combination occupied the opposing compartment (trials 8b and 9a). females clearly exhibited a particular preference for male grouping of both 3 tuxedo: 2 red and 2 tuxedo: 3 red (p < 0.001; trials 4b, 5b, 6a, 6b, 7a and 7b). total five-second time allotments for female guppies in preference areas a and b, when confronted with groupings of five tuxedo males and combinations of tuxedo and red males (trials 3 – 8), are presented in table 2. with the exception of trial sets 8b and 9a (1 tuxedo: 4 red occupying a and 5 tuxedo occupying b; 5 tuxedo occupying a and 1 tuxedo: 4 red occupying b, respectively), where females exhibited an affinity towards the front view of the experimental aquarium aerial view of the experimental aquarium figure 1. front and top aerial views of the experimental aquarium.overall dimensions were 74 cm long x 20 cm wide x 30 cm tall. male compartments are labeled sections a and b. the female preference areas, a and b, are juxtaposed to the male compartments. the female no preference area, labeled section d, is in the center of the aquar ium and contains the holding column. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 215 table 2. total time allocations per trial (in 5 second intervals) by females (n = 11) courting heterogeneous male groupings of red tuxedo and red color morphs and all-red tuxedo groupings, expected ratios, degrees of freedom (df), chi-square values, and probabilities. trial time allocations courting red tuxedo/ red time allocations courting all-red tuxedo expected ratio df chi-square value probability 3a. 4:1/5 184 222 1:1 1 3.557 0.059 3b. 4:1/5 170 190 1:1 1 1.111 0.292 5a. 3:2/5 204 224 1:1 1 0.935 0.334 5b. 3:2/5* 270 110 1:1 1 67.368 <0.001 7a. 2:3/5* 299 199 1:1 1 20.080 <0.001 7b. 2:3/5* 305 220 1:1 1 13.762 <0.001 9a. 1:4/5* 153 215 1:1 1 10.446 0.001 9b. 1:4/5 160 195 1:1 1 3.451 0.063 2a. 4:1/5 237 227 1:1 1 0.216 0.643 2b. 4:1/5 205 185 1:1 1 1.026 0.311 4a. 3:2/5* 224 258 1:1 1 2.938 0.123 4b. 3:2/5* 225 145 1:1 1 17.297 <0.001 6a. 2:3/5* 262 88 1:1 1 86.503 <0.001 6b. 2:3/5* 200 95 1:1 1 37.373 <0.001 8a. 1:4/5 193 191 1:1 1 0.010 0.919 8b. 1:4/5* 205 255 1:1 1 5.435 0.002 total 16 270.508 <0.001 pooled 3496 3019 1:1 1 34.924 <0.001 heterogeneity 15 235.584 <0.001 *trials with significant chi-square values (p < 0.05). numbers in red and blue denote the number of red and red tuxedo morphs used within the compartment. compartment trial a/b time allocated in a time allocated in b expected ratio df chi-square value probability 1 5/5 230 245 1:1 1 0.474 0.491 2a. 5/4:1 227 237 1:1 1 0.216 0.643 2b. 5/4:1 185 205 1:1 1 1.026 0.311 3a. 4:1/5 184 222 1:1 1 3.557 0.059 3b. 4:1/5 170 190 1:1 1 1.111 0.292 4a. 5/3:2 258 224 1:1 1 2.938 0.122 4b. 5/3:2* 145 225 1:1 1 17.297 <0.001 5a. 3:2/5 204 224 1:1 1 0.935 0.334 5b. 3:2/5* 270 110 1:1 1 67.368 <0.001 6a. 5/2:3* 88 262 1:1 1 86.503 <0.001 6b. 5/2:3* 95 200 1:1 1 37.373 <0.001 7a. 2:3/5* 299 199 1:1 1 20.080 <0.001 7b. 2:3/5* 305 220 1:1 1 13.762 <0.001 8a. 5/1:4 191 193 1:1 1 0.010 0.919 8b. 5/1:4 255 205 1:1 1 5.435 0.020 9a. 1:4/5 153 215 1:1 1 10.446 0.001 9b. 1:4/5 160 195 1:1 1 3.451 0.063 10a. 5/5 246 253 1:1 1 0.098 0.754 10b. 5/5 210 205 1:1 1 0.060 0.806 *females preferentially courting male groups of 3 red: 2 red tuxedo or 2 red: 3 red tuxedo over all-red tuxedo groupings. numbers in red and blue denote the number of red and red tuxedo morphs used within the compartment. table 1. total time allocations per trial (in 5 second intervals) by females (n = 11) courting male groupings of red tuxedo and/or red color morphs, expected ratios, degrees of freedom (df), chi-square values, and probabilities. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) all-tuxedo grouping, they did not show a preference for either all-tuxedo or 1:4 or 4:1 groupings. further, while females were clearly attracted towards those male groupings with combinations of 3:2 and 2:3, they overwhelmingly selected heterogeneous groupings in general, regardless of compartment location (p < 0.001). table 3 illustrates the comparison of total five second allotments spent within preference areas a or b as compared to those observed for females in the no-preference area d. here, clear indications of choice were apparent, with females spending significantly more time outside of the no preference section d in 16 of 19 trial sets. there are many potential explanations for the coexistence of color polymorphisms within populations. in practice, however, it has proven difficult to determine which mechanisms explain the presence of multiple color variants within any particular population (munday et al., 2003). female mate choice has been proposed as a means of increasing diversity where a strong preference for rare or novel color patterns is indicated. however, this “rare male” advantage will be detectable only when phenotype frequencies are perturbed from their equilibrium values (hughes et al., 2013). the results obtained from this investigation of mate choice in p. reticulata provide strong evidence towards female mating preference for groupings of males exhibiting multiple phenotypes. data compiled from this study clearly indicates that when female guppies are presented with a choice between males that exhibit a single phenotype or multiple phenotypes, they exhibit some preference towards the latter. further, in 16 of the 19 complete trials, female time allocations towards preference groups (i.e. making a courting decision) exceeded the time spent for making no courting choice (i.e. remaining in section d) and is consistent with findings in other mate selection studies (kodricbrown, 1985; baer et al., 1994; maclaren and fontaine, 2012). as trials extended beyond the initial all-tuxedo groupings, females showed no preference for male phenotypic combinations of 4:1 (trials 2a, 2b, 3a, 3b) or 1:4 (trials 8a, 8b, 9a, 9b). in fact, females exhibited an affinity towards the all-tuxedo male grouping when confronted with that homogeneous grouping and the 1 tuxedo: 4 red combination (trials 9a and 8b; p < 0.05) and occurred whether the all-tuxedo grouping occupied section a or b. in effect, for trials where male groupings had a phenotypic frequency of 0.20 for the “rare” color morph, females showed no statistically significant preference for those groups. as the phenotypic frequency of the initially rare color morph increased to 0.40, the affinity of female guppies for those male groupings became evident. of the eight trial sets comprised of color ratios of 2:3 or 3:2, six resulted in statistical significance of female mate choice for these combinations. this clear affinity of females towards male groupings exhibiting 40 % rarity for either tuxedo or red color morphs may, in part, be due to a phenotypic threshold for frequency-dependent selection for this trait. roff (1998) has provided evidence that certain levels of phenotypic variation may be maintained in a population at some “threshold level” and suggests that frequency-dependent selection can give rise to, and maintain, polymorphisms in a population. this may speak to an equilibrium state where male groups 216 compartment trial a/b time allocated to a or b time allocated to d expected ratio df chi-square value probability 1. 5/5 475 185 1:1 1 127.424 <0.001 2a. 5/4:1 464 196 1:1 1 108.824 <0.001 2b. 5/4:1 390 275 1:1 1 21.818 <0.001 3a. 4:1/5 406 254 1:1 1 35.006 <0.001 3b. 4:1/5 360 300 1:1 1 5.455 0.020 4a. 5/3:2 482 178 1:1 1 140.024 <0.001 4b. 5/3:2 370 290 1:1 1 9.697 0.002 5a. 3:2/5 428 232 1:1 1 58.206 <0.001 5b. 3:2/5 380 280 1:1 1 15.152 <0.001 6a 5/2:3 350 310 1:1 1 2.424 0.120 6b. 5/2:3 295 365 1:1 1 7.424 0.006 7a. 2:3/5 498 162 1:1 1 171.055 <0.001 7b. 2:3/5 525 135 1:1 1 230.455 <0.001 8a. 5/1:4 384 276 1:1 1 17.673 <0.001 8b. 5/1:4 460 200 1:1 1 102.424 <0.001 9a. 1:4/5 368 292 1:1 1 8.752 0.003 9b. 1:4/5 355 305 1:1 1 3.788 0.052 10a. 5/5 499 161 1:1 1 173.097 <0.001 10b. 5/5 415 245 1:1 1 43.788 <0.001 numbers in red and blue denote the number of red and red tuxedo morphs used within the compartment. table 3. time allocations per trial (in 5 second intervals) by females (n = 11) courting males in preference areas a or b, or making no decision and remaining in no preference section d, expected ratios, degrees of freedom (df), chi-square values, and probabilities. 217 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) of ~20% for a novel phenotype represent a threshold level with the common phenotype (hughes et al., 2013). therefore, evidence exists that traits being maintained through frequencydependent mating strategies may be not only driven by these disruptive mating strategies, but also maintained by them. in the research presented here, the affinity of females towards 2:3 and 3:2 heterogeneous male groupings when the color morphs reach frequencies between 0.40 and 0.60 may flag a threshold for female mate choice for these particular color morphs in p. reticulata. acknowledgements the authors wish to extend their appreciation to george middendorf and hemayet ullah, department of biology, howard university, washington, dc, and to tyler frankel, department of animal and avian sciences, university of maryland, college park, md, for their suggestions during the course of this investigation. references baer fc, m danitzker, and mj ryan (1994) a test for preference of association in a color polymorphic poeciliid fish: laboratory study. environmental biology of fishes 43: 207-212. baldauf sa, h kullmann, sh schrotth, t thunken, and tc bakker (2009) you can’t always get what you want: size assortative mating by mutual mate choice as a resolution of sexual conflict. bmc evolutionary biology 9: 129-138. basolo a (1990) female preference for male sword length in the green swordtail, xiphophorus helleri (pisces: poeciliidae). animal behavior 40: 332-338. bazin e, g sylvain, and n galtier (2006) population size does not influence mitochondrial genetic diversity in animals. science 312: 570-572. bierbach d, v sassmannshausen, b streit, l arias-rodriguez, and m plath (2013) females prefer males with superior fighting 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(2012) female bias for male lateral projection area in poecilia reticulata. environmental biology of fishes 93 (1): 105-119. munday pl, pj eyre, and gp jones (2003) ecological mechanisms for coexistence of colour polymorphism in coral-reef fish: an experimental evaluation. oecologia 137: 529-526. pilastro a, e giacomello, and a bisazza (1997) sexual selection for small size in male mosquito fish (gambusia holbrooki). proceedings of the royal society biological sciences 264: 1125-1129. punzalan d, fh rodd, and ka hughes (2005) perceptual processes and the maintenance of polymorphism through frequency-dependent predation. evolutionary ecology 19: 303-320. pryke sr, and sc griffith (2007) the relative role of male vs. female mate choice in maintaining assortative pairing among discrete colour morphs. journal of evolutionary biology 20 (4): 1512-1521. roff da (1998) the maintenance of phenotypic and genetic variation in threshold traits by frequency-dependent selection. journal of evolutionary biology 11: 513-529. royle nj, j lindström, and nb metcalfe (2008) context-dependent mate choice in relation to social composition in green swordtails, xiphophorus helleri. behavioral ecology 19 (5): 998-1005. atlas journal of biology 2 (2): 147–153, 2013 doi: 10.5147/ajb.2013.0128 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) use of food additives to control postharvest citrus blue mold disease askarne l.*, h. boubaker, e. h. boudyach, and a. ait ben aoumar laboratoire de biotechnologies et valorisation des ressources naturelles, université ibn zohr, faculté des sciences, b.p 8106, agadir, morocco. received: may 30, 2013 / accepted: july 21, 2013 __________________________________________________ * corresponding author: as.latifa@gmail.com 147 abstract the aim of this study was to find an alternative to the chemical fungicide currently used in the control of postharvest citrus diseases. the antifungal activity of 10 salt compounds, considered as common food additives was assayed in in vitro and in vivo trials against penicillium italicum, causal agent of citrus blue mold. among the 10 tested salt compounds, sodium carbonate, ammonium carbonate, copper sulfate, sodium edta and sodium metabisulfite completely inhibited mycelial growth of penicillium italicum at 20 mm. colony growth of p. italicum on ph adjusted medium was evaluated. results indicate that p. italicum can grow on both acidic and alkaline ph, with the optimum growth occurred in the range of 4.0 and 8.0. results of the in vivo trials with tested salt compounds indicate that sodium metabisulfite (100 and 200 mm), boric acid (400 mm), sodium salicylate and sodium sulfite (200, 300 and 400 mm) completely inhibited blue mold development on citrus fruit. boric acid (400 mm) and sodium metabisulfite (100 mm) gave the best results as they completely inhibited the fungus development without damaging fruit rind. such healthy products therefore may represent a sustainable alternative to the use of chemical fungicides for controlling postharvest diseases of citrus fruit. key words: citrus, food additives, blue mold, penicillium italicum. introduction citrus fruit cultivation is very important in morocco, being the first exporting agricultural sector and playing a major role in the national economic development. the largest volume of citrus fruit for fresh fruit consumption and export is grown and shipped from packing houses in souss-massa-draa (smd) valley (boubaker et al., 2009). postharvest green mold, caused by penicillium digitatum (pers.:fr.) sacc. and blue mold, caused by p. italicum wehmer are the most important postharvest diseases that cause commercially significant losses, in morocco (elkhamass et al., 1994) and worldwide (eckert jw and il ears 1989, holmes gj and jw eckert, 1999; zhu et al., 2006). these penicillium species are strict wound pathogens, they are ubiquitous and produce profuse amount of asexual conidia that are readily disseminated by air current (boubaker et al., 2009; holmes and eckert, 1995; holmes and eckert, 1999). therefore they can infect the fruit in the grove, the packinghouses and marketing, through wounds occurred during harvest and subsequent handling (boubaker, et al., 2009; brown and miller, 1999). blue mold is more harmful because it spreads in the box and healthy fruits are directly attacked, regardless of injury. this disease is, also, more important under cold storage conditions. currently, such fungal diseases are commonly controlled worldwide by applying chemical fungicides that are usually incorporated into waxes before fruit storage (boubaker et al., 2009; smilanick and sorenson, 2001). however, the use of fungicides is becoming increasingly restricted due to stringent regulation, pathogen resistance development and growing public concern about chemical residues a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 148 in fruit (palou et al., 2008; zhang and swingle, 2003). these issues have resulted in an intensive search for non polluting control methods. various alternative measures such as the application of microorganisms (el-ghaouth et al., 2000; lahlali et al., 2011; taqarort et al., 2008), plant extracts (ameziane et al., 2007; askarne et al., 2013) or the use of effective natural substances like food additives that have minimal adverse effect on the environment and health (arslan et al., 2009) and which exhibit a broad-spectrum antifungal activity (corral et al., 1988), in combination or in a replacement for fungicide have been developed. several studies have dealt with the use of different salt compounds to control various post-harvest diseases of citrus and other crops (arslan et al., 2006; arslan et al., 2009; nigro et al., 2006). treatment of fruit with carbonate or bicarbonate salts was shown to reduce the incidence of post-harvest diseases of citrus fruit caused by penicillium digitatum, penicillium italicum or geotrichum candidum (smilanick et al., 2006; smilanick et al., 2008; zhang and swingle, 2003). they have also been used to control the blue mold caused by penicillium expansum and the gray mold caused by botrytis cinerea in apple fruit (droby et al., 2003; palou et al., 2009). sodium metabisulfite was shown to reduce potato silver scurf caused by helminthosporium solani (hervieux et al., 2002; mills et al., 2006; olivier et al., 1998) and potato dry rot caused by fusarium sambucinum (mecteau et al., 2002). sodium edta was used to control p. digitatum on oranges (valencia-chamorro et al., 2008) and b. cinerea on apple fruit (droby et al., 2003). the present work was performed to evaluate the efficacy of 10 salt compounds, considered as common food additives, for in vitro and in vivo control of penicillium italicum the causal agent of blue mold of citrus fruit. materials and methods food additives and fungal species the food additives tested for their antifungal activity were listed in table 1. the p. italicum isolate used in this study was obtained from naturally decayed orange fruit. small pieces of fruit tissue, previously surface-disinfected with 90% ethanol, were aseptically excised from the advancing edge of the rot and transferred to petri plates containing potato dextrose agar (pda) acidified with 1 ml of lactic acid (80%) per litre. after a 4-day incubation period at 25°c, plates were examined under a stereomicroscope to determine colony identities. the isolate used in this work was the most aggressive one in our collection and produced the largest lesions on inoculated fruit. this fungus was purified and maintained on pda and stored at 4°c, with periodic transfers through citrus fruit to maintain its aggressiveness (taqarort et al., 2008). fruit fruit of mandarin (citrus reticulata blanco) cv. clementine were used. fruit were harvested from orchards of the m’brouka cooperative, in the souss-massa valley, morocco. only healthy and commercially mature fruit were used in the experiments. freshly harvested or briefly stored (no longer than 2 days) fruit were used in the screening tests. in vitro antifungal tests the inhibitory effects of 10 food additives on mycelial growth of penicillium italicum were tested in vitro using the agar dilution technique. an aqueous solution of each compound was prepared in sterile distilled water and was added aseptically to autoclaved and cooled pda medium at 50°c to achieve final concentrations of 2, 5, 10, 15, 20, 50,75, 100, 150 and 200 mm. the food additive-amended medium was dispensed (15ml/ plate) aseptically into 9-cm-diameter petri plates. chemical unamended plates served as control. hyphal plugs (5 mm diameter) were cut from the periphery of actively growing colonies (7 to 10 day-old) and transferred aseptically, mycelium down, to three replicate petri plates containing pda medium supplemented with chemicals. the plates were sealed with parafilm and incubated in the dark at 25°c. radial growth was measured daily at two perpendicular colony diameters until the growth in the control reached the edge of the petri plates. the antifungal activity was expressed in terms of percentage of reduction of mycelial growth calculated according to the following formula: reduction (%) = [(diameter in control– diameter in treatment) / diameter in control] × 100. the concentrations of food additives that caused 50% reduction (ec50) of mycelial growth were calculated using probit a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. chemicals used in this study. food additive chemical formula molecular weight boric acide h3bo3 61.83 ammonium carbonate (nh4)2co3 96.09 copper sulfate cuso4, 5h2o 249.68 sodium edta c 10 h 14 n 2 na2 o 8, 2h2o 372.24 potassium carbonate k2co3 138.21 sodium bicarbonate nahco3 84.01 sodium carbonate na2co3 105.99 sodium metabisulfite na2s2o5 190.1 sodium salicylate c7h5nao3 160.11 sodium sulfite na2so3 126.04 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 149 analysis (polo software). the minimum inhibitory concentration (mic) and minimum fungicidal concentration (mfc) were also determined in parallel experiment. the nature of toxicity (fungistatic/fungicidal) of the food additives was determined by following the method of tripathi et al. (2004). the inhibited fungal discs showing no growth were taken from the food additive treated petri plates, and then re-inoculated separately into the fresh medium and revival of their growth was observed for the next 9 days at 25°c. effect of ph on mycelial growth of p. italicum since some food additives could affect the ph of pda medium, the effects of ph on p. italicum colony growth was examined on adjusted pda at ph 2, 4, 6, 8, 10 and 12 with 1n hcl or naoh. hyphal plugs (5 mm diameter) cut from the periphery of actively growing colonies (7 day-old) were transferred aseptically, mycelium down, to three replicate petri plates containing pda at different ph. radial growth was determined daily, by measuring colony size along two perpendicular axes. percentage of colony growth which is the ratio of colony growth at various food additive concentrations compared with that of control was determined. effects of food additives on blue mould development in artificially wounded and inoculated fruit the in vivo test was conducted as previously described by (askarne l, et al. 2012). briefly, fruit were wounded (2 mm deep and 3 mm wide) using a sterile needle at the equatorial side. the wounds were treated with 40 µl of food additive solutions at concentrations of 50, 100, 200, 300 and 400 mm. controls were treated with the same volume of sterile distilled water under the same conditions. after 2-h incubation at room temperature, each wound was inoculated with 20 µl of an aqueous suspension of conidia of p. italicum adjusted to 106 spores ml-1 (palou et al., 2002). treated fruit were placed on plastic tray in cardboard boxes and stored at 20°c and ~95% relative humidity (rh) for 5 days. the number of infected wounds and lesion diameters of the overall treated fruit were determined daily. all treatments were arranged in a complete randomized block design. sixteen oranges constituted a single replicate, and each treatment was replicated three times. the experiment was conducted twice, obtaining consistent results. the reported values are the average of the measurements. the incidence and severity of disease were calculated as follows: disease incidence (%) = [(number of rotten wounds/ number of total wounds)] x 100. disease severity (%) = [(average lesion diameter of treatment/ average lesion diameter of control)] x 100. in all experiments, the possible phytotoxic effect on orange fruit was examined. statistical data analysis all data were subjected to statistical analysis of variance (anova) using statistica software, version 6, statsoft, 2001, france. percentage values were subjected to arcsinesquare root transformation before analysis of variance. duncan multiple range tests were used to segregate treatments which were significantly different at p<0.05. the ec50 values were calculated for each compound by probit analysis using polo software. results in vitro antifungal tests the in vitro experiments (table 2) show that among the ten food additives tested, only ammonium carbonate, copper sulfate, sodium edta, sodium carbonate and sodium metabisulfite completely inhibited the growth of p. italicum. among these salt compounds only ammonium carbonate that showed a fungicidal effect at 20 mm, the remaining salts were fungistatic. the lowest ec50 values against tested fungus were recorded in sodium food additive reduction (%) ec 50 (mm) mic (mm) mfc (mm) ammonium carbonate 100 ± 0 a 3.43 10 20 boric acid 68.78 ± 2.67 b 16.14 50 > 200 copper sulfate 100 ± 0 a 3.69 10 20 sodium edta 100 ± 0 a 2.67 10 150 potassium carbonate 55.66 ± 5.22 c 25.96 150 > 200 sodium bicarbonate 48.88 ± 3.75 d 21.63 75 > 200 sodium carbonate 100 ± 0 a 12.07 20 > 200 sodium metabisulfite 100 ± 0 a 1.69 5 5 sodium salicylate 44.47 ±3.48 e 27.29 150 200 sodium sulfite 42.41 ± 2.01 e 24.52 150 200 values expressed are mean of three replicates; means followed by the same letter do not differ significantly according to duncan multiple range tests at p<0.05. table 2. reduction (%) at 20mm, ec50, minimum inhibitory concentration (mic) and minimum fungicidal concentration (mfc) values of various compounds tested against p. italicum. metabisulfite (1.69 mm), sodium edta (2.67 mm), ammonium carbonate (3.43 mm ) and copper sulfate (3.68 mm) (table 2). similar results were recorded in mic values. the mic value of sodium metabisulfite was lower than that of copper sulfate, sodium edta, and ammonium carbonate. the lowest mfc value was recorded in sodium metabisulfite too. the mfc values of ammonium carbonate and copper sulfate were lower than that of sodium edta. boric acid and potassium carbonate reduced the mycelial growth by more than 50 %. the remaining compounds inhibited mycelial growth by less than 50%. ph tests the obtained results demonstrate that p. italicum grew on both acidic and alkaline ph (fig.1). the data indicate that the optimum growth of tested fungus was obtained between ph 4 and ph 8 as the colony diameter is not significantly affected after 7 days of incubation at 25°c. bellow ph 4 and above ph 8, p. italicum grew at a reduced rate. 150 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 1. effect of ph on in vitro mycelial growth of p. italicum. medium ph was adjusted with naoh or hcl. bars represent standard deviations of means. 0 20 40 60 80 100 120 0 2 4 6 8 10 12 14 m yc el ia l gr ow th ( % ) ph values table 3. blue mold incidence on oranges fruit ‘cv. clementine‘ treated with various concentrations of salts and stored at 20°c and ~95% relative humidity for 5 days . salts disease incidence (%) concentration (mm) 50 100 200 300 400 control 100,00 c 100,00 c 100,00 d 100,00 d 100,00 d sodium carbonate 100,00 c 100,00 c 100,00 d na* 31,25 b sodium metabisulfite 27,08 a 0,00 a 0,00 a na na boric acid na na 33,33 b na 0,00 a sodium sulfite 83,33 b 35,42 b 0,00 a 0,00 a 0,00 a sodium salicylate 97,92 bc 39,58 b 0,00 a 0,00 a 0,00 a potassium carbonate na na 83,33 c 66,67 b 58,33 b ammonium carbonate na na 100,00 d 91,67 cd 91,67 cd sodium bicarbonate na na 91,67 cd 83,33 bc 83,33 c copper sulfate 91,67 bc 83,33 c 41,67 b na na sodium edta 100,00 c 100,00 c 91,67 cd na na values were the mean of three replicates. means followed with different letters in each column are statistically different according to duncan multiple range tests (p<0.05) applied after an analysis of variance of the arcsine of the square root of the proportion of decayed fruit. non transformed data are shown. *: not applied. 151 effect of salts on blue mold development the data presented in table 3 shows that tested food additives reduced the incidence of blue mold on citrus in a dose dependent manner, with the exception of sodium metabisulfite (100 and 200 mm), boric acid (400 mm), sodium salicylate and sodium sulfite (200, 300 and 400 mm) which completely inhibited rot development on fruit. sodium metabisulfite (50 mm), sodium carbonate (400 mm), boric acid (200 mm), sodium sulfite (100 mm), sodium salicylate (100 mm), copper sulphate (200 mm) reduced significantly the incidence of the decay compared with the control. the percentage of rot incidence ranged between 27.08 and 41.67%. regarding severity of the disease, which is the ratio of lesion diameter at various food additive concentrations compared with that of control, we found that boric acid (400 mm) and sodium metabisulfite (100 mm) that gave the best results as they completely inhibited the development of the fungus without damaging fruit (table 4). the other treatments that completely inhibited the fungus in in vivo experiments, lead to a drying of the rind of the fruit around the wounds which could be accompanied by browning that increases with increasing concentration. treatment with sodium carbonate (400 mm), sodium metabisulfite (50 mm), boric acid (200 mm), sodium sulfite (100 mm), sodium salicylate (100 mm), potassium carbonate (300 and 400 mm) and sodium edta (200 mm) significantly reduced the severity of the decay without damaging the fruit. the percentages of severity varied between 19.9 and 55.22% (table 4). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) discussion the obtained results demonstrate that several food additives can inhibit significantly the growth of p. italicum. in our study, we found that sodium metabisulfite completely inhibited mycelial growth of p. italicum. several previous studies demonstrate that sodium metabisulfite has been shown to completely inhibit in vitro mycelial growth of h. solani (hervieux et al., 2002), fusarium sambucinum (mecteau et al., 2002), geotrichum candidum (talibi i, et al. 2011) and a wide range of potato postharvest pathogens (mills et al., 2004). the present study shows that copper sulfate completely inhibited the mycelial growth of p. italicum. however, mills et al. (2004) reported that copper sulfate had only a reduced effect on mycelial growth of phytophthora erythroseptica. sodium carbonate completely inhibited the mycelial growth of p. italicum. it has also a strong effect on geotrichum candidum as reported by talibi et al. (2011). palou et al. (2001), demonstrated that sodium carbonate had fungistatic rather than fungicidal activity against p. italicum which is consistent with our data. considering that several salt compounds could influence medium ph, the effect of ph on p. italicum growth was determined. the results showed that p. italicum grew at acidic ph as well as at alkaline ph, which agree with the results of talibi et al. (2011) concerning geotrichum candidum. the optimum growth of p. italicum was obtained in the range of 4.0 and 8.0 as colony growth was not significantly affected by ph modifications. panasenko (1967) reported that most of penicillium species could develop even at ph 2.0 and they are generally fruit contaminants. byrde and willets (1977) studtable 4. blue mold severity on oranges fruit ‘cv. clementine‘ treated with various concentrations of salts, and stored at 20°c and ~95% relative humidity for 5 days . salts disease severity (%) concentration (mm) 50 100 200 300 400 control 100,00 d 100,00 d 100,00 g 100,00 d 100,00 d sodium carbonate 94,88 cd 96,18 d 88,60 ef na* 23,81 b sodium metabisulfite 28,20 a 0,00 a 0,00 a na na boric acid na na 19,90 b na 0,00 a sodium sulfite 71,99 b 33,58 b 0,00 a 0,00 a 0,00 a sodium salicylate 86,12 bc 27,35 b 0,00 a 0,00 a 0,00 a potassium carbonate na na 75,50 de 52,98 b 44,70 b ammonium carbonate na na 98,35 fg 80,54 c 83,92 c sodium bicarbonate na na 94,02 fg 64,42 b 76,72 c copper sulfate 73,50 b 72,45 c 36,42 bc na na sodium edta 92,80 cd 60,14 c 55,22 cd na na values were the mean of three replicates. means followed with different letters in each column are statistically different according to duncan multiple range tests (p<0.05) applied after an analysis of variance of the arcsine of the square root of the percentage of lesion diameters. non transformed data are shown. *: not applied. ied the effect of ph on monilinia sp. growth and found that the specie can grow at varied ph (from 1.5 to 9.00) with optimum growth occurred under acidic ph. this ability of pathogens to grow over a wide range of ph, shows that differences in mycelial growth in salts amended medium (table 2) cannot be only due to the effect of ph. in addition, we have got a total inhibition in the radial growth of p. italicum both in the case of treatment with salt solutions at acidic or alkaline ph. hervieux et al. (2002) reported also that differences in the behavior of fungi toward salts could not be only due to the effect of ph. yaganza (2005) explained the adaptation of pathogens to a wide range of ph by several mechanisms known as “ph homeostasis” (white, 2000). these mechanisms exist in the cell membranes of pathogens. they maintain the stability of the macromolecules such as enzymes and therefore the growth and metabolism of these microorganisms. their mode of action is based on the regulation of ion transport across membranes, even when the extracellular ph varies significantly and this by means of the selectivity and energy coupling to the translocation of solutes (booth, 1988). sofos et al. (1986) reported that the inhibitory effect of salts to microorganisms could be due to the altered function of the transport cells and enzymes involved in the glycolytic pathway. for the in vivo tests, the results showed that sodium metabisulfite (100 and 200 mm), boric acid (400 mm), sodium salicylate and sodium sulfite (200, 300 and 400 mm) completely inhibited the development of blue mold on treated citrus fruit (table 3). talibi et al. (2011) also reported that boric acid and sodium salicylate applied at a concentration of 3% significantly reduced the incidence and severity of sour rot caused by g. candidum. although sodium salicylate was effective against both citrus blue mold and citrus sour rot, it was phytotoxic to fruit rind at all most tested concentrations. talibi et al. (2011) reported also that sodium edta significantly reduced the incidence and severity of sour rot. this is consistent with previous studies that have shown that sodium edta is effective against citrus green mold, caused by p. digitatum (valencia-chamorro et al., 2008), and against gray mold of apples caused by b. cinerea (droby et al., 2003). the present study showed that sodium edta significantly reduced the severity of postharvest citrus blue mold, but he had only a limited effect in reducing the incidence of the disease. in the current study, we found that sodium carbonate and potassium carbonate (400 mm) reduced significantly the incidence of citrus blue mold compared with the control. palou et al. (2009), reported that sodium carbonate and potassium carbonate reduced also the incidence of the decay caused by monilinia fructicola, botrytis cinerea, geotrichum candidum, and penicillium expansum in many stone fruit. however, treatment of citrus fruit with ammonium carbonate showed a high decay incidence of blue mold (current data), while talibi et al. (2011) reported that ammonium carbonate at 3% (312,2 mm) reduced the incidence of sour rot caused by g. candidum in postharvest citrus fruit by more than 51% and the severity by more than 74% . these behavioral differences of ammonium carbonate toward post-harvest fungi could be explained by changes in the 152 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ph of the environment of the wounds. indeed, g. candidum is a post-harvest fungus that leads to an increase in the ph of the medium. furthermore, palmer et al. (1997) reported that ammonium salts are effective under alkaline rather than acidic conditions, where the production of ammonia gas (nh3) is favored over the form (nh4+) which is inefficient. montesinos-herrero et al. (2011) reported that they effectively controlled post-harvest green and blue molds on lemons and oranges by applying ammonium in its active form, which is fumigation of fruit with a dose of ammonia gas not exceeding 6000 µl/l for 6 h at 22°c. these authors found also that germination of p. italicum conidia was more sensitive to the treatment compared with those of p. digitatum, and fumigation with ammonia gas could even control an isolate of p. digitatum resistant to the treatment with imazalil. conclusion the result of this study showed that among tested salt compounds boric acid, sodium metabisulfite, sodium sulfite, sodium salicylate, sodium carbonate and copper sulfate showed high antifungal activity against citrus blue mold in both in vitro and in vivo tests. the use of these compounds can be considered a useful strategy to be included in an integrated approach for controlling postharvest diseases of citrus fruit. however, the potential use of salt compounds to control postharvest diseases requires a detailed examination of their biological activity in vivo and the development of formulation which inhibits the growth of the pathogens at non-phytotoxic concentrations. acknowledgements we are grateful to the m’brouka cooperative for providing technical assistance and citrus fruits. references ameziane n, h boubaker, h boudyach, f msanda, a jilal and a ait benaoumar (2007) antifungal activity of moroccan plants against citrus fruit pathogens. agronomy for sustainable development 27(3): 273-277. arslan u, k ilhan and oa karabulut (2006) evaluation of food additives and low-toxicity compounds for the control of bean rust and wheat leaf rust. journal of phytopathology 154(9): 534-541. arslan u, k ilhan, c vardar and oa karabulut (2009) evaluation of antifungal activity of food additives against soilborne phytopathogenic fungi. world journal of microbiology and biotechnology 25(3): 537-543. askarne l, i talibi, h boubaker, e boudyach, f msanda, b saadi and a ait ben aoumar (2013) use of moroccan medicinal plant extracts as botanical fungicide against citrus blue mould. letters in applied microbiology 56(1): 37-43. askarne l, i talibi, h boubaker, eh boudyach, f msanda, b saadi, ma serghini and a ait ben aoumar (2012) in vitro and in vivo antifungal activity of several moroccan plants against penicillium italicum, the causal agent of citrus blue mold. crop protection 40(0): 53-58. booth i (1988) control of proton permeability: its implications for energy transduction and ph homeostasis. in: whittenburry r., banks j. g., gould g. w. and b. r. g. 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on florida citrus fruit using bicarbonate salts. pp. 375-378. zhu j-w, q-y xie and h-y li (2006) occurrence of imazalil-resistant biotype of penicillium digitatum in china and the resistant molecular mechanism. journal of zhejiang university science a 7(0): 362365. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 153 atlas journal of biology 2017, pp. 376–383 doi: 10.5147/ajb.2017.0153 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) additional polymorphisms linked to soybean cyst nematode resistance at the rhg4 locus oleksandra zatserklyana1,2, khalid meksem3, and david a. lightfoot2,3,* 1 department of biochemistry and molecular biology, southern illinois university carbondale, 1245 lincoln dr., mail code 4413, carbondale, il 62901; 2 genomics core facility; department of plant soil and agricultural systems, and center for excellence in soybean research, teaching and outreach, southern illinois university at carbondale, carbondale, il 62901; 3 department of plant, soil, and agricultural systems, southern illinois university carbondale, 1205 lincoln dr., mail code 4415, carbondale, il 62901. received: march 29, 2017 / accepted: april 20, 2017 __________________________________________________ * corresponding author: ga4082@siu.edu 376 abstract glycine max l. merr., (soybean) is one of the major crops in the united states, south america and asia. yet, the seed yield of soybean is significantly reduced due to heterodera glycines (ichinohe), the soybean cyst nematode (scn). scn is one of the most destructive pests and pathogens of soybean because the main methods for control have proven difficult. lineages of soybean that are resistant to scn have been developed but it is yet unknown what all the molecular causes of this resistance are. rhg1 and rhg4 loci are two of about 10 loci that underlie resistance. the rhg4 locus, is required for resistance to scn race 3 in ‘peking’–type derived resistances. one gene in the locus has been isolated and a causative link shown, but the surrounding regions have not been fully analyzed. focusing on the rhg4 locus, this study was aimed at uncovering other potential causes of resistance of soybean to scn. in a bioinformatic analysis of the rhg4 locus, alleles of nine genes were analyzed. the set of large intergenic regions have key regulatory elements in them. since partial resistances are often multigeneic, some of these nine genes could be candidates for causing and regulating resistance. keywords: soybean; nematode; resistance; rhg4. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction scn or soybean cyst nematode (heterodera glycines ichinohe,) is one of the most destructive pests of glycine max l. merr. (soybean) worldwide (matthews, 2004; kurle et al., 2015; yu q. 2015). due to the importance of g. max as an agricultural crop in the united states, the need for better management of scn-infested fields is pronounced, as crop yield can be reduced by up to 80% in the presence of scn in a field (smolik and draper, 2007; kazi et al., 2017). two loci involved in resistance to scn, are rhg1 and rhg4 (liu et al., 2012; liu et al., 2011). both rhg1 and rhg4 combine to encode resistance to the same type of scn race 3, hgtype0 (afzal et al., 2012; 2013; liu et al., 2012). rhg4 encodes resistance (brucker et al., 2005; liu et al., 2012) only in the ’peking’ – type of resistance that is the chief subject of this study. among the resistant lines that have been developed, none have been fully sequenced (schmutz eet al., 2010). among the partially sequenced resistant cultivars is ‘forrest’ (unpublished) that was used extensively in this study. the cultivars used that were susceptible to h. glycines were ‘williams 82’ [genbank: ef623856] and ‘asgrow 3244’ [genbank: gp062386.1]. deletions in these sequences can be seen (zatserklyana, 2015; tuteja and vodkin 2008; hauge et al., 2009a,b), introducing the problem of three sequences possibly not aligning correctly during genome mapping and recombination events. the origin of the forrest cultivar was from the peking lineage of soybeans (hartwig and epps 1973; liu et al., 2009). peking is a semi-wild, or semi-domesticated, black seeded g. max. the forrest cultivar was derived from peking lineage by introgression, of cnvs, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) snps, insertions and deletions at both the rhg1 and rhg4 loci (cook et al., 2012; liu et al 2012; srour et al., 2012). forrest cultivar sequence derived from two bac libraries using bamhi and hindiii enzymes (meksem et al., 2000). the construction of bac libraries and the physical map helped put the fragmented genome sequence of forrest together (shultz et al., 2006). although the forrest cultivar library and the bac libraries for the asgrow 3244 and the williams 82 susceptible cultivars were available, complete bac libraries did not exist for either cultivar (wu et al., 2004). wu et al. (2004) reported a complete physical map from three bac libraries derived from the forrest cultivar. as the bac libraries created complement each other, they expanded the number of clones used soybean genome analysis. the williams 82 susceptible cultivar, in contrast, arose largely through intra-cultivar genetic heterogeneity following or during introgression of phythophtora resistances (haun et al., 2011). not all the genes at or near or in the rhg4 locus have had their functions discovered (yi et al., 2010; center for integrative genomics 2017; liu et al., 2017). among those that have is chs7, which is the seventh gene in the chalcone synthase family, a hydroxyl-proline serine transferase and the i gene for seed coat color. the difficulty in fully mapping the rhg4 locus and the molecular markers associated with it arises out of hgtype variability, genomic variability in the soybean and the very small distance between this locus and i locus of less than 0.01cm (weisemann et al., 1992; lewers et al., 2001; wu et al., 2011). this relatively small genomic distance between the loci makes it difficult to map the rhg4 locus relative to the i locus at high resolution (weisemann et al., 1992). for rhg1 and rhg4 loci, both mapping and annotation are necessary to fully elucidate the full set of causes of resistance to all hgtypes of scn. genetic mapping alone may not be sufficient to show this (lewers et al., 2002). the annotation to date has shown extensive duplications that have taken place in bac 56g2, the bac where the williams 82 rhg4 susceptible sequence is located (lewers et al., 2002). here, nine genes were found in a bac derived from forrest that aligned poorly with williams 82 sequences, some of which could be candidates for causing and regulating resistance. further, large variations in intergenic regions were present that could have key regulatory elements within them. materials and methods bac sequences from forrest, asgrow 3244, and williams 82 soybean cultivars, and blastn, tblastx, tblastn, blastp, orf finder, and genemark.hmm were used. sequence from forrest cultivar was provided by dr. lightfoot from a bac sent to jcvi for sequencing. the other cultivar sequences were obtained through blast searches from ncbi (used for asgrow 3244) and phytozome (for williams 82). the nucleotide blast program at ncbi was run to differentiate between the two files containing the forrest sequence. the alignment of each of the files containing the forrest sequence separately gave the accession number of the bac williams 82 sequence. it was the accession number of the bac williams 82 sequence that was used in the alignments. blast and clustalw were used to do multiple alignments. with the blastn program, the alignment was being shown as broken up into sections. after the large gaps were found, needleman-wunsch was used to complete the alignment. completing the alignment included trimming the williams 82 56g2 bac sequence to the length of the forrest bac (92 kbp) and doing alignments with the trimmed williams 82 56g2 bac sequence. pairwise alignments and orf finder were used to search for genes and for what could be genes in the rhg4 region. the pairwise alignments were done using blastn, tblastx, and tblastn (mcginnis and madden, 2004; ye et al., 2006; johnson et al., 2008) using the forrest b100b10 bac dna sequence and the nr database, limiting the search to g max. the reason for this was to see what known genes, if any, could align with the forrest b100b10 bac dna sequence. the orf finder at ncbi (wheeler, 2003) complemented these blast searches by showing what other genes may be encoded in the forrest b100b10 bac dna sequence and where they may be located. genemark.hmm and blastp were used in annotating the forrest b100b10 bac sequence. genemark.hmm was used to predict the genes that are located on the forrest b100b10 sequence using the gff mechanism of the program and having genemark.hmm give the protein sequences as part of the output. blastp was then used to look for the possible amino acid changes in the protein sequence. results indels and snps all the insertions and deletions were looked at in forrest in comparison to both susceptible cultivars and to either of the susceptible cultivars as was the case in the region of alignment where williams 82 and asgrow 3244 overlap (supplementary table 1). this was the region from 850 bp to 7,466 bp. longer insertions and deletions the region of alignment between the forrest and williams 82 sequences showed a protein coding region (1,284 – 5,720 bp) in the deletions in forrest (supplementary table 1). out of the 1603 amino acids in this protein, nineteen amino acids were deleted from forrest in the process of the forrest cultivar becoming resistant to scn. sixteen of the nineteen deleted amino acids were nonpolar and hydrophobic, suggesting that this protein lies somewhere in one of the membranes in the cells. more than sixty percent of the deletions where codons have been deleted from the forrest cultivar were more at rich than gc rich (table 1). outside of the protein-coding region in the stretch of dna where forrest and williams 82 align, this becomes a pattern. 99.8% of the deletions in this region are at rich. it is possible these deletions result from forrest cultivar undergoing mutation while being strongly selected for resistance to scn during backcrossing. table 2 shows the deletions and how at rich they are over the entire forrest-williams 82 region. 377 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 378 snps the distribution of snps forms a more or less normal distribution through the forrest-williams 82 and the forrest-asgrow 3244 aligned dna. in the same region (through the 92.5% of the region of dna where the williams 82 sequence and the asgrow 3244 sequence align), the number of the insertions and deletions in snps differ by whether a nucleotide is a purine or a pyrimidine. a few snps that are insertions are found in pyrimidine nucleotides, the opposite is seen with the snps that are deletions (table 3). however, when the alignment of forrest b100b10 bac dna sequence and williams 82 56g2 bac dna sequence was analyzed, there were 508 snps that are result of a single nucleotide substitution. there is great diversity between the forrest b100b10 bac dna sequence and the williams 82 56g2 bac dna sequence. graphically, in the alignment between the forrest b100b10 bac dna sequence and the williams 82 56g2 bac dna sequence the two sequence appear to align well in small regions (figure 1-2). analysis of this alignment, by blast, showed the alignment as sections with gaps. hence the need for needleman-wunsch alignments. table 1. here are listed the deletions in the forrest-williams 82 protein-coding region and the percentage of at or gc that is present in each deletion. the deletions, which are indicated in column 1 by number, all come from the forrest sequence compared to the williams 82. columns 2 and 3 show the starting and ending base position of each deletions and while the numbers in columns 2 and 3 correspond to the base positions in asgrow, they are of use as this sequence overlaps with williams 82 in this region. columns 4 and 5 show the percentage of at and gc, respectively, and the last column shows codon. number start end length % at codon 1 96623 96625 3 33 tgg 2 96634 96636 3 66.7 gaa 3 96749 96751 3 66.7 atg start 4 96877 96879 3 33 ggt 5 96985 96987 3 67 ttg 6 97123 97125 3 67 cta 7 97168 97170 3 67 att 8 97274 97276 3 67 gta 9 97725 97727 3 66.7 not a codon 10 98135 98137 3 66.7 tac 11 98409 98411 3 66.7 cat 12 98554 98556 3 33.3 cac 13 98680 98682 3 33.3 cag 14 99114 99116 3 33.3 agc 15 99127 99129 3 100 ttt 16 99379 99381 3 33.3 gtg 17 100089 100091 3 33.3 gga 18 100655 1000657 3 33.3 gga 19 101159 101161 3 66.7 ttc 20 101256 101258 3 33 gag 21 101322 101324 3 100 att 22 101337 101339 3 66.7 gta 23 101436 101438 3 66.7 ctt 24 101556 101558 3 100 taa stop table 2. here is listed the deletions in the entire forrest-williams 82 region and the percentage of at or gc that is present in each deletion. the deletions, which are indicated in column 1 by number, all come from the forrest sequence in comparison to the williams 82. columns 2 and 3 show the starting and ending base position of each deletions and while the numbers in columns 2 and 3 correspond to the base positions in asgrow, they are of use as this sequence overlaps with williams 82 in this region. column 4 shows the length of each deletion and was the basis of sorting the values of the table for the graph. column 5 shows how at rich each of the deletions is while the percentage of how gc richness each deletion is shown in column 6. number start end length % at 1 96623 96625 3 33 2 96634 96636 3 66.7 3 96749 96751 3 66.7 4 96877 96879 3 33 5 96985 96987 3 67 6 97123 97125 3 67 7 97168 97170 3 67 8 97274 97276 3 67 9 97725 97727 3 66.7 10 98135 98137 3 66.7 11 98409 98411 3 66.7 12 98554 98556 3 33.3 13 98680 98682 3 33.3 14 99114 99116 3 33.3 15 99127 99129 3 100 16 99379 99381 3 33.3 17 100089 100091 3 33.3 18 100655 100657 3 33.3 19 101159 101161 3 66.7 20 101256 101258 3 33 21 101322 101324 3 100 22 101337 101339 3 66.7 23 101436 101438 3 66.7 24 101556 101558 3 100 25 101999 102001 3 100 26 102046 102048 3 66.7 27 102253 102255 3 100 28 102727 102729 3 67 29 103201 103203 3 66.7 30 101890 101893 4 50 31 96436 96439 4 25 32 96792 96795 4 50 33 97640 97644 4 60 34 97903 97906 4 75 35 98216 98219 4 25 36 98424 98427 4 25 37 98647 98650 4 50 38 98662 98665 4 75 39 98938 98941 4 50 40 99183 99186 4 60 41 99800 99804 4 40 42 100034 100037 4 75 43 101092 101095 4 50 44 101105 101108 4 50 45 101293 101296 4 100 46 101924 101927 4 50 47 101946 101949 4 50 48 102124 102127 4 50 49 102231 102234 4 50 50 103126 103129 4 50 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 379 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) needleman-wunsch with the subrange for the query and subject sequences set to 10 kbp for the first nine sub-regions, and then to 1336 bp the tenth sub region, the needleman-wunsch algorithm was able to make the alignment. between 1 and 200 bp, 201 and 400 bp, 1 and 1000 bp, 90,001 and 94,336bp there were many gaps in table 2. continued. number start end length % at 51 96314 96318 5 40 52 96447 96451 5 100 53 97284 97288 5 60 54 97315 97319 5 60 55 97411 97415 5 80 56 97864 97868 5 80 57 97965 97969 5 60 58 98330 98334 5 0 59 98704 98708 5 60 60 98752 98756 5 80 61 98881 98885 5 60 62 100834 100838 5 40 63 101391 101395 5 60 64 101527 101531 5 60 65 101690 101694 5 60 66 102389 102393 5 80 67 102423 102427 5 80 68 102990 102994 5 100 69 97196 97201 6 83.3 70 97970 97975 6 83.3 71 97985 97990 6 66.7 72 98715 98720 6 66.7 73 103174 103179 6 66.7 74 97253 97259 7 85.7 75 97336 97342 7 57.1 76 97813 97819 7 57.1 77 101303 101309 7 100 78 101580 101586 7 100 79 96378 96385 8 75 80 96465 96472 8 75 81 97385 97392 8 75 82 97710 97717 8 50 83 98381 98388 8 25 84 100609 100616 8 50 85 101673 101680 8 87.5 86 101982 101989 8 75 87 96402 96410 9 22.2 88 96981 96989 9 87.5 89 98121 98129 9 55.6 90 98617 98625 9 27.8 91 100780 100788 9 66.6 92 101265 101273 9 88.9 93 101281 101289 9 66.7 94 97268 97277 10 60 95 100710 100719 10 90 96 101444 101453 10 60 97 97678 97688 11 81.2 98 101535 101545 11 81.8 99 102951 102962 12 75 100 96330 96343 14 46.2 101 102885 102898 14 14.3 102 97474 97488 16 50 103 96850 96866 17 68.8 104 98584 98600 17 68.8 105 99412 99429 18 94.1 figure 1. the alignment between the forrest b100b10 bac dna sequence (y axis) and the williams 82 56g2 bac dna sequence (x axis) that had been trimmed to the length of the forrest b100b10 bac dna sequence of 93.7bp. figure 2. the alignment of the rhg4 gene between the forrest b100b10 bac dna sequence (x axis) and the williams 82 56g2 bac dna sequence (y axis) from 1-200 bp. figure 3. the alignment of the rhg4 gene between the forrest b100b10 bac dna sequence (x axis) and the williams 82 56g2 bac dna sequence (y axis) from 201-400 bp. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 380 gene # gene name strand start end snps amino acid changes 1 serine hydroxymethyltransferase 1441 2757 1593 a to t gniyrigslpsgfdll qmsinltcslcdcfa 2 adenosylhomocysteinase + 10044 12138 no substitutions v238f, m239d, s240n, f242y, p243g, crhsl, l24g6, v247l, sflc 3 predicted heat stress transcription factor a-5-like isoform x2 20983 21828 no substitutions no amino acid changes 4 unknown glycine max gene + 25127 25432 no substitutions r10f, k11p, t12q, y13t, v14i, g15d, a19s, a22t, k24e, v25i, l28f, r32g, e33k, k34e, e35r, a36d, s37g, g40s, r41d, p43g, h44d, e46k, v49t, g50a, g51t, r54t, n55d, v57r, v58i, g59s, g60v, g61a, i62v, v63f, l66i, g68t, l69f, e70f, k71q, r75h, t7r6, t77n, g79t, l80r, w81t, r82v, t83l, h85r 5 predicted putative pentatricopeptide repeat-containing protein at1g12700 mitochondrial isoform x2 35243 36895 no substitutions no amino acid changes 6 predicted microfibrillar-associated protein 1 + 37980 39182 38375 g to a, 38436 g to c, 38459 g to a no amino acid changes 7 receptor-like kinase, leucine-rich repeat receptor-like kinase 73853 74395 73866 g to a no changes 8 leucine-rich receptor-like kinase, rhg4-like receptor kinase, receptor-like kinase 75072 77252 76596 a to t, 77124 c to a g722a 9 predicted anthocyanidin 3-o-glucoside 5-o-glucosyltransferase 1-like 83975 85411 84209 t to c, 84232 c to t, 84239 g to c, 84242 c to t, 83245 c to a, 84251 g to t, 84257 c to t, 84260 a to g, 84265 t to a, 84279 g to a, 84287 t to g, 84290 a to c, 84302 a to c, 84308 t to c, 84326 c to a, 84335 t to c, 84337 t to c, 84340 a to c, 84386 c to a, 84395 t to c, 84397 t to a, 84416 t to c, 84504 c to g, 84520 g to t, 84534 a to g, 84552 t to a, 84563 a to c, 84574 c to t, 84584 c to g, 84589 t to c, 84590 c to g, 84611 t to c, 84621 t to c no amino acid changes table 3. snps that result from single nucleotides substitutions and the changes in amino acids they cause, along with the genes these snps are expected to be located in. red letters indicate insertion and blue letters indicate deletion. (the information in this table is up to date as of august 14, 2015). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 381 the alignment (figures 3-5). alignments inferred the exact (already known) location of rhg4 gene (liu et al., 2012). using the trimmed and untrimmed version of the williams 82 bac sequence, the query coverage percentages were compared. for the alignment of forrest as the query sequence and the trimmed williams 82 bac sequence as the subject sequence, the query coverage was 97% while for the alignment of forrest as the query sequence and the untrimmed williams 82 bac sequence (starting from the point in the williams 82 bac sequence where forrest sequence would end) as the subject sequence it was only 3%. orf finder an unknown was how many of the deleted or inserted codons (table 1) were in the coding region of proteins. in total, in the forrest b100b10 bac sequence the orf finder found 327 orfs (open reading frames), six of which are greater than 1,000 bp in length (figure 6). one open reading frame was similar to serine hydroxyl-methyl serine transferase mrna (1,317 bp for the open reading frame and 1,416 bp for the mrna) but was incomplete on the bac. annotation another unknown was which snps, caused changes in the amino acid sequences. genemark.hmm predicted ten genes within the forrest b100b10 bac dna sequence, but manual annotation inferred there were nine genes in the forrest b100b10 bac dna sequence, as genes 2 and 3 may be one gene. in contrast to the large number of snps that were found in the forrest b100b10 bac dna sequence that are the result of single nucleotide substitutions, the number of amino acid changes and the number of snps in the nine coding regions were 56 and 40, respectively. the results of the genemark.hmm prediction of genes in the forrest b100b10 bac dna sequence and blastp, along with the snps and amino acid changes were shown in table 3. discussion forrest cultivar sequence resistant to h. glycines and the susceptible asgrow 3244 and williams 82 cultivar sequences were used in this project. deletions in these sequences can be seen (tuteja and vodkin 2008; hauge et al., 2009a,b; zatserklyana, 2015) which can caused the cultivar sequences to not align correctly, partly because the cultivars retain heterologous regions (haun et al., 2011) and partly because bacs are single alleles cloned from within that variation. multiple sequence alignments and needleman-wunsch global alignments were the key tools in the project. a deleted peptide was noted and the size of insertions and/ or deletions was notably larger compared to the alignment around rhg1 (hemmati et al., 2017). transposons often cause indels and may be responsible for altering the degree and type of scn resistance. in the rhg4 region are predicted to be nine genes, four of which are genes with predicted functions (heat stress transcription factor a-5-like isoform x2, putative pentatricopeptide repeat-containing protein at1g12700 mitochondrial isoform x2, microfibrillar-associated protein 1, and anthocyanidin 3-o-glucoside 5-o-glucosyltransferase 1-like; figure 6). figure 4. the alignment of the rhg4 gene between the forrest b100b10 bac dna sequence (x axis) and the williams 82 56g2 bac dna sequence (y axis) from to 1-1000 bp. figure 5. the alignment of the rhg4 gene between the forrest b100b10 bac dna sequence (x axis) and the williams 82 56g2 bac dna sequence (y axis) from 89 kbp to 91 kbp. figure 6. the overview of the rhg4 region showing the seven genes, regulatory elements and extent of the rhg4 region in relation to forrest b100b10 bac dna sequence. if we look at these genes as though they would be located on one strand, if transcription would occur, we would see ahc, unknown g. max gene, and map 1 genes being transcribed to the right and shtm (rhg4 gene) , hstf a-5-like isoform x2, pprcp at1g12700 ml isoform x2, rlk, lrr-rlk, and ac 3-o-g 5-o-g 1-like genes being transcribed to the left. (the information in this figure is up to date as of august 14, 2015). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 382 the major gene at the rhg4 region was at the end of the forrest bac sequence, a serine hydroxymethyltransferase. it is a gene proven to be involved in the resistance of soybean to scn (liu et al., 2012; lakhssassi et al., 2012; 2017; liu et al., 2017). this study confirmed those earlier findings and showed the complexity of the introgressed region. strong selection pressures on wild and semi-domesticated soybeans to develop resistance to scn were inferred (han et al 2016). authors’ contributions dal and oz did the analysis, with oz actively carrying out the bioinformtics (dal generated the genemark.hmm file) and dal guided the work. km isolated the bac and wasco-pi on the grant that sequenced it. the text of this article was based on the ms thesis written by oz that dal helped edit. acknowledgements we thank dr. khalid meksem for creating the clone. he persisted, away from home, at tamu, to pick the 100th plate of the second library. we thank dr. hogbin zhang for hosting khalid and teaching the bac library creation methods. we thank dr. naghmeh hemmati for her help with downloading the ‘williams 82’ sequence and submitting ‘forrest’ sequence to genbank as hq008938.1. the sanger sequencing was paid for by the united soybean board. dr. christopher town of jcvi is thanked for supervising the sequencing and assembly. dr. matthew jb geisler is thanked for providing lists of newly discovered cre sequences prior to his own publications. conflicts of interest none exist for any co-author. references afzal aj, a srour, a goil, s vasudaven, t liu, r samudrala, n dogra, p kohli, a malakar, and da lightfoot (2013) dimerization, ligand binding and structural prediction validation with a leucine rich repeat domain associated with soybean resistance to cyst nematodes and fusarium sp. bmc plant biology 13:43-53 afzal aj, a srour, n saini, n hemmati, ha el shemy, and da lightfoot (2012) recombination suppression at the dominant rhg1/rfs2 locus underlying soybean resistance to the cyst nematode. theor appl genet 124:1027-1039 doi10.1007/s00122-011-1766-6. brucker e, s carlson, e wright, t niblack, and b diers (2005) rhg1 alleles from soybean pi 437654 and pi88788 respond differentially to isolates of heterodera glycines in the greenhouse. theor appl genet 111:44-49. center for integrative genomics (2017) genetic genome browser, version 2.39. in: phytozome. http://www.phytozome.net/cgi-bin/ gbrowse/soybean/. accessed 5 may 2015. grant d, rt nelson, ma graham, and rc shoemaker (2008) bioinformatic resources for soybean genetics and genomic research. in: stacey g, editor. genetics and genomics of soybean. new york: springer-verlag llc, new york, pp144-63. han y, x zhao, d liu, y li, da lightfoot, z yang, l zhao, g zhou, z wang, l huang, and z zhang (2016) domestication footprints anchor genomic regions of agronomic importance in soybeans. new phytologist, 209 (2): 871-884 hartwig ee and jm epps (1973) registration of ‘forrest’ soybeans. crop. sci. 13:287-287doi 10.2135/cropsci1973.001183x00130 00200047x. hauge bm, ml wang, jd parson, and ld parnell (2009a) methods of introgressing nucleic acid molecules associated with soybean cyst nematode resistance into soybean. united states patent us7485770-a 4. hauge bm, ml wang, jd parson, and ld parnell (2009b) sequence 4 from patent us 7485770. http://www.ncbi.nlm.nih.gov/nuc leotide/223623398?report=genbank&log$=nucltop&blast_ rank=1&rid=jgtug796016. in: ncbi genbank, accessed 22 june 2015. haun wj, dl hyten, ww xu, dj gerhadt, tj albert, t richmond, ja jeddeloh, g jia, nm springer, cp vance, and rm stupar (2011) the compositions and origins of genomic variation among individuals of the soybean reference cultivar williams 82. plant physiol 155: 645-655. hemmati nh, mjb geisler and da lightfoot (2017) annotation of cultivar variations at the multigeneic rhg1/rfs2 locus: polymorphisms underling alterations of root development and pest resistance. teor appl genet (in revision). johnson m, i zaretskaya, y raytselis, y merezhuk, s mcginnis, and tl madden (2008) ncbi blast: a better web interface. nucleic acids res 36:w5-w9 doi 10.1093/nar/gkn201. kazi s, j shultz, j afzal, y-c lee, and da lightfoot (2017) identification of loci underlying seed yield in recombinant inbred and near isogeneic soybean lines derived from flyer by hartwig. atlas journal of biology 2017, pp. 355-363. doi: 10.5147/ajb.2017.0149. kurle j, d malvick, b potter, and j orf (2015) soybean cyst nematode management guide. http://www.extension.umn.edu/agriculture/soybean/soybean-cyst-nematode/efans-soybean-soybeancystnematode-webquality.pdf accessed 13 october 2015. lakhssassi n, s liu, s bekal, z zhou, v colantonio, k lambert, a barakat, and k meksem (2017) characterization of the soluble nsf attachment protein gene family identifies two members involved in additive resistance to a plant pathogen. scientific reports 7, 45226. lakhssassi n, vg doblas, a rosado, ae del valle, d pose, aj jimenez, ag castillo, v valpuesta, o borsani, and ma botella (2012) the arabidopsis tetratricopeptide thioredoxin-like gene family is required for osmotic stress tolerance and male sporogenesis. plant physiol 158: 1252-1266. lewers k, r heintz, h beard, l marek, and b matthews (2002) a physical map of a gene-dense region in soybean linkage group a2 near the black seed coat and rhg4 loci. theor appl genet 104: 253260. lewers ks, sd nelmalgoda, al warner, ht knap, and bf matthews (2001) physical mapping of resistant and susceptible soybean genomes near the soybean cyst nematode resistance gene rhg4. genome 44: 1057-1064. liu s, pk kandoth, n lakhssassi, j kang, v colantonio, r heinz, g yeckel, z zhou, s bekal, j dapprich, b rotter, s cianzio, mg mitchum, and k meksem (2017) the soybean gmsnap18 gene underlies two types of resistance to soybean cyst nematode. nature communications, 8, 14822. liu s, pk kandoth, n lakhssassi, j kang, v colantonio, r heinz, g yeckel, z zhou, s bekal, j dapprich, b rotter, s cianzio, mg mitchum, and k meksem (2017) the soybean gmsnap18 gene underlies two types of resistance to soybean cyst nematode. nature communications 8, 14822. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 383 liu s, pk kandoth, sd warren, g yecke, r heinz, j alden, c yang, a jamai, t el-mellouki, ps juvale, j hill, tj baum, s cianzio, sa whitham, d korkin, mg mitchum, and k meksem (2012) a soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens. nature 492: 256–260. liu x (2009) molecular characterization of soybean resistance to soybean cyst nematode https://mospace.umsystem.edu/xmlui/ bitstream/handle/10355/9861/research.pdf?sequence=3. accessed 2012. liu x, s lui, a jamai, a bendahmane, da lightfoot, mg mitchum, and k meksem (2011) soybean cyst nematode resistance in soybean is independent of the rhg4 locus lrr-rlk gene. funct integr genomics doi 10.007/s10142-011-0225-4. matthews b (2004) genomic approaches for developing soybeans with resistance to pests (soybean cyst nematode) in stalker te, brummer c, and wilson rf, eds., legume crop genomics, aocs press. p. 312-25. mcginnis s and tl madden (2004) blast: at the core of a powerful and diverse set of sequence analysis tools. nucleic acids res 32: w20-w25 doi 10.1093/nar/gkh435. meksem k, k zobrist, e ruben, d hyten, t quanzhou, hb zhang, and da lightfoot (2000) two large-insert soybean genomic libraries constructed in a binary vector: applications in chromosomes walking and genome wide physical mapping. theor appl genet 101: 747-755. shultz jl, d jayaraman, kl shopinski, mj iqbal, s kazi, k zobrist, r bashir, s yaegashi, n lavu, aj afzal, cr yesudas, ma kassem, c wu, hb zhang, cd town, k meksem, and da lightfoot (2006) the soybean genome database (soygd): a browser for display of duplicated, polyploid, regions and sequence tagged sites on the integrated physical and genetic maps of glycine max. nucleic acid research 34: d758-765. smolik jd and ma draper (2015) soybean cyst nematode. http:// pubstorage.sdstate.edu/agbio_publications/articles/fs902a.pdf. revised 2007. accessed 8 july 2015. srour a, aj afzal, n saini, l blahut-beatty, n hemmati, dh simmonds, h el shemy, cd town, h sharma, x liu, w li, and da lightfoot (2012) the receptor like kinase transgene from the rhg1/rfs2 locus caused pleiotropic resistances to soybean cyst nematode and sudden death syndrome. bmc genomics 13:368-377 tuteja jh and lo vodkin (2008) structural features of the endogenous chs silencing and target loci in the soybean genome. crop sci 48: s49-s68 doi:10.2135/cropsci2007.10.0542tpg. weisemann jt, bf matthews, and te devine (1992) molecular markers located proximal to the soybean cyst nematode resistance gene, rhg4. theor appl genet 85: 136-138. wheeler dl, dm church, s federhen, ae lash, tl madden, ju pontius, gd schuler, lm schriml, e sequeira, ta tatsunova, and l wagner (2003) database resources of the national center for biotechnology. nucleic acids res 3: 28-33. wu cc, p nimmakayala, fa santos, r springman, c scheuring, k meksem, da lightfoot, and hb zhang (2004) construction and characterization of a soybean bacterial artificial chromosome library and use of multiple complementary libraries for genome physical mapping. theor appl genet 109: 1041-1050. doi: 10.1007/s00122004-1712-y. wu x, td vuong, jl leroy, jg shannon, da sleper, and ht nguyen (2011) selection of a core set of rils from forrest × williams 82 to develop a framework map in soybean. theor appl genet 122: 1179-1187. doi 10.1007/s00122-010-1522-3. ye j, s mcginnis, and tl madden (2006) blast: improvements for better sequence analysis. nucleic acids res. 34: w6-w9. doi: 10.1093/nar/gkl164. yi j, mr derynck, l chen, and s dhaubhadel (2010) differential expression of chs7 and chs8 genes in soybean. planta 231: 741753 doi 10.108/s00425-009-1079-z. yu q (2015) soybean cyst nematode (heterodera glycines ichinohe) (2015) http://cdn.intechopen.com/pdfs/22782/intech-soybean_ cyst_nematode_heterodera_glycines_ichinohe_.pdf. accessed 1 october 2015. zatserklyana o, k meksem, and da lightfoot (2015) glycine max cultivar forrest clone bac 73p06 genomic sequence. in: ncbi genbank. http://www.ncbi.nlm.nih.gov/nuccore/hq008938.1. accessed 10 september 2015. kbm journal of biology (2010) 1 (2): 34-40 doi: 10.5147/kbmjb.2010.0018 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) risk factors affecting the phenotypic expression of heterozygous familial hypercholesterolemia in pakistani population fauzia imtiaz1, wajahatullah khan2*, and ahsan a. vahidy3 1 department of biochemistry, dow international medical college, dow university of health sciences, baba-eurdu road, po box 942,943, karachi 74200, pakistan. 2 biochemistry department, king saud university, po box 2455, riyadh 11451, saudi arabia. 3 department of genetics, university of karachi, karachi pakistan. received: february 16, 2010 / accepted: may 27, 2010 abstract familial hypercholesterolemia (fh) is a genetic disorder of lipoprotein metabolism recognized by high plasma concentrations of low-density lipoprotein cholesterol (ldlc), tendon xanthomas, and high risk of early coronary heart disease. the phenotypic expression of fh is variable and several genetic and other risk factors contribute to such variability in clinical expression of the disease. fh is an autosomal disorder characterized by increased levels of total cholesterol (tc), low density lipoprotein cholesterol (ldl-c) and tendinous xanthmata (tx). clinical phenotype of fh has previously been shown to be associated with increased coronary heart disease (chd) and premature death. the present study was carried out in a pakistani population to examine the contribution of environmental factors on the expression of this disease. a detailed examination of the physical and biochemical features of the fh was performed in a cohort of 335 individuals (202 males, 133 females) from pakistan. our results indicate that pakistani females with fh had high levels of tc and ldl-c as compared to pakistani males. overall the concentrations of high density lipoprotein cholesterol (hdl-c) were significantly lower for both sexes as compared to the controls (normal). we have also found that high cholesterol levels were associated with increased incidence of chd in fh pakistani population where both male and female individuals showed 69% and 31% chd, respectively. the mean onset age for coronary symptoms was about 38 years in males as compared to 45 years in females. a greater risk of developing chd has been linked with levels of tc along with a history of smoking in males and presence of hypertension (htn) in females. our results indicate that these risk factors possibly affect and contribute in the phenotypic expression of fh in pakistani population. __________________________________________________ * corresponding author: wkhan@ksu.edu.sa keywords: phenotypic expression, familial hypercholesterolemia, risk factors, coronary heart disease, smoking, hypertension, total cholesterol, ldl-c introduction familial hypercholesterolemia (fh) is a genetic disorder associated with lipoprotein metabolism, where elevated levels of low-density lipoprotein cholesterol (ldl-c) occur due to a mutation in the ldl receptor gene (austin et al., 2004; kwiterovich, 2008) to which ldl-c bind and remove it from the bloodstream. however, people with fh have less ldl receptors which result in increased than normal levels of ldl-c in the blood. transmitted in an autosomal dominant fashion, fh is also characterized by the presence of tendinous xanthomas (tx) and premature atherosclerosis. (alonso et al., 2009). fh is also one of the most common inherited diseases in the world, with a worldwide frequency of 1 in 500 for heterozygotes and 1 per million for homozygotes or compound heterozygotes (goldstein et al., 2001). however, a higher incidence rate can be found in certain populations, for example the afrikaners, christian lebanese, finns, and french-canadians, because of the founder effects (goldstein et al., 2001). patients with heterozygous fh express high levels of plasma ldl-c at a young age, causing atherosclerosis and an increased risk of cardiovascular diseases (austin et al., 2004). heterozygous fh patients show about twoto three-fold increase in ldl-c concentrations with a decrease in high-density lipoprotein (hdl-c) levels, along with tendinous xanthomatosis and premature heart disease (chd) between the age of 35 and 55 years (gagné et al., 1979; civeira, 2004). about half of the offspring of an affected parent will have an increased level of cholesterol in the plasma and both genders are equally 34 35 affected. although, a large number of fh cases are due to the mutations in the ldl receptor gene (ldlr), additional contributory genes have also recently been reported for fh. nevertheless, the mutations in these genes appear to be rare in the populations that have so far been investigated (abifadel et al., 2003; rader et al., 2003; damgaard et al., 2004; graham et al., 2005). advancing age and male gender are strongly associated with chd risk, with men typically developing disease symptoms 10–15 years earlier than women who generally do not show such symptoms until after menopause (aha, 2001). however, the course of chd symptom in fh is different, with some patients expressing events earlier than others inspite of similar increased ldl-c levels. hence other factors may play a part in chd risk in these individuals. earlier studies in european and north-american populations have established the influence of risk factors like age, smoking and hypertension (htn) in fh individuals (civeira, 2004). among the environmental risk factors, smoking is the major contributor and has been associated with a nearly two fold higher risk (hill et al., 1991). lack of exercise and the related adiposity, including high intake of saturated fats and a reduced intake of certain vitamins have also been linked with increased risk (aha, 2001). the mechanism of action of these factors is by determining the differences in the plasma levels of lipids and lipoproteins that are atherogenic and high levels of ldl-c and low levels of hdl-c have been associated with chd (castelli et al., 1986). evidence for strong genetic component for chd risk is supported by the association between a family history of early chd and a personal increased risk (kardia et al., 2003) in the order of 1.7-fold higher (hawe et al., 2003). the clinical phenotype for homozygotes is more severe than heterozygotes; however the expression of heterozygous fh is highly variable probably due to other risk factors in addition to the genes affecting the lipoprotein metabolism (bertolini et al., 2004). we have previously reported that the cause of fh in pakistani population is partly due to familial resemblance contributing due to the genes but other risk factors may also play a role in the progression of the fh disease in pakistani population (imtiaz, 2009). in the present study, we studied and assessed the contribution of risk factors on the phenotypic expression of fh disease in a pakistani population. materials and methods the study, upon written informed consent approval, included a group of 1523 unrelated patients with the clinical diagnosis of heterozygous fh. a detailed examination of the physical and biochemical features of fh was obtained in this cohort. clinical examination physical fh characteristics the physical characteristics that were examined in fh patients included presence of tendinous xanthoma (tx), xanthelesma, arcus cornea (ac) and polyarthritis. a xanthelasma is a clearly distinguishable yellowish collection of cholesterol underneath the skin, typically on or in the region of the eyelids. the xanthelasma is a distinctive condition and is called a xanthoma when it becomes bigger and nodular. the tx is clinically characterized by papules and nodules found in the tendons of the hands. the ac is due to the white arc appearance on the cornea as a result of abnormal deposits of phospholipids and cholesterol. the polyarthritis is a condition when any type of arthritis involving five or more joints of the body was present in patients. three hundred thirty-five (335) subjects fitting to “definite fh” criteria had increased levels of ldl-c, presence of early heart attacks in the family and tendon xanthomatosis. five probands were examined and their family pedigree trees were constructed to observe the dominant inheritance pattern of the disease. biochemical analysis the biochemical tests were performed following overnight (12–14 hrs) fasting. about 5 ml blood samples were drawn to analyze the total cholesterol (tc) levels, triglyceride (tg), hdl and ldl. the concentration of serum cholesterol, hdl-c, and tg were determined enzymatically by the chod-pap and gpopap methods (boehringer mannheim, germany) (tietz, 1986). the hdl-c was isolated from serum by heparin manganese precipitation of the other lipoproteins. all the lipid profile analysis of tc, hdl-c, ldl-c and tg concentrations were observed in the serum of all 1523 pakistani individuals diagnosed with fh, cholesterol content of the serum ldl-c was estimated using friedewald formula (friedewald et al., 1972). statistical analysis the obtained data were analyzed for correlation among different risk factors using the statistical package for social sciences version 12 (spss, inc). statistically significant differences between groups or among groups for continuous variables were evaluated using student’s t test for unpaired data and anova respectively. the pedigree was made by using the computer based software cyrillic version 2.10 (oxford, uk). results our previous study on the heritability of pakistani fh population showed that the genetic factors are the major determinant of fh, however because heritability is the proportion of variation due to additive familial effects of genetic and nongenetic sources, therefore we concluded that other non-genetic risk factors may also contribute in the progression of the disease (imtiaz, 2009). the present study was conducted to examine the involvement of risk factors such as age, sex, htn, smoking and tc concentrations in the phenotypic expression of heterozygous familial hypercholesterolemia in a cohort of pakistani population living in the city karachi. we have studied a total of 1523 unrelated patients with the clinical diagnosis of heterozygous fh. out of these 1523, only 335 subjects with “definite fh” diagnosis belonged to five probands with cholesterol level >300 mg dl-1 (normal range 140-240 mg dl-1). among them kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 36 202 were males and 133 were females. upon clinical physical examination, out of these 335 individuals (202 males and 133 females) 224 (70%) showed tx. a total of 146 males (72%) and 78 females (58%) aged between 30-40 showed tx symptoms (table 1). xanthelesma was detected in 258 individuals (80%), i.e. 148 males (73%; aged 40-50) and 110 females (82 %; aged 30-40). the ac was observed in a total of 224 patients (70%), where 63% males (128 individuals) aged 40-50 and 72% females (96 individuals) aged 30-40 showed the ac symptoms. the polyartheritis symptoms were less common among the observed individuals and only 7 individuals (2%) have this condition. among the 202 males (aged 40-50), only 2 males (0.9%) and 5 females (3.7%) showed the clinical symptoms of polyartheritis (table 1). hence, the occurrence of xanthoma (i.e. tendinous xanthomata and xanthelesma) in the present study in general was about 70% in the patients diagnosed with fh. a detailed examination of the physical and biochemical features of fh of cohort of 208 females and 156 males showed that females with fh had higher levels of total ldl-c (196 to 358 mg dl-1) compared to the male subjects (201 to 301 mg dlkb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 1) (figure1), while having lower hdl-c levels (25-55 mg dl-1) in both genders compared to the normal (figure 1). our results also showed that the mean age of fh showing the symptoms in males was earlier than normal, i.e. 38 years compared to 45 years in females. the fh disease prevalence rate was 69% in males compared to 31% in females. the risk factors such as total cholesterol (tc), smoking and htn were found to be highly correlated with fh appearance. we observed that smoking and htn were significantly (p < 0.05) correlated. similarly, a high correlation (p < 0.05) was found between smoking and tc in the individuals with fh. however, tc and htn were even more strongly correlated with fh at p < 0.01 (table 2). the risk factors such as smoking and htn exhibited significant effects in the appearance of the disease. our study showed that smoking and htn were significantly correlated with the level of total cholesterol at p<0.05 (0.047) and p<0.01 (0.000), respectively (table 2). the smoking in males had profound effect in observed individuals, where we noticed that 70% male smokers were positive for chd. i:1 (2 60 m g/ dl ) i:2 ii:1 (3 58 m g/ dl ) ii:2 (2 70 m g/ dl ) ii:4 (2 51 m g/ dl ) ii:3 (2 00 m g/ dl ) ii:6 (2 41 m g /d l ) ii:5 ( 2 01 m g/ dl ) ii:7 (2 50 m g/ dl ) ii:8 ii:9 (2 58 m g/ dl ) ii:10 i:3 i:4 ii:11 (1 96 m g/ dl ) ii:12 (2 09 m g/ dl ) ii:13 (2 00 m g/ dl ) ii:15 (3 01 m g/ dl ) ii:14 (2 42 m g/ dl ) ii:16 (2 19 m g/ dl ) iii:1 (2 90 m g/ dl ) iii:2 (2 41 m g/ dl ) iii:3 (2 28 m g/ dl ) iii:4 (2 60 m g/ dl ) iii:5 (2 18 m g/ dl ) iii:6 (2 39 m g/ dl ) iii:7 (2 11 m g/ dl ) iii:9 (2 40 m g/ dl ) iii:10 (2 48 m g/ dl ) iii:8 (2 22 m g/ dl ) iii:11 (2 30 m g/ dl ) iii:12 (2 14 m g/ dl ) iii:13 (2 21 m g/ dl ) iii:14 (2 00 m g/ dl ) iii:16 (2 12 m g/ dl ) iii:15 (2 00 m g/ dl ) iii:17 (2 14 m g/ dl ) iii:18 (2 20 m g/ dl ) iii:19 (2 58 m g/ dl ) iii:20 (2 40 m g/ dl ) iv:3 (2 00 m g/ dl ) iv:1 (2 12 m g/ dl ) iv:2 (2 16 m g/ dl ) iv:5 (1 96 m g/ dl )iv:4 (2 09 m g/ dl ) figure 1. fig. 1. pedigree showing the presence of fh in every generation of the proband confirming the dominant inheritance pattern. the amount of ldl-c is mentioned as mg/dl. 37 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) discussion fh is a public health problem throughout the world and many heterozygous fh remain undetected until adulthood. the present study was conducted to study the contribution of risk factors (age, sex, smoking and hdl-c concentrations) in the phenotypic expression of heterozygous fh in a pakistani population. fh may also cause premature coronary artery disease and atherosclerotic plaque formation and is considered as one of the first inherited disorders as being a cause of myocardial infarction (heart attack). in the present study, out of a total of 1523 unrelated patients with the clinical diagnosis of heterozygous fh, only 335 individuals diagnosed with fh belonged to five probands (202 males and 133 females). similar criteria have previously been used to identify patients with fh based on high plasma levels of total and ldl cholesterol, family history of hypercholesterolemia, tendon xanthomas or corneal arcus, and personal and family history of premature chd (goldstein et al., 2001). the fh patients have ldl-c levels almost twice than the normal population (190 to 400 mg dl-1). the clinical examination of the 335 individuals with fh showed that 72% males and 58% females had tx at ages between 30-40 years. xanthelesma was found in 80% males (aged 40-50) and in 82 % females (aged 30-40). the ac was observed in 63% males (aged 40-50) and 72% females (aged 30-40). a very few individuals (2% i.e. 7 individuals) showed polyartheritis symptoms among them were only 2 males (0.9%) aged 40-50; while 5 females (3.7%) showed the disease symptoms. the tx is pathognomonic of fh but its identification is not always straightforward and to some extent considered insensitive diagnostic markers. however, other criteria should be taken into consideration for the diagnosis of fh including personal and familial ldl-c levels, history of chd, and presence of ca before the age of 45 years and xanthomas (fernando, 2004). overall, the incidence of xanthoma (i.e. tendinous xanthomata and xanthelesma) in the present study was quite high i.e. 7 out of 10 (70%) cases with fh had it. recently, junyent et al. (2005) has reported that the occurrence of tx is very much linked to the fh incidences in individuals with family history of hypercholesterolemia, primary hypercholesterolemia and premature coronary disease. although tx is pathognomonic of fh but a high variability of xanthoma may occur in fh patients (descamps et al., 2001). xanthelasmas occur commonly in heterozygotes, and are rare in homozygotes. the presence of xanthelasmas is not specific for fh and may also occur in individuals with normal lipid levels (goldstein et al., 2001). the frequency of fh among caucasians is 1:500, however in some populations there is high heterozygous fh frequency such as french canadians, lebanese christians, south african afrikaners, lithuanian ashkenzai jews, druze and finns (lehrman et al., 1987; kotze et al., 1991; meiner et al., 1991; leitersdorf et al.,1990; landsberger et al., 1992; koivisto et al.,1992). the heterozygote fh individuals have about two fold increase in their plasma cholesterol. the fh subjects show 4-5 time higher age-sex standardized mortality ratios in the general populations (castro-orós et al., 2010). the mean age of males that showed the symptoms of fh in the present study was 38 years, compared to 45 years in females. the incidence rate in the observed males was 69% compared to 31% in females. similar results have previously been reported where the observed age was strongly associated with high cholesterol levels in men, whereas women had significantly lower non-hdl-c level (gardner et al., 2000). for women, the age range was 25 to 64 years, whereas for men it ranged from 25 to 54 years. non-hdl concentration was higher in individuals with increasing age. however, this observation seemed to be not influenced by age as the total cholesterol levels were found to be higher in all age groups. the mean age of males, showing symptoms of fh was 38 ± 5 years, which is earlier as compared to females (45±5 years). the fh should immediately be treated upon diagnosis and if left untreated approximately 85% of males and 50% of females will develop a coronary problem before the age of 65 (civeira, 2004). it is striking that up to 9% of the total premature chd in eastern germany and table 1. clinical features of 335 participants with cholesterol level >210 mg/dl in pakistani population. table 2. correlation studies showing relations among smoking, hypertension (htn) and total cholesterol levels in pakistani population. significant at *p< 0.05, ** p< 0.01. observed features gender age (years) total observed cases percentage (%) tandinous xanthomata male 30-40 40-50 50-60 202 0 0 146 0 0 72 0 0 female 30-40 40-50 50-60 133 0 0 78 0 0 58 0 0 xanthelesma male 30-40 40-50 50-60 0 202 0 0 148 0 0 73 0 female 30-40 40-50 50-60 133 0 0 110 0 0 82 0 0 arcus cornea male 30-40 40-50 50-60 0 202 0 0 128 0 0 63 0 female 30-40 40-50 50-60 133 0 0 96 0 0 72 0 0 polyarthritis male 30-40 40-50 50-60 0 202 0 0 2 0 0 0.9 0 female 30-40 40-50 50-60 0 0 133 0 0 5 0 0 3.7 smoking htn total cholesterol smoking 1.000 0.32* 0.47* htn 1.000 0.000** total chlesterol 1.000 38 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) finland has been linked with fh (koivisto et al., 1993; baron et al.,1996; schuster, 2002). hence, the major cause of death in fh patients has been linked with the occurrence of chd (mabuchi et al., 1989; miettinen and gylling, 1988). in the present study, we found that the high levels of ldl-c (> 190 mg dl-1) were detected in the proband and in the related family members (figure 1). the clinical diagnosis of heterozygote fh is associated not only with the high plasma level of ldl-c but also with the incidence of other coronary risk factors (hill et al, 1991). we have found that the females with fh had higher levels of total ldl-c (196-358 mg dl-1) as compared to the male subjects (201-301 mg dl-1) and with lower levels of hdl-c (25–55 md dl-1). the average age of onset of coronary symptoms was delayed in females, with a mean age of 55 years compared with 48 years for males (p <0.05). previous studies have shown an association between clinical heterozygous fh and coronary heart disease (kalina et al., 2001; umans-eckenhausen et al., 2002). however, the heterogeneity in fh patients with regards to plasma ldl-c and chd has also been suggested (castroorós et al., 2010). multivariate analyses have revealed that in fh heterozygotes and controls, the hdl-c levels may contribute to a greater proportion of the variation in tc to hdl-c ratio than tc (torres et al., 1996). a number of factors, including age, sex, smoking and hdl-c concentrations have previously been identified as risk factors for chd in fh in cross-section studies worldwide (jansen et al., 2004). there are 100 million people with fh worldwide, mainly heterozygotes, and approximately 85% of males and 50% of females with fh will suffer a coronary event before they are 65 years old, if appropriate preventive measures are not implemented (fernando, 2004). the risk factors such as smoking and htn showed a significant correlation in the appearance of the disease. our study showed that a strong correlation between the risk factors, smoking and htn, was present in the individuals with fh. the tc and smoking were significantly correlated (p <0.05) in the fh pakistani population. similarly, smoking and htn were also significantly (p <0.05) correlated. but a very strong correlation (p <0.001) between htn and tc was observed in the studied individuals. smoking in males had profound effect, where 70% males with positive chd were smokers. however, the htn in females was associated with higher incidence of chd. the htn has been considered as an independent risk factor for females with fh (hill et al., 1991). it has been reported that the level of total cholesterol was positively related to the prevalence of systolic htn and obesity, in addition to smoking (rywik et al., 1999). a meta-analysis was conducted by rywik et al. (1999), where they concluded that for every 10% reduction in tc, there was 2.5% decrease in the incidence of chd. a multivariate analysis performed on a canadian population indicated that the low hdl-c and smoking were the best predictors of risk and the average age of onset of coronary symptom were delayed in females (55 years) compared to males (48 years) (weber et al., 1997). the risk of developing heart related problems has also been connected with the lower levels of hdl-c and a history of smoking; however, in women, the heart related problems were mainly associated with elevated tg levels and the presence of htn (hill et al., 1991; neil et al., 2004). our results are in agreement with earlier published reports (vuario et al., 1997; jansen, 2004) on the risk factors like age, sex, smoking and htn. our results are consistent with accumulating evidence from a number of cross-sectional population and case control studies that have assessed the role of established and emerging risk factors in individuals with fh. this study on a pakistani population has shown that fh is linked to risk factors such as age, smoking and htn in addition to total cholesterol levels and to a larger extent was associated with the development of premature heart diseases as well as in the phenotypic expression in heterozygous fh. upon diagnosis the fh patients should immediately be treated and if left untreated, they have a high chance (about 8 times) of early coronary heart disease (chd). cholesterol can be accumulated in the coronary arteries of the heart causing angina or heart attacks. other factors that increase the risk of chd are smoking and high blood pressure. however, fh is a very treatable condition which can be controlled with the use of cholesterol-lowering drugs combined with a healthy lifestyle. it is now known that people with fh who are treated can expect to have a life expectancy which is the same as the general population. therefore it is vitally important to diagnose 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haw, v seagroatt, dj betteridge, m p cooper, p n durrington, j p miller, m seed, r p naoumova, g r thompson, r huxley, and s e humphries (2004) established and emerging coronary risk factors in patients with heterozygous familial hypercholesterolemia. heart 90: 1431 – 1437. rader dj, cohen j, hobbs hh (2003) monogenic hypercholesterolemia: new insights in pathogenesis and treatment. j clin invest. 111: 1795–1803. rywik sl, am teri, p andrzej, p walerian, ed clarence, bb grazyna and k ewa (1999) association of lipids and lipoprotein level with total mortality and morbidity caused by cardiovascular and cancer disease (poland and united states collaborative study on cardiovascular epidemiology). am j cardiol 84: 540-548. schuster h (2002) high risk/high priority: familial hypercholesterolemia –a paradigm for molecular medicine. atheroscler suppl. 2:27–30. tietz nw (1986) textbook of clinical chemistry, philadelphia, pa, wb, saunders co. pp. 888. torres al, s moorjani, mc vohl, c gagne, b lamarche, ld brun, pj lupien, and jp despres (1996) heterozygous familial hypercholesterolemia in children: low-density lipoprotein receptor mutational analysis and variation in the expression of plasma lipoprotein-lipid concentrations. atherosclerosis 126 (1): 163-71. umans-eckenhausen ma, ej sijbrands, jj kastelein, and jc defesche 40 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) (2002) low-density lipoprotein receptor gene mutations and cardiovascular risk in a large genetic cascade screening population. circulation 106: 3031–3036. vuario af, h turtola, and km piilahti (1997) familial hypercholesterolemia in the finnish north karelia. a molecular, clinical and genealogical study. arterioscler thromb vasc biol 17: 3127-3138. weber m, s mcnicoll, m marcil, p connelly, s lussier-cacan, j davignon, y latour, and j genest jr (1997) metabolic factors clustering, lipoprotein cholesterol, apolipoprotein (a) and apolipoprotein e phenotypes in premature coronary artery disease in french canadians. can j cardiol 13 (3): 253260. kbm journal of biology (2010) 1 (2): 18-25 doi: 10.5147/kbmjb.2010.0008 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) genetic diversity analysis of the natural populations of mediterranean mussels [mytilus galloprovincialis (lmk.)] in agadir bay: assessment of the molecular polymorphism and environmental impact amal korrida¹,²,*, sami jamil jadallah², hassan izaabel¹, and said benhissoune³ ¹ equipe de génétique et ecologie des populations humaines (geph), laboratoire de biologie cellulaire et génétique moléculaire (lbcgm), université ibn zohr, faculté des sciences agadir, bp 8106 cité dakhla, agadir, morocco. ² hrh prince sultan bin abdul aziz al saud international foundation for conservation & development of wildlife, department of genetics, po box 116, agadir, morocco. ³ laboratoire des substances naturelles. equipe d’océanographie biologique, département de biologie, université ibn zohr, faculté des sciences agadir, bp 403, agadir, morocco. received: february 16, 2010 / accepted: may 20, 2010 absract mediterranean mussel (mytilus galloprovincialis lmk) has a great environmental and economic importance for morocco. this work studies the genetic structure and impact of chemical pollution on three different marine populations of mytilus galloprovincialis that live within agadir bay. three collections were made at two clean sites (cape ghir and cape aglou) and at an impacted site exposed to intense boating and industrial activities (anza). a 300-bp portion of the mitochondrial dna coding-region cytochrome c oxidase subunit 1 (coi) was studied by polymerase chain reaction (pcr) and dna sequencing reactions to assess and evaluate amounts of polymorphism in each site. genetic analysis using coi for 64 individuals showed no significant differentiation between the three subpopulations. amova demonstrated that only 2.83% of variation exists between populations. besides the genetic evidence presented herein, mussel’s adaptation mechanisms and strategies to marine pollution are also discussed. key words: mytilus galloprovincialis, cytochrome c oxidase gene, marine pollution, mussels’ genetic structure. __________________________________________________ * corresponding author: amalkorrida11@aol.com introduction mytilus galloprovincialis is a cosmopolitan and an invasive species with antitropical distribution that occurs in the atlantic coasts from agadir bay to the british isles, the whole mediterranean basin, south africa, new zealand and california. (mcdonald et al. 1991 and naciri 1998). in the northern hemisphere, m. edulis constitutes a complex comprising three species: m. galloprovincialis, m. edulis and m. trossulus. genetic studies on mussels using morphometric characters, allozymes and the mitochondrial 16s rdna gene (mcdonald et al.1991 and hilbish et al. 2000) suggested that in the southern hemisphere, mussels are in general closely related to m. galloprovincialis, except for mytilus spp. from kerguelen islands and south america that might be related to m. edulis (gérard et al. 2008). the taxonomic status of the genus mytilus is still unclear and in morocco, where hybridization is the most likely common, at least four species belonging to the family mytilidae can be found: perna perna (abouabdellah et al. 2008), mytilus galloprovincialis (daguin et al. 1999 and kaimoussi et al. 2001), mytilus edulis (dardignac-corbeil 1986), and perna picta (shafee et al. 1989 and wood et al. 2007). the coastal and marine environment of agadir bay, which stretches for about 8 km, is exposed to intensive urban rejections at anza area, as a consequence of industrial, agricultural, and boating activities that characterise this area. the majority of pollutants are directly discharged in 18 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) the atlantic ocean with no preliminary treatment. we chose to work on the mediterranean mussel species for several reasons: (i) it has social, economic and ecological interest (ii) as a sentinel species, sensitive to xenobiotic substances and bio-accumulating marine pollutants, it constitutes a good indicator of water quality (iii) its sampling is straightforward, especially during low-tide periods (iv) except for the studies done by (jaziri et al. 2002 and 2003), the genetics of mytilids is not well-studied in morocco, whereas their biology, population dynamics and ecology is much better known (bouhaimi et al. 1999-2000, mimouni et al. 2002, moustaid et al. 2005, maanan et al. 2008). we examined a part of the mitochondrial dna (mtdna) cytochrom c oxidase subunit i (coi) to study the genetic structure of mytilus galloprovincialis species and to assess the impact of pollution or environmental contamination on this important component of the moroccan marine community. materials and methods study sites and sample collection three stations on the atlantic coast of morocco were the subject of this environmental and genetic study: (a) cape ghir station (30°38 n and 9°53 w) is situated 50 km north of agadir city. this clean area with an important ecological interest is considered as the southern limit of the coastal upwelling (benomar et al. 2006). in april 2008, 16 females were collected and a seawater ph of 7.7 was measured, (b) anza station (30°26 n and 9°38 w) is located 8 km north of agadir city. this site is exposed to intensive boating and industrial activities and is classified as polluted (trace metals, inorganic elements, polycyclic aromatic hydrocarbons …etc.) (mimouni et al. 2002). a total of 32 individuals were sampled in june 2008. the seawater ph was 7.2, and (c) cape aglou site (29°48 n and 9°49 w) is approximately 100 km south of agadir city at the foot of the anti-atlas mountains. in april 2008, a total of 16 females were collected from afetass aglou village. the seawater ph was 8.8. the whole sampling process was carried out during the low-tide periods and all samples were put on ice during their transportation to the laboratory, and then stored at -20°c. dna extraction, primers design, amplification and sequencing genomic dna was extracted from the adductor muscle using phenol-chloroform (sambrook et al. 1989) and chelex-100 methods. a part of the mitochondrial coi gene (300 bp) were amplified using (folmer et al. 1994) primers, which we modified and re-designed in this study: lcoi1490-m13f: 5-agg gtt ttc cca gtc acg acg ttg gtc aac aaa tca taa aga tat tgg-3’and hco2198-m13r: 5’-gag cgg ata aca att tca cac agg taa act tca ggg tga cca aaa aat ca-3’. gradient pcrs were used during the optimisation phase and for a final volume of 20µl. the pcr master mix contained 1.5µl of adn, 10µm of dntps, 2.5µm of mgcl2, 0.5µm of forward and reverse primers, 0.0375 units of taq polymerase and 10.75µl of milliq h2o. pcr reactions were carried out using a biometra t1 thermal cycler under the following conditions: one cycle at 94°c for 4 min, 30 cycles at 94°c for 15s, 60°c for 30s and 72°c for 30s, and one final cycle at 72°c for 10 min. pcr products were loaded in a 1% low melting point agarose gel and visualised under uv light. using a sterile scalpel, the bands of interest were cut from the agarose gel and purified using the traditional freeze-squeeze method (diethard & renz 1983). 8.15µl of each purified pcr product were then used as a template for bidirectional sequencing using the fmol® dna cycle sequencing system (promega). the same pcr profile was used as described above except for the addition of 10% dmso as an adjuvant. sequencing pcr products were electrophoresed on a li-cor 4000l sequencing device with a 6.5% li-cor kb plus gel matrix. data analysis sequences were aligned and edited using the software proseq v 2.91 (filatov d.a 2002). tcs software (clement et al. 2000) was used to infer the genealogical relationships among haplotypes of the 3 populations and the construction of the minimum spanning network. analysis of molecular variance (amova), genetic variance partitioning among and within populations, pairwise fst and p-value, and the assessment of the gene flow amount were carried out using arlequin package (excoffier et al. 2006). the distance method considered was the kimura 2-parameters (1980). neutrality tests, haplotype and nucleotide diversities, genetic code assignment, dna polymorphism, and divergence were estimated using dnasp v. 5.10.01 (librado and rozas 2009). results intra-population variation a total of 300 bp of m. galloprovincialis’ coi was successfully sequenced and generated 13 haplotypes (8 hplotypes in anza site with 3 shared and 5 private, 4 haplotypes in aglou site with 3 shared and 1 private and 6 in cape ghir site with 2 shared and 4 private). the haplotypes distribution within the whole population of mussels is given in (table 3). were carried out also, 10 segregating or polymorphic sites with 5 in anza site at nucleotide positions (109, 112, 224, 235, and 265), 5 table 1. dna sequences variation of the 13 observed haplotypes in the three populations of mytilus galloprovincialis at agadir bay. 19 haplotypes 6 54 88 109 112 170 224 235 265 270 hap 1 (anza 1) a t t t c t c t t c hap 2 (c.ghir 26) . c . . . a . . . . hap 3 (c.ghir 13) . . c . . . . . c . hap 4 (anza 32) . . . c t . . . . . hap 5 (anza 19) . . . . t . . c . . hap 6 (anza 21) . . . . t . . . . . hap 7 (anza 31) . . . . t . t . . . hap 8 (anza 30) . . . . . . t c c . hap 9 (anza 14) . . . . . . . c c . hap 10 (c.ghir 28) . . . . . . . . . a hap 11 (anza 4) . . . . . . . c . . hap 12 (c.ghir 14) . . . . . . t . . . hap 13 (c.aglou 11) c c . . . . . . c . kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) in aglou site at positions (6, 54, 112, 235, and 265), and 7 in cape ghir at positions (54, 88, 170, 224, 235, 265, and 270) (table 1 and 2). the nucleotide and protein sequences of the 13 haplotypes were deposited in genbank under accession numbers fj638333, fj638334, fj638335, fj638336, fj638337, fj638338, fj638339, fj638340, fj638341, fj638342, fj638343, fj638344, and fj172994. the genetic code was first assigned assuming the invertebrates mitochondrial code as described by (hoffmann et al. 1992) in mytilus. five parcimony sites (54, 112, 224, 235, and 265), six sites with synonymous or silent substitutions (88, 109, 112, 224, 235, and 265) and four sites with non-synonymous substitutions or amino acid altering mutations (6, 54, 170, 270) were determined at the first and/or the second position of codons (table 1). the 4 latter substitutions changed the amino acid sequences of the studied exon (table 6). diversity parameters are presented in (table 2). haplotype diversity (h) ranged from 0.6750 to 0.8044 and nucleotide diversity (π) from 0.0035 to 0.0043. tajima’s selective neutrality test (1983) generated the following results: d = -0.96266 for cape aglou population, d = -1.42180 for cape ghir population, and d = 0.03136 for anza population. all tests were not statistically significant (p > 0.10). 20 study sites n no. of segregating sites no. of haplotypes h π anza 32 5 8 0.8044 ± 0.0431 0.0043 ± 0.0031 cape aglou 16 5 4 0.6750 ± 0.0853 0.0035 ± 0.0027 cape ghir 16 7 6 0.7667 ± 0.0839 0.0042 ± 0.0031 table 2. summary of the dna polymorphism inferred from 64 mtdna sequences of m. galloprovicialis in agadir bay. (h): gene or haplotype diversity ± standard deviation (π): nucleotide diversity ± standard deviation. haplotype anza (32) c. aglou (16) c. ghir (16) hap1 11 8 7 hap4 1 0 0 hap6 5 2 0 hap7 1 0 0 hap8 1 0 0 hap9 2 0 0 hap5 3 0 0 hap11 8 5 4 hap13 0 1 0 hap2 0 0 1 hap3 0 0 1 hap10 0 0 1 hap12 0 0 2 table 3. haplotype distribution in the mediterranean mussel among the three study sites. anza c. aglou c. ghir anza 0.33496 0.02637* c. aglou 0.00562 0.70801 c. ghir 0.06792* 0.01922 table 4. estimates of pairwise p-value (above the diagonal) and fst (below the diagonal) for the three populations of the mediterranean mussel. * p < 0.05. source of variation degrees of freedom sum of squares variatio n % of variation among populations 2 1.972 0.01816 2.83 within populations 61 37.993 0.62283 97.17 total 63 39.964 0.64098 100 table 5. analysis of molecular variance (amova) at the interand intra-population levels. figure 1. minimum spanning network constructed from the 300 bp sequences of the mtdna cytochrome c oxidase gene. figure 2. the canary current along the atlantic coast of morocco (as described by machín et al. 2006). lp: la palma, ac: azores current, cc: canary current, cuc: canary upwelling current, and nec: north equatorial current. kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) inter-population variation the mean genetic distance within study sites and for the whole study area, using the kimura 2-parameters model that takes in consideration transversions and transitions, was estimated as d = 0.4%. the mean genetic distance between anza and cape aglou populations is equal to that between aglou and cape ghir (0.4%), and between anza and cape ghir is equal to 0.5%. the coefficient of differentiation is of 0.032. in (table 4), the values of pairwise fst and p-values are shown. genetic structure and population differentiation were examined using the analysis of molecular variance method (amova) (excoffier et al. 2006) and this revealed that 97.17 % of variation exists within populations and only 2.83 % between them (table 5). the fixation index (wright 1951) was estimated at the 5% level as fst = 0.0283 (p-value = 0.1214). gene flow appears to be more common between anza and cape aglou populations (nm = 88.49), weaker between anza and cape ghir (nm = 6.86), and absent between cape aglou and cape ghir populations (nm <0). 21 table 6. distribution of non-synonymous substitutions. four amino acids were changed: tyrosine (y) to serine (s), valine (v) to alanine (a), tyrosine (y) to asparagine (n), and threonine (t) to lysine (k). kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) the minimum spanning network showed no distinct clades among the 3 wild populations and the 13 otus (operational taxonomic units) (fig. 1). four haplotypes were private in cape ghir (hap 12, hap 3, hap 10 and hap 2), one in cape aglou (hap 13) and five in anza site (hap 4, hap 7, hap 5, hap 8 and hap 9). the common ancestor is represented by a central rectangle (anza1). the rest of haplotypes was shared between two or three populations (hap 1, 6, and 11 between anza and cape aglou, hap1 and 11 between anza and cape ghir, and between cape aglou and cape ghir. discussion the choice of the gene coi in an early stage, we carried out several tests on the mtdna hypervariable region. these tests failed to amplify the d-loop region, most probably due to polymorphism in the species at the priming sites targeted by the primers we essayed. indeed, the genome of mussels is considered one of the most complex genomes in the animal kingdom. their mtdna organization can undergo several genetic events such as double uniparental inheritance (dui) (zouros et al. 1992, garrido-ramos et al. 1998, zbawicka et al. 2003, mizi et al. 2005), masculinization event (hoeh et al. 1997, sirna et al. 2007), gene rearrangements, recombination (hoffmann et al. 1992, ladoukakis et al. 2001, mizi et al. 2005, burzyński 2007 et al. and venetis et al. 2007), and males heteroplasmy and females homoplasmy (hoeh et al. 1991, wenne et al. 1995, cao et al. 2004). the mtdna coi gene was chosen as an alternative for two reasons: (i) its fast mode of evolution (wares et al. 2001, riginos et al. 2004 and gérard et al. 2008), and (ii) to our knowledge, the fact that no genetic study has used this gene to survey the moroccan populations of mytilus galloprovincialis. the studies undertaken on dna barcodes, especially focusing on the coi gene, were very useful and facilitated this population study. in addition to the fact that the coi gene constitutes the core of a global bio-identification system for animals, it also allows unambiguous identification of more than 200 species and remains thus, the most accessible and practical molecular tool for populations studies (herbert et al. 2002). genetics of populations in population genetics, the distribution of genetic variation between populations and individuals is deduced from the study of their genetic structure. the latter is often under the action of various evolutionary forces like selection, migration, genetic drift and mutation. according to (table 5), the intra-population genetic diversity is very high in the three zones (97%). in spite of the observed mutations, amova failed to reveal a significant difference between the three populations and showed that the essential haplotype variation is essentially due to the intra-population diversity. mussel mtdna may also be subject to different selective pressures (zbawicka et al. 2003 and riginos et al. 2004). the frequency and strength with which selection shapes patterns of genetic variation is unknown. whereas all loci should be roughly equally affected by demography and population history, selected loci may exhibit increased or decreased genetic differentiation relative to neutral loci (cavalli-sforza 1966 and riginos et al. 2002). neutrality results suggested that cape ghir and aglou populations are under the action of a directional and purifying selection since the values of tajima’s test were negative. on the other hand, anza population might be under the action of balanced and stabilizing selection that favours the extreme individuals on this impacted site. despite the potential pollutioninduced mutations at anza site, and according to amova, fst analysis, and gene flow estimates, it seems that there is a genetic homogeneity across the three sites, despite inter-site distances that can be over 150 km. the observed homogeneity could be explained by the marine canary currents characterizing this coastal area (fig. 2). many oceanographic and environmental studies have examined these currents, which constitute a natural extension of the main azores current. furthermore, (mittelsstaedt 1991), (jaziri et al. 2002), (stevens et al. 2003), and (machín et al. 2006) confirmed that at cape ghir, the canary current hugging the atlantic coast changes completely its direction and continues towards the canary archipelago. this change of direction suddenly cuts and stops the pelagic larval dispersal and, consequently, the gene flow in this region, characterized also by the formation of the upwelling filaments. mediterranean mussel: genetics and impact of pollution mussels adjust their functions to ordinary environmental changes such as, temperature fluctuations and emersion-related hypoxia and react to various contaminants (venier et al. 2006). during the sampling phase, we noticed that the phenotype of the mussels is slightly different. mussels sampled from the impacted area were small and their numbers seem to be depleted. cape aglou samples were medium-sized and abundant, and cape ghir samples were big and apparently abundant. also, during the dna extraction phase, the majority of the tissues of anza mussels showed spotted patterns and viscous haemolymphs. could this be considered as a manifestation of the pollution impact at anza region? in his evaluation of the moroccan marine environment, (berraho 2006) stated that the fundamental ecological parameters like species richness and the abundance have been affected by the high quantities of heavy metals at the pollution epicentres. it were also reported, the dramatic impacts of the discharge by the jorf lasfar industrial and phosphate processing complex on the marine fauna (e.g. mussels showing shell deformations). at the genetic level, no significant differentiation between cape ghir and cape aglou populations was found. however, a shallow significant differentiation is observed between anza and cape ghir populations (fst = 0.0672 and p-value = 0.002637). unless the connectivity factor is taken into consideration, this differentiation might not be explained by marine pollution at anza region as the 4 non-synonymous substitutions occurred in 3 individuals that lived out of this impacted region. our results on the whole distribution of the genetic variation among and within 22 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) populations demonstrated the absence of structuring between the three populations (table 5). the calculation of the global fixation index confirmed this (fst = 0.02832). in a similar study, a lack of genetic structure and likely high gene flow, among littorina brevicula populations was also found by (kim et al. 2003) while working on polluted and non polluted sites around the korean coastlines. according to the food and agriculture organization, only few studies have dealt with the genetic changes potentially associated to marine pollution. investigations made by (lavie et al. 1982) and (hvilsom 1983) on molluscs and crustaceans showed different survival rates of distinct allozymes genotypes to heavy metals pollution. (fevolden et al. 1986) studied the exposition of mytilus edulis to low oil contents in norwegian fjords and did not detect evidence of a genotypic selection. additionally, (nevo et al. 1986), while studying pairs of species exposed to marine pollutants, stated that the species having the highest level of genetic diversity had the longest survival potential. our investigation on mussel’s mechanisms and strategies of adaptation to marine pollution, led us to a study conducted by (duchemin 2007) on marine immunotoxicology. duchemin’s study, carried out on two bivalve species (mussel and oyster), showed that the immune system adopts a seasonal variation in narrow correlation with the function of reproduction (thus, with the genetic function). moreover, it was demonstrated that females possess a significantly higher immune competence than males during all seasons. so, most likely, through this control of the immune system by sex and reproduction cycle, bivalves manage to somewhat counter the immunotoxic effect of chemical and organic pollutants. conclusion and perspectives mytilus galloprovincialis constitutes a biological resource with ecological and economic interests. certainly, the species plays an important role for the intertidal communities. for example, with its filtrating capacity of the marine material in suspension, it can influence the dynamics of many coastal systems, and consequently, the local marine biodiversity (seed 1996). mussels are also regarded as structural and functional entities because, via their beds along the marine rocky zone, they provide refuges, shelters and habitats for other invertebrates such as polychaetes and amphipods (peake et al. 1993). the results of the studies cited above are mostly confirmed by our findings. however, for a broader evaluation and assessment of the impact of pollution on mussels’ populations living in agadir bay, or in the other moroccan coastal regions, supplementary data and records from the monitoring and surveillance of the marine areas’ quality, human exploitation and the surrounding marine ecosystems, prove to be necessary. environmental parameters like temperature, salinity, sites exposure to wave action, sites geography and food availability, also have a great impact on mytilus galloprovincialis survival and biology (spawning and reproduction periods, growth, filtration rate…etc.), and should be further investigated in this region of concern. the estimation of genetic variation showed that in spite of the pollution impact, particularly at anza zone, genetic diversity has not been lost yet, a factor that might enable the mussels living at agadir bay to survive and cope with the different environmental pressures and stresses. since mussel farming is nascent in morocco, we do hope that this preliminary study might add to the valorization of the marine resources and the development of this practice at agadir bay. for the next phase, we will study male specimen and also will increase our samples to cover more atlantic and mediterranean regions, so that we can contribute to a more informed coastal planning in morocco. acknowledgements the authors are very grateful to the institut national de recherche halieutique (inrh), agadir, morocco, his royal highness prince sultan bin abdul aziz al saud international foundation for conservation and development of wildlife, morocco, and pr. roman wenne from the polish academy of sciences. for their technical assistance, we thank, pr. hassan jaziri, jonathan gardner, ann wood, gerardo zardi, and manuel manchado. we are indebted to fatima el aamri, m. naoufal tamsouri, badiâa iazza, m. taha moutaoufik, and subhash krishnan either for their assistance in the field or help. thanks must also go to pr. cynthia riginos, pr. wiebe kooistra, carlos fernandes and anonymous referees for their helpful comments on the manuscript. a special thank to m.h miouz for the inspiration during this study. references abouabdellah r, taleb h, bennouna 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gardner jpa (2007) a molecular phylogeny of the marine mussel genus perna (bivalvia: mytilidae) based on nuclear (its1&2) and mitochondrial (coi) dna sequences. molecular phylogenetics and evolution 44: 685–698 wright s (1951) the genetical structure of populations. ann eugen 15: 323–354. zbawicka m, skibinski dof, wenne r (2003) doubly uniparental transmission of mitochondrial dna length variants in the mussel mytilus trossulus. mar. biol. 142, 455–460. zbawicka m, wenne r and skibinski d.o.f (2003) mitochondrial dna variation in populations of the mussel mytilus trossumus from the southern baltic. hydrobiologia, 499, n° 1-3, pp. 1-12(12). zouros e, freeman kh, ball ao, pogson gh (1992) direct evidence for extensive paternal mitochondrial dna inheritance in the marine mussel mytilus. nature 359:412–414. 25 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) atlas journal of biology 2 (1): 84–87, 2012 doi: 10.5147/ajb.2012.0062 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) inheritance of humeral spotting in the croaking gourami (osphronemidae: trichopsis vittatus) jack s. frankel*, lisa adler-golden, and andre n. porter department of biology, howard university, washington, dc 20059, usa received: january 28, 2012 / accepted: march 11, 2012 __________________________________________________ * corresponding author: jfrankel@howard.edu 84 abstract the croaking gourami (trichopsis vittatus) exhibits two phenotypes associated with humeral spotting. fish possess a prominent, dark humeral patch or spot located behind the operculum or lack this spotting pattern. segregation patterns observed from the progenies of eleven different crosses support the hypothesis that the inheritance of humeral spotting in t. vittatus is controlled by the action of a single autosomal locus, with complete dominance of the allele controlling the spotted phenotype. key words: trichopsis vittatus, croaking gourami, humeral spotting, osphronemidae. introduction labyrinth fishes of the teleost families osphronemidae, helostomatidae, belontiidae, and anabantidae comprise the traditional anabantoids, a group of approximately 80 relatively small african and southeast asian species (linke, 1991). the anabantoids are quite popular with aquarium hobbyists for their interesting reproductive behaviors, with males of most species brooding eggs in their mouths or in a floating bubblenest (linke, 1991; axelrod & vorderwinkler, 1995). the croaking gourami, trichopsis vittatus (cuvier, 1831), is named for its ability to produce an audible chirping noise, accomplished via the utilization of specialized adaptations of the pectoral fins (henglmuller & ladich, 1999). these chirping sounds are produced by both sexes during breeding and well-mated individuals can continue producing sounds for a few hours. males alone may also produce such sounds during the establishment of dominance hierarchies (ladich, 1998; ladich et. al., 1992). the croaking gourami is an asian anabantoid with a widespread distribution throughout sumatra, the sunda islands, malaysia, thailand, and vietnam (linke, 1991; axelrod & vorderwinkler, 1995). this species occupies bodies of water ranging in size from pond-like accumulations of water to small rivers. in addition to its unique sound-producing ability, both female and male fishes in natushort communication a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 85 ral populations of t. vittatus typically exhibit a black patch or spot located caudally to the operculum. while this humeral spot is a feature of most individuals, there is an alternate phenotype lacking this humeral spot. the mode of inheritance of this spot is of particular interest, as it likely serves as an eyespot to confuse predators. as a continuation of our interest in the inheritance of both banding and spotting patterns in freshwater teleosts (frankel, 1985, 1991, 1998, 2001, 2002, 2004, 2009, 2011), the present study was undertaken to ascertain the mode of inheritance of humeral spotting in the croaking gourami. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. probable genotypes (pg), observed phenotypic numbers, expected ratios, degrees of freedom (df), chi-square values (x2) and probability of fit (p) for crosses amongst humeral spotted and unspotted trichopsis vittatus. * (h) = humeral spotted parental fishes; (n) = unspotted parental fishes; (f) = f1 offspring. + the probability for all x2 tests is > .05; thus, results fit expected ratios according to mendelian inheritance. cross no. parents* phenotypic numbers exp. ratio df x 2 p + ♀ (pg) x ♂ (pg) humeral spotting no spotting 1 hi (aa) x h1 (aa) 24(fi&f1) 0 1:0 2 hii (aa) x h2 (aa) 17(fii&f2) 0 1:0 3 hiii (aa) x h3 (aa) 12 0 1:0 4 hiv (aa) x h4 (aa) 26 0 1:0 5 hv (aa) x h5 (aa) 20 0 1:0 pooled 99 0 1:0 6 ni (aa) x n1 (aa) 0 22 0:1 7 nii (aa) x n2 (aa) 0 21(fiii&f3) 0:1 8 niii (aa) x n3 (aa) 0 19(fiv&f4) 0:1 9 niv (aa) x n3 (aa) 0 14(fv&f5) 0:1 pooled 0 76 0:1 10 hi (aa) x f1 (aa) 25 0 1:0 11 fi (aa) x h1 (aa) 27 0 1:0 12 hiv (aa) x f2 (aa) 16 0 1:0 13 fii (aa) x h4 (aa) 18 0 1:0 pooled 86 0 1:0 14 nii (aa) x f3 (aa) 0 12 0:1 15 niv (aa) x f5 (aa) 0 10 0:1 16 niii (aa) x f4 (aa) 0 15 0:1 17 fiii (aa) x n2 (aa) 0 11 0:1 18 fv (aa) x n3 (aa) 0 22 0:1 pooled 0 70 0:1 19 hii (aa) x n1 (aa) 21(fvi&f6) 0 1:0 20 hiii (aa) x n2 (aa) 32(fvii&f7) 0 1:0 21 hv (aa) x n3 (aa) 10(fviii&f8) 0 1:0 22 ni (aa) x h2 (aa) 12(fix&f9) 0 1:0 23 niii (aa) x h5 (aa) 15(fx&f10) 0 1:0 pooled 90 0 24 fvi (aa) x f9 (aa) 15 6 3:1 1 0.1429 0.7054 25 fvii (aa) x f8 (aa) 19 7 3:1 1 0.0513 0.8208 26 fviii (aa) x f7 (aa) 12 5 3:1 1 0.1765 0.6743 27 fix (aa) x f6 (aa) 16 5 3:1 1 0.0159 0.8996 28 fx (aa) x f10 (aa) 15 4 3:1 1 0.1579 0.691 29 fvi (aa) x f10 (aa) 22 7 3:1 1 0.0115 0.9146 30 fvii (aa) x f6 (aa) 21 8 3:1 1 0.1034 0.7477 total 7 0.6593 0.9986 pooled 120 42 3:1 1 0.0741 0.7854 heterogeneity 6 0.5852 0.9966 31 fvi (aa) x f4 (aa) 18 15 1:1 1 0.2727 0.6015 32 fvii (aa) x f5 (aa) 14 15 1:1 1 0.0345 0.8526 33 fiv (aa) x f7 (aa) 12 10 1:1 1 0.1818 0.6698 34 fv (aa) x f8 (aa) 9 11 1:1 1 0.2 0.6547 total 4 0.689 0.9526 pooled 53 51 1:1 1 0.0385 0.8444 heterogeneity 3 0.6505 0.8847 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 86 materials and methods healthy adult specimens of t. vittatus were obtained from a wholesale distributor in maryland, usa, and maintained in separate 76 liter holding tanks equipped with aerators at 25o c. male and female fishes exhibiting either the characteristic humeral spotting phenotype or an aberrant, unspotted phenotype were selected at random from stock specimens, placed in separate 76 liter tanks, and allowed to develop at 25oc until sexually mature. optimal water conditions were provided for all fish (i.e. low water hardness of 5o dgh, ph 7.5, and temperature 25oc). sexually mature fishes were transferred to 36 liter tanks with temperature raised to 30oc (linke, 1991). all progeny for this study were obtained utilizing artificial fertilization techniques. artificial fertilizations were performed at a constant temperature of 30oc. eggs were collected and inseminated in fish ringer’s solution prepared with 6.50 g nacl, 0.250 g kcl, 0.20 g nahco3, and 0.30 g cacl2 in one liter of distilled water. gametes were obtained by cycling individual females through at least one natural breeding period in the presence of three males. each gravid female was transferred to a 250 ml fingerbowl containing ringer’s solution. a small piece of nylon mesh netting was used to secure the female, bellyside down, against the side of the fingerbowl. gentle and even pressure was then applied to the abdominal flank, resulting in the release of eggs. sperm was obtained by gently compressing the abdomen of a male fish against the side of a 500 ml fingerbowl containing tank water. the milt, clearly visible at the urogenital aperture as a thin white stream, was collected using a narrow-mouthed pipette and expelled over the eggs. the total volume of sperm suspension, collected as “dry” as possible, was approximately 0.5 ml. the time interval from egg collection to sperm addition was between 20 and 40 seconds. parental fishes, exhibiting either the spotted (h) or unspotted (n) phenotype, along with f1 progeny (f), were used in a series of 34 crosses (table 1). embryos from all crosses were transferred to 250 ml fingerbowls containing tank water and incubated at 30oc. dead or developmentally arrested individuals were removed daily until fry were free-swimming. progeny groups then were placed in separate 36 liter rearing tanks, fed initially on pure aquatic brand premium fry food, and allowed to develop until their phenotype could be visually determined. since humeral spotting is more defined in young adults, determination of spotting was only scored for those individuals. phenotypic data of all progeny were recorded and subjected to chi-square analysis. pooled and heterogeneity chi-square tests were also performed, treating the progenies from reciprocal f1 x f1 crosses as single large progenies in an analysis of overall goodness of fit. results and discussion table 1 presents data for the proposed genotypes of parental fishes, observed phenotypic numbers, expected ratios, and probability of fit for t. vittatus analyzed for the mode of inheritance of humeral spotting. parental fishes and progeny from all crosses clearly displayed either the humeral spotted or unspotted phenotype. females and males exhibiting a humeral spot hi, hii, hiii, hiv, hv and h1, h2, h3, h4, h5, respectively, were scored as homozygous dominants, as crosses involving these individuals always resulted in spotted progeny (crosses 1-5, 10-13, 19-23). parental fishes lacking a humeral spot (females ni, nii, niii, niv and males n1, n2, n3) were scored as homozygous recessives, as crosses amongst these individuals consistently bred true (crosses 6-9). further, when fiii and fv females, and f3, f4, and f5 males were mated with their parent, the resulting offspring consistently lacked a humeral spot (crosses 14-18). in addition, reciprocal crosses between spotted and unspotted parental fishes always resulted in spotted progeny (fvi-fx and f6-f10) (crosses 19-23). crosses amongst f1 fishes resulting from parental spotted (h) and unspotted (n) matings always resulted in a satisfactory fit to a 3:1 phenotypic ratio of f2 progeny (crosses 24-30), commensurate with the action of a single autosomal gene locus exhibiting complete dominance for humeral spotting; the a_ genotype results in expression of humeral spotting. this mode of mendelian inheritance was further supported by crosses between presumptive f1 homozygous recessives (fiv, fv, f4, f5) and f1 heterozygotes (fvi, fvii, f7, f8) (crosses 31-34). both spotted and unspotted fry resulted from these crosses and, based on chi-square analyses, conformed to the expected 1:1 phenotypic ratio. results of this study support the hypothesis that humeral spotting in the croaking gourami is controlled by a single locus, with dominance at this locus required for the expression of the spotted phenotype. segregation patterns for the spotted and unspotted phenotypes of t. vittatus clearly fit an autosomal pattern of inheritance, as chi-square tests do not deviate significantly from expectations. the data also suggest complete dominance for humeral spotting, since there is no discernable difference in the appearance of spotting between presumptive heterozygous (aa) and homozygous (aa) fishes. further, results of heterogeneity tests also support the acceptance of the null hypothesis for these data. this prominent, dark humeral spot in the croaking gourami most certainly serves as an eyespot and, therefore, would give a selective advantage to those individuals possessing this feature by providing them with an interspecific marking to minimize predation. a monogenic mode of inheritance has also been reported for the expression of a caudal peduncle marking in the labyrinth fish pseudosphromenus cupanus (frankel, 2001). here, the presence of a caudally located band is also controlled by a dominant allele determining this eyespot phenotype. it is interesting to note, however, that markings serving to provide fishes with disruptive patterns are not always the preferred or prominent phenotypic alternative. indeed, studies investigating a melanic (mottled-black) body spotting pattern in the mosquitofish (gambusia holbrooki) (bisazza & pilastro, 2000; horth, 2006), have shown that this phenotype is expressed in very low frequency or is completely absent from populations of this poeciliid. references axelrod hr, and w vorderwinkler (1995) encyclopedia of tropical fishes with special emphasis on techniques of breeding. t.f.h. publications. pp. 174-184. bisazza a, and a pilastro (2000) variation of female preference for male coloration in the eastern mosquitofish (gambusia holbrooki). behavior genet 30:207-212. frankel js (1985) inheritance of trunk striping in the sumatran tiger barb, barbus tetrazona. j hered 76:478-479. frankel js (1991) inheritance of body marking patterns in the halfbanded barb, barbus semifasciolatus. j. hered. 82:250-251. frankel js (1998) monogenic inheritance of trunk banding patterns in the sumatra barb, barbus tetrazona. j fish biol 53:1357-1359. frankel js (2001) inheritance of caudal peduncle banding in the spiketailed paradisefish. j fish biol 59:1095-1097. frankel js (2002) caudal spotting in the beacon fish (hemigrammus ocellifer characidae). j. hered. 93:285-286. frankel js (2004) inheritance of trunk banding in the tetra (gymnocora tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ymbus ternetzi characidae). j hered 95:262-264. frankel js (2009) inheritance of shoulder spotting in the tetra, hyphessobrycon bentosi characidae. open fish sci. j. 2:39-41 frankel js (2011) inheritance of shoulder spotting in the red-base tetra (characidae: hemigrammus stictus). altas j biol 1:62-65. henglmuller sm, and f ladich (1999) development of agonistic behavior and vocalization in croaking gouramis. j fish biol 54:380-395. horth l (2006) a sex-linked allele, autosomal modifiers and temperature-dependence appear to regulate melanism in male mosquitofish (gambusia holbrooki). j exp biol 209:4938-4945. ladich f (1998) sound characteristics and outcome of contests in male croaking gourami (teleostei). ethology 104:517-529. ladich f, w brittinger, and h kratochvil (1992) significance of agonistic vocalization in the croaking gourami (trichopsis vittatus, teleostei). ethology 90:307-314. 87 atlas journal of biology 2 (1): 78–83, 2012 doi: 10.5147/ajb.2012.0059 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) lichens of the hassan tower monument (rabat, morocco) ilham nattah1, amina ouazzani touhami1, rachid benkirane1, mohamed el kortbi2, and allal douira1 1 laboratory of botany and plant protection, university ibn tofail, faculty of sciences, p. o. box 133, 14000 kénitra, morocco; 2 laboratoire des matériaux et génie civil, université hassan ii, casablanca, morocco received: august 22, 2011 / accepted: february 1, 2012 __________________________________________________ * corresponding author: douiraallal@hotmail.com introduction the imperial city of rabat (capital city of morocco) hosts famous monuments built during the almohad era (1128 to 1269ad). the hassan tower, oudayas, chellah, are some notable examples of the arab-islamic architecture of the xii-xiii centuries. hassan tower is a symbolic feature representing the city (el azhari, 2009). according to caillé (1954), hassan tower is the minaret of an incomplete mosque in rabat. the construction of the tower in 1195 ad, was intended to be the largest minaret in the world along with the mosque. in 1199, sultan yacoub al-mansour died and the construction on the mosque stopped. at the time, the tower only reached 44 m, about half of its intended 86 m height. instead of stairs, the tower is provided with ramps which would have enabled the muezzin to ride a horse to the top of the tower to call for prayers. the tower, made of red sandstone along with the remains of 78 abstract this paper describes the lichen species that colonize the walls of the hassan tower, a historical monument of morocco. the lichen species identified as caloplaca flavescens, c. vitellinula, rocella phycopsis and xanthoria calcicola are distributed differently on the walls of the tower. in addition to other environmental factors, such lichen species encountered are involved in one way or another in the deterioration of building materials of the hassan tower. keywords: morocco, hassan tower, monument, lichens, deterioration. the mosque and the modern mausoleum of mohammed v, forms an important historical and tourist complex in rabat. the historical monuments of rabat show many signs of weathering. the scientific works on the weathering process of these monuments all agreed that the causes of deterioration are anthropogenic origin (bellitir 1987, alaoui et al., 2006, zaouia et al., 2006, asebriy et al., 2007, 2009). the climate of rabat is characterized by high humidity and high salt content of marine origin. added to climatic, the exhausts of vehicles on the arterial roads of the city induce further deterioration (el azhari, 2009). lichen species also play an important role in the process of deterioration of a wide range of building materials. this is attribute to their capacity to grow on a variety of substrate under a wide range of environmental conditions (nimis et al., 1987, 1996). the present work focuses on lichens of hassan tower. materials and methods the study of the lichens involved their identification both in the field and in the laboratory. the lichen samples collected from the walls of hassan tower were examined using light microscopes. free-hand sections were made using a razor blade and mounted in water and cotton blue. tissue and ascospore measurements were made in water. granulation of tissues was observed in polarized microscopes. spot test reactions of thalli, apothecial margins, medulla and discs were tested with the standard reagents, potassium hydroxide 10% (k) and sodium hypochlorite (c). a comparison was then made with the information available in various analytical keys and guides (harmand, 1913; wade, 1965; mayrhofer and poelt, 1979; steiner and poelt, 1982; laudon, 1992; arup, 1994; wetmore and karnfelt, 1998; lindblom et al., 2005; soun and vondrak, 2008; gaya et al., 2008). the specimens are kept in the herbarium of the laboratory of botany and plant protection, faculty of sciences, university ibn tofail, kénitra, morocco. results the results of this study showed the presence of four lichen species: caloplaca flavescens, caloplaca vitellinula, roccella phycopsis and xanthoria calcicola detailed as follows: a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 79 caloplaca flavescens (huds) laundon: thallus crustose, forming closely adpressed rosettes, yellow to orange up to 1-2 cm in diameter. rosettes of thalli single or grouped, whitish at the centre which often disintegrates and falls out with age (fig. 1 a). lobes at the margin of thallus 0.5 -1.5 mm long and 1 mm wide, convex and sometimes overlapping. cortex present and a continuous layer visible in polarized light (fig. 1c). apothecia up to 0.8-1mm in diameter, orange, mainly confined to the centre of the thallus; disc flat to convex. spore broad, lemon, up to 10 µm long and 6 µm wide (fig. 1e), 8 per ascus (fig. 1d). reaca tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. caloplaca falavescens: thallus (a), k+: redpurple (b), thallus transversal section visualized under polarized light (c), ascus (d), spore (e). a b c d e 13,32 μm 0.57 cm 0.8 cm tions of thallus and apothecia k+: red purple. (fig. 1b). habitat: saxicolous on earth of the hassan tower. caloplaca vitellinula (nyl.) h. olivier: thallus crustose, reduced to little orange-yellow granules (fig. 2a). apothecia yolk; they are rare or sometimes many, scattered or grouped in the center of the thallus; they are large below 0.5 mm, thin margin, the disc more or less covex, epithecium golden yellow. paraphyses simples to branched, with the top, 2 or 3 short articles and unevenly swollen (fig. 2c). ascus ellipsoids; spores 8 per ascus, polaribilocular, ellipsoid, hyalines, sometimes very close together in boxes, spores 8.9 µm long 5.49 µm wide (fig. 2d). test color: k+: redpurple (fig. 2b). habitat: saxicolous on the tower’s wall. rocella phycopsis (ach.) ach.: thallus fruticulose, up to 5 cm long, grey branches round or slightly compressed, 1 to 2 mm thickness, smooth, pruinose, subsimple or dichotomously divided (fig. 3a, 3b and 3c) ; medulla of the spike yellow (fig. 3e); soralia many, whitish, tubercled to globose; apothecia absent. cortex: c+ red (fig. 3d) then turns into orange, then yellow and disappears; medulla and soralia c-, koh -. habitat: saxicolous on the tower’s wall of hassan (substrate type: calcarenite). xanthoria calcicola oksner: thallus foliose, rigid, up to 3 cm long, upper side yellow orange, granular, warty (presence of large convex isidia towards the center of thallus), underside simple white rhizines; lobes overlapping and corrugated (fig. 4a). apothecia scarce or absent; disc orange, 0.8 mm across with, frequently notched warty edge; ascus spores, hyaline, polariloculaire, up to 10.82 µm long and 6.82 µm wide (fig. 4c). test color: k+: redpurple (fig. 4b). habitat: saxicolous on the tower’s wall. discussion and conclusion calcareous stones, known as beige calcarenite (“stone of salé”), plio-quaternary age (el azhari, 2009), were used in the construction of the hassan tower. the calcarenite is richer in quartz grains and shows a very high porosity (el amrani, 2009). the high porosity of the rock salt facilitates the infiltration of fluids whose speeds are altered (el azhari, 2009). this process of rock weathering facilitates the development of some lichen species (kofler, 1954). the lichen species found on this monument are calcicolous. they were reported in the north-west and central region of morocco (egea, 1996; alonso and egea, 1997). they were not described and sometimes their substrates were not specified; many factors (historical, topographic, temporal, access, edaphic, climatic, ecological) influence the establishment of mural vegetation. however, lisci et al. (2003) reported that the distribution of the encountered lichen species depends on humidity. roccella a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 80 figure 2. caloplaca vitellinula: thallus attached to the wall tower hassan (a), k+: red purple ( b), paraphyse (c), spore (d). a 0.71 cm b 11.55 µm c d a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 81 phycopsis represents the most abundant species. it is very common on the exposed face northwest of the tower and covers several meters above the ground. rocella phycopsis is almost always fixed vertically to the substrate (lisci et al., 2003). in morocco this species was reported by alonso and egea (1997) on pistacia lentiscus (al hoceima), tetraclinis articulata (dar-mter) and argania spinosa (agadir) caloplaca vitellinula is less abundant and forms an association with rocella phycopsis whereas caloplaca flavescens abounds on the cement ground near the tower. the latter is considered as more frequent not only on pure limestone but also on other calcareous or alkaline rocks and artificial substrata such as cona tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 1 cm d a 1.2 cm c e b figure 3. roccella phycopsis: thallus attached to the wall tower hassan (a), (b) & (c), spot test reaction of thallus c +: red (d), medulla of the spike: yellow (e). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 82 crete, mortar, roofing tiles and monuments but very rarely on the dusty bark of trees near quarries (laundob, 1992). xanthoria calcicola colonizes the walls of the terrace of the tower. it was reported on calcarenite rocks (anonymous, 2005) accompanying rocella phycopsis (kinalioglu, 2007) and is well known for its accumulation of trace metal at high ratio (uluoglo et al., 2007). although lichens are excellent atmospheric pollution bioindicators (rigamonti, 2008). they may produce much worse damage, such as major alterations of stone surfaces through biogeophysical and biogeochemical processes. biogeophysical alterations are caused by the penetration of fungal hyphae beneath the stone surface and by the contraction and expansion of the lichen subsequent to desiccation and rehydration (seaward, 1988). the chemical alterations lichens produce are due to three substances they secrete (nimis, 2001) which carbon dioxide, lichen compounds with complex properties, and oxalic acid carbon. in fact, according to rigamonti (2008), if on the one hand lichens can be considered as being a disturbance that must be eliminated because chemical-physical deterioration and chromatic alteration that some species cause. on the other hand, following a naturalistic approach, they can act as a useful indicator as to the condition of the place. references alaoui m, l asebriy, c bucci , ei el amrani, r franchi, f guerrera, m martin martin, c patamia, g raffalli, mp robles, j tejera de leon, and l tentoni (2006) gis and new technologies in geological analysis for restoration of islamic monuments of the city of rabat (morocco). 5th european congress on regional geoscientific cartography and earth information and systems water, barcelona, catalonia, a b c 13.32 µm0.75 cm figure 4. xanthoria calcicola: thallus attached to the wall tower hassan (a), k+: redpurple (b), spore (c). spain, june 13-16. anonymous ( 2005) www.mousses-lichens-haut-languedoc.org/xanthoria_calcicola.htm. alonso fl and jm egea (1997) líquenes epifíticos de algunas localidades costeras de marruecos. acta botanica malacitana 22: 13-18. arup u (1994) the genus caloplaca on seashore rocks in eastern north america. the bryologist 97 (4): 377-392. asebriy l, c bucci, i el amrani, r franchi, f guerrera, m martin martin, c patamia, g raffaelli, p robles martin, j tejera de leon, and l tentoni (2007) étude intégrée de la dégradation des monuments historiques romains et islamiques de la ville de rabat (maroc): proposition de solutions durables de prévention et restauration. science and technology for cultural heritage 16 (1-2): 45-65. asebriy l, c bucci, te cherkaoui, ei el amrani, r franchi, f guerrera, m martin martin, c patamia, g raffaelli, p robles marin, j tejera de leon, and fj. alcala (2009) deterioration processes on archaeological sites of chellah and oudayas (world cultural heritage, rabat, morocco): restoration test and recommendations. ital. j. geosci., 128 (1): 157-171. bellitir d, r nijs, l asebriy, and m aberkane (1987) evolution de la calcarénite dans les constructions, vitesse d’altération en fonction des facteurs naturels et industriels. mines, géologie & énergie 57: 83-88. caillé j and j hainaut (1954) la mosquée de hassan à rabat. publications de l’institut 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d pinna and o salvadori (1996) licheni e conservazione dei monumenti. clueb ed., bologna, pp. 165. nimis pl, m monte, and m tretiach (1987) flora e vegetazione lichenica di aree archeologiche del lazio. studia geobotanica 7: 3-161. nimis pl (2001) artistic and historical monuments: threatened ecosystems. in frontiers of life, part 2: discovery and spoliation of the biosphere, sect. 2: man and the environment. san diego: academic press, s diego, pp. 557-569. rigamonti m (2008) experimental ecology and geobotany lichens and monuments: searching for the balance between the necessity of restoration and preservation, and the improvement of the naturalisticenvironmental aspects of the historical-architectural heritage. scientifica acta 2 (2): 93-96. seaward mrd (1988) lichen damage to ancient monuments: a case study. lichenologist 20 (3): 291–295. šoun j, and j vondrák (2008) caloplaca aurantia and caloplaca flavescens (teloschistaceae, lichen-forming fungi) in the czech republic; with notes to their taxonomy and nomenclature. czech mycology 60: 275–291. steiner m and j poelt (1982) caloplaca sect. xanthoriella sect. nov.: unterschungen über die “xanthoria lobulata gruppe “( lichenes, teloschistaceae). pl. syst. evol. 140: 151-177. uluozlu od, k kinalioglu, m tuzen, and m soylak (2007) trace metal levels in lichen samples from roadsides in east blak sea region, turkey. biomedical and environmental sciences 20: 203-207. wade ae (1965) the genus caloplaca th fr. in the britsh isles. the lichenologist 3: 1-28. wetmone cm, and ei kärnefelt (1998) the lobate and subfruticose species of caloplaca in north and central america. the bryologist 101 (2): 230-255. 83 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) kbm journal of biology (2010) 1: 10-17 doi: 10.5147/kbmjb.2010.0003 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) molecular epidemiology of nosocomial infection: analysis of chromosomal restriction fragment patterns by pulsed-field gel electrophoresis afaf i shehata1*, alwaleed a. al-aidan2, and buthainah al-shahrani1 1 department of botany and microbiology, college of science, king saud university, p.o. box 2455, riyadh 11451, saudi arabia; 2 molecular virology & infectious disease laboratory, research centre, king faisal specialist hospital and research centre, saudi arabia received: march 4, 2010 / accepted: april 9, 2010 abstract acinetobacter baumannii is a species of non-fermentative gram-negative coccobacilli commonly found in soil, water and other environmental samples. this bacterium is defined as being strict aerobes, nonmotile, catalase-positive and oxidase-negative. this organism was susceptible to most antibiotics in the 1970s. a. baumannii is an opportunistic pathogen that may be an important threat due to its increasing multidrug resistance and is involved in nosocomial infections that are often severe. the objective of this study was undertaken to elucidate the molecular epidemiology of a. baumannii using the most widely applicable dna – based typing methods namely pulsed-field gel electrophoresis (pfge). these strains comprised isolates from environmental samples, blood, wound, urine, cerebrospinal fluid and tracheal aspirates. pfge analysis of 81 clinical isolates has been carried out by using chef–dr iii systems from bio – rad and following the protocol of gautom with some modifications. a 2.00% band tolerance and an optimization of 4.00% were selected for use during comparisons of generated fingerprints or pulsotypes after digestion with apa i restriction enzyme. similarity values have been generated using bionumerics software, cluster analysis was performed by the unweighted pair – group method using arithmetic averages and dna relatedness was calculated based on dice coefficient. an interlinkage homology level of 80% between patterns was assumed as the cutoff for defining a close genetic relationship between strains and was used to define the cluster. as per the generated dendogram, isolates were categorized into 18 major groups designated as strain i to strain xviii. overall, pfge was able to discriminate the 81 different acinetobacter baumannii isolates with similarity levels of 63.63%. __________________________________________________ * corresponding author: a.shib@hotmail.com introduction bacteria of the genus acinetobacter are increasingly being implicated in numerous outbreaks and have become a growing concern in hospitals, identifying a. baumannii as the most predominant species involved. acinetobacter spp. can cause a wide range of clinical conditions, including pneumonia, septicemia, urinary tract infections, wound infections, endocarditis, and meningitis (bergogne–berezin and towner, 1996; mandell, 2000). contaminated hospital equipment or colonized hands of hospital staff have previously been identified as reservoirs of this organism in epidemics (aygun et al., 2002). resistance to multiple antibiotics is a frequent finding with this organism (van looveren et al., 2004). risk factors for acquisition of this organism include prolonged hospital stay, serious underlying disease, intravascular and intravesical catherization, and treatment with broad-spectrum antibiotics (danes et al., 2002). characteristics of acinetobacter spp. may contribute to their epidemic behavior, such as the ability to acquire multiple antibiotic resistance and the ability to survive on inanimate and dry surfaces for prolonged periods of time (dijkshoorn et al., 1987, 1996). however, it should be noted that acinetobacters are ubiquitous organisms that can also be isolated readily from non clinical sources such as soil, drinking and surface waters, sewage, and a variety of different foodstuffs (hanlon, 2005). in many cases, the true source of infection cannot be traced, because members of the genus acinetobacter are widespread in the hospital environment and can be isolated from sinks, tap water, and dust or can be present as commensal organisms of human skin and respiratory tract (gorbach et al., 1998). in order to understand the epidemiology of acinetobacter spp., in hospitalized patients and in the hospital environment, it is therefore vital that the organism be identified to the genomic species level and then typed before epidemiological conclusions are drawn (zarrilli et al., 2004) . the objective of this study was undertaken to elucidate the molecular epidemiology of a. baumannii using the most widely applicable dna – based typing methods namely pulsed field 10 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) gel electrophoresis (pfge). eighty – one clinical a. baumannii isolates were included in this study collected from year 2001 to 2006. the strains were originally isolated from different clinical and environmental specimens by the microbiology laboratory of the king faisal specialist hospital and research centre, riyadh, saudi arabia. analysis by pfge of restriction fragment length polymorphisms generated from intact chromosomal dna has been used to compare fingerprints obtained from acinetobacter strains following restriction with apai, smai, and nhei (luey et al., 2007). these studies have indicated considerable dna polymorphism in the clinically important genomic species 2 (a. baumannii), even within biotypes, and good correlation between strains from within defined outbreaks or multiple isolates from single patients. equipment for pfge is costly, while the preparation of intact chromosomal dna and subsequent digestion and electrophoresis require several days. nevertheless, pfge seems to provide highly discriminatory results and extremely useful epidemiological information (bergogne-berezin and towner, 1996). material and methods sample collection a total of 81 acinetobacter isolates were investigated in this study. bacterial strains tested were obtained from the microbiology laboratory of the king faisal specialist hospital and research centre (riyadh, saudi arabia), collected from year 2001 till 2006. the strains were originally isolated from different clinical and environmental specimens, e.g., blood, cerebrospinal fluid, sputum, tracheal aspirate, urine and wound. they were preserved in tryptic soy broth (tsb) supplemented with 20% (v/v) glycerol. pure isolates were stored at -800c until used in the research centre, kfsh&rc. confirmation of bacterial identification samples were already identified as acinetobacter sp. in microbiology laboratory, department of pathology and laboratory medicine, king faisal specialist hospital & research centre. frozen bacterial suspension was streaked in tryptic soy agar (tsa) and incubated overnight at 370c (jawad et al., 1994 acinetobacter baumannii grows at 440c after consecutive incubation. for gram’s staining, reagents and gram stain kit were used from bd were used (cat. no. 8820191; becton, dickinson and company, md, usa). dna extraction two methods have been carried out to check which will give a better amplifiable dna. first, using the classical proteinase k treatment and the second method using a commercial kit, genomicprep cells and tissue dna isolation kit (catalog no. 27 – 5237 – 01; amersham biosciences, usa). dna extraction using genomic prep cells and tissue dna isolation kit the inoculated tsb was placed overnight at 370c shaker incubator. centrifugation was performed for 5 minutes at 13,000 rpm at room temperature. the supernatant was decanted and the pellet was collected; and extracted using the commercially available genomicprep cells and tissue dna isolation kit (amersham biosciences, usa; catalog no. 27 – 5237 – 01). for rnase treatment, 3 ul of rnase a solution (4 mg/ml) was added to the cell lysate. mix the sample by inverting the tube 25 times and incubate at 370c for 15 – 60 minutes. for protein precipitation, samples should be allowed to cool at room temperature. protein precipitation solution was added to the rnase a-treated cell lysate and dna was precipitated using 70% ethanol. pulsed–field gel electrophoresis (pfge) bacterial extraction and purification were carried out as reported previously with some modification (gautom, 1997). bacterial isolates on tsa plate were incubated overnight at 37ºc. a single bacterial colony is grown overnight in 3 ml tryptic soy broth at 37ºc in a shaker incubator. cells are harvested by centrifugation at 3000 ug for 10 minutes and the supernatant are then discarded. resuspend cells in se buffer (25 mm edta [ph 8.0], 75 mm nacl [ph 8.0]). adjust the optical density of the cells at wavelength of 610 nm to 1.40 with se buffer. mix 0.5 ml aliquot of the bacterial suspension with 0.5 ml of 2% low melting point (lmp) agarose in te buffer (10 mm tris-hcl, 0.1 mm edta). pipette mixture into reusable (300 µl) large size 11 m 1 2 3 4 5 m figure 1. patterns obtained by pfge for acinetobacter baumannii for samples 1 to 5 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. m 6 7 8 9 10 11 14 15 16 17 18 81 m figure 2. patterns obtained by pfge for acinetobacter baumannii for samples 6 to 11, 14 to 18 & 81 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) plug mold and allow to solidify at 4ºc for 30 minutes. release the plugs into 15ml tubes containing 1 ml of lysis buffer (50 mm tris – hcl [ph 8.0], 50 mm edta [ph 8.0], 1% sodium lauroyl sarcosine). proteinase k (1 mg/ml) is added fresh on the day of the experiment. incubate plugs overnight at 55ºc in a waterbath. replace the lysis buffer with 5 ml of sterile distilled water and incubate at room temperature for 5 minutes. replace with 3 ml of te [10 mm tris – hcl [ph 8.0], 1 mm edta [ph 8.0]) for 5 minutes at room temperature. final sets of four washes, 30 minutes each, are done with 3 ml of te (10 mm tris hcl [ph 8.0], 1 mm edta [ph 8.0]) at room temperature. place a plug slice of 3 x 5 mm wide in a 200 µl of 1x restriction buffer 4 and incubate for 30 minutes at 4ºc with gentle agitation. place the plugs in 200 µl of 1x restriction buffer 4 containing 50 units of apa i restriction enzyme and incubate the mixture overnight at room temperature. wash plugs with 0.5 ml of 0.5x tbe for 37ºc for 30 minutes. insert the plugs into the wells of 1% agarose gel dissolved in 0.5x tbe. overlay all the wells with 1% lmp agarose dissolved in 0.5x tbe and allow to solidify at 4oc for 30 minutes. 0.5x tbe running buffer was allowed to re – circulate on ched – dr iii (bio – rad) at least 2 hours before running the gel to maintain a temperature of 14ºc. dna restriction fragments were separated for 22 hours at 200 v, with pulse times ranging from 2.2 to 54.2 seconds at 120. gel is then soaked in 300 ml of deionized water containing 1 µg/ml of ethidium bromide for 30 minutes, and visualized the bands using an ultraviolet illuminator. results pfge analysis pfge analysis of 81 clinical isolates has been carried out by using chef – dr iii systems from bio–rad and following the protocol of gautom with some modifications. figure1 to 10 show fingerprints or pulsotypes using apa i restriction enzyme. pfge patterns were analyzed by both computer – assisted program (bionumerics software) and by manual or visual comparison of each banding patterns. figure 11 shows dendogram based on computer – assisted comparison of pfge profiles of 81 acinetobacter baumannii isolates using bionumerics software. a 2.00% band tolerance and an optimization of 4.00% were selected for use during comparisons of dna profiles. table 2 shows generated similarity values using bionumerics software, cluster analysis was performed by the unweighted pair – group method using arithmetic averages and dna relatedness was calculated based on dice coefficient. an interlinkage homology level of 80% between patterns was assumed as the cutoff for defining a close genetic relationship between strains and was used to define the cluster. as per the generated dendogram, isolates were categorized into 18 major groups designated as strain i to strain xviii. overall, pfge was able to discriminate the 81 different acinetobacter baumannii isolates with similarity levels of 63.63%. a total of 4 isolates were categorized into strain i: strain i-1, i-2, i-3 and i-4 represented by isolates number 10, 54, 56 and 37, respectively. strains ii-1, ii-2 and ii-3 were represented by isolates number 6, 8 and 3, respectively. strain iii-1, iii-2 and iii-3 represented by sample number 50, 53 and 27, respectively. strain iii-4 was 12 figure 3. patterns obtained by pfge for acinetobacter baumannii for samples 12 & 13 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. figure 4. patterns obtained by pfge for acinetobacter baumannii for samples 19 to 25 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. m 12 13 m m 19 20 21 22 23 24 5 m m 26 27 28 29 30 31 m 32 33 34 35 36 37 m figure 5. patterns obtained by pfge for acinetobacter baumannii for samples 26 to 37 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) comprised of 5 isolates (32, 33, 34, 35 & 36) that were indistinguishable from each other. strain iii-5 is represented by sample number 78. strains iv-1, iv-2 and iv-3 were represented by sample numbers 63, 64 and 13, respectively. strain v is represented by sample number 20. strain vi, which is considered one of the biggest cluster in this analysis was comprised of 13 isolates (sample number 76, 77, 65, 66, 71, 73, 75, 72, 74, 79, 80, 82 and 83). strain vi-1 is represented by sample number 76. strain vi-2 is represented by sample number 77. strain vi-3 is represented by samples number 65 and 66 that are indistinguishable from each other. strain vi-4 is represented by sample number 71. strain vi-5 is represented by samples number 73 and 75 (indistinguishable). strain vi-6 represented by samples number 72, 74 and 79 (indistinguishable). strain vi-7 represented by sample number 80. strain vi-8 represented by samples number 82 and 83 (indistinguishable). strain vii represented by sample number 5. strain viii-1 to viii-3 represented by sample number 42, 68 and 69, respectively. strain ix-1 is represented by samples number 47, 48 and 49 (indistinguishable). strain ix-2 represented by samples number 26 and 27 (indistinguishable). strain ix-3, ix-4 and ix-5 represented by sample number 61, 30 and 55, respectively. x-1 and x-2 represented by sample number 70 and 84, respectively. strain xi is represented by sample number 17. xii-1 and xii-2 are represented by sample number 1 and 2, respectively. strain xiii-1 is represented by sample number 15 and 18 (indistinguishable). xiii-2 is represented by sample number 7. strain xiii-3 is represented by sample number 21 and 24 (indistinguishable). strains xiii-4 and xiii-5 were represented by samples number 29 and 62, respectively. strain xiv represented by sample number 4. strain xv, together with strain vi, which is also considered one of the biggest clusters in this analysis consists of 13 isolates (sample number 25, 28, 38, 39, 41, 44, 46, 52, 81, 58, 59, 60 and 57). strains xv-1 and xv-2 were represented by samples number 25 13 figure 6. patterns obtained by pfge for acinetobacter baumannii for samples 38 to 45 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. figure 7. patterns obtained by pfge for acinetobacter baumannii for samples 42, 46 to 55 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. figure 8. patterns obtained by pfge for acinetobacter baumannii for samples 56 to 67 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. figure 9. patterns obtained by pfge for acinetobacter baumannii for samples 68 to 80 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. m 38 39 40 41 42 43 44 45 m m 42 46 47 48 49 50 m 51 52 53 54 55 m m 56 57 58 59 60 61 m 62 63 64 65 66 67 m m 68 69 70 71 72 73 74 75 76 77 78 79 80 m kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) and 28, respectively. strain xv-3 was represented by sample numbers 38, 39, 41, 44 and 46, which are indistinguishable. strains xv-4 and xv-5 were represented by sample number 52 and 81, respectively. xv-6 is represented by samples number 58, 59, 60 and 57 (indistinguishable). strains xvi-1 and xvi2 are represented by samples number 31 and 51, respectively. xvi-3 represented by samples number 11 and 14 (indistinguishable). strains xvi-4 and xvi-5 represented by samples number 16 and 45, respectively. strain xvii is represented by sample number 22. strain xviii-1 is represented by samples number 12 and 19 (indistinguishable). strain xviii-2 is represented by sample number 9. for visual analysis, the tenover classification (tenover et al. 1995) was used to interpret the banding patterns generated. results were summarized in table 10, which generates 25 strains. wherein strain a and strain b were considered the biggest in the group comprising of 13 isolates per group. strain a were divided into 7 (seven) subgroups, a1 to a7. strain a1 was represented by sample numbers 38, 39, 41, 44 and 46. strain 14 figure 10. patterns obtained by pfge for acinetobacter baumannii for samples 81 to 84 after running in 1% agarose gel, staining with ethidium bromide and illuminated under uv light. 50 – 1000 kb lambda ladder was used as a standard size marker. figure 11. dendogram based on pulsed – field gel electrophoresis (pfge) of 81 samples of acinetobacter baumannii. the dendogram is shown on the left with the percent – homology score indicated on top (0% 100%). the actual pfge banding patterns are given on the immediate right of the dendogram. dice (opt: 4.00%) (tol 2.0%-2.0%). m 81 82 83 84 m kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) represented by sample number 80. strain b5 was represented by samples number 65 and 66. strains b6 to b8 were represented by samples number 71, 77 and 76, respectively. strain c was classified into 3 (three) subgroups: c1 represented by samples number 32, 33, 34, 35 and 36. strains c2 and c3 were represented by samples number 78 and 27, respectively. strain d was classified into 4 (four) subgroups: d1 represented by samples number 15 and 18. d2 represented by sample number 7; d3 represented by samples number 21 and 24; and d4 represented by sample number 29. strain e were divided into 3 (three) groups: e1 represented by samples number 47, 48 and 49; e2 represented by samples number 26 and 67; and e3 represented by sample number 61. strain f was classified into 5 (five) subgroups: f1 represented by samples number 11 and 14; f2 to f5 were represented by samples number 16, 31, 51 and 45, respectively. strain g was classified into 2 (two) groups: g1 represented by samples number 12 and 19; g2 is represented by sample number 9. strain h was classified into 3 (three) subgroups: h1 to h3 were represented by samples number 10, 54 and 56, respectively. strain i was classified into subgroup i1 to i3, that are represented by samples number 6, 8 and 3, respectively. strain j was classified into subgroups j1 to j3, which are represented by samples number 63, 64 and 13, respectively. strain k was classified into subgroups k1 to k3, which are represented by samples number 42, 68 and 69, respectively. strain l was classified into 2 (two) subgroups: l1 and l2, which are represented by samples number 70 and 84, respectively. the rest of the 13 samples were considered unique, and assigned to strain m to strain y that are represented by samples number 1, 2, 20, 5, 17, 4, 22, 30, 50, 55, 62, 53 and 37, respectively. numerical index of the discriminatory ability of typing system was calculated using the simpson’s index of diversity (hunter and gaston et al. 1988) and is given by the following equation: where n is the total number of strains in the sample population, s is the total number of types described, and nj is the number of strains belonging to the jth type. the equation can be applied both to a direct comparison of the discriminating power of typing methods and to analysis of the discriminating power of combined typing schemes. table 1 shows the discriminating indiex for pfge. it can be seen that the discriminatory power of pfge (0.914). typability (100%) was also obtained for this methods used in this study (table 2). this is done by dividing the samples that is typeable against the total number of samples. table 2. showed t groupings of 81 samples for pfge analysis . discussion acinetobacter spp. has unique characteristics among nosocomial gram – negative bacteria that favor their persistence in the hospital environment. this organism spreads easily in the environment of infected or colonized patients and can persist in that environment for many days, a factor that may explain their propensity for causing extended outbreaks. however, it should be noted that acinetobacters are ubiquitous organisms that can also be isolated readily from nonclinical sources such as soil, drinking and surface water, sewage, and a variety of different foodstuffs. there appears to be a significant population differences between the genomic species found in clinical specimens and those found in other environments, and it is therefore 15 a2 is represented by samples number 58, 59 and 60. strains a3 to a7 were represented by samples number 52, 81, 28, 25 and 57, respectively. strain b was classified into 8 (eight) subgroups; b1 represented by samples number 72, 74 and 79. strain b2 represented by samples number 73 and 75. strain b3 is represented by samples number 82 and 83. strain b4 is table 1. discrimination indiex for typing method used in this study pfge. method no. of types typability discrimination index pfge 18 100% 0.914 table 2. groupings of 81 samples for pfge analysis. kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) vital that acinetobacters be identified to the genomic species level and then typed before epidemiological conclusions can be drawn. the increased incidence of acinetobacter baumannii infection required clarification of a possible mode of transmission. the predominant of one genotype in patient or environmental specimens seemed to suggest transmission from common sources. bacterial typing schemes based on genotypic analysis of multiple isolates within a particular species to identify characteristics that may subdivide the strains into smaller groupings. such analyses have several uses – to investigate outbreaks that may in turn influence or focus epidemiological investigations, to examine sequential isolates from a single patient to determine whether infection is recurring or the patient has suffered a relapse, to establish whether certain strains are associated with specific clinical syndromes, and therefore, have unusual pathogenic mechanisms, and in a wider context, to increase our understanding of the epidemiology of infection. nevertheless, typing is most often used to differentiate dissimilar isolates rather than to confirm a relationship between two different strains. the basic premise of all typing schemes is that strains isolated from an epidemiological cluster arise from a common precursor and therefore, that these strains will share certain characteristics that can distinguish them from epidemiologically unrelated strains of the same species. at least three criteria are necessary for the evaluation of typing schemes: (1) typability – the ability to obtain a definite result for each isolate tested, (2) reproducibility – the ability to achieve the same result whenever and wherever the same strain is tested, and (3) discrimination – the ability to distinguish between epidemiologically unrelated strains. pfge technique was shown to be most suitable method for differentiating strains from hospital outbreaks. because pfge fingerprints are highly reproducible, interpretation is fairly straightforward. however, major disadvantage of pfge is the difficulty of comparing results obtained from different laboratories. in addition to the expense of the pfge apparatus, the total time required to perform the test is a disadvantage of this method. plugs containing the dna extraction procedures take about 2 to 3 days, although more rapid methods have been developed. the electrophoresis time is also lengthy, 24 hours is a typical running time, and a fair amount of technical expertise is necessary. to prevent mechanical breakage of chromosomal dna, all extraction steps must be carefully performed with preparations embedded in agarose. in this study, evaluation of genomic fingerprinting methods performed by computerized comparison of digitized fingerprinting patterns (in pfge analysis) is easier and gives an accurate analysis for large numbers of samples tested. data analysis by computer offers the possibility of comparison of large numbers of patterns, formation of databases, and cluster analysis. although visual analysis for pfge is also possible, however for pfge analysis, a guest commentary (tenover et al., 1995) proposes a set of guidelines for interpreting dna restriction patterns generated by pfge. the authors are investigators from the united states who, over the last several years, have correlated epidemiologic data from dozens of outbreaks with strain typing results produced by pfge. these guidelines are intended to be used by clinical microbiologists in hospital laboratories to examine relatively small sets of isolates (typically, < 30) related to putative outbreaks of disease. in this study, to give an assessment in whichpfge typing method is the most efficient several factors must be considered that includes: reproducibility, typability and discrimination. reproducibility is the percentage of strains that give the same result on repeated testing. typability of a method is the percentage of distinct bacterial strains which can be assigned a positive typing marker. pfge give 100% typability. the discriminatory power of a typing method is its ability to distinguish between unrelated strains. numerical index of discriminatory ability of typing system was calculated using simpson’s index of diversity. it can be seen that the discriminatory power of pfge (0.914) . in conclussion, our data suggest that pulsed – field gel electrophoresis (pfge) can cluster epidemiologically unrelated strains of acinetobacter baumannii into distinct populations; and the three factors or criteria for an effective assessment of typing method has been met. at this stage, having a properly optimized laboratory protocol for pfge technique that can generate comparable and reproducible results is a necessary first step. in choosing a typing scheme for epidemiological studies, one should aim for as large discriminatory index as possible. the acceptable level of discrimination will depend on a number of factors, but an index of greater than 0.90 would seem to be desirable if the typing results are to be interpreted with confidence. references aygun g, o demirkiran, t utku, b mete, s urkmez, m yilmaz, h yasar, y dikmen, and r ozturk (2002) environmental contamination during a carbapenem-rresistant acinetobacter baumannii outbreak in an intensive care unit. j hosp infect 52: 259–62. bergogne-berezin e and kj towner (1996) acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. clinical microbiology reviews. 9: 148–165. danes c, mm navia, j ruiz, f marco, a jurado, mt jimenez de anta, and j vila (2002) distribution of b-lactamases in acinetobacter baumannii cclinical isolates and the effect of syn 2190 (ampc inhibitor) on the mics of different b-lactam antibiotics. j antimicrob chemother 50: 261–264. denton m, nj todd, kg kerr, pm hawkey and jm littlewood (1998) molecular epidemiology of stenotrophomonas maltophilia isolated from clinical specimens from patients with cystic fibrosis and associated environmental samples. j of clin microbiol 36: 1953–1958. dijkshoorn l, h aucken, p gerner-smidt, p janssen, me kaufmann, j garaizar , j ursing and tl pitt (1996) comparison of outbreak and non-outbreak acinetobacter baumannii strains by genotypic and phenotypic methods. j clin microbiol 34: 1519–1525. dijkshoorn l, w van vianen, j degener, and m michel (1987) typing of acinetobacter calcoaceticus strains iisolated from hospital patients by cell envelope proteins profiles. epidemiol. infect 99: 659–667. erdenizmenli m and a grossato (2007) rapid pulsed–field gel electrophoresis for typing of acinetobacter species. department of histology, microbiology and medical biotechnology. university of padua, padova, italy. gautom rk (1997) rapid pulsed–field gel electrophoresis for typing of escherichia coli o157:h7 and other gram negative organisms. j clin microbiol 35: 2911–2980. gerner–smidt p and i tjernberg (1993) acinetobacter in denmark ii. molecular sstudies of the acinetobacter calcoaceticus acinetobacter baumannii complex. acta pathol microbiol immunol scand 101: 826–832. goering rv (1993) molecular epidemiology of nosocomial infection: analysis of chromosomal restriction fragment patterns by pulsed– field gel electrophoresis. infection control and hospital epidemiology 14: 595–600. 16 gorbach sl, jg bartlett, and nr blacklow (1998) infect diseases (2nd eds.). pp. 1877–1878. hanlon gw (2005) under the microscope: the emergence of multidrug resistant acinetobacter species: a major concern in the hospital setting. letters in appl microbiol 41: 375–378. hunter pr and ma gaston (1988) numerical index of the discriminatory ability of typing systems: an application of simpson’s index of diversity. j clin microbiol 26: 2465–2466. jawad a, p hawkey, j heritage and a snelling (1994) description of leeds acinetobacter medium, a new selective and differential medium for isolation of clinically important acinetobacter spp., and comparison with herellea agar and holton’s agar. j clin microbiol 32: 2353–2358. kropec a, j hubnerand, and f daschner (1993) comparison of three typing methods in hospital outbreaks of acinetobacter calcoaceticus infection. j hosp infect 23: 133–141. kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 17 luey ck, yw chu, tk cheung, cc law, my chu, dt cheung and km kam (2007) rapid pulsed-field gel electrophoresis protocol for subtyping of streptococcus suis serotype 2. j microbiol methods 68: 648–650. mandell gl, je bennett, and rd dolin (2000) principles and practice of infectious diseases, fifth edition; pp. 2339. tenover fc, rd arbeit, rv goering, pa mickelsen, be murray, dh persing, and b swaminathan (1995) interpreting chromosomal dna restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. j clin microbiol 33: 2233–2239. van looveren m, h goossens and the arpac steering group (2004) antimicrobial resistance of acinetobacter spp. in europe. clin microbiol infect 10: 684–704. zarrilli r, m crispino, m bagattini, e barretta, a di popolo, m triassi and p villari (2004) molecular epidemiology of sequencial outbreaks of acinetobacter baumannii in an intensive care unit shows the emergence of carbapenem resistance. j clin microbiol 42: 946–53. kbm journal of biology (2010) 1 (2): 26-29 doi: 10.5147/kbmjb.2010.0009 kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) survey of macrofungi (including truffles) in qatar roda f. al-thani department of biological and environmental sciences, college of arts and sciences, qatar university, qatar received: february 16, 2010 / accepted: may 27, 2010 introduction qatar is a limestone peninsula predictive northward into the arabian gulf. it is approximately 160 km long by 80 km wide, consisting mostly of low-lying, arid, stony desert. average temperatures vary between 12 ºc – 21 ºc in january, and 35 ºc 49 ºc from june to september. the climate during autumn and spring is moderate, while winter can be surprisingly cool. average rainfall is 5-9 cm per year, although the actual amount varies considerably from one year to the next. macrofungi are fungi that form large fruiting bodies visible without the aid of a microscope. the distribution of macrofungal species is low in hot and dry seasons while they are abundant in spring and autumn due to the humid climate as well as the richness of the flora at this time (sibounnavong et al., 2008). macrofungal studies have long been of interest to scientists in qatar and elsewhere as well as the community due to their significant roles in human life, their use in the pharmaceutical industry, and the mass production of cultivated fungi in the food industry (lindequist et al., 2005), as well as their vital role in biodegradation (de boer et al., 2005). __________________________________________________ * corresponding author: ralthani@qu.edu.qa some of the different species of desert truffle that grow in arid and semi arid areas and that prefer high ph calcareous soils have been found in qatar. traditionally, desert truffles are used by some people for food, as a cash crop, and as medicine for a variety of ailments (mandeel and al-laith, 2007). surveys of macrofungi were reported by different researchers but little information is reported about the macrofungi of qatar (moubasher, 1993). the aim of this study was to determine and provide more data on the hyper off macrofungi and desert truffle species in the harsh environment of qatar. materials and methods visually conspicuous macrofungal species were collected from different natural sites (natural areas, damp places of public gardens, on decayed roots of the dead trees and under trees), photographed and kept for laboratory identification. materials were examined and morphological characteristics were recorded and identified to species level by comparison to literatures (moustafa, 1995; segedin and pennycook, 2001; 26 abstract fourteen macrofungal genera belonging to 11 families and 7 orders were identified. these were isolated from different habitats (semidesert, gardens, park, decayed roots of dead trees and under trees) in qatar. macrofungi are defined here as ascomycetes and basidiomycetes with large, conspicuous spore-bearing structures that form above or beneath ground. this study indicated that the distribution of macrofungi is dependent on the plant community and the environmental conditions. key words: macrofungi, desert truffles, biodiversity, qatar. kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) 27 figure 1. some macrofungi and truffle species reported for qatar. agaricus sp. podaxus sp. ulostoma album volvariella volvacea terfezia claveryi tirmania nivea ganoderma lucidum montagnea arenaria kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) jean lodge et al., 2004; abou-zeid and altalhi, 2006; helfer, 2008). results and discussion in this study, 14 macrofungal genera belonging to 11 families and 7 orders were identified. the data presented in table 1and plate 1, document the identified macrofungi . two genera of the family terfeziaceae occur in qatar. these are terfezia and tirmania. terfezia spp. have spherical and ornamented spores, while tirmania spp. have smooth spores and amyloid asci. the fruitbodies are round, tan to brown, and look like small, sandy potatoes. the three well known varieties of truffles, namely, phaeangium lefebvrei (hoper) (pyronemataceae), terfezia claveryi (khlasi) (terfeziaceae), and tirmania nivea (zabidi) (pezizaceae) are common in the qatar desert (table 1). there is rising interest in mapping the macrofungal flora of many areas to obtain distribution records similar to those already existing for flowering plants (mueller et al., 2007). however, unlike plants, the identification of macrofungi relies on the collection of fruiting bodies, which in turn is largely dependent upon the availability of moisture, that is, in most cases, seasonal rains (arora, 1991). hayes (1976) reported that natural geographic areas of mushrooms extend all over the northern hemisphere outside the tropic and the arctic. gray (1997) reported that agaricus campastris is common wild mushrooms in europe and america. abou-zeid and altalhi (2006) recorded that the highest frequency occurrence of wild mushrooms in al-taif governorate in saudi arabia was for agaricus bisporus; lepiota procera and l. rhacodes. al-fatimi et al. (2006) on an ethnobotanical study on fungi in yemeni ethnomedicine found that the fungus podaxis pistillaris (podaxales, podaxaceae, basidiomycetes) exhibits antibacterial activity against staphylococcus aureus, micrococcus flavus, bacillus subtilis, proteus mirabilis, serratia marcescens and escherichia coli. in china, mushrooms can be used to solve several problems such as human nutrition and avoiding waste pollution by degrading waste products of animals (chen li and nicholas, 2005). chang and lee (2004) observed that species of local macrofungi are utilized not only for food, but also as medicine and for spiritual purposes, including discouraging certain undesirable behavior in children. genera terfezia and tirmania in the family terfeziaceae, order pezizales, grow in arid and semi-arid areas of the mediterranean region, the arabian peninsula, and north-africa. some have been found in south africa and china. species of terfezia and tirmania prefer high ph calcareous soils, typical of desert soils. although the genera terfezia and tirmania are primarily ectomycorrhizal (forming a sheath around the roots of their host plant), they are highly adaptable (diez et al., 2002). some species, like terfezia arenaria, terfezia claveryi, and tirmania pinoyi, form endomycorrhizal associations in phosphate-poor soils and ectomycorrhizal associations in phosphate-rich soils. species of both genera form mycorrhizas on roots mainly of members of the genus helianthemum (family cistaceae) (see fig. 1). heavy rains, thunder and soil type are the most important reasons in truffle formation. the role of desert truffles on traditional, socioeconomic, ecology and socio-cultural in qatar and others gulf states is well known. in qatar two truffle species were identified; tirmania nivea “zubaidi” was found to be the most preferred expensive and common type of truffle in the region due to its pleasant light smell, delicacy, and soft white tissues. this was followed by terfezia claveryi “ikhlasi”. the lists of macrofungi in this paper provide the baseline data needed for ongoing assessment of changes in biological diversity in qatar. it is an important first step towards a “checklist of macrofungi in qatar” and a source of baseline data for the scientific community. acknowledgments i wish to thank prof. eklas abd elbary for her expert advice in this studies and prof. malcolm potts for his helpful comments on the manuscript. references abou-zeid am and altalhi ae (2006) survey of some mushrooms in al-taif governorate of saudi arabia. world journal of agricultural sciences 2 (1): 01-05. al-fatimi m, ju lich wd, jansen r and lindequist u (2006) bioactive components of the traditionally used mushroom podaxis pistillaris. ecam 3(1): 87–92. arora d (1991) all that the rain promises and more. a hip pocket guide to western mushroom. ten speed press, 261 pp. chang ys and lee ss (2004) utilization of macrofungi species in malaysia. fungal diversity 15: 15-22. chen li and nicholas ho (2005) the most widely recognized mushroom: chemistry of the genus amanita. life science 78:532-538. de boer w, folman lb, summerbell rc and boddy l (2005) living in a fungal world: impact of fungi on soil bacterial niche development. fems microbiology review 29: 795–811. 28 figure 2. tirmania nivea “zubaidi” form mycorrhizas on roots of helianthemum sp. kb m j ou rn al o f bi ol og y is sn 1 94 858 83 . p ub lis he d by k bm s ci en tifi c pu bl ish in g, l p (w w w .k bm -s ci en tifi cpu bl ish in g. or g) diez j, manjon jl and martin f (2002) molecular phylogeny of the mycorrhizal desert truffles (terfezia and tirmania), host specificity and edaphic tolerance. mycologia 94 (2): 247-259. gray w (1997) the use of fungi as food processing. crc press, new york, usa., pp: 30. hayes wa (1976) new looks at mushrooms. j nutrition and food sciences, 42: 2-6. helfer s (2008) mycota of south-west asia. turkey journal of botany. 32: 481-484 jean lodge d j, ammirati jf, o’dell te and mueller gm (2004) collecting and describing macrofungi. in: g mueller, g bills and m foster (eds) biodiversity of fungi inventory and monitoring methods. elsevier academic press, pp. 128-158. lindequist u, niedermeyer thj and ju¨lich wd (2005) the pharmacological potential of mushrooms. ecam 2: 285–299. mandeel q a and al-laith a a (2007) ethnomycological aspects of the 29 desert truffle among native bahraini and non-bahraini peoples of the kingdom of bahrain. journal of ethnopharmacology. 110 (1): 118-129. moubasher ah (1993) soil fungi in qatar and other arab countries, university of qatar press, doha, qatar, pp. 566. moustafa af (1995) taxonomic studies on the fungi of kuwait. iii. ascomycotina (plectomycetes and discomycetes). journal of university of kuwait 12: 79-100. mueller gm. et al. (2007) global diversity and distribution of macrofungi. biodiversity and conservation. 16: 37–48 segedin bp and pennycook sr (2001) a nomenclatural checklist of agarics, boletes and related secotioid and gasteromcetous fungi recorded from new zealand. new zealand journal of botany 39: 285–348 sibounnavong p, cynthia cd, kalaw sp, reyes rg and soytong k (2008) some species of macrofungi at puncan, carranglan, nueva ecija in the philippines. journal of agricultural technology 4 (2): 105-115. atlas journal of biology 1 (3): 62-65, 2011 doi: 10.5147/ajb.2011.0049 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) inheritance of shoulder spotting in the red-base tetra (characidae: hemigrammus stictus) jack s. frankel* department of biology, howard university, washington, dc 20059, usa received: january 26, 2011 / accepted: may 8, 2011 __________________________________________________ * corresponding author: jfrankel@howard.edu introduction fishes in the teleost genus hemigrammus (characidae) exhibit a broad spectrum of body colorations and marking patterns (axelrod and vorderwinkler, 1995; riehl et al., 1997; frankel, 2000, 2002). the red-base tetra (h. stictus), so named due to the prominent red coloration found on both the caudal peduncle and caudal fin, is a south american characin that is easily maintained and bred in captivity. while not a commonly imported species, h. stictus routinely appears in catches of the more popular south american characins collected from venezuela, guyana, and brazil and, as such, is usually available to aquarists. in addition to this strikingly red caudal region, both female and male fishes in natural populations of h. stictus characteristically exhibit a prominent black spot or patch located caudally to the operculum. while this shoulder spot is a feature of virtually all individuals of h. stictus, a rare alternate phenotype is occasionally found which lacks this shoulder spot. the inheritance of this spotting pattern is of particular interest, since it most probably serves a prominent role in protecting individuals from predation by acting as an eye-spot. as a result of our interest in the 62 short communication abstract the red-base tetra (hemigrammus stictus) exhibits two phenotypes associated with shoulder spotting. fish either possess a prominent black shoulder spot located directly behind the operculum or lack this spotting pattern. segregation patterns observed from the progenies of eleven different crosses suggest that the inheritance of shoulder spotting is controlled by the action of two autosomal loci acting in a complementary recessive fashion, with dominance at either locus resulting in the expression of the spotted phenotype. key words: hemigrammus stictus, red-base tetra, shoulder spotting, characidae. 63 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. probable genotypes (pg), observed phenotypic numbers, expected ratios, degrees of freedom (df), chi-square values (x2) and probability of fit (p) for crosses amongst spotted and unspotted hemigrammus stictus. * (s) = spotted parental fishes; (n) = unspotted parental fishes; (f) = f1 offspring. + the probability for all x2 tests is > .05; thus, all observed results fit the expected ratio according to mendelian inheritance. cross no. parents*__ _ ♀ (pg) ♂ (pg) _ phenotypic numbers___ spotting no spotting exp. ratio df x2 p+ 1 si (aabb) x s1 (aabb) 40(fi&f1) 0 1:0 2 sii (aabb) x s2 (aabb) 51 0 1:0 3 siii (aabb) x s3 (aabb) 50 0 1:0 4 siv (aabb) x s4 (aabb) 38 0 1:0 5 sv (aabb) x s5 (aabb) 42(fii&f2) 0 1:0 6 si (aabb) x s4 (aabb) 46 0 1:0 pooled 267 0 1:0 7 ni (aabb) x n1 (aabb) 0 35(fiii&f3) 0:1 8 nii (aabb) x n2 (aabb) 0 46(fiv&f4) 0:1 9 niii (aabb) x n3 (aabb) 0 47 (fv&f5) 0:1 pooled 0 128 0:1 10 si (aabb) x f1 (aabb) 32 0 1:0 11 fi (aabb) x s1 (aabb) 30 0 1:0 12 sv (aabb) x f2 (aabb) 41 0 1:0 13 fii (aabb) x s5 (aabb) 44 0 1:0 pooled 147 0 1:0 14 ni (aabb) x f3 (aabb) 0 43 0:1 15 nii (aabb) x f4 (aabb) 0 29 0:1 16 niii(aabb) x f5 (aabb) 0 47 0:1 17 fiv (aabb) x n2 (aabb) 0 33 0:1 18 fv (aabb) x n3 (aabb) 0 35 0:1 pooled 0 187 0:1 19 si (aabb) x n1 (aabb) 37(fvi&f6) 0 1:0 20 sii (aabb) x n2 (aabb) 32(fvii&f7) 0 1:0 21 siii (aabb) x n3 (aabb) 44(fviii&f8) 0 1:0 22 n1 (aabb) x s4 (aabb) 31(fix&f9) 0 1:0 23 nii (aabb) x s5 (aabb) 35(fx&f10) 0 1:0 pooled 179 0 24 fvi (aabb) x f6 (aabb) 38 2 15:1 1 .107 .7440 25 fvii (aabb) x f7 (aabb) 37 5 15:1 1 2.292 .1300 26 fviii (aabb) x f8 (aabb) 48 3 15:1 1 .012 .9136 27 fix (aabb) x f9 (aabb) 37 1 15:1 1 .849 .3568 28 fx (aabb) x fx (aabb) 33 4 15:1 1 1.314 .2518 29 fvi (aabb) x f8 (aabb) 28 1 15:1 1 .389 .5331 30 fvii (aabb) x f9 (aabb) 60 6 15:1 1 .909 .3404 total 7 5.872 .5547 pooled 281 22 15:1 1 .528 .4673 heterogeneity 6 5.344 .5005 31 fix (aabb) x f3 (aabb) 28 8 3:1 1 .148 .7003 32 fx (aabb) x f4 (aabb) 35 10 3:1 1 .185 .6670 33 fiv (aabb) x f8 (aabb) 30 9 3:1 1 .077 .7815 34 fv (aabb) x f9 (aabb) 15 7 3:1 1 .545 .4602 total 4 .955 .9165 pooled 108 34 3:1 1 .085 .7713 heterogeneity 3 .870 .8326 64 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) inheritance of disruptive banding and spotting patterns in several genera of freshwater teleosts (frankel, 1985, 1991, 1998, 2001, 2002, 2004, 2005, 2009), the present study was undertaken to ascertain the mode of inheritance of shoulder spotting in h. stictus. materials and methods healthy juvenile specimens of h. stictus were obtained from a wholesale distributor in maryland, usa, and maintained in separate 76 liter holding tanks at 26oc. male and female fishes exhibiting either the characteristic shoulder spotting phenotype or the unspotted phenotype, were selected at random from stock specimens, placed in separate 76 liter tanks, and allowed to develop at 26oc until sexually mature. optimal water conditions for hemigrammus were provided for all fish (i.e. low water hardness of 5o dgh, ph 6.5, and temperature 26oc) (riehl et al., 1997). all progeny for this study were obtained from artificial fertilizations as previously described (frankel, 1985). parental fishes, exhibiting either the spotted (s) or unspotted (n) phenotype, along with f1 progeny (f), were used in a series of 34 crosses (table 1). embryos from all crosses were incubated at 26oc in 250 ml fingerbowls containing tank water. dead or developmentally arrested embryos were removed daily. fry hatched 24-36 hours post-fertilization and were free-swimming 72-96 hours post-hatching. progeny groups were placed in separate 36 liter rearing tanks, fed initially on rotifers and allowed to develop until their phenotype could be visually determined. since spotting is more defined in mature individuals, determination of spotting was only scored for mature individuals. phenotypic data of all progeny were recorded and subjected to chi-square analysis. pooled and heterogeneity chi-square tests were also performed, treating the progenies from reciprocal f1xf1 crosses as single large progenies in an analysis of overall goodness of fit. results and discussion table 1 presents data for the proposed genotypes of parental fishes, observed phenotypic numbers, expected ratios, and probability of fit for h. stictus analyzed for the mode of inheritance of shoulder spotting. parental fishes and progeny from all crosses clearly displayed either the spotted or unspotted phenotype. spotted females si, sii, siii, siv, sv and males s1, s2, s3, s4, s5 were scored as homozygous dominants, as crosses involving these individuals always resulted in spotted progeny (crosses 1-6, 10-13, 19-23). parental fishes lacking shoulder spots (ni, nii, niii, females and n1, n2, n3 males) were scored as homozygous recessives, as crosses amongst these individuals consistently bred true (crosses 7-9). further, when fiv and and fv females, and f3, f4, and males were crossed with their parents, the resulting offspring consistently lacked a shoulder spot (crosses 14-18). in addition, reciprocal crosses between spotted and unspotted parental fishes always resulted in spotted progeny (crosses 19-23). crosses amongst the f1 fishes resulting from parental matings always resulted in a satisfactory fit to a 15:1 phenotypic ratio of f2 progeny (crosses 24-30), commensurate with a modified 9:3:3:1 ratio resulting from recessive complementary gene action; the aabb genotype resulting in fishes absent shoulder spotting. this mode of inheritance was further substantiated by matings between presumptive f1 homozygous recessives and f1 heterozygotes (crosses 31-34). both spotted and unspotted fry resulted from these matings and, based on chi-square analyses, conformed to the expected 3:1 phenotypic ratio. results of this study support the hypothesis that shoulder spotting in h. stictus is controlled by two loci acting in a complementary fashion, with dominance at either locus required for the expression of the spotted phenotype. segregation patterns for the spotted and unspotted phenotypes of h. stictus clearly fit an autosomal pattern of inheritance, as chi-square tests do not deviate significantly from expectations. results of heterogeneity tests also support the acceptance of the null hypothesis for this data. further, observations of the extent of spotting in parental, f1, and f2 fishes also suggest that these loci do not act in an additive fashion, since there is no perceptible difference in the appearance of spotting in presumptive aabb spotted parentals (s) with either presumptive aabb spotted f1 progeny (fvi-fx and f6-f10) or spotted f2 fishes, some of which would be heterozygous at one of the two loci involved (i.e. aabb or aabb individuals). a digenic mode of inheritance has also been reported for shoulder spotting in the tetra hyphessobrycon bentosi characidae (frankel, 2009), although in this species shoulder spotting results from a pair of autosomal loci exhibiting dominant complementary gene action. as in h. bentosi, the prominent dark shoulder spot in the red-base tetra most certainly serves as an “eye-spot” and, therefore, gives a selective advantage to those individuals possessing this feature by providing them with an interspecific marking to minimize predation. indeed, the mode of inheritance suggested here for shoulder spotting in h. stictus would clearly provide for individuals in populations of this species to exhibit the spotted phenotype (i.e. a_bb, aab_, and a_b_ all result in the expression of shoulder spotting). interestingly, spotting is not always the preferred or prominent phenotypic alternative in fishes. for instance, studies on the mosquitofish (gambusia holbrooki) (bisazza and pilastro, 2000; horth, 2006), have shown that the melanic (mottled-black) body spotting pattern is inherited as a y-linked trait with autosomal modifiers and is either expressed in very low frequency or is completely absent from certain populations of this poeciliid. references axelrod hr, and w vorderwinkler (1995) encyclopedia of tropical fishes with special emphasis on techniques of breeding. tfh publications. pp. 174-184. bisazza a, and a pilastro (2000) variation of female preference for male coloration in the eastern mosquitofish (gambusia holbrooki). behavior genet 30: 207-212. frankel js (1985) inheritance of trunk striping in the sumatran tiger barb, barbus tetrazona. j hered 76: 478-479. frankel js (1991) inheritance of body marking patterns in the half-banded barb, barbus semifasciolatus. j hered 82: 250-251. frankel js (1998) monogenic inheritance of trunk banding patterns in the sumatra barb, barbus tetrazona. j fish biol 53: 1357-1359. frankel js (2000) monogenic control of iris coloration in the january tetra (hemigrammus hyanuary characidae). j hered 91: 411-412. frankel js (2001) inheritance of caudal peduncle banding in the spiketailed paradisefish. j fish biol 59: 1095-1097. frankel js (2002) caudal spotting in the beacon fish (hemigrammus ocellifer characidae). j hered 93: 285-286. frankel js (2004) inheritance of trunk banding in the tetra (gymnocorymbus ternetzi characidae). j hered 95: 262-264. frankel js (2005) digenic control of colouration in the two-spot gouraa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) mi, trichogaster trichopterus trichopterus. j genet 84: 101-103. frankel js (2009) inheritance of shoulder spotting in the tetra, hyphessobrycon bentosi characidae. open fish sci j 2: 39-41 horth l (2006) a sex-linked allele, autosomal modifiers and temperature-dependence appear to regulate melanism in male mosquitofish (gambusia holbrooki). j exp biol 209: 4938-4945. riehl r, h baensch, h smith, e schulze, and b behme (1997) baensch aquarium atlas: 6th ed. steven simpson books. pp. 266-280. 65 atlas journal of biology 3 (2): 224–235, 2015 doi: 10.5147/ajb.2015.0137 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) genetic mapping of qtl associated with seed macronutrients accumulation in ‘md 96-5722’ by ‘spencer’ recombinant inbred lines of soybean nacer bellaloui1, laila khandaker2, masum akond2, stella k. kantartzi3, khalid meksem3, alemu mengistu4, david a lightfoot3, and my abdelmajid kassem2* 1 crop genetics research unit, agricultural research service, u.s. department of agriculture, 141 experiment station road, p.o. box 345, stoneville, ms, usa; 2 plant genomics and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc, usa; 3 department of plant, soil and agricultural systems, southern illinois university, carbondale, il, usa; 4 crop genetics research unit, usda-ars, jackson, tn, usa; 5 current address: the school of arts and sciences, american university of ras al khaimah, ras al khaimah, p.o. box 10021, uae. received: february 7, 2015 / accepted: march 15, 2015 __________________________________________________ * corresponding author: mkassem@uncfsu.edu 224 abstract research of quantitative trait loci (qtl) for macronutrient accumulation in soybean seed is limited. therefore, the objective of this research was to identify qtl related to macronutrients (n, c, s, p, k, ca, and mg) in seeds in 92 f5:7 recombinant inbred lines (rils) developed from a cross between md 965722 (md) and spencer using a total 5,376 single nucleotide polymorphism (snp) markers. a genetic linkage map based on snp markers was constructed using the illumina infinium soysnp6k beadchip array. the rils were genotyped using 537 polymorphic, reliably segregating snp markers. a total of 8 qtl for k (qpot001-qpot008) were identified on lgs d1b (chr 1), n (chr 3), a1 (chr 5), o (chr 10), f (chr 13), b2 (chr 14), and j (chr 16). four qtl for mg (qmag001qmag004) were identified on lgs n (chr 3), a1 (chr 5), j (chr 16), and g (chr 18). one qtl for p (qpho001), one for c on lg j (chr 16), one for n (qnit001) and s (qsul001) on the same lg j (chr 16), and one qtl for ca (qcal001) on lg g (chr 18). k and mg qtl were clustered together on lg a1 (chr 5) with a peak position of 9.50 cm and lod support interval of 8.50-9.50 cm. similar observation was noticed for p, k, mg, c, n, and s, where the qtl were clustered on lg j (chr 16) with peak position of 11 cm for k, p, and s, and 10 introduction macronutrients such as n, c, s, p, k, mg, and ca are essential for plant growth, development, and production, and their deficiency in soil leads to yield loss, disease infection, and poor seed quality. metabolic and physiological roles of these nutrients were well documented (mengel and kirkby, 1982; barker and this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. cm for c and n, and 12 cm for mg. the lod support intervals for all these clustered qtl were between 8.90 and 12.30 cm. the qtl clustering of these nutrients suggests possible common physiological and genetic relationships, suggesting possible similar metabolic processes and pathways for these nutrients. the inverse relationships between n:s ratio and all nutrients suggest possible use of n:s ratio as a measure for higher nutrients accumulation in seed. since most of qtl identified in this study were not previously reported, this research will further help breeders to improve nutrient accumulation in seeds and contribute to our understanding of the physiological and genetic bases of seed nutrition quality. keywords: snps markers, rils, nutrients, minerals, genetic mapping, seed composition. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 225 pilbeam, 2007; marschner, 2012). for example, n is involved in protein synthesis and protein storage, nucleic acid (dna and rna), phytohormones, co-enzymes, and energy transfer systems. sulfur is involved in enzymes, co-enzymes, s-containing amino acids such as cysteine and methionine (two amino acids that are deficient in soybean cultivars). phosphorus role in plants includes its involvement in cell membrane, lipid synthesis, energy transfer as atp and nadp-h, and phosphorylation reactions, nucleic acids (dna and rna), carbohydrate metabolism, and nutrient active uptake processes. magnesium is important for chlorophyll molecule, osmoregulation, cell development, activation of atpase and enzyme molecules (balke and hodges, 1975), activation of ribulose bisphosphate carboxylase (lilley and walker, 1975), and co2 assimilation and carbohydrate production. calcium is an important mineral for membrane permeability, cell structure, cell membrane integrity, and stabilization. calcium deficiency leads to a leaky and impaired membrane (marinos, 1962; van steveninck, 1965). it has a role in germination and growth of pollen, cell nucleus matrix (wunderlich, 1978), interaction with protein bound-membrane enzymes to make ca-protein. the process of ca-protein complex formation is regulated by cellular flux of ca. potassium is essential for osmoregulation, stomatal closure, carbohydrate movement, nutrient mobility and uptake, photorespiration and co2 fixation, activation of enzymes, translocation and mobilization of stored metabolites, and atpase activity stimulation. macronutrient accumulation in seeds determines the nutritional value of seeds. for example, the major storage form for p in seeds of plants is myo-inositol-1,2,3,4,5,6-hexakisphosphate (ip6, or phytic acid), and this compound often chelates metal ions such as ca, mn, zn, and fe (raboy, 1997), and the relationship between the accumulation of p in seed and minerals such as ca, mg, fe, mn, cu is not well known. calcium in seeds determines the quality of soyfoods such as tofu, natto, and miso (mullin and xu, 2001; zhang et al., 2009). the accumulation of nutrients in seed involves complex physiological and metabolic process starting from their uptake through translocation and distribution, to their movement from leaves to seed. in spite of the enormous efforts devoted to plant nutrition research, mechanisms controlling the movement of nutrients within plants and from leaves to seeds are still not well understood. this is due to a complex process of nutrient metabolism that are affected by several biotic and abiotic stress factors such as drought, heat, disease, and nutrients levels in the soil and their availability. therefore, understanding the physiological and genetic bases of nutrients accumulation in seeds and genetic factor controlling the accumulation of nutrients in seeds is critical for breeding selection for higher seed nutrition qualities. mapping qtl on the genome is becoming increasingly important in modern breeding programs through marker-assisted selection (mas) and gene discovery (price, 2006). there are many successful examples of using qtl to facilitate crop cultivar development, especially for disease resistance (beaver and osorno, 2009; swarbrick et al., 2009) and quality improvement (blair et al., 2009; sabouri, 2009). however, information on genetic mapping for macronutrients accumulation in seeds is scares (zhang et al., 2009), and what is available deals with other species such as seed p qtl in andean bean (cichy et al., 2009); k qtl related to k efficiency in wheat (kong et al., 2013); nutrients qtl in arabidopsis (chardon et al., 2014); qtl associated with p in seed in mungbean (sompong et al., 2012); qtl related to p in seed in common bean (blair et al., 2009); qtl in seed minerals in brassica napus) (ding et al., 2010) mostly deals with macronutrients and their status in soybean leaves, shoot, or roots, but not in seeds. for example, qtl of p nutrition in relation to root morphology and physiology have been reported (beebe et al., 2006; li et al., 2007; chen et al., 2009; cichy et al., 2009; li et al., 2009). liang et al. (2010), using a population of 106 f9 recombinant inbred lines (rils) derived from a cross between bd2 and bx10 and 296 simple sequence repeat (ssr) markers, found a total of 31 qtl controlling root traits and p efficiency in soybean. among them 13 qtl were found for root traits and 18 for p efficiency parameters. yuste‑lisbona et al. (2014) studied the epistatic qtl using a population of 185 rils of a cross involved an adapted common bean (pmb0225 parent) and an exotic nuña bean (pha1037 parent), and they found 59 qtl on all linkage groups (lgs), 18 of them had individual additive effects, 27 had epistatic effects, and 14 had both effects. it was also found that the detected qtl explained from 8 to 68 % and 2 to 15 % of the additive and epistatic effects, respectively. zhang et al. (2009) studied qtl associated with seed calcium content (hardness) in 178 f2:3 and 157 f2:4 lines derived from a cross of ss-516 (low calcium) x camp (high calcium) using 148 simple sequence repeat markers (ssr). they found four qtl (ca 1, ca 2, ca 3, and ca 4) on lgs a2, i, and m, identified by both single-marker analysis and composite-interval mapping, and found that the qtl accounted for 10.7%, 16.3%, 14.9%, and 9.7% of calcium content variation, respectively. hard seeds do not absorb water during the soaking process and cause problems for soybean sprouting and natto manufacturing (mullin and xu, 2001) and hard seed determine soyfoods texture (trumbull, 1992). studying ca levels in seeds determines the quality of tofu, natto, and miso, which make up nearly 90% of the food quality soybean market (maughan et al., 2000). also, it was found that ca in the seed coat to be positively correlated with water absorption (saio et al., 1973; saio, 1976). three qtl for calcium uptake were identified and accounted for 51.8% of the phenotypic variation, and 3 survivorship qtl were found to explain 78% of the calcium uptake (lexer et al., 2003). because of the limited information on genetic mapping of soybean seed minerals qtl (zhang et al., 2009; king et al., 2013; ramamurthy et al., 2014), the objective of this research was to identify qtl for macronutrient concentrations in seed in 92 f5:7 recombinant inbred lines developed from a cross between md 96-5722 and spencer using a total 5,376 single nucleotide polymorphism (snps) markers. the genetic mapping of seed nutrients by identifying qtl controlling seed nutrient levels in seeds and their associated molecular markers on the genome could lead to an efficient soybean breeding to select for appropriate seed nutrient levels using marker-assistant selection (mas) (chang et al., 2009). the information obtained from this research will provide a fundamental basis for future research on genetic improvement of soybean seed nutrition qualities. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 226 materials and methods plant material and growth conditions a recombinant inbred lines (rils) population of 92 f5:7 was developed by a cross between md 96-5722 (md) and spencer to obtain phenotypic and genotypic data. the cross was made in 2004 by southern illinois university at carbondale (siuc) breeding program and advanced to the f5:7 generation by single‑pod descent method. soybean lines were grown in a field at fayetteville state university (fsu) campus, fayetteville, nc in 2012 with row spaces of 25 cm and seeding rate of 160,000 seeds ha-1. no fertilizers were added to soil and no insecticide applications were made. the soybean population was developed as previously reported by akond et al. (2013). soybean seed were collected at maturity (r8) for macronutrient (n, c, s, p, k, mg, p, and ca) quantification as described below. phosphorus measurement the concentrations of p in seeds were determined at seed maturity (r8). phosphorus concentration was measured spectrophotometrically using the yellow phosphor-vanado-molybdate complex according to cavell (1955). briefly, a dried ground seed sample of 2 g was ashed to completely destroy organic matter, and after ashing, 10 ml of 6 m hcl was added and the sample was placed in a water bath to evaporate the solution to dryness. after drying, 2 ml of 36% v/v hcl were added; the sample was boiled under the heat. then, 10 ml of distilled water was added, and the solution was then boiled for a few seconds, transferred to a 50‑ml volumetric flask, diluted to 50 ml with distilled water, and filtered. a volume of 2 ml of filtrate was first discarded and the remainder was kept for p analysis. a volume of 5 ml of 5 m hcl and 5 ml of ammonium molybdate–ammonium metavanadate reagent were added to 5 ml of the filtrate, and the solution was diluted with distilled water to 50 ml. the solution was then allowed to stand for 30 minutes before measurement. ammonium molybdate–ammonium metavanadate was made by dissolving 25 g of ammonium molybdate and 1.25 g of ammonium metavanadate in 500 ml of distilled water. the standard curve was made from p standard solutions (0–50 μg ml-1 of p) using dihydrogen orthophosphates. phosphorus concentrations were determined using a beckman coulter du 800 spectrophotometer at 400 nm. seed minerals, n, s, and c analysis seed samples were collected at maturity stage (r8) and were analyzed for minerals, n, s, and c by digesting 0.6 g of dried, ground plant materials in hno3 in a microwave digestion system. samples were ground using a laboratory mill 3600 (perten, springfield, il usa), and the concentration of k, ca, mg was determined using inductively coupled plasma spectrometry at (?)the university of georgia’s soil, plant, and water laboratory, athens, ga, and detailed by bellaloui et al., 2011, 2014. for n, c, and s measurements, a 0.25 g ground-dried sample was combusted in an oxygen atmosphere at 1350 ºc, converting elemental n, s, and c into n2, so2, and co2, respectively. these gases were then passed through infrared cells and n, s, and c are determined by an elemental analyzer using thermal conductivity cells (lecocns-2000 elemental analyzer, leco corporation, st. joseph, mi usa) at the university of georgia’s soil, plant, and water laboratory, athens, ga, as detailed by bellaloui et al. (2011, 2014). genetic map construction and qtl identification the genetic linkage map of the md 96-5722 by spencer was constructed using soysnp6k illumina infinium beadchip array (akond et al., 2013) and used to identify qtl for seed macronutrients quantification. the rils were genotyped using 5,376 single nucleotide polymorphism (snp) markers using the illumina infinium soysnp6k beadchip array (akond et al., 2013). a genetic linkage map was constructed based on 537 polymorphic snp markers (akond et al., 2013). the analysis of qtl was conducted using the composite interval mapping (cim) of winqtlcart 2.5 (http://statgen.ncsu.edu/qtlcart/wqtlcart. htm) (wang et al., 2014). the model 6 with four parameters for forward and backward stepwise regression, 10 cm window size, 1 cm step size and five control markers were selected for running winqtlcart (wang et al., 2014). permutations of 1,000 were used as threshold. analysis of means (cv, maximum and minimum values, and sd) were carried out using proc means in sas. coefficient of correlation was conducted by sas using proc reg. results and discussion variation and correlations the range of variations of nutrient accumulation in seeds in the population is dependent on the nutrient type (table 1). the lowest coefficient of variation was noticed for c and n, and the highest was in ca and s, k, mg (table 1). the concentrations of nutrients in md parent fell within the range of the population lines. however, for the parent spencer, the nutrient accumulation of ca, k, mg, and p fell within the range of the population lines, but n, c, and s did not. minerals (ca, k, mg, and p) were significantly (p<0.0001) correlated compared with non‑minerals (c, n, and s) (table 2), and the pattern of this correlation was positive either for minerals or non-minerals (figures 1 and 2). rils parents nutrient mean sd minimum maximum differences (%) cv md spencer ca 0.39 0.06 0.23 0.56 143.5 14.13 0.54 0.53 k 1.77 0.19 1.12 2.17 93.8 10.73 1.58 1.56 mg 0.25 0.03 0.16 0.33 106.3 10.33 0.26 0.25 p 0.52 0.05 0.32 0.68 112.5 9.86 0.60 0.63 c 50.84 0.63 49.4 52.20 5.63 1.24 49.22 49.2 n 6.29 0.31 5.53 6.79 22.8 4.92 6.74 6.82 s 0.25 0.03 0.16 0.31 93.8 14.09 0.29 0.33 table 1. mean, standard deviation (sd), minimum and maximum values of macronutrient concentrations, and coefficient of variation (cv) of soybean ril population and its parents (maryland, md, and spencer). 227 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. patterns of correlation between seed macronutrient and ca (a), k (b), and mg (c) concentrations in a population of 92 f5:7 recombinant inbred lines (rils). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 2. patterns of correlation between seed macronutrient and p (a), c (b), and n (c) concentrations in a population of 92 f5:7 recombinant inbred lines (rils). frequency distribution showed, generally, a normal distribution (figures 3, 4). the wide range of nutrient concentrations in seeds of individual lines was expected as the accumulation of nutrients in seed was reported to be affected by genotype differences (white and broadley, 2009; bellaloui et al., 2011), and could be due to the uptake, transport, accumulation, and distribution of nutrients within the plant. the wide range of nutrient accumulation would give breeders an opportunity to select for more efficient genotypes for macronutrients. the differences of coefficient of variation between nutrients in the population indicated the different level of sensitivity of each nutrient to intrinsic and extrinsic factors. genetic analysis (figure 5) showed that 8 qtl for k (qpot001‑qpot008) were identified on lgs d1b (chr 1), n (chr 3), a1 (chr 5), o (chr 10), f (chr 13), b2 (chr 14), and j (chr 16). four qtl for mg (qmag001-qmag004) were identified on lgs n (chr 3), a1 (chr 5), j (chr 16), and g (chr 18). one qtl for p (qpho001), n (qnit001), and s (qsul001) on the same lg j (chr 16), and one qtl for ca (qcal001) on lg g (chr 18) (table 3). qtl for k and mg were clustered together a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 228 table 2. correlation between seed nutrient concentrations in 92 f5:7 recombinant inbred lines developed from a cross between maryland (md) 96‑5722 and spencer. level of significance was at p≤0.05. figure 3. frequency distribution for seed ca (a), k (b), mg (c), and p (d) in the md 96-5722 by ‘spencer’ ril population in soybean. nutrients ca k mg p c n s n:s ratio ca 1 k 0.451 1 <.0001 mg 0.733 0.500 1 <.0001 <.0001 p 0.550 0.83489 0.749 1 <.0001 <.0001 <.0001 c -0.0066 0.0946 -0.0049 0.12531 1 ns ns ns ns n 0.06546 -0.03978 -0.0216 -0.0660 0.2097 1 ns ns ns ns ns s 0.43941 0.56612 0.25681 0.5341 0.340 0.221 1 <.0001 <.0001 0.0157 <.0001 0.001 0.039 n:s ratio -0.452 -0.640 -0.329 -0.623 -0.291 0.129 -0.924 1 <.0001 <.0001 0.0017 <.0001 0.006 0.231 <.0001 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 229 on lg a1 (chr 5) with a peak position 9.50 cm and lod support interval of 8.50-9.50 cm. similar observation was noticed for p, k, mg, c, n, s where the qtl were clustered on lg j (chr 16) with peak position of 11 cm for k, p, and s, and 10 cm for c and n, and 12 cm for mg. the lod support intervals for all these qtl were between 8.90-12.30 cm (table 3). the identification of qtl for macronutrients is very limited, and almost non-existent for nutrient accumulation in soybean seed. most of the researchers identified qtl associated with leaves, roots, and shoot, but very few on seeds. searching grin soybean base, it was found that zhang et al. (2009) detected four qtl for seed calcium (ca 1-ca 4) on a2, i, m with the optimum position of 16.4 on a2; 9.2 cm on i; 96.7 on m, and 92.7cm on m, with the position of flaking markers on the consensus map positioned at 116.3-154.11 cm on a2; 36.59-46.22 cm on i; 5.6–1.0 cm on m; and 75.6–1.0 cm on m. for example, li et al. (2005) found two qtl for leaf p in soybean on lgs f at genomic map position between 11.37-16.08 and 0-3.86 cm. liang et al. (2010) found 7 qtl associated with shoot and root p in soybean on lgs d2, b1, and f; specht et al. (2001) found 5 qtl related to carbon isotope for drought tolerance in soybean on lgs c2, d2, f, and l; and panthee et al. (2004), using a population of 101 f6:8 recombinant inbred lines (ril), found 17 qtl for soybean seed n on lgs a2, b2, d1b, e, g, m, d1a, and g. they found 3 qtl on lgs a2, 2 on b2, 2 on d1b, 5 on e, 3 on g, 1 on m, and 1 on d1a. they found that phenotypic variation explained by an individual qtl ranged from 5 to 11.6% and concluded that these qtl and markers associated with them may be useful for soybean seed protein improvement. the qtl reported by panthee et al. (2004) were detected at developing stages of seeds (7 qtl were detected at r5; 3 qtl at r6 stage; and 7 qtl at r7 stage). in the current study, one qtl was detected on lg j (chr 16) and it is different from those reported by panthee et al. (2004) and ramamurthy et al. (2014) as qtl found by panthee et al. were detected at r5, r6, r7 stages, which characterize developing seeds and not mature seed at r8 stage at which the full seed‑fill and seed physiological maturity was reached. in addition, the qtl for n, reported here, was found clustered with s, ca, p, c, mg, and k, suggesting close relationships between these nutrients and n. the close relationship between these minerals was reflected with the highly significant positive correlation (table 2) as the increase pattern indicated the increase of one nutrients leads to the increase in the other (figures 1, 2). the qtl clustering of these nutrients suggests similar physiological and metabolic processes. ramamurthy et al. (2014) found 2 qtl for s on lg j (16) and lg g (chr 18). we detected one qtl for s on lg j (chr 16) at a peak position of 11.00 cm with lod support interval of 9.90-11.10 cm and marker interval of ss248983974ss248977568. the current qtl for s could be different from that detected by ramamurthy et al. (2014) as the peak position of their qtl was 66.3 cm. zhang et al. (2009) found twenty-two markers on 8 lgs associated with seed ca content among which six markers on each of lgs a2 and i, 3 markers on lg l, 2 markers on each of lgs b1 and m, and 1 marker on each of lgs d2, f, and g, and one marker on each of lgs d2, i, and m. they reported that the qtl found in this study can be considered as preliminary research on mas for low ca content in natto soybean breeding. we were able to detect one ca qtl on lg g (chr 18) with a peak position of 13.20, interval position of 10.70-15.60 cm, and markers intervals of ss249715368ss249830195. this qtl was not previously reported as zhang et al. (2009) detected four qtl for seed calcium (ca 1-ca 4) on a2, i, m with the optimum position of 16.4 on a2; 9.2 cm on i; 96.7 on m, and 92.7cm on m, with the position of flaking markers on the consensus map of 116.3-154.11 cm on a2; 36.59-46.22 cm on i; 5.6–1.0 on m, and 75.6–1.0 on m. also, we were able to detect four qtl for mg on lgs n, a, j, and g, which were different from those reported by ramamurthy et al. (2014), who found one qtl on lg n (chr 3). therefore, the current reported qtl for mg are new findings. potassium qtl were previously reported by ramamurthy et al. (2014) who found four qtl on lgs c1, e, j, and g. we detected 8 qtl for seed k on 7 lgs (d1b, n, a1, o, f, b2, and j). except for qtl on lg j, the rest of qtl were not previously reported, contributing to our understanding of the genetic bases of seed k nutrition. one c qtl was detected in the current research on lg j (chr 16) with peak position of 10.00 cm and lod support interval of 9.00-10.00 cm and marker interval of ss248983974ss248977568. no seed c qtl were previously figure 4. frequency distribution for seed c (a), n (b), and s (c) in the md 96-5722 by ‘spencer’ ril population in soybean. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) reported, hence the current qtl is new finding. it must be noted here 4 qtl associated with c isotopes related to drought trait and water efficiency use were reported (specht et al., 2001). no qtl for seed k, mg, or s were found in soybean base (http://www.soybase.org/search/). recently, ramamurthy et al. (2014) identified qtl for seed mineral concentrations in three soybean recombinant inbred line mapping populations derived from crossing williams 82 × dsr-173, williams 82 × nks19-90 and williams 82 × vinton 81. using a joint linkage map from these populations, ramamurthy et (2014) detected forty qtl for 18 traits including seed mg, s, k, ca, n:s; one qtl for mg on chr 3, two for s on chr 16 and 18, two for k on chr 4, 15, 16, and 18, one for ca on chr 6, two for n on chr 14, 15, 16, and 18, and three for n:s on chr 16, 20, and 3. they found an inverse relationship between n:s ratio and most nutrients, and suggested that the n:s ratio can be used as an indirect measure of seed mineral concentration in soybean breeding programs. our finding agreed with ramamurthy et al. (2014) in that we found an inverse relationship (correlation) between n:s ratio and all nutrients investigated (table 2), and qtl detected in the current research for seed nutrients were different from theirs for the reasons previously explained above. genetic mapping for p was reported by king et al. (2013), who identified candidate gene(s) involved in phosphate metabolism and transport, and p accumulation in soybean seed. they were able to identify one putative qtl region on chr 12, and this region contained a phosphate transporter gene. also, they identified two additional suggestive qtl on chr 7 and 17, with chr 7 having both a phosphate transport gene and a zip transporter gene in the region of the qtl. king et al. (2013) reported, using combined total p qtl, 6 qtl on lgs o (chr 10), g (chr 18), i (chr 20), m (chr 7), h (chr12), d2 (17). we were able to identify one qtl for seed p accumulation on lg j (chr 16) that was not previously reported, suggesting that the current p qtl is a new finding. no qtl for seed k, mg, p, and s were reported in soybean base (http://www.soybase.org/search/), indicating further research is needed in the area of genetic mapping for seed mineral nutrition (king et al., 2013; ramamurthy et al., 2014). trait qtl lg /chr †peak position (cm) ‡2-lod support interval (cm) marker interval §peak lod #r2 (%) ††additive effects potassium qpot001 d1b /chr_1 4.50 4.40-4.60 ss244503611-ss244513054 2.63 0.02 -0.04 potassium qpot002 d1b /chr_1 12.10 11.10-13.10 ss244525143-ss244552583 2.81 0.02 0.03 potassium qpot003 n /chr_3 16.40 16.00-16.40 ss245025187-ss245021991 9.88 0.04 0.01 magnesium qmag001 n /chr_3 9.00 8.10-9.10 ss244947608-ss244967332 8.26 0.91 0.13 potassium qpot004 a1 /chr_5 9.50 8.50-9.50 ss245747167ss245786667 3.43 0.69 0.75 magnesium qmag002 a1 /chr_5 9.50 8.50-9.50 ss245747167ss245786667 8.68 0.02 -0.02 potassium qpot005 o /chr_10 5.30 4.30-6.30 ss247085505ss247098566 11.82 0.03 6.36 potassium qpot006 f /chr_13 2.70 2.50-2.80 ss247942156ss247937719 8.55 0.01 0.05 potassium qpot007 b2 /chr_14 8.00 5.80-9.20 ss248293401ss248275088 7.25 0.57 0.91 potassium qpot008 j /chr_16 11.00 9.90-12.90 ss248983974ss248977568 3.24 0.05 0.34 magnesium qmag004 j /chr_16 12.00 11.00-12.30 ss248983974ss248977568 8.49 0.94 0.25 phosphorus qpho001 j /chr_16 11.00 9.90-11.00 ss248983974ss248977568 9.49 0.94 0.25 carbon qcar001 j /chr_16 10.00 9.00-10.00 ss248983974ss248977568 10.01 0.94 24.93 nitrogen qnit001 j /chr_16 10.00 8.90-10.00 ss248983974ss248977568 9.85 0.94 3.25 sulfur qsul001 j /chr_16 11.00 9.90-11.10 ss248983974ss248977568 9.73 0.94 0.15 calcium qcal001 g /chr_18 13.20 10.70-15.60 ss249715368ss249830195 2.72 0.66 0.01 magnesium qmag005 g/chr_18 2.20 2.10-2.20 ss249623816ss249632893 8.59 0.95 0.13 †position of peak lod value on composite maps described previously (coles et al., 2010);‡the positions that define the two lod intervals around the position of peak likelihood for the qtl; §the log of odds (lod) value at the position of peak likelihood of the qtl; #r2 estimates the proportion of ril mean variance (%) explained by the detected qtl; †† a positive number in additive effect of the qtl indicates that the allele for susceptibility was derived from the line indicated and a negative number means that the allele for resistance was derived from the line indicated. table 3. chromosomal locations and parameters associated with the quantitative trait loci (qtl) for seed mineral accumulation in 92 f5:7 recombinant inbred lines developed from a cross between md 96-5722 and spencer. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 230 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 5. locations of qtls and snp markers associated with seed macronutrients accumulation. 231 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 5. continued. 232 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 5. continued. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 233 conclusions most of the research conducted on the genetic mapping for plant nutrition was associated with leaves, roots, or shoot, but not on nutrient accumulation in seeds. our current research contributes to the limited available information (king et al., 2013; ramamurthy et al., 2014) on genetic mapping of nutrient accumulation in soybean seeds. the current research showed that several qtl for macronutrients were detected. the genetic analyses resulted in 8 qtl for k (qpot001-qpot008), four qtl for mg (qmag001-qmag004), one qtl for p (qpho001), one qtl for n (qnit001), one qtl for s (qsul001), and one qtl for c (qcal001). most of these qtl are new, contributing to the previously reported qtl. qtl clustering between k and mg, and between p, k, mg, c, n, and s indicates physiological and genetic relationships between these nutrients, suggesting possible similar metabolic processes. information obtained from this research would help breeders to select for higher seed mineral quality, and physiologist to further understand the physiological and genetics based of seed mineral accumulation. acknowledgements the authors would like to thank the department of defense (dod) 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j, dl jamar, p lou, y wang, j wu,x wang , g bonnema, m koornneef, and d vreugdenhil (2008) quantitative trait loci analysis of phytate and phosphate concentrations in seeds and leaves of brassica rapa. plant cell environ 31: 887–900. 235 atlas journal of biology 1 (3): 52-61, 2011 doi: 10.5147/ajb.2011.0048 a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) impacts of silver nanoparticle ingestion on pigmentation and developmental progression in drosophila s. catherine silver key1, denise reaves1, fran turner1, john j. bang2* 1 department of biology, north carolina central university, durham, nc 27707, usa; 2 departments of environmental, earth, and geospatial science, north carolina central, university, durham, nc 27707, usa received: february 12, 2011 / accepted: may 14, 2011 __________________________________________________ *corresponding author: jjbang@nccu.edu introduction since the discovery of fullerene and carbon nanotubes, the use of engineered nanomaterials has exponentially increased for various applications. among the nanomaterials used, silver (ag) in the form of nanoparticle and nanopowder (np) is one of the most popular for various applications including antibacterial uses (sharma et al., 2009). in fact, the use of silver for therapeutic and industrial purposes traces back to the time period long before nanotechnology (chen and schluesener, 2008). today, because ag nps have well-documented beneficial effects due to their unique physicochemical properties that larger counterparts do not carry, ultrafine and nanopowder forms of silver penetrate tissues more effectively. thus, ag np is not only used in burn and diabetic ulcer creams, but is also used in common household items such as first aid bandages, cosmetics, appliances, and clothing (liu et al., 2010; kumari et al., 2010). these products are presumed safe once in consumer hands. however, industrial workers, physicians, and researchers are increasingly exposed to a plethora of nanoparticles, the impact of which is currently unknown. while the intended uses of these materials are to benefit human health, comprehensive toxicological profiles for nano materials are yet to come. the toxicity of silver nanoparticles (ag np) has been documented in a large number of in vitro studies using a number of cell lines including but not limited to human hepatoma cells (kim et al., 2009), pc12 neuronal cells (powers et al., 2010), brl3a rat liver cells, germline stem cells (braydich-stolle et al., 2005), human lung fibroblasts, and gliablastoma cells (asharani et al., 52 abstract in recent years, the advent of nanomaterial use has increased exposure rates and raised health concerns. however, the toxicology profiles of many nanomaterials are far from complete for various reasons. in this study, drosophila melanogaster, commonly called fruit flies, were exposed to one of the most widely used nanomaterials, silver nanopowder (ag np), to assess its toxicity and determine if d. melanogaster would be a good model organism for nanotoxicology studies. comparison of developmental progression amongst groups of flies ingesting different ag np concentrations (0.05%/~90 ppm-5.0%/~9000 ppm), revealed that hatch rates were unaffected, but that larval progression was impeded at any dosage of ag np. at 0.3% ag np an approximate ld50 was observed. additionally, a distinctive phenotype was observed among emergent adults (f1 generation) that arose from larvae exposed to ag np which included reduced body pigmentation accompanied by shortened life span and abnormal climbing behavior. the phenotype prompted speculation that ag nps may affect the dopamine and/or the stress response pathway(s). keywords: silver nanoparticles, exposure, development, pigmentation, drosophila. abbreviations and acronyms: ag=silver, np=nanoparticle or nanopowder, p=parental generation, f1=adult flies of the first filial generation, l=larvae of f1 generation. 53 2009). however, a fewer number of in vivo studies using animal models has been conducted (choi et al., 2009; ahamed et al., 2010b; posgai et al., 2009; kumari et al., 2009; li et al., 2010; wu et al., 2009). the results from previously conducted studies provided consistent data indicating that ag np exposure elicits both oxidative stress and apoptotic responses, and that the particles can accumulate in organs including the brain. drosophila melanogaster, commonly known as the ‘fruit fly’, is an excellent organism for answering many questions regarding human health because drosophila shares some 60-70% homology to human genes including those that encode molecules essential for carcinogenesis, pigmentation, and the nervous system (rubin, 2000; rand, 2010). because of a well-established genetics, short life cycle (10-14 days from embryo to adulthood), and highly conserved genes, many developmental discoveries have been made in drosophila that apply to humans. in addition, the average cost for conducting an in vivo exposure experiment by using drosophila is a lot lower than that of other animal models. for these reasons, this study aims to assess the feasibility of using drosophila as an in vivo model for future exposure-induced toxicity experiments on engineered nanomaterials including ag np. the three major entry modes of nanoparticles or nanomaterials typically include inhalation, epidermal absorption, and ingestion. currently, ag np is used in food packaging as well as in medical treatment procedures (johnston et al., 2010). ag np also has been shown to accrue in waste, and the silver particulates could contaminate consumer’s food and drinking water (fernandez et al., 2010; erickson, 2009; kim et al., 2010). further, ag nps have been shown to accumulate in animal and human tissues and organs (jonas et al., 2007; larese et al., 2009; ahamed et al., 2010; tang et al., 2010a). given the great potential for human ingestion of ag np, this study aims to understand the impacts of ag np exposure through the ingestion mode on phenotypic outcomes and behavioral characteristics of drosophila. the implications for ag np effect on pigmentation, life span, and behavioral response are discussed. materials and methods experimental design silver nanoparticles (ag np) of different concentrations ranging between 0.05% (89.3 ppm) and 5.0% (8930 ppm) were prepared to determine the exposure effects at different developmental stages. a manageable number of newly emerged parental flies were either exposed immediately to a given ag np concentration (p+, exposed parents) or placed in food vials that contained control food (p-, unexposed parents). the eggs laid by these parents were collected and transferred to either ag np-containing food (l+, larvae exposure) or to food lacking ag np (l-, larvae unexposed) as described in the drosophila nanopowder exposure section below. progression through all developmental stages was monitored: egg hatching into the f1 generation of larvae, larval progression, pupal stage development, and eclosion of adult flies. groups of f1 adult flies were then monitored for the presence or absence of pigment and longevity for up to 20 days. experiments were conducted by using triplicate samples with each set comprised ~200 eggs. during each developmental stage, observation of overt phenotypes was documented. f1 generation adults that had emerged from larvae exposed to ag np (l+) were assessed for climbing behavior as described below. data from all replicate experiments was assessed using a student t-test shown elsewhere. statistical analysis of developmental progression, maternal versus larval exposure analysis, and life span assessment all calculations for average, standard deviation, and t-test were performed using microsoft excel version 2007 formulas. triplicate samples were used during the study unless otherwise specified. for the developmental progression analysis in figure 2, three replicates of ~200 eggs per condition were run. as the percent eggs laid was arbitrarily set to 100%, the average percent and standard deviation of eggs that progressed to each developmental stage (larval, pupal, and adult (shown in both figure 2 and table 1)) were calculated. t-tests were performed for each stage (larval, pupal, and adult) using the unexposed group as a control. for the analysis on exposure timing effect as shown in figure 3b, five sets of 30 eggs per condition were run. the average and standard deviation were calculated using microsoft excel 2007 formulas. a two-tailed t-test was run comparing the following data sets for maternal effect assessment: p-/lversus p+/land p-/l+ versus p+/l+ and the following sets for larval effect assessment: p-/lversus p-/l+ and p+/l versus p+/l+, where p=parent, l=f1 larval generation, ‘-‘ indicates unexposed and ‘+’ indicates ag np exposed. a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 1. ag np under transmission electron microscopy. the ag np suspension was put on a cu grid for a tem exam. a smaller number of ag np is bigger than the average values presented by the dls. agglomeration of smaller particles occurs during the drying process and there are limitations of the manufacturing process. however, the majority of the particles are within the average values presented by the dls. 54 for the life span analysis, replicate runs of at least 50 flies per conditions were observed for 20 days after eclosion from pupal cases. because flies in each group died at different times during each run the weighted average was calculated to assess approximate length of survival for the group as a whole. statistical comparisons between the control group and each ag np condition were assessed using a one-tailed t-test. silver nanopowder (ag np) the tem image of the silver nanopowder (ag np) used in this ingestion exposure study is shown in figure 1. commercially available ag np was used (sun innovations corp., fremont, ca). ag np was added to filtered water (nanopure diamond from barnstead). a suspension of ag np in purified water was prepared at different concentrations so that their converted final concentration after mixing with food would be equivalent to the concentration levels used for the study (i.e., 0.05, 0.1, 0.3, 0.5, 1.0, 2.0, and 5.0% in dry ag np to dry food weight). for example, for 0.5% ag np food (dry weight of ag np in dry food weight), 125 mg of ag np was mixed in 140 ml of water. this makes up a 893 ppm concentration of ag np in prepared food which is equivalent to 8.3 mm ag np (125 mg/108 mg/ mole/0.14l of water). before ag np was mixed with the fly food, the ag np suspension was sonicated for at least 30 minutes to obtain a homogeneous dispersion, and the physical dimensions of the ag np in suspension were evaluated by using a dynamic light scatter (dls using a cgs 3 spectrometer (alv gmbh, germany) equipped with a helium-neon laser (633.4 nm) and goniometer). the average radius of the ag np used was 16.7±0.30 nm. the average width was 11.3±0.30 nm. dls measures the hydrodynamic diameter particles in suspension and presents the average values of the measured particles. drosophila nanoparticle exposure a commonly used control fly strain, w1118 (bloomington stock # 3605), was chosen to initiate nanoparticle exposure experiments. fifty virgin female and 30 male flies were collected and placed (within 24 hours) into cages capped with grape juice agar plates for egg collection. each plate contained 1 gram of nutri-flytm bf food (catalog# 66-112, genesee scientific) with a different concentration of silver nanoparticles (0.05%, 0.1%, 0.3%, 0.5% and 1.0%) or control food (0% ag np). four different exposure settings were created. 1) newly emerged parental flies (p generation) were exposed to ag np, their eggs collected and transferred to hatch in food containing ag np for the larval (l) exposure (p+/l+). 2) newly emerged p generation flies were exposed to ag np and the larvae were unexposed (p+/l-). 3) exposure-free p generation flies and their progeny larvae exposed to ag np (p-/l+), and 4) exposure-free p generation flies and then progeny larvae were also unexposed (p/l-). forty-eight hours after flies were transferred to food, eggs were collected for 24 hours at 25oc. because w1118 lacks eye pigmentation, the oregon rs strain (bloomington stock #4269) with wildtype eye color was used to look at the impacts of ag a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) percent silver nanopowder (ag np) % embryos develop to adults 1 survival time (in days)2 f2 generation produced? abdominal/thoracic pigmentation3 5.0 7 ± 1.8 1 ± 0.3 no none 2.0 1 ± 0.2 1 ± 0.3 no none 1.0 1 ± 0.2 2 ± 0.2 no none 0.5 20 ± 6.0 18 ± 2.0 no none to very light 0.3 45 ± 3.7 19 ± 2.4 yes light to normal 0.1 60 ± 4.6 20 ± 1.6 yes light to normal 0.05 45 ± 6.7 20 ± 0.8 yes light to normal control 83 ± 7.3 20 ± 1.0 yes normal table 1. outcomes of exposure to silver nanopowder on developmental progression, life expectancy, reproductive ability, and pigmentation. 1 the standard deviations are shown (±). t-tests indicate significant differences in the percent of ag np-exposed embryos progressing to adulthood as compared to control: 0.3% (p = 0.044), 0.5% (p-value = 0.012), 1.0% (p-value = 0.003), 2.0% (p-value =0.003) and 5.0% (p-value = 0.005). 2 standard deviations are shown (±). t-test calculations indicate that survival time differences for 5.0% (p-value=0.0004), 2.0% (p-value = 0.0006) and 1.0% (p-value = 0.0011) were significant compared to control. survival length in days was not significantly different for all other concentrations. adults were observed for a 20-day period. 3 adult pigmentation was determined at the day of death. emergent f1 generation adult flies were maintained on a continuous concentration of silver nanoparticle-containing food during parental and larval stages (p+/l+ condition). f1 generation adults were observed every 2-3 days for up to twenty days. 55 np exposure (0.05% and 1.0%) on eye color. all stages were kept at 25oc. all experiments as describe above (p+/l+, p+/l-, p+/land p-/l-) were completed in triplicate. collected eggs on plates containing the fly food with each concentration of silver (ag) nanoparticle powder were counted immediately and then 24 hours later to determine the hatch rate. the numbers of unhatched eggs, larvae, pupae, and adults were counted and recorded. all data was entered into excel spreadsheets, with averages, and standard deviations calculated using excel formulas (microsoft office excel 2007). for the longevity study, the filial generation adult flies (i.e. f1), which resulted from larvae fed ag np, were maintained on food that either had ag np or lacked ag np for approximately 3 weeks and observations recorded in table format. assessment of pigmentation emergent flies (f1 generation) from all four exposure groups (p+/l+, p+/l-, p-/l+, p-/l-) were evaluated for pigmentation by using a leica lz6 steromicroscope. flies were photographed using a finepix f20 se digital camera and xd-picture card (fuji photo film, ltd. tokyo, japan) temporarily mounted to the scope using the orion steadypixtm deluxe stand (orion telescopes & binoculars, watsonville, ca). three independent experiments were completed. climbing behavior analysis climbing assays were performed on f1 generation flies as previously described (jimenez-del-rio et al., 2010; chaudhuri et al., 2007) with the exceptions that the height of the climb was 7 cm and the age of the flies in this study was specified at 9-days old. the flies (f1 generation) emerging from both groups of larvae, either ag np exposed or non-exposed, were tapped down to induce climbing behavior and video recorded (sharp viewcam liquid display camcorder model vl-e760u). video recordings were transferred to digital format using a dvd maker usb 2.0 (usb2800d, kworld (usa) computer co., ltd, irvine, ca). windows movie maker software was used to generate snapshots for figure 6b and to create the video in supplementary materials. video play-back was halted using the ‘still’ function to measure the rate of climbing for the first 5 a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 2. impacts on developmental progression by silver nanopowder (ag np) ingestion. newly emerging p generation flies were fed on ag np containing food; subsequently, their larvae were exposed to ag np-containing food (p+/l+ condition) and assessed for progression through larval, pupal and adulthood (f1 generation). in panels a and b, results are shown as percentages of live organisms at each developmental stage (egg, larvae, pupae, or f1 adult) in the form of dose response curves for each concentration of ag np as indicated. panel a, non-exposed, control fed organisms ((p-/l-), blue diamonds), 0.05% ag np (red squares), 0.1% ag np (green triangles), and 0.3% ag np (purple xs). panel b, results from different ag np concentration exposures; 0.5% ag np (blue diamonds), 1.0% ag np (red squares), 2.0% ag np (green triangles) and 5.0% ag np (purple xs) are directly comparable to panel a. a total of three replicate experiments were run using approximately 200 eggs per experimental condition. since the number of eggs laid per condition was equivalent, the percent of eggs laid was arbitrarily set at 100% for each condition and the standard deviation (bars) is shown for larval, pupal, and adult stages. one-tailed t-test analysis was completed by comparing percent larvae, pupae, or adult flies in each ag np condition to the percent of larvae, pupae, or adult flies surviving in the control condition. for 0.5% (p-value=0.033), 1.0% (p-value=0.028), 2.0% (p-value = 0.009) and 5.0% (0.009) ag np exposure, the percent of pupal cases observed was significantly fewer than in the control condition. further, the percent of adult eclosion was also significantly lower among the f1 generation flies exposed to higher 0.3% ag np concentrations and higher. a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) flies that ascended to the top (6-7 cm for control flies) and the time recorded. locomotion speed was calculated by dividing the distance by the time elapsed. trials were run three times for each concentration from 0.05% (89.3 ppm) to 0.5% (893 ppm) of ag np. statistical analysis on climbing behavior statistical analysis on climbing behavior data was accomplished using microsoft excel 2007 two-tailed t-test for 15 independent flies per condition. results impairment of developmental progression by ingested silver nanoparticles to determine the developmental stage(s) at which fruit flies may be most sensitive to dietary silver nanoparticles (ag np) exposure, drosophila melanogaster adult flies and their progeny were exposed to non-coated ag np and monitored throughout development from egg hatching to adult ecolosion. newly emerged, unexposed male and female parental flies (p generation) were collected and immediately exposed to food with ag 56 fig. 3. stage dependent effects of ag np exposure on larval progression. five plates of 30 eggs were collected from flies consuming a diet consisting of either 1.0% ag np or control food (no exposure) on gja plates. eggs were transferred to gja plates containing either control or 1.0% ag np food. in both panels a and b, (p-/l-) group represents no exposure for parents or larvae; (p+/l-) represents exposed parents with non-exposed larvae; (p-/l+) indicates non-exposed parents with exposed larvae; (p+/l+) indicates both parents and larvae were exposed. panel a. image of larval size in p-/lexperimental condition versus two concentrations of ag np: 0.05% and 1.0%. panel b. graph indicating the average number of larval progressing to each length: 1st instar size 0.5<1 mm (blue bars) 2nd instar size (red bars) = 1<1.5 mm, and 3rd instar (green bars) = 1.5-2 mm in length. standard deviation are shown (thin black bars). two-tailed t-test comparisons between parental exposure and non-exposure groups indicated the difference was not significant with p-values greater than 0.05 (p-/l+ versus p+/l+ and p+/lversus p+/l+). comparison of larval exposure groups indicated a significant difference for the 3rd instar stage only for the following groups: p+/lgroup versus the p+/l+ group (p-value = 0.008) and p-/lversus p-/l+ group (p-value = 0.007). np (p+) or a control food without ag np (p-). the number of eggs laid by the parental (p) adult flies with a history of exposure of any amount of ag np during their adult stage (i.e., p+) was comparable to the number of eggs laid by the flies fed non-exposed food (p-) (~200±50 per overnight plate; p-value = 0.05 for all concentrations versus unexposed control). in addition, the hatch rates of eggs laid by both p+ mothers (at 1.0% ag np exposure) and pmothers were also comparable at nearly 95±5% (pvalue = 0.14). however, when the f1 generation larvae were exposed to any amount of ag np, developmental progression of the larvae to the pupal stage presented a different picture. in the group of larvae exposed to ag np at concentrations of 1.0% and above, only a small percentage of the f1 generation embryos progressed through the larval stages to reach the pupal stage (~13-40%, figure 2b) while around 92±8% of non-exposed larvae reached the pupal stage (figure 2a). compared to the non-exposed group in which 87±7% of the f1 generation embryos progressed to adulthood, the eclosion rates of larvae exposed to higher ag np concentrations are dramatically reduced to 1-7% (1.0% and above, figure 2b, p-values less than 0.05: i.e., 0.003, 0.003 and 0.005 respectively). at the 0.5% ag np concentration (893 ppm), about 39±6% of embryos progressed to the pupal stage with only 20 ±6% emerging as adults (figure 2b, p-value = 0.030). for larvae fed less than 0.3% ag np, eclosion rates appeared to plateau at 65±2% and never achieved an eclosion rate equivalent to control fed f1 generation flies (87±7%) under our experimental conditions (figure 2a, compare 0.1% and control). when parental flies and their larvae (l) were exposed to a diet containing 0.3% ag np (p+/l+), then more than 65% of eggs developed to the pupal stage with 45±4% eclosing as adults (compare 2a to 2b). thus, with about half the embryos eclosing as adults, the 0.3% ag np conditions approximated a lethal dose 50 (ld50). it was apparent that f1 generation larvae from the two groups differed in size (fig 3a). while the non-exposed larvae grew to a size of ~2 mm, the majority of the 1.0% ag np fed larvae remained at a length of 1mm or less over the same 5-day period (figure 3a). in order to determine if timing of exposure to ag np is a critical factor for developmental progression, a combination of four exposure conditions was tested as shown in figure 3b (i.e., p+/l+ (parent exposed/larvae exposed), p+/l(parent exposed/larvae non-exposed), p-/l+ (parent non-exposed/larvae exposed), p-/l(parent non-exposed/ larvae non-exposed)). when the f1 generation of larvae were not exposed to ag np, the parental (p) exposure history, either p+ or p-, did not influence the numbers of larvae reaching the 3rd instar stage as much as the other cases where the larval history of exposure, either l+ or l-, was compared (i.e. p+/l+ vs. p+/lor p-/l+ vs. p-/l-). when larvae are exposed to ag np, noticeably fewer larvae progressed to 3rd instar, regardless of parental exposure (figure 3b). two-tailed t-tests indicate that the effect of ag np exposure at the parental stage (p) does not significantly affect larval progression at all three larval stages (i.e. p-value greater than 0.05). rather, larval progression to the 3rd instar stage appears significantly impacted if exposure happens during their larval stage. for exposed parental groups (p+/lvs p+/l+), for example, the history of exposure at the larval stage showed a statistically significant difference in reaching the 3rd instar stage (p-value = 0.008). in a similar way, when parents do not have an exposure history, the ag np exposure at a larval stage showed a difference at a statistically significant level in progressing to the 3rd instar stage (p-value = 0.007). thus, it appears that maternal exposure to the ag np does not significantly impact larval development. instead, ag np exposure during larval stages seems to negatively affect larval progression. exposure timing and concentration dependent pigmentation level in adult flies the few w1118 adult flies (f1 generation) that emerged at the initial tests of 1.0% ag np appeared to completely lack fig. 4. inverse correlation between the level of melanization and exposure level of ag np. f1 generation w1118 flies emerging from pupal cases resulting from parental (p) and larval (l) exposure to a continuous diet of control (no exposure (p-/l-)) or silver nanoparticles (ag np, (p+/l+)). white arrows point to the third abdominal stripe in each female adult. control, 0.05% ag, 0.3% ag and 0.5% ag flies were less than 8 hours old, while the 1.0% ag np fly was 24 hours old. 57 a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) body pigmentation. to investigate whether this phenomenon of loss of pigmentation is a gradual event or if there is a threshold exposure level, experiments were conducted with doses ranging from 0.05% to 0.5 %, retaining 1.0% as a point of reference. the flies emerging from larvae exposed to a dose of 0.5% dietary ag np, regardless of parental exposure, also lacked detectable body pigmentation. head, thorax, abdomen, legs, and fig. 5. impacts of ag np exposure targeting on specific body parts sparing eyes. images of the oregon r drosophila strain f1 generation adults emerging from either a vial with control food (p-/l-) or 1.0% ag np-containing food (p+/l+). panel a. male flies emerging from the nonexposed conditions are either 24 hrs old or newly emerged virgin which are naturally less pigmented (9 and 6 o’clock positions, respectively), the 0.05% ag np (12 o’clock position), or 1.0% ag np (3 o’clock position) in panels a and b were digitally captured as indicated in materials and methods. panel b. higher magnification of eyes and bristles (black arrows). bristles appeared devoid of melanin and almost appeared to have a metallic sheen (figure 4). the flies exposed to 0.3%, 0.1%, and 0.05% ag np also showed a concentration dependent reduction in pigmentation (figure 4 and table 1). because many of the flies regained pigmentation as they aged beyond 14 days post-eclosion, the reduced pigmentation resulting from exposure to ag np at concentrations of 0.05-0.3% is transient. notably, pigmentation was only affected if larvae were exposed to ag np, regardless of parental exposure. furthermore, flies reared on dietary zinc nanoparticles (10-100 nm size) in concurrent experiments have never exhibited the de-pigmentation phenotype seen with ag np (data not shown). these data suggest that ag np exposure during larval development specifically affects pigmentation in adult flies. 58 a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 6. impedance in climbing behavior after exposure to ag np. panel a, climbing behavior was monitored using video analysis of 9-day old w1118 f1 generation adults emerging from continuous developmental exposure to control (p-/l-) or ag np-containing food ((p+/l+) 0.05%, 0.1%, 0.3%, and 0.5%) as described in materials and methods. panel b, snapshots of the first 10 s of the 0.5% ag np (vial on left, marked ‘0.5%’) versus control (vial on right, marked with ‘c’). analysis was completed by using triplicate sets of data with each set using 15 flies. the bars indicate standard deviation. t-test comparison between the 0.1% agnp group and control showed a significant difference with p=5.1x10-07. t-test comparisons between control and 0.05% ag np were not significantly different. because the w1118 strain of flies lack eye pigmentation, the impact of ag np exposure on eye color was tested by using the oregon r adult flies. comparing the eye color of control-fed f1 generation males (24 hr old and virgin) to 1.0% ag np f1 generation reared males (24 hrs old), we observed that the flies from the exposed parental/larval (p+/l+) group lacked body pigmentation while the eye color was virtually unaffected (figure 5a and b). in this strain, the contrast between the pigmented bristles on control-fed and the unpigmented bristles of the ag np fed flies is more pronounced (figure 5b, arrows). this result suggests that the observed de-pigmentation due to ag np exposure is specifically affecting body or cuticular pigmentation. taken together, the observations support the hypothesis that the phenotypic characteristic of losing cuticular pigment is exposuretiming specific (larval stages) and ag np dependent. stage specific exposure related to life span reduction the f1 generation larvae exposed to high levels of ag np (l+) suffered reduced longevity. f1 generation flies exposed to1.0%-5.0% ag np-containing food during their larval stage only lived at most 2±0.3 days post-eclosion without producing progeny, (f2 generation, table 1). however, f1 generation flies exposed to lower ag np concentrations during their larval stage lived longer, 18-20 days, with life spans comparable to the control group, 20±1days, and successfully reproduced (f2 generation, table 1). only the 1.0-5.0% exposed flies lived significantly fewer days than control flies (all p-values less than 0.0011, table 1 legend). the role of ag np exposure during the larval stages of drosophila development became even more clear when the f1 generation adults arising from non-exposed larvae (l-) were transferred to food vials with various ag np concentrations: these flies retained their pigmentation and all of the flies lived as long as control-fed flies in a 20-day exposure time (80±10% flies survived in all groups, p<0.05). further, exposure of the parental (p) generation to ag np did not significantly affect the life span of the f1 generation adult flies (data not shown). all these observations indicate the significance of exposure timing, especially during the larval stage, on the longevity of adult flies. compromised climbing behavior among drosophila reared on the ag np diet because pigmentation defects can be linked to developmental abnormalities including reduction in locomotor ability (walter et al., 1991; wright et al., 1976; drapeau et al., 2003; neckameyer et al., 2001; suh and jackson, 2007), we wanted to assess whether f1 generation adult flies arising from larvae reared on a ag np diet exhibited decreased locomotor behavior. to accomplish this we used the climbing behavior assay as previously described (jimenez-del-rio et al., 2010; chaudhuri et al., 2007). briefly, groups of ten f1 generation, ag npreared flies (p+/l+) versus control f1 generation flies (p-/l-) were compared for their ability to climb a vial wall approximately 7cm in height after being tapped down. since flies arising from the food with 1.0% ag np were few in number, climbing analysis was carried out on f1 generation flies emerging from an exposure concentration of 0.5% ag np and lower. the rate of locomotion of the exposed adult flies seemed to be inversely proportional to the ag np concentration. analysis of digital videography confirmed that the rate of negative geotaxis is inversely proportional to the amount of ag np in the diet. comparing the 0.05% ag np group to the 0.5 % group, the observed decrease in the rate of climbing is from 2.25 cm s-1 to 0.55 cm s-1 (figure 6a). a t-test comparison of the climbing rates of f1 generation adult flies in the unexposed group versus the exposed group (0.1% ag np) suggested that the difference is statistically significant (at a p-value less than 0.05, figure 6a). in figure 6b is an example taken from the video recordings, illustrating that the organisms fed the high ag np concentration (0.5% ag np), climb at a slower rate than unexposed, control flies at the time marks of 0s, 5s, and 10s post tap-down (figure 6b, compare 0.5% ag np-fed flies on left to control (c) flies on right). regardless of the exposure concentration levels, the multiple sets of one-minute videos revealed that once the ag np-fed flies reached the apex of their climb, their movement essentially ceased (see supplementary video footage). in general, the higher the ag np exposure level, the shorter the distance traversed in the allotted 60 second time period. for example, the majority of flies emerging from the larvae exposed to 0.3% and 0.5% ag np diets were observed to climb for 10-20 seconds, halt at the 0-4 cm mark, and appear to tremble in place for the remainder of time. in contrast, the majority of flies resulting from developmental doses of 0.1% and 0.05% ag np climbed to the apex of ~6.5 cm at rates approximating control rates (within 20s or 10s respectively), but again exhibited reduced lateral movement and jumping activity compared to control fed flies. taken together, the observations suggest that exposure to dietary ag np during larval development appears to reduce climbing behavior in emergent drosophila melanogaster adult flies. discussion in this study, we have exposed drosophila melanogaster to dietary silver nanopowder (ag np) at different developmental stages to determine the overall effects on the fly life cycle. the results suggest that exposure during the parental stage (p generation) does not significantly affect developmental progression of the f1 generation larval progeny (l) and that ag np-exposure levels of 0.3% resulted in an overall survival rate that approximated an ld50. specifically, ag np-exposure during f1 generation larval development appeared to negatively impact larval progression, decreased f1 generation adult longevity, altered f1 generation adult locomotor behavior, and decreased f1 generation adult pigmentation. in humans, exposure to silver has been documented to cause medical conditions such as argyria, an irreversible grayish discoloration of the skin (payne et al., 1992; chen and schluesener, 2008; kwan et al., 2009). as levels of ag np are now detectable in the human environment (kim et al., 2010; ahamed et al., 2010; chen and schluesener, 2008), it is imperative to investigate the entire spectrum of impacts on health. 59 a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 5. p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) de-pigmentation may be due to induction of a stress response. previous reports indicate that increased stress levels can effect pigmentation, and may be associated with increased expression of heat shock protein 70 (hsp70) (denman et al., 2008; galvan and alonso-alvarez, 2009; glassman, 2011). posgai et al showed that 3rd instar larvae feeding on 50 mg/ml and 100 mg/ml coated ag np of 10 nm in size have increased levels of proteins responding to stress including glutathione s transferase (gst), superoxide dismutase (sod) and heat shock protein 70 (hsp70) (posgai et al., 2009). exposure to zinc nanoparticles (zn np) also induces a stress response and increases the level of hsp70 in marine organisms (heng et al., 2010; wong et al., 2010). however, in the present study drosophila exposed to zn np in comparable amounts to the ag np exposures did not lose pigmentation (data not shown). from these observations and previous findings, the mechanism(s) through which ag np exposure is associated with de-pigmentation in fruit flies seems to be exposure agent specific. the mechanisms through which ag np exposure during f1 larval stages may cause de-pigmentation, reduced longevity, and reduced locomotor activity in fruit flies could be multi-factorial. while eye pigmentation involves a myriad of genes performing various functions (lloyd et al., 1998), pigmentation of the adult cuticle (or body) results predominantly from the defined biochemical synthesis of melanins via the melanization/dopamanine pathway. this pathway has been well described and involves a number of enzymes encoded by genes such as: pale, yellow, dopa decarboxylase (ddc), and ebony (sugumaran, 2009; sugumaran et al., 1992; wittkopp and beldade, 2009; wittkopp et al., 2003; tang, 2009; carroll, 2005; han et al., 2002). dopamine metabolism is known to generate reactive oxygen species (ros) which can affect the nervous system and overall negatively affect longevity (perez and hastings, 2004). in a previous report, it was shown that fly strains with short life spans appeared to have increased pigmentation compared to long-lived fly strains and that deeper pigmentation was associated with increased dopamine levels, increased oxygen consumption, and increased locomotor activity which indirectly assesses ros levels (vermeulen et al., 2006). in contrast, our study suggested that loss of pigmentation is associated with decreased longevity and decreased locomotor activity. thus, phenotypes arising from ag np exposure during larval development may be due to a combination of factors in both the pigmentation and the stress response pathways. ag nps may act through one of the following mechanisms: 1) ag nps may decrease dopamine levels directly by targeting dopamine pathway genes, enzymes, or localization of melanins; or 2) ag nps may elicit the stress response, increasing hsp70 expression and generating ros, or 3) a combination of both mechanisms. future studies on the effect of ag nps in whole organisms should focus on delineating relevant pathways related to dopamine synthesis, stress response, or some combination. acknowledgements the authors thank dr. mitch mcvey at tufts university for manuscript critique, dr. eric spana at duke university for oregon r flies, bloomington stock center for w1118 flies, the reviewers for constructive feedback, dr. mark wiesner and dr. appla r badireddy at the center for environmental implications of nanotechnology at duke university, 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deoxynivalenol jiazheng yuan1,2, michelle zhu3, khalid meksem4, matt geisler2, patrick hart5, and david a. lightfoot1,2* 1 department of plant, soil sciences, and agriculture system, southern illinois university at carbondale, carbondale, il 62901 usa; 2 department of plant biology, southern illinois university at carbondale, carbondale, il 62901 usa; 3 department of computer science, southern illinois university at carbondale, carbondale, il 62901 usa; 4 plants and microbes genomics and genetics lab, department of plant, soil sciences, and agriculture system, southern illinois university at carbondale, carbondale, il 62901 usa; 5 department of pathology, michigan state university, east lansing mi , 48824 usa. received: june 30, 2013 / accepted: august 22, 2013 __________________________________________________ * corresponding author: ga4082@siu.edu 154 abstract mycotoxin deoxynivalenol (don), produced by gibberella zeae (schwein.) petch (teleomorph of fusarium graminearum schwabe) was known to be both a virulence factor in the pathogenesis of triticum aestivum l. (wheat) and an inhibitor of arabidopsis thaliana l. seed germination. fusarium graminearum causes both gibberella ear rot in maize (zea mays l.) and fusarium head blight (fhb) in wheat and barley. arabidopsis thaliana was also a host for the related root rot pathogen f. virguliforme aoki. a. thaliana seedling growth was reduced by the pathogen in a proportional response to increasing spore concentrations. here, the changes in transcript abundances corresponding to 10,560 a. thaliana expressed sequence tags (ests) was compared with changes in 192 known plant defense and biotic/abiotic stress related genes in soybean roots after infestation with f. virguliforme. a parallel comparison with a set of resistance pathways involved in response to the don toxicity in a. thaliana was performed. a. thaliana data was obtained from the afgc depository. the variations of transcript abundances in arabidopsis and soybean treated with pathogen suggest that both plants respond to the pathogen mainly by common, possibly global responses with some specific secondary metabolic pathways involved in defense. in contrast, don toxin appeared to impact central metabolisms in arabidopsis plants with significant alterations ranging from the protein metabolism to redox production. several new putative resistance pathways involved in responding to both pathogen and don infestation in soybean and a. thaliana were identified. keywords: glycine max, arabidopsis thaliana, fusarium, deoxynivalenol, don, pathway, interaction. introduction in the field, many fungal species cause plant diseases, several of which produce specific mycotoxins (bai et al., 2002). mycotoxins are diverse secondary metabolites formed by fungi through terpenoid, polyketide, and other biosynthetic pathways. one of the important groups of fusarial mycotoxins, named the trichothecenes, that include deoxynivalenol (don) and t-2 toxin, are commonly found in cereal grains as a result of fungal infection. trichothecenes inhibit protein synthesis. don inhibits a wide set of steps in gene expression but has been shown to reduce the activity of eukaryotic ribosomes (60s subunit) and impairs the initiation or elongation and termination steps of protein synthesis (ehrlich and daigle, 1987; feinberg and mclaughlin, 1989). sudden death syndrome (sds) of soybean (glycine max l. merr.) caused by fungal pathogen fusarium virguliforme (aoki; ex. f. solani mart. sacc., ex f.sp. glycines roy) produces root cell infection as well as leaf scorch, which mycotxins produced in roots translocated into soybean leaves and resulted in disease symptom. both root rot and leaf scorch can cause significant yield losses. iqbal et al. (2005) measured changes in transcript abundance (ta) of 192 known plant defense and biotic/abiotic stress related genes from a soybean root cdna library within a time course study. they found at least two-fold ta increase among 36 of these resistance genes from day 3 to day 10 after the pathogen was infested in the soil surrounding the roots of a resistance genotype. arabidopsis thaliana (l). is compatible with many types of phytopathogens (bai et al., 2002). arabidopsis is a host for f. virguliforme and the growth responses to the pathogen were correlated with the spore concentration (yuan et al., 2008). it is unclear whether f. virguliforme releases just don or also other trichothecenes (baker and nenec, 1994). similar responses to a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 155 the concentration of don were seen for arabidopsis root elongation (shin et al., 2012). transgenic expression of a barley udp-glucosyltransferase provided resistance to don. masuda et al. (2007) demonstrated that seedlings showed dwarfism with morphological changes such as petiole shortening, curled dark-green leaves, and reduced cell size under t-2 toxin treatment. when t-2 toxin was applied into the leaves of arabidopsis, it showed an elicitor-like activity in plant cells (nishiuchi et al., 2006). don tends to be harmful to the translational machinery in plants but may also inhibit transcription and elicitor-like signaling pathways in arabidopsis cells (masuda et al., 2007; shin et al., 2012). studies showed that pathogens with toxicity (tox) gene mutations were often nonvirulent (graniti, 1991), or caused less symptoms than the wild-type counterparts, suggesting that trichothecenes were pathogenesis factors of fusarium graminearum (desjardins and hohn, 1997; awad et al., 2008). genetic engineered defense response genes provide an alternative strategy to reduce f. graminearum infection. overexpression of a barley class ii chitinase (ec 3.2.1.14) gene increased the resistance against f. graminearum in wheat (mackintosh et al., 2007; shin et al., 2008) and thereby reduced head blight and don toxicity. don and glutathione formed a glucoseconjugate to reduce don toxicity in vivo and wheat transgenic plants carrying barley udp-glucosyltransferase converted don into a don-glucoside and displayed an enhanced tolerance to don (shin et al., 2012). to date, there is no a single model of plant vs. toxin interaction and no simple or common resistance mechanism to explain the mechanism of plant resistance to tricothecene toxins like don and t2. whether there is a close correlation between don toxicity and fusarium infection is still not fully understood. microarray experiments allow the dissection genome wide patterns of mrna abundances and improve understanding of the molecular basis of the plant defense responses. these global and simultaneous analyses of ta profiles enable variations in mrna abundances under specific treatments to be compared. in order to compare the plant resistance mechanism to don toxicity and infection of fusarial pathogens, microarray data from arabidopsis thaliana cells challenged with the mycotoxin don was compared to the data from the plants responding to f. virguliforme infestations herein. the cdna microarray chips used contained over 10,000 different ests (afgc set 2001) was employed in both analyses. the first objective of this approach was to identify genes that were transcriptionally regulated when plants were treated with the toxin. the second objective using the microarray data was to identify resistance pathways where these co-regulated genes were positioned. the parallel comparison on transcriptional activities among arabidopsis and soybean after fungal pathogen f. virguliforme pathogenesis and arabidopsis with don treatment was also performed. materials and methods arabidopsis thaliana seed germination, rna isolation and microarray procedure a. thaliana ecotype ‘columbia’ seeds were germinated and rna was extracted from young seedlings treated with don (for 6 hours or 24 hours) or water (ftp://smd-ftp.stanford.edu /smd/organi sms/at), respectively. microarray (21124.xls and 22172.xls at ftp://smd-ftp.stanford.edu/smd/ organisms/at/) platform for the gene expression analysis was performed in this experiment. the mrna samples corresponding to don treatment and non-treated control were labeled using cy3or cy5 labeled dutp. one technical replicate was applied by using reversed dye labels compared to the first hybridization (ftp:// smd-ftp.stanford.edu/smd/). dr. patrick hart at michigan state university carried out the microarray experiment. microarray gene expression data analysis the cdna microarray data were normalized by local (local background value was subtracted from the intensity value of each spot) metrics. stringent quality control measures were applied to all stages of data analysis. the normalization procedure for the microarray data followed the method described by pevsner (2003) to adjust for differences in the intensity of the two labels. coefficients of means, variances of the signal intensities in each channel, and ratios of signals from two replicates were calculated by a c++ program written for the purpose. the c++ program was also applied to handle the missing and extra data values. mapmantm allows the visualization of transcriptomic and metabolomic data simultaneously (http://gabi.rzpd.de/projects/ mapman/; thimm et al., 2004). classification of image annotator in the software was also used to diagram the data display. after the arabidopsis est-based microarray data were converted into affymetrix 22k array annotation, all the genes that corresponded from both reverse labeled arrays were subjected to pathway construction using the mapman platform (http://gabi. rzpd. de/projects/mapman). transcripts that were increased in abundance were denoted as blue, and transcripts that were decreased in abundance were denoted as red. the identified metabolic clusters and their relationships in metabolism by the mapman platform were displayed as bins (1-100) and each bin represented a set of related pathways. in the scale used for the visualized data, a 2-fold (1 on log2) change was required to produce a visible coloration. a computer platform, arabidopsis interactions viewer (renamed interactome 2.0, http://bar.utoronto.ca/interactions/cgi-bin/arabidop sis_interactions_viewer. cgi) was employed to predict protein-protein interactions (interologs, geisler-lee et al., 2007). output of interlogs was plugged into the cytoscape software environment (shannon et al., 2003) for network visualization and modeling to against each other in order to catalog all of their conserved pathways and gene interaction networks. the program was equipped with a plug-in architecture for customizing applications. visual data were displayed on log 2 scale and saved as a cytoscape graph. in the cytoscape diagram of a transcriptional network, nodes represented structural genes and their regulatory elements and links represented protein-protein (transcription factor dna binding) interactions. regulatory genes appeared as ‘hubs’ in a network and possessed many interactions. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 156 results overview of ta alterations after don treatment the microarray analysis clearly indicated that deoxynivalenol (don) treatment resulted in major responses among transcript abundances (tas; figure 1; table 1). tas in cell cultures derived from arabidopsis seedlings were altered differently by don over time. six hours (6h) after don treatment, 391 transcripts were increased in abundance by more than 2 fold (displayed as 1 fold on log 2 scale) but 169 transcripts were decreased in abundance by more than 2 fold (displayed as -1 fold on log 2 scale) and 119 of them were reduced by more than 3 fold. however, only 120 transcripts appeared to be increased in abundance by more than 2 fold 24 hours (24h) after don was applied (figure 1; table 2). conversely, 181 transcripts were decreased in abundance by more than 2 fold at this time point. moreover, the transcripts that could be detected by the microarray experiment were significantly reduced by nearly 40% suggesting that the toxicity to transcription and subsequent metabolic perturbations might occur in the process of the time course experiment (data not shown). description of significantly regulated pathways in the mapman platform the arabidopsis transcript abundances that were significantly increased (log 2 ≥ 1) and decreased (log2 ≥ -1) were visualized within the mapman platform to identify metabolic clusters and their relationships in metabolism. the preliminary analysis displayed by the mapman platform indicated that the transcript abundances following the don treatment had been dramatically altered in many metabolic clusters. by 6 hours after don treatment, the 560 genes that had significantly altered ta (≥1 or ≤-1 on log2 scale) in the two reversed labeled slides were assembled into 12 major bins and each bin represented a set of related pathways (table 1). within these 12 bins, more than 41 sub-bins were differentially regulated by don. the toxicity of don had a major effect on the pathways of the protein synthesis and degradation. there were 12 sub-bins in bin29 (protein metabolism) and 4 sub-bins in bin27 (rna synthesis) being strongly affected by don, which suggested that don toxicity also targeted to the gene transcription (table 1a, b). moreover, ta changes in signaling and hormone metabolism bins were also altered. at 24h after don treatment, 301 genes that had tas significantly altered were assembled into 16 major bins, within which were a total 1,945 genes (table 2). there were more than 35 sub-bins that contained tas that were significantly affected by the don toxin (p<0.05). tas in bin27 (rna transcription) and bin29 (protein metabolism) were reduced significantly at this stage. in total 1,045 genes encompassed by the bin27 were affected by don 6h after treatment but only 776 genes remained in the bin 24h after the treatment. the bin29 also showed clearly an inhibition in protein metabolism indicating a suppressed responsiveness by the toxin 24h post don treatment (figure 1b). only 3 of the 12 sub-bins altered at 6h were still preserved at 24h after don treatment. the number of genes encompassed by these affected sub-bins were reduced from 410 to 79. therefore, a large proportion of genes involved in protein metabolism that were poisoned by the mycotoxin at 24h were normally abundant at 6h after treatment. in parallel, several other bins such as transporter genes and the genes related with lipid metabolism and redox reactions were altered at 24h, whereas they were not at 6h. furthermore, about half of the transcripts altered at 24h had unknown functions (713 genes) and can now be annotated as invilvoed in responses to don. whether the plants attempt to reconfigure and bypass the inhibited enzymes and damaged pathways still need to be determined. unlike f. virguliforme infestation of soybean and arabidopsis, none of the crucial gene transcripts that potentially involved in secondary metabolism (bin 16) had been increased in abundance after don toxin was applied. no pathway leading to isoprenoid, phenylpropanoid, and lignin biosynthesis was shown increased in ta at either 6 or 24 h after treatment. therefore, don inhibition does not appear to trigger all of the same biotic and abiotic stress response pathways as f. virguliforme infestation. networks of protein-protein interaction bin29 contained many genes involved in plant defense schema (wang et al., 2006) and transcripts in this bin were greatly affected by don treatment. the 417 genes encompassed in this bin (from the stage of 6 hours after don treatment) were visualized using the arabidopsis interactions viewer (geisler-lee et al., 2007) to investigate the potential molecular protein-protein interactions among genes with ta changes. the output of the interlogs from the arabidopsis interaction viewer (geisler-lee et al., 2007) was plugged into the cytoscape software environment (shannon et al., 2003) for network visualization and modeling. in these diagrams of protein –protein interaction networks, nodes represented structural genes and their regulatory elements while links denoted protein-protein interactions. regulatory genes like transcription factors appeared to be ‘hubs’ in a network and had many interactions. four hubs, at2g38560 (tfiif (-3.258/log2)), at3g55620 (emb1624 (1.310/log2)), at1g36730 (etif5 (1.025/log2)), and at2g47020 (pcrf (1.079/log2)) were identified 6h after don was applied (figure 3). the multiple network characteristics among several hubs (highlighted in red) inferred resistance related protein-protein interactions and displayed a correlation between the complexity of a network and the property of resistance. four major hubs, at5g35980 (protein kinase (-1.269/log2)), at5g57020 (nmt1 (-3.727/log2)), at3g59950 (apg4a (-1.716/log2)), at5g45900 (apg7 (1.051/log2)), at3g11830 (chaperonin (-5.442/log2)) were observed 24h after don treatment. the metabolic networks in bin29 were represented as a linear relationship among the hubs and nodes (figure 3). however, plants may attempt to avoid don toxicity by reconfiguration of the metabolic networks. the great number of genes with unknown functions that were identified 24h post don treatment might be involved in that response. the increased response among genes of unknown function was in contrast to the reduction in the number of tas that were involved in other metabolisms in arabidopsis plants at this stage. whether or how the increased ta of these genes of unknown function provides a bypass for damaged enzymes and compartments to escape the toxin selection is still not known. 157 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. transcript abundance changes in responses of arabidopsis cells to don treatments. the crucial metabolic pathways in the mapman program were marked in red rectangles . the small blue and red squares denoted increased (positive) and decreased (negative) transcript abundances (tas) of individual genes. the bar was shown on log 2 scale (1=two fold change). a. 6 hours after don was applied. b. 24 hours after don was applied. b. 24 hours after don applied a. 6 hours after don applied 158 discussion a number of fusarial species cause infections that both reduce yield and grain quality on major cereal crops such as wheat, barley, and maize. mycotoxins produced by these pathogens lead to feed-born intoxications in farm animals (awad et al., 2008). mycotoxins have also been considered as aggressiveness factors in pathogens in both wheat and maize. cumagun et al. (2008) identified loci associated with pathogenicity and aggressiveness in a f. graminearum population derived from a cross between a niv producer lineage 6 from japan and a don producer lineage 7 from kansas. lineage 7 was the dominant race that caused fhb and gibberella ear rot in crops (o’donnell et al., 2000; xu and nicholson, 2009). mycotoxin trichothecenes (that include don and t2 toxin) appeared to possess multiple a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) inhibitory effects ranging from protein, dna, and rna synthesis to signaling and membrane functions in eukaryotes (rocha et al., 2005). the toxins enhance the spread of f. graminearum fungal pathogen (lori et al., 1997). a close relationship between the concentration of don and fhb severity and 1,000 kernel weight was observed suggesting that the trait was affected by the production of the toxin (lori et al., 1997; hestbjerg et al., 2002). don also affected root elongation and the roots were less organized compared with the control in a. thaliana (hart, unpublished data, aafc). evidences demonstrated that reduction of toxin production by the pathogen or removal/degradation of the toxin by the host reduced aggressiveness of some pathogens (foroud and eudes, 2009). pathogens with toxicity (tox) gene mutation appeared to be non-virulent (graniti, 1991) or caused lesser disease symptoms than the wild-type (desjardins and bin name elements p-value 1 ps 26 0.015 1.2 ps.photorespiration 4 0.046 2.1 major cho metabolism.synthesis 4 0.019 2.1.2 major cho metabolism.synthesis.starch 3 0.041 3.1 minor cho metabolism.raffinose family 4 0.013 3.5 minor cho metabolism.others 5 0.048 3.6 minor cho metabolism.callose 3 0.02 3.1.2 minor cho metabolism.raffinose synthases 2 0.023 3.1.2.2 minor cho metabolism.raffinose synthases.putative 2 0.023 8 tca / org. transformation 10 0.035 8.2.4 tca / org. transformation.idh 2 0.035 10 cell wall 79 0.027 10.3 cell wall.hemicellulose synthesis 4 0.039 10.8.1 cell wall.pectin*esterases.pme 10 0.032 11 lipid metabolism 67 0.014 11.8 lipid metabolism.'exotics' (steroids, squalene etc) 18 0.037 13 amino acid metabolism 61 0.026 13.1 amino acid metabolism.synthesis 43 0.045 13.1.4 amino acid metabolism.branched chain group 8 0.038 13.1.6.1 amino acid metabolism.aromatic aa.chorismate 6 0.016 19.99 tetrapyrrole synthesis.unspecified 2 0.039 20.1 stress.biotic 60 0.035 27 rna 544 0.004 27.3 rna.regulation of transcription 453 0.009 27.3.27 rna.regulation of transcription.nac domaintf 16 0.007 27.3.32 rna.regulation of transcription.wrky domain tf 12 0.022 27.3.35 rna.regulation of transcription.bzip tf 20 0.009 29.2.1 chloroplast/mito plastid ribosomal protein 9 0.033 29.2.1.1 chloroplast/mito plastid ribosomal protein.plastid 5 0.005 29.2.2.50 misc ribososomal protein.brix 4 0.023 29.2.5 protein.synthesis.release 2 0.033 29.3 protein.targeting 37 0.04 29.3.4 protein.targeting.secretory pathway 17 0.009 29.3.4.99 protein.targeting.secretory pathway.unspecified 7 0.036 29.4.1.57 postranslational modification.rlck vii 11 0.028 29.5.11.4 protein.degradation.ubiquitin.e3 166 0.014 29.5.11.4.3 protein.degradation.ubiquitin.e3.scf 74 0.004 29.5.11.4.3.2 protein.degradation.ubiquitin.e3.scf.fbox 69 0.002 29.5.11.5 protein.degradation.ubiquitin.ubiquitin protease 9 0.045 30.2.6 signalling.receptor kinases.leucine rich repeat vi 4 0.021 30.7 signalling.14-3-3 proteins 2 0.038 table 1. description of the mapman bins (metabolic clusters) with significant changes in tas by 6 hours after don treatment. the bin numbers were denoted from 1-100. sub-bins were listed as decimals. only bins encompassing genes with significant changes in transcript abundance (p<0.05) were listed. the number of elements in each bin present on the array was listed. the five bins highlighted in light blue and two bins in brown contained many tas highly affected by the mycotoxin. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) hohn, 1997) also suggesting that there was a biological role for the trichothecenes in the virulence of f. graminearum. here don toxin was shown to have a profound and pathogen-like impact on the transcript abundance of genes encoding several crucial metabolic pathways in arabidopsis with a scope from the protein metabolism to redox control. in contrast, some of the responses might not be related to pathogenicity. for example, many of the 1,045 genes distributed among bin27 of the rna transcription related bins were altered in ta by don. this result contrasted to that of f. virguliforme infestation where there was no bin27 pathway effect was observed (yuan et al 2008). similarly, during f. virguliforme infection, a large proportion of altered tas that were in protein metabolism related genes assigned to bin29 (protein metabolism) were not concentrated in the same sub-bins of protein synthesis and posttranslational modification altered by don (yuan et al., 2008). only a few of transcripts altered were involved in protein activation, posttranslational modification, degradation, and folding. . this was a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 159 consistent with the known activities of don as an inhibitor of translation and a pathogenesis factor (ueno et al., 1969; feinberg and mclaughlin, 1989; ehrlich and daigle, 1987; harris et al., 1999; cumagus et al., 2008; shin et al., 2012). the toxic function of don is an inhibitor for the protein synthesis on the 60s subunit of eukaryotic ribosomes and inhibits the initiation or elongation and termination steps of protein synthesis (feinberg and mclaughlin, 1989; ehrlich and daigle, 1987). from 6 to 24h after don treatment, the number of affected transcripts in bin27 and bin29 was reduced significantly. there were only 3 sub-bins left in bin29 24h after don treatment. this suggested that a large proportion of genes involved in protein metabolism were affected by the mycotoxin initially but metabolism was restored later. however, several other new bins emerged, such as transporter genes and the genes related with lipid metabolism and redox reactions at this stage. about half of the transcripts altered were among bins encompassing genes with unknown functions (713 genes). the results were in table 2. description of the mapman bins (metabolic clusters) with significant changes in tas by 24 hours after don treatment. the bin numbers were denoted from 1-100. sub-bins were listed as decimals. only bins encompassing genes with significant changes in transcript abundance (p<0.05) were listed. the number of elements in each bin present on the array was listed. the bins highlighted by light blue and brown colors contained many tas highly affected by the mycotoxin. (p<0.05). bin name elements p-value 1.1.30 ps.lightreaction.state transition 2 0.024 1.3 ps.calvin cyle 5 0.042 3.1.2 minor cho metabolism.raffinose synthases 2 0.04 3.6 minor cho metabolism.callose 2 0.033 8 tca / org. transformation 15 0.01 8.1.1 tca / org. transformation.tca.pyruvate dh 2 0.017 8.2.10 tca / org. transformation 2 0.026 11.2 lipid metabolism.fa desaturation 5 0.036 13.2 amino acid metabolism.degradation 14 0.008 16.7 secondary metabolism.wax 3 0.022 17.8 hormone metabolism.salicylic acid 6 0.049 17.8.1 hormone metabolism.sa.synthesis-degradation 6 0.049 20.2.99 stress.abiotic.unspecified 17 0.001 21 redox.regulation 29 7.00e-04 21.6 redox.dismutases and catalases 3 0.005 23.1 nucleotide metabolism.synthesis 9 0.016 23.1.2 nucleotide metabolism.synthesis.purine 6 0.03 26.19 misc.plastocyanin-like 2 0.048 27 rna 394 0.015 27.3 rna.regulation of transcription 333 0.008 27.3.11 rna.regulation of transcription.c2h2 zinc finger family 18 0.029 27.3.15 rna.regulation of transcription. hap3 2 0.033 27.3.21 rna.regulation of transcription.gras ta 5 0.029 27.3.25 rna.regulation of transcription.myb domain ta 19 0.01 27.3.69 rna.regulation of transcription.set-domain ta 5 0.024 29.2.3 protein.synthesis.initiation 23 0.016 29.4.1.57 protein.postranslational modification.kinase.rlck vii 7 0.042 29.5.11.4.3 protein.degradation.ubiquitin.e3.scf 49 0.042 33 development 81 8.00e-04 33.99 development.unspecified 70 0.0002 34.12 transport.metal 13 0.048 34.21 transport.calcium 4 0.018 35.1.41 not assigned.no ontology.hydroxyproline rich proteins 14 0.014 35.1.5 not assigned.no ontology.pentatricopeptide (ppr) p. 65 0.0005 35.2 not assigned.unknown 713 0.011 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 160 concordance with earlier studies of fusarium infested in arabidopsis thaliana (yuan et al., 2008). if plants rather attempt to reconfigure those inhibited enzymes and bypass the damaged pathways via a backup system is still unknown and functions for these genes can be inferred. unlike in soybean f. virguliforme infestation (yuan et al., 2008), none of the crucial gene transcripts that were classified as potentially being involved in secondary metabolism (bin 16) had been increased in abundance when don toxin was applied. no pathway leading to isoprenoid, phenylpropanoid, and lignin biosynthesis was shown increased in the tas. f. virguliforme infection of a. thaliana only increase pal and c3h suggesting phenolics rather than flavonoids were being produced. therefore, it appears that flavonoids (anthocyanins, proanthocyanidins, flavones, and flavonols) in arabidopsis may indeed not play a significant role in defense. dissection of gene transcript abundance regulation may be one of the main strategies used to decipher resistance gene function because many transcript changes may be involved in plant defense processes (journot-catalino et al., 2006). by comparison of species, organ, and time specific transcript abundances of thousands of genes simultaneously, investigations of alterations in these transcripts have provided unique opportunities to delve into gene function. to analyze the dynamics of ta change during don mycotoxin treatments, arabidopsis seedlings were treated with don toxin and transcript abundances were evaluated in a time course study. moreover, in specific metabolic pathways, the transcripts that could be detected by the microarray experiment were significantly reduced and a large number of genes of unknown function were emerged 24 hours after don treatment suggesting that the toxicity to transcription and subsequent metabolic perturbations might have largely occurred in the later stage of the time course experiment. the reasons for the antiparallel reduction in one pathway and induction of the unknowns still need to be investigated. if a transcription factor or factors underlay the inhibition of groups of genes, each individual metabolic pathway should be interrogated for a common cis-acting element. in contrast, if toxicity occurred in the transcriptome, investigations of adaptive changes in the metabolic transcriptome should be performed. the similarities and differences inferred among the metabolic responses of arabidopsis thaliana to don and fusarium virguliforme might be attributed to nature of the pathogenesis factors. the resistance regulation and signaling pathways in arabidopsis plants to these pathogenesis factors may share similar components. based on results here, don toxin appeared to be a remarkable pathogenesis factor with effects on the crucial metabolic pathways ranging from the protein metabolism to redox reactions with significant metabolic alterations in arabidopsis plants. therefore, the results presented here may provide basic knowledge and an alternative strategy for developing more cultivars with resistance/tolerance to the toxin. overexpression of defense response genes such as a barley class ii chitinase (ec 3.2.1.14) gene that broke chitin, a key component of the cell wall in fungi enhanced the resistance against fusarium graminearum (mackintosh et al., 2007; shin et al., 2008) and thereby reduced fhb (fusarium head blight) and don toxicity. shin et al. (2012) showed that don conjugated with glutathione into a glucose-conjugate in vivo was no longer toxic. barley udp-glucosyltransferase converted don into a don-glucoside and therefore, transgenic arabidopsis carrying the barley gene displayed an enhanced tolerance to don. therefore, the effective neutralization of don and other trichothecene toxins can be used to provide resistance/tolerance to the fusarium pathogens and this strategy should be also explored in genetic engineering soybean plants. tfiis pcrf emb162 4 etif5 figure 2. network of predicted protein-protein interactions among genes from bin 29 in a. thaliana 6 hours post don treatment. the networks were generated by cytoscape (shannon et al. 2003) and visual displays were saved as cytoscape graphs. major interaction hubs were marked by red. figure 3. network predicted protein-protein interactions from bin29 in a. thaliana treated with don after 24 hours. the networks were generated by cytoscape (shannon et al. 2003) and visual displays were saved as cytoscape graphs. major interaction hubs were marked by yellow. protein kinase apg4a apg7 chaperonin a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 161 acknowledgements this research was funded in part by grants from the national science foundation, the illinois soybean association and the united soy board. references aoki t, k o’donnell, y homma, and ar lattanzi (2003) sudden death syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the fusarium solani species complex—f. virguliforme in north america and f. tucumaniae in south america. mycologia 95: 660–684. bai gh, ae desjardins, and rd plattner (2002) deoxynivalenol nonproducing fusarium graminearum causes initial infection, but does not cause disease spread in wheat spikes. mycopathologia 153: 91-98. baker ra and s nemec (1994) soybean sudden death syndrome: isolation and identification of a new phytotoxin from cultures of the causal agent, fusarium solani. 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yuan jz, mx zhu, da lightfoot, mj iqbal, j yang, and k meksem (2008) in silico comparison of transcript abundances during arabidopsis thaliana and glycine max resistance to fusarium virguliforme. bmc genom s2: 1-15. atlas journal of biology 2 (2): 142–147, 2013 doi: 10.5147/ajb.2013.0087 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) practical use of nitrogen gas as a method for insect control in herbaria miriam kritzer van zant1*, don ugent2, and david a lightfoot1* 1 department of plant soil and agricultural systems, southern illinois university, carbondale, il 62901-4415; 2 southern illinois university, carbondale, il 62901. in memorium. received: april 18, 2013 / accepted: may 19, 2013 __________________________________________________ * corresp. authors: mkvzant@siu.edu and ga4082@siu.edu 142 abstract herbaria are libraries of dried mounted plants used for plant identification, research vouchers and teaching. herbarium specimens are subject to damage from insects, fungi and bacteria, and must be protected by treatments that kill damaging organisms. naphthalene, the most common chemical currently used in herbaria, is a class c carcinogen and potential allergen. the aim was to develop an affordable alternative to treatment with persistent hazardous toxins for maintaining dried herbarium specimens. the new method uses ambient temperature nitrogen gas and widely available, valved, nylon, oxygen barrier bags. nitrogen gas treatment has been shown to be less expensive than freezer storage and safer than treatment with naphthalene and other toxins. the lower hazard of nitrogen treatment compared to naphthalene offers a practical option for k-12 and institutions of higher education to initiate, reinstate or strengthen herbarium collections for teaching and/or research. this is the first report of the use of inexpensive valved oxygen barrier bags for herbarium pest control. keywords: nitrogen; herbaria; method; specimens; plants; napthalene. introduction herbarium specimens in archives are mounted on large sheets of acid-free paper, with the intention that they will last for hundreds of years (bridson and forman, 1989, 1998). plant specimens are flattened to save space, dried, then sewn or glued onto paper and stacked on shelves inside tightly sealed protective cabinets. detailed labels containing information on specimen origin and identity are attached to each sheet. drying of specimens, at low temperatures prior to mounting on paper, is necessarily thorough. drying reduces growth of molds and bacteria and decreases the likelihood of attack from insects. specimens are stored in sealed cabinets except when in use. on rare occasion, invasion of storage cabinets by insects from the surrounding environment occurs, requiring treatment. however, most insects are introduced from contact with improperly treated specimens, often during storage. certain plant families are very susceptible to insect infestation (jessup, 2005). cabinets are sometimes monitored with pheromone traps. most of these traps are specific for cigarette beetle (lasioderma serricorne fabricus; jessup, 2005). pheromone traps are especially important for large collections where not all cabinets receive regular attention. pheromone traps should be replaced about every three months and cost about $11 per cabinet (herbarium supply, 2012). sprinkling boric acid powder every few months around cabinets is less expensive than using traps, as boric acid is not volatile and is known to kill beetles. boric acid should not be applied directly to specimens, so is only useful to prevent infestation by crawling insects from the vicinity of cabinets. traps may still be needed, particularly short methods article a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 143 for monitoring susceptible plant families. toxicity from boric acid requires ingestion (a.d.a.m., inc., 2012; harper et al. 2012). diatomaceous earth is considered non-toxic to mammals (hope, 2011). diatomaceous earth works in a similar manner to boric acid. hower, it is not as fast-acting as boric acid. in the past, harsh chemicals containing mercury or naphthalene were applied to herbarium sheets, to improve the chances of specimens surviving attack by other organisms. mercury is a neurotoxin, a serious human health hazard in quantities as small as 100-300 ng per kg of body weight per day (us epa, 1997; ziff and ziff, 2001; clarkson, 2002). naphthalene displaced mercury as the standard for protection of herbarium specimens until about 20 years ago. acute exposure to naphthalene can result in liver and neurological damage in humans (us epa, 1999, 2004; preuss et al., 2003). chronic exposure may result in cataracts and retinal damage. naphthalene has been classified by the epa in group c, among possible human carcinogens. the group c ranking indicates that data is still insufficient for absolute classification as a carcinogen. students, after brief periods in herbaria where naphthalene is in use, often complain of headaches, nausea and burning eyes and skin. following the us epa (1997, 1999) assessments, many herbaria have purchased walk-in or chest freezers (bridson and forman, 1989, 1998). freezers cost more to obtain and operate than naphthalene application. smaller herbaria, and individuals maintaining cabinets, are often unable to buy and/or maintain freezers. freezers require more space than cabinets. freezer malfunction allows rapid growth of mold. freezers may cause freeze-thaw damage to specimens. the moisture in freezers can reduce the mildew protection resulting from complete drying. freezing may not kill all insect eggs. nonetheless, curators at the association of systematic collections (zycherman and schrock, 1988) and kew herbarium (bridson and forman, 1989, 1998) endorse the use of either freezers or nitrogen to kill insects as an alternative to naphthalene and other harsh chemicals. bridson and forman (1998) recommend that nitrogen be used at temperatures above 20ºc and that it is most effective between 30-38ºc. further, they (bridson and forman, 1998) recommend treatment time for nitrogen exposure of 1 week for most insects but 1-3 weeks for wood-borer infested samples. the hong kong special administrative region of the people’s republic of china (2006, 2012) has a nitrogen chamber used for curation of national treasures in the form of wooden furniture. the hong kong website states termites, woodborers and furniture beetles can be killed in controlled conditions of 55% (v/v) humidity between 0.28-0.30% (v/v) oxygen, at regulated room temperature. nitrogen sterilizes by displacing oxygen, preventing aerobic metabolic activities (watanabe and morita, 1998). hence nitrogen discourages most molds, kills most insect eggs (morin et al., 2005), and halts the oxygen promoted breakdown of paper. nitrogen is 80% (v/v) of the atmosphere (raven et al., 2005). therefore, the environmental impact of use of room temperature nitrogen for curation is generally considered to be very low. to date few specifics have been offered for controlling nitrogen for herbarium curation. we first considered existing systems for utilizing nitrogen. nitrogen tents have been designed for high altitude training of athletes (pedlar et al., 2005). the tents are not air tight to prevent suffocation. therefore, altitude training tents require expensive regulators which mix air with nitrogen. nitrogen tents used for plant research on effects of atmosphere are left open at the bottom (holton et al., 2003). air-tight nitrogen tents would stop plant respiration, defeating the purpose of those studies. partially open tents require continuous free-flowing nitrogen, more costly than filling a sealed area once. sealing an existing design would have been haphazard. designing a new kind of tent would have required considerable time and expense. nasa utilizes nitrogen storage systems to store moon rocks for the long term in hard shell glove boxes (hoversten, 2000). producing a hard shell container also requires tools, skills, time and funding. worse, hard shell containers could implode when oxygen containing atmospheric gases are removed, prior to filling the container with nitrogen. again expensive regulation equipment would have been needed to precisely control removal and input of gases. hard bubble-type or room nitrogen chambers, like the one made for curation in hong kong, and their accompanying gas regulation equipment, are very expensive. here we report a low cost, low hazard method, for treating samples with nitrogen using space bags® (s.c. johnson & son, racine, wi., usa), and simple equipment. materials and methods space bags® are sold for storing clothing and bedding. the bags contain mylar® (dupont teijin films, usa) polyester. mylar® has oxygen barrier properties and is also found in the walls of helium balloons. patented one-way valves on space bags® provide a reliable way to remove air, including oxygen (spacebag® company, 2004). sliding air-tight seals on space bags® operate similarly to those on zip-lock® (s.c. johnson & son, racine, wi., usa) sandwich bags. space bags® are inexpensive and available in various sizes. manufacturers sell the bags in assorted size groupings through commercial outlets. in order to purchase bags of a single size we were required to order an assorted size grouping for about $20 directly from the spacebag® company (https://www.spacebag.com/spacebag), to establish an account. this may have changed since the company was acquired by s.c. johnson. four original jumbo 36” x 49” bags should hold the contents of a full herbarium cabinet. three original jumbo bags and one medium 18” x 22.5” bag held all of the specimens from an almost completely full standard herbarium cabinet (fig. 1). equipment used included: vacuum cleaner with hose; tank containing room temperature nitrogen; standard gas regulator for the nitrogen tank; anchored position and belts for securing the nitrogen tank; tubing; boxes and box lids to protect herbarium specimens from crushing either under their own weight or due to creation of a vacuum during withdrawal of oxygen; sufficient flat surface to retain filled bags for at least one week for herbaceous material (fig. 1). different sized bags worked well with different sized boxes. lids from cartons that had held packages of copy machine paper (fig. 1), hold standard (11” x 17”; 27.9 x 43.2 cm) herbarium sheets. breaking down one side of the lid makes them the right size for holding most longer a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 144 herbarium sheets. by staggering these box lids, weight on the lower specimens was reduced. the largest bags available when we started our trials (original jumbo, 36 x 49”; 91.4 x 124.5 cm) held eight box lids full of specimens, paired into four slightly staggered piles. the valve on the bag used for extraction always faced upward during our study. open boxes were used inside the bags, as they are best suited to air exchange. room temperature nitrogen gas was used in the experiment. it is much less expensive than liquid nitrogen and widely available. a standard vacuum cleaner with a hose (figure 1a) was used to remove air through the bags’ one-way valves. a standard gas regulator is needed for the nitrogen tank. most institutions already have such regulators or they can be purchased for around $100.00. nitrogen tanks cost about $2.00 per month per tank to keep full. there can also be a first time fee of about $75.00 when service is started. one tank was sufficient to treat the contents of an herbarium case, with 20 of its 22 shelves filled with specimens. the amount of gas used included enough to refill bags that were improperly sealed or leaked. bags must be monitored for potential leaks, especially for the first two days to make sure they are not deflating. the inflation or deflation of each bag provided an indicator for successful removal of air and filling with nitrogen. sufficient table or other space was needed to leave the bags undisturbed for at least one week (figure 1d). air was collected from the valve positioned at the top (figure 1), using the vacuum cleaner and hose. tubing was used, long enough to deliver the nitrogen from the tank deeply into the back bottom of the bags (figure 1b). in the typical protocol, specimens were set in the bags, arranged in the open boxes with the bag’s valve on top. tubing attached to the nitrogen tank was placed as deeply as possible into the bottom back end of the bag. seals were closed tightly against the tubing, using the provided plastic slider. next the entire length of the seal was reinforced with the slider. prior to inflation, the snap on valve lid was removed. the vacuum was turned on to remove enough air to cause the bag to collapse slightly around the boxes of specimens, but not enough to allow the vacuum to crush boxes or dried plant materials. immediately after the vacuum cleaner was shut off, the nitrogen regulator valve was opened to gently inflate the bag. care was taken to not over-fill the bag nor open the seal. next, at least 1/3 of the gas in the bag was again removed from the top, until the bag began to collapse again against the boxes. more nitrogen was let into the bag, to re-inflate it. the process was repeated for a third time. the final step was to rapidly withdraw the tubing and immediately seal the bag. bags that were properly sealed stayed visibly inflated. after the third filling, the valve lid was gently twisted until it snapped into position over the valve to prevent accidental leaks. if the seal opened on the bag, the whole process was repeated. a b c d figure 1. use of nitrogen with valved mylar containing bags for the preservation of herbarium specimens. panel a shows removal of air from a bag containing boxed specimens. panel b shows the use of tubing to deliver nitrogen gas into the bottom of the bag. panel c shows a bag inflated with nitrogen with the blue seal facing the viewer. panel d shows the contents of a standard herbarium cabinet under treatment with room temperature nitrogen. if moving a bag is necessary, the seal should be checked and the slider passed across the seal at the destination site. as long as the bag still appears inflated, nitrogen must remain in the bag. if any air containing oxygen were to remain in the bag, it would be pushed to the very bottom, requiring insects, bacteria and mold spores to remain at the bottom of the bag itself for the entire week to avoid asphyxiation. at the end of the treatment period, the bag should be opened facing a moving draft or ventilation system. specimens should then immediately be placed into a clean cabinet and the door sealed. safety precautions must be taken when applying this method. nitrogen is not a fire hazard but since it displaces oxygen it is an asphyxiation hazard. though nitrogen is not a long term environmental hazard, there are some safety issues to consider for its use and release. a proper anchor and belts for the tank are necessary. any improperly secured gas tank can become a torpedo and destroy a room, if the valve is ruptured by a fall. securing the tank to an anchored work-bench with tank belts is essential. the gas regulator must work and be properly connected to the tank. seals on bags can wear or pop open when gas is added so it is important to check that gas remains in the bag. nitrogen should only be released in a ventilated area. if allowed to build up in a room, nitrogen could be hazardous to small children, pets, maintenance workers or crawlers during a fire. nitrogen buildup could be deadly for someone who loses consciousness in the gas for a sustained period. any plastic bag and especially an oxygen barrier bag full of nitrogen is a smothering hazard, so access by children and pets should be tightly controlled. direct release of nitrogen into the operators face is unpleasant as it momentarily deprives lungs of air. for that reason nitrogen should not be released directly out of a window. during this study, nitrogen was mixed back into the atmosphere by releasing it into a ventilation system leading outdoors. releasing nitrogen into a fan directed outward would give a similar result. moisture can build up in closed bags when they are not in use, so stored bags should be checked for molds before re-use. though nitrogen will kill most molds, mold exposure to specimens and workers is unnecessary. mold can be prevented by placing unused bags inside a single large bag filled with nitrogen, and/or by leaving silica dry packs in each bag. results eighty seven percent of space bags® tested held nitrogen with no difficulty for one week or longer (fig. 1). fifty percent of the bags were still fully inflated with nitrogen at the end of three months. full bags were carefully and successfully transported on rolling carts and even by car for short distances on several occasions. bags that lost nitrogen, as evidenced by partial collapse visible by the second day, were refilled and stayed inflated. in these cases seals may have been jarred during filling, transport or sealing. occasionally, bags popped open during filling, causing the operator to inhale nitrogen. inhalation caused dry irritation to the throat, but no lasting consequences. the asphyxiation hazard is small (watanabe and morita, 1998) if the work is carried out in a well-ventilated area. one hundred percent of specimens treated with nitrogen in the study remained free from insects and molds for at least three years when their observation for this purpose was discontinued. discussion and conclusions sufficient space bags®, a new gas regulator, an inexpensive vacuum, tubing and sufficient nitrogen including a startup fee, to maintain the contents of one or several herbarium cabinets for one to several years, were purchased for under $400 dollars in 2004. if a regulator and vacuum were already available and tank service established, start-up costs drop to about $120. yearly maintenance costs to treat specimens filling a single cabinet were between $25 for nitrogen under a monthly contract and $125.00 for nitrogen and replacement of all bags if needed. pheromone traps for cigarette beetles add no more than $44 to costs per standard cabinet per year and are optional for most teaching collections. sufficient boric acid for periodic dusting of the floor of a typical school laboratory or classroom would cost under $7.00 per year if purchased from a pharmacy. if kept dry, boric acid should keep in its container for several years. there was a one-year replacement warranty on the bags. bags may outlast the warranty, depending on frequency of use. the same bags can be used to treat more than one cabinet and for incoming dried material, either from the field or borrowed from other institutions. there are other less expensive brands of valved oxygen barrier plastic storage bags besides space bag®, but mylar® contents vary among brands, and other valves may not be as effective as those patented for one way air flow. acidity transfer from the cardboard boxes should not be an issue while the specimens are under inert conditions in nitrogen, especially as cardboard boxes are already being used to hold herbarium specimens in freezers. boxes or lids can be lined with acid free paper if there is concern. though we used open boxes, closed boxes may also be used inside of the bags as long as they are not air tight. this room temperature nitrogen method for controlling invasive organisms on herbarium sheets does not result in freezethaw damage to cells. because nitrogen halts aerobic metabolic activity, it may not prove useful for storage of propagules or pollen intended for germination. however, that remains to be tested. freezing and thawing can alter protein structure through crystallization (morin et al., 2005). chemicals like naphthalene leave residues on specimens that can alter the results of pharmacognosy research and other biochemical assays. nitrogen does not leave any residue. oxygen (15.999) is slightly heavier than nitrogen (14.007) according to the periodic table. air contains close to 80% nitrogen and 20% oxygen. pure oxygen would therefore be slightly heavier and pure nitrogen slightly lighter than air for the same volume. therefore, nitrogen seeps upward, displacing the room air containing oxygen, which will sink to the bottom inside the bag. it may be better in the future to place the bags on racks with valves pointing downward. this would allow air and gas extraction from underneath, and may allow the downwardly seeping oxygen and air to be more completely extracted from the bags. though nitrogen gas is not available everywhere, it is avail145 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 146 able in major cities throughout the world and can be obtained almost anywhere there is a road in developing areas. the infrastructure for transporting gas tanks exists anywhere propane is available or where acetylene torches are used. as 80% (v/v) of the atmosphere is nitrogen, there is no known long-term environmental damage from preparation, use or release. there may be environmental issues with disposal of the bags. however, there are greater problems with the disposal of freezers and their chemicals. in addition, this method may be used for short-term maintenance of material that is not dried. it would be interesting to see if some kinds of biological specimens currently maintained in costly frozen nitrogen or in formaldehyde or acetic acid (zycherman and schrock, 1988) would retain their usefulness if stored in room temperature nitrogen. others have used variations on this method for long-term specimen preservation. fort lee virginia is home to the u.s. army facility for storage of archeological artifacts (fort lee regional archeological curation facility, 2006). the facility uses nitrogen to store artifacts for the long term. curators there place artifacts in oxygen barrier bags, withdraw most of the oxygen containing air from the valve, fill the bags with nitrogen sometimes adding silica to keep specimens dry, and set them in a cabinet where they can be stored for up to 30 years (jason j. huggan, carol l. anderson, personal communication 2006). spacebags® were specifically recommended by the fort lee curators. treatment of herbarium material usually requires only one week of exposure to nitrogen. researchers at the university of hawaii have used nitrogen with dry packs in plastic bags available from herbarium supply companies for ethnobotanical specimens (will mcclatchey, personal communication 2005). however, this kind of bag is not equipped with a valve to facilitate removal of oxygen and is made of polyethylene (www.herbariumsupply.com) polyethylene can be made to have oxygen barrier properties but this requires additional chemicals. oxygen barrier properties were not attributed to the bags specified on the herbarium supply website. preservation equipment® company (2006), in the uk, sells heavier specimen preservation bags than those from herbarium supply company, and also roles of film made from the same material, specified as containing nylon. unfortunately the uk bags require special sealing machines, that cost between £1,295.00 to £1,795.00 in 2004, and their bags still lack valves for air removal. spacebags® cost less to buy and use, have simple to use seals and valves which allow easy access and extraction of displaced oxygen from the top with nothing more expensive than a vacuum cleaner, making them overall more adaptable for herbarium insect control. they also only take up much space when actually in use and there are no expensive costs for installation or removal as for freezers. further, maintenance costs for nitrogen tanks are lower than monthly electric bills for freezers processing comparable numbers of specimens, and power outages are less likely to affect treatment with nitrogen. replacement costs for the bags are much lower than maintenance costs for freezers, and bags take up less space than freezers a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) in landfills, even when replacements are considered. there are concerns that nitrogen might cause brittleness in specimens over long periods (will mcclatchey, personal communication 2005). mcclatchey pointed out that dry nitrogen from tanks may be preferable to the sometimes wet nitrogen made with portable units. these issues require further scrutiny for long-term storage. there may also be residual oxygen present in our system, either due to incomplete removal, or re-contamination in the moment when the tubing to deliver the nitrogen is pulled from the bags, prior to completion of sealing the opening. only testing with insects or oxygen detectors will clarify this fully. however it is clear from our study that as bags were deflated and re-inflated, air was successfully removed and nitrogen successfully deposited into and held within the bags. the method described here may also prove practical for protecting other insect susceptible materials such as woolen clothing, replacing chemicals and high maintenance low temperature storage in regions where storage bags alone do not provide sufficient protection. the method described was practical for maintaining enough material to fill a single standard herbarium cabinet. it may prove less practical for large national collections as more effort is needed to organize material to fit into the bags and to fill them and release the nitrogen, than to move a box into or out of a freezer. this method may still prove useful at larger institutions for maintenance of a small number of specimens away from the main herbarium, such as in a lab or office in a distant building. the method also makes a good back up at times when need exceeds freezer space or during a break down or maintenance period for freezers. if biologists interested in conservation do not find economically realistic, environmentally safe or at the very least safer methods to conduct research, the result will be a weakening of their disciplines. the impetus and authority for biologists to make the case for using environmentally cleaner methods to other segments of society must be prescient to the investment of time and money in finding less harmful substitutes. at the very least the method described here allows institutions to accommodate naphthalene sensitive or concerned students and provides a simple and relatively inexpensive way for small herbaria to comply with epa recommendations concerning naphthalene in the work place. acknowledgements thanks are due to the curatorial staffs at missouri botanical garden, the united states national herbarium, field museum herbarium, fort lee regional archaeological curation facility and to the office of disability support services at southern illinois university for advice and encouragement. thanks for cooperation are due to dr. stephen ebbs and the southern illinois university herbarium. dr brian klubek is thanked for his help. this article is dedicated to the memory of dr. donald ugent, a true gentleman and scholar. references a.d.a.m., inc. (2012); (eds.) d. zieve, and d.r. eltz. boric acid poisoning. u.s. national library of medicine website. nih http://www.nlm.nih. gov/medlineplus/ency/article/002485.htm downloaded mar 11 2013. bridson d and l forman (1989) the herbarium handbook. revised edition, 303 pp. royal botanic gardens, kew, uk. bridson d and l forman (1998) the herbarium handbook. 3rd edition, 334 pp. royal botanic gardens, kew, uk. clarkson tw (2002) the three modern faces of mercury. environ. health perspect. 110: suppl. 1:11-23. fort lee regional archeological curation facility (2006) personal communication with curators jason j. and carol l. anderson website www.lee.army.mil/dpw/curation.htm. downloaded 2006. harper, b. j.a. gervais, k. buhl, and d. stone (2012). boric acid technical fact sheet. national pesticide information center, oregon state university extension services http://npic.orst.edu/factsheets/borictech.pdf downloaded mar 11 2013. herbarium supply company (2006, 2013) website http://www.herbariumsupply.com/ downloaded jan 17 2006, and feb 5 2012. holton mk, lindroth rl, and nordheim (2003) foliar quality influence tree-herbivore-parasitoid interactions: effects of elevated co2, o3, and plant genotype. oecologia 137:233-244. hong kong special administrative region of the people’s republic of china (2006, 2012) website http://www.lcsd.gov.hk/ce/museum/conservation/eng/speciality/ organics.htm. downloaded jan 21 2006, and feb 5 2012. hope, b. 2011. diatomaceous earth. http://www.deq.state.or.us/er/ docs/lowerbridge/diatomaceousearthfactsheet.pdf deq (department of environmental quality state of oregon). downloaded mar 11 2013. hoversten p. (2000) 30 years later, moon rocks retain their secrets. http://www.space.com/scienceastronomy/solarsystem/moon_rock_ analysis_000522_mb_.html downloaded may 5 2004. jessup wc. (2005) integrated pest management a selected bibliography for collections care. http://palimpsest.stanford.edu/byauth/ jessup/ ipm.html downloaded may 8 2005. morin p jr, mcmullen dc, and kb storey (2005) hif-1alpha involvement in low temperature and anoxia survival by a freeze tolerant insect. mol cell biochem 280 (1-2): 99-106. pedlar c, whyte g, emegbo s, stanley n, hindmarch i, and r godfrey (2005) acute sleep responses in a normobaric hypoxic tent. med sci sports exerc 37: 1075-1079. preservation equipment company (2006) website https://www2.preservationequipment.co.uk/. downloaded jan 17, 2006. preuss r, j angerer, and h drexler (2003) naphthalene--an environmental and occupational toxicant. int. arch. occup. environ. health. 76 (8): 556-576. raven, p.h., r.f. evert, s.e. eichhorn (2005). biology of plants 7th edition. w.h. freeman and co., ny, ny, pp. 817. space bag® company (2004) website https://www.spacebag.com/ spacebag downloaded 2004. us environmental protection agency (1997) mercury report to congress office of air quality and standards. washington dc: us environmental protection agency. us environmental protection agency (1999) integrated risk information system (iris) on naphthalene. national center for environmental assessment, office of research and development, washington, dc. us environmental protection agency (2004) technology transfer network air toxics website 91-20-3 naphthalene http://www.epa. gov/ttn/atw/hlthef/naphthal.html. downloaded 2004. watanabe t and m morita (1998) asphyxia due to oxygen deficiency by gaseous substances. forensic sci int 96 (1): 47-59. ziff s and mf ziff (2001) dentistry without mercury. bio-probe, inc. florida, pg. 96. zycherman l and jr schrock (1988) a guide to museum pest control. the foundation of the american institute for conservation of historic and artistic works and the association of systematics collections washington, dc. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 147 atlas journal of biology 2 (2): 100–115, 2012 doi: 10.5147/ajb.2012.0092 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a mutated yeast strain with enhanced ethanol production efficiency and stress tolerance naghmeh hemmati1*, david a. lightfoot1,2, and ahmed fakhoury3 1 department of plant soil and agricultural systems, southern illinois university at carbondale, carbondale, il 62901-4415, usa; 2 department of biochemistry and molecular biology, southern illinois university at carbondale, carbondale, il 62901usa; 3 department of plant soil and agricultural systems, southern illinois university at carbondale, carbondale, il 62901 usa received: july 15, 2011 / accepted: may 6, 2012 __________________________________________________ * corresponding author: naghmeh@siu.edu 100 abstract one of the strategies to improve and optimize bio-ethanol production from new feed stocks is to develop new strains of saccharomyces cerevisiae with tolerance to stresses. the main objectives here were to; generate s. cerevisiae mutants tolerant to high ethanol concentrations; test for their ability to ferment maize starch; and partially characterize the mutations responsible for the new phenotypes. a combination of mutagenesis, selection and cross-stress protection methods were used. ems (ethyl methanesulfonate) was used to mutagenize one s. cerevisiae strain. the mutagenized yeast strain was exposed to high concentrations of ethanol and tolerant mutants were isolated. mutants showed improved ethanol yield (0.02-0.03 g/g of maize) and fermentation efficiency (3-5%). finally, aflp (amplified fragment length polymorphism) was performed to identify polymorphisms in the mutants that might underlie the strains ethanol tolerance. the best performing mutant isolate had four altered gene transcripts encoding; an arginine uptake and canavanine resistance protein (can1); mitochondrial membrane proteins (sls1); a putative membrane glycoprotein (vth1); and cytochrome c oxidase (cox6; ec 1.9.3.1) among about 1,000 tested. it was concluded these mutations might underlie the improved ethanol production efficiency and stress tolerance. keywords: aflp, mutagenesis, polymorphism, snps, ethanol tolerance, can1, sls1, vth1, cox6, ethanol yield, fermentation efficiency. introduction modern civilization has been greatly dependent on the oxidation of fossil fuel reserves over the past 150 years for energy production (hambourger et al., 2009; stephenson et al., 2011). fossil fuel reserves are limited and their current oxidation rate is a major global and environmental concern, with complex and severe impacts on the climate of the planet (decc, 2009; stephenson et al., 2011). several factors have contributed to a revival of interest in bio-fuels, both in the united states and worldwide (bothast et al., 1999; jeffries and jin, 2004; fargione et al., 2008). these include; a steep increase in the price of crude oil and crude oilbased products, an increase in the awareness of the detrimental effects of burning fossil fuels, release of greenhouse gases; a renewed political awareness of the need for decreasing the reliance of the economy on foreign oil and the availability of land set aside from food crop production ideal for biofuel crops (bothast et al., 1999; jeffries and jin, 2004). saccharomyces cerevisiae has been broadly used for fuel ethanol production due to its ability to produce high concentration of ethanol from simple sugars. ethanol counts as a toxin for yeast cells and tolerance to it is closely related to ethanol productivity which is a major factor in industrial ethanol production (jones, 1989). improving and increasing understanding of the impact of ethanol toxicity on yeast cells will assist enhancing yeast ethanol tolerance and higher ethanol production (demain, 2009; stanley, 2010). earlier studies showed that the acquired tolerance to formerly lethal stress levels has been linked to the activation of specific a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 101 stress response mechanisms during pre-exposure to the sub-lethal stress (plesset et al., 1982; sanchez and lindquist, 1990; coote et al., 1991). the effects of pre-exposure to stress conditions have been studied in ethanol stress (vriesekoop and pamment, 2005) and other stress conditions such as osmotic (trollmo et al., 1988; varela et al., 1992) and oxidative stresses (davies et al., 1995). stanley et al. (2010) proposed that pretreated yeast cultures showed a 70% reduction in the stress adaptation period when exposed to higher ethanol concentrations. therefore, the isolation of stress resistant strains should include a pretreatment phase. however, improving the efficiency of ethanol production in engineered strains is not trivial. selection for small scale batch culture growth and the numbers of background mutations are the major issues that have been hard to overcome. the main objectives of the research reported here were to; generate s. cerevisiae mutants with improved toleranance to high concentrations of ethanol; assess the ability of the mutants to produce bio-ethanol in larger scale cultures; and to partially characterize the mutations. materials and methods strains the yeast strain (ncyc-1681 brewing strain) that was developed for fermentation was chosen for this experiment. the strain was; round-oval in colony shape; cream in color; had a shiny surface; and showed a smooth texture on agar. cell viability was retained during fermentation to final ethanol concentrations of 15% (v/v). the strain was obtained from the national collection of yeast cultures, institute of food research, norwich research park, norwich, united kingdom, nr4, 7ua. media the complete growth medium for s. cerevisiae consisted of ypd liquid and solid medium (ypd broth, fisher). yeast storage stock solutions were prepared by adding 15% (v/v) glycerol to complete growth medium of yeast after 24 hours and were stored at -80°c. mutagenesis and screening for survival rate a modified version of burke et al. (2000) was used. briefly, the cells were incubated at room temperature in 200 μl sterilized distilled water containing 8% (w/v) ems for 5-20 min with constant vortexing. the optimum lethal dose (ld) of each ems concentration was calculated. the aim was to identify 70% to 80% lethality and was obtained by plating and incubating an appropriate dilution of cells at 30°c overnight. the growing colonies were isolated after 24 hours. selection of ethanol-tolerant mutants previous researchers have demonstrated that pre-exposure of yeast to a sub-lethal amount of stressing agent such as ethanol or heat can stimulate molecular responses resulting in resistance to higher levels of the same stress condition compared to control cells, cells without pre-exposure (plesset et al., 1982; sanchez and lindquist, 1990; coote et al., 1991). in the current research study, pre-treatment to stress condition (ethanol stress) and selection were performed after the mutagenesis step. briefly, about 0.1 ml of the cell suspension (2 × 108 cell/ml) was added to 20 ml ypd liquid and cultivated at 30°c overnight. the cells were exposed to gradual additions of absolute ethanol (99.9% with 0.1% (v/v) methanol) to cultures. concentrations were raised from 5% to 35%-40% (v/v) within 5 days (additions were 5% ethanol for the first day; 5% for the second day; 10% for the third day, 10% for the fourth day; and 5%10% for the fifth day). positive controls (with 0% ethanol) were cultured under identical conditions. after the fifth day the cells were pelleted, washed and suspended in 2 ml sterilized distilled water. the resulting suspensions were plated on ypd-agar and cultured at 30°c overnight for mutant selection. this procedure was repeated more than 20 times and each time there were 3 replications. the growing colonies showed that the mutant strain was able to tolerate ethanol up to 40% (v/v). testing fermentation abilities the mutants (two mutants) and the parent strain were sent to national corn to ethanol research center (ncerc) in edwardsville, il to test for their fermentation ability (ncerc, 2007). the process was divided into three steps; preparation of yeast strains, liquefaction of starch substrate and fermentation. the ethanol yields and fermentation efficiencies for yeast strains (one control and two mutants) were calculated from the ethanol concentrations measured by hplc after 64.5 hours of fermentation. the statistical significance of the differences between samples was evaluated using analysis of variance (anova). dna extraction genomic dna was obtained following chung, (1996). dna quality was assessed by spectrophotometer (spectronic, madison, wi) by calculating the a260/a280 nm ratios and the a260 nm values were used to determine dna concentrations. the dna was stored at -20°c for the subsequent experiments. aflp (amplified fragment length polymorphism) and transformation aflp analysis was carried out using the aflp microorganism primer kit (invitrogen, carlsbad, ca). polyacrylamide gel electrophoresis (page) was divided to five major steps; preparation of glass plates; assembling and pouring the gel; electrophoresis for 2 h at 200v; fixing and silver staining gels; and extracting the bands with altered intensities in mutants or control strains from the gel. pcr was performed using band dna as a template with the appropriate selective aflp primers. amplified bands were separated and purified by agarose gel electrophoresis. qiaquick gel extraction kit (qiagen, hilden, germany) was used to extract the bands from the agarose gel. pgem®-t or pgem®-t a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 102 easy vectors kit from promega (madison, wi) was used for ligation. transformations were made into e.coli as follows. the tubes containing the ligation reactions were centrifuged to collect contents at the bottom of the tubes. just 1μl of the ligation reaction was added to 10 μl of thawed jm109 high efficiency competent cellstm. the reaction was mixed gently. the tubes were placed on ice for 20 minutes. the cells were placed on a heat plate for 45–50 seconds at exactly 42°c and immediately after that tubes were returned to ice for 2 minutes. about 900 μl of lb medium was added to each tube and was incubated at 37°c for 1.5 hours. about 100 μl of each transformation reaction was plated on lb agar (0.625% (w/v)) with 100 μg/ ml ampicillin, 100 μg/ml x-gal and iptg (80 μg/ml). the plates were incubated at 37°c overnight. white and blue colonies were identified after incubation. plasmid dna purification and sequencing two or three white colonies from each sample were cultured in separate tubes containing 5 ml lb medium and incubated in a shaker and incubator (150 rpm) at 37 °c overnight. wizard® plus sv minipreps dna purification system kit (promega) was used for isolation and purification of plasmid dna. a pcr reaction was performed on the plasmid dna and dna quality was assessed and the size of each fragment was estimated after agarose gel electrophoresis. dna sequencing was carried out at the iowa state university dna facility. the dna sequences were analyzed by vecscreen (vector screen), blast (basic local alignment search tool) at ncbi (national center for biotechnology information) and sgd (saccharomyces genome database). snps (single nucleotide polymorphisms) in the sequences were recognized and recorded. results and discussion fermentation two mutants were isolated that could grow when ethanol concentrations were 35-40% (v/v) in media. in contrast the parent strain did not grow at concentrations above 15 %. medium scale fermentation data indicated mutants had better ethanol yields (figure 1) and fermentation efficiency than the parent strain (figure 2). polymorphic aflp band isolation overall, 10 bands were identified polymorphic from the primer pairs tested (figure 3). the primer pairs generated about 10 discernible bands. of those, 8 were dominant bands in the mutant. they were labeled as m1 to m8. only two dominant polymorphic bands were found in control and absent from the mutant. they were labeled as c1-c2. the estimated size of the dna fragments that showed polymorphism varied from 250 to 495 bp. from the 10 dna fragments identified, 8 (c2, m2, m3, m4, m5, m6, m7 and m8) were successfully isolated. after transformation, minipreps and pcr steps, 6 dna fragments were successfully figure 1. ethanol yield for all yeast strains that were tested, the parental control and the two mutants. the error bars represent one standard deviation of three independent replicate fermentations. the ethanol yields were calculated for each yeast strain from the ethanol concentrations measured by hplc after 64.5 hours of fermentation. the statistical significance of the differences between hybrids was evaluated using analysis of variance (anova). statistically significant differences were detected among the ethanol yields for the yeast strains that were tested (p = 1.79 x 10-5), where p is the probability that the ethanol yield for all yeast strains is the same. strains with same letter were not statistically significantly different from each other. figure 2. fermentation efficiency for all yeast strains that were tested, the parental control and the two mutants. the error bars represent one standard deviation of three independent replicate fermentations. the fermentation efficiency was calculated for each yeast strain from the ethanol concentrations measured by hplc after 64.5 hours of fermentation. the statistical significance of the differences between hybrids was evaluated using analysis of variance (anova). statistically significant differences were detected among the ethanol yields for the yeast strains that were tested (p = 1.79 x 10-5), where p is the probability that the ethanol yield for all yeast strains is the same. strains with same letter were not statistically significantly different from each other. isolated and dna sequence analyzed (c2 464 bp; m2 468 bp; m3 450 bp; m4 446 bp; m5 457 bp and m7 327 bp). gene identification blast searches indicated that three fragments, c2, m4, and m5, showed high similarity (c2 98%; m4 98%; m5 99%) to the can1 gene,yel063c (supplementary figure 1, section a). can1 gene family members encode membrane proteins with about 590 amino acids that function as arginine permeases (sgd # can1/yel063c ). the can1 gene is required for arginine uptake by yeast cells and canavanine resistance by selective exclusion (rak et al., 2007). two snps were found between the mutant and the wild type allele in the alignment between c2 and can1 gene (table 1; supplementary figure 1, section c). both snps, were in coding regions (supplementary figure 1, section b). one of the snps changed the amino acid sequence and produced nonsynonymous amino acid but the other one did not change the amino acid sequence and produced synonymous change in amino acid sequence (annotation table). one snp was found between the mutant and the wild type allele in the alignment between m4 insert and the expected can1 gene sequence (supplementary figure 1, section e). the only snp was in coding regions (supplementary figure 1, section d). this snp did not change the amino acid sequence and produced synonymous amino acid (annotation table). two snps were found between the mutant and the wild type allele in the alignment between m5 insert and the expected can1 gene sequence (supplementary figure 1, section g). both snps, were in coding regions (supplementary figure 1, section f). one of the snps changed the amino acid sequence and produced nonsynonymous amino acid but the other one did not change the amino acid sequence and produced synonymous change in amino acid sequence (annotation table). the transportation systems for many amino acids are proton symports that an amino acid molecule enters the cell along with a proton (h+). the h+ ion entry to the cell reduces cellular ph. the cell preventive reaction from acidification of cytoplasm, maintaining cell ph and secondary transport mechanisms, is exporting the proton into the outside medium by a membrane bound atpase that acts as a transporter which actively pumps hydrogen ions. during the fermentation for ethanol production, amino acid transport in yeast cells was strongly inhibited by the significant amounts of ethanol. the reason that yeast cells prevent the uptake of amino acids along with protons would be related to stopping too many protons from entering the cell (dharmadhikari, 2007, aguilera et al., 2006, monteiro and sa´correia, 1998; ogawa et al., 2000; rosa and sa´-correia, 1991). many research studies presented that membrane structure and function are the predominant target of ethanol stress. exposure of yeast to ethanol results in increased membrane fluidity and consequential decrease in membrane integrity (mishra and prasad, 1989). teixeira et al. (2009) demonstrated a detailed physiological and molecular study that illustrates changes in plasma membrane organization and function of yeast cells as a response to ethanol stress. the results agreed with the previous researches (meaden et al., 1999; rosa and sa´-correia, 1991; salgueiro et al., 1988). the desired phenotype, tolerance to high concentration of ethanol, might be related to mutations that have been observed in can1 gene. the polymorphism observed in the can1 gene may have resulted in molecular modifications (via ems mutagenesis and pre-treatment to stress condition) that improved the uptake of arginine (amino acid) in the presence of high concentration of ethanol. the higher tolerance to ethanol in mutants might be related to improved amino acid up-take during fermentation. m2 fragment showed high similarity (99%) to the sls1 gene. two snps were found between the mutant and the wild type allele in the alignment between the m2 insert and sls1 gene sequence (table 1; supplementary figure 2, section c). both snps were in coding regions (supplementary figure 2, section b). both snps did not change amino acid sequence and resulted in synonymous amino acid (annotation table). the sls1 gene is located on crick strand of chromosome xii and encoded for mitochondrial membrane proteins with 643 amino acids, (supplementary figure 2, section a). sls1 gene is one of the required factors for assembly of respiratory-chain enzyme complexes. it also coordinates in expression of mitochondria-encoded genes and delivery of mrna to membrane-bound translation machinery (sgd, 2008). the finding here is further evidence that the major targets of ethanol are membrane structure and function as described by earlier researches (mishra and prasad, 1989; teixeira et al., 2009). benitez and codon (2003) also indicated that the destructive effect sites of ethanol activity in yeast are the plasma membrane, hydrophobic proteins of the cell, mitochondrial membranes, nuclear membrane, vacuolar membrane, endoplasmic reticulum, and hydrophobic proteins in the cytoplasm. previous research showed respiratory deficient (rd) mutants had lower growth rate, fermentation rate and respiration rate than aerobically growing wild strain of yeast. rd mutants were anaerobically grown cells which did not have active mitochondria and oxidative metabolism. the results illustrated the functional mitochondria was necessary for a greater tolerance to ethanol in yeast cells. it was suggested that the fac103 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. list of geneic non-synonymous mutations detected by dna sequence of bands showing polymorphisms by aflp of genomic dna (figure 3). gene name gene id segment snp amino acid can1 856646 c2 c453t a117a can1 856646 c2 t627g s176a can1 856646 m4 , m5 g566t v155v can1 856646 m5 t637a i178n sls1 850830 m2 g645a k98k sls1 850830 m2 g675c l108l vth1 854634 m3 insertion361ata a102d,t vth1 854634 m3 t369a n104k vth1 854634 m3 t372c y105y vth1 854634 m3 t374a f106y vth1 854634 m3 a445g n130d cox6 856448 m7 t732c a94a tor responsible for the improvement in ethanol tolerance was not respiratory metabolism itself but majorly was more about the differences in cellular components, membrane lipids and the physiology derived the ability of yeast cell to respire (aguilera and benitez, 1985; van uden, 1989). teixeira et al (2009) indicated that the 30 common mitochondrial genes were also yeast resistant genes to high concentrations of ethanol, suggesting mitochondrial functions were essential for ethanol tolerance even in the presence of glucose. these results were also reconfirmed the previous findings that ethanol tolerance depends on the stability of the mitochondrial genome (jimenez and benitez, 1988). the desired phenotype, tolerance to high concentration of ethanol, might be related to mutations that have been observed in sls1 gene. the polymorphism observed in the sls1 gene may have resulted in molecular modifications (via ems mutagenesis and pre-treatment to stress condition) that improved cellular components, membrane lipid compositions and modified membrane fluidity which are required for higher ethanol tolerance during anaerobic fermentation (aguilera and benitez, 1985; van uden, 1989; teixeira et al., 2009). the m3 sequence showed high similarity (96%) to the vth1 gene, yil173w. four mutations and indel, three nucleotide insertions, were detected between the mutant and the wild type allele in the alignment between the m3 fragment and the vth1gene sequence (table 1; supplementary figure 3, section c). all four snps and indel, three nucleotide insertions, were in coding region (supplementary figure 3, section b). three of four snps changed the amino acid sequence and produced nonsynonymous amino acid. one of them did not change the amino acid sequence and produces a synonymous amino acid. the three nucleotide insertions, indel, changed the amino sequence and produced two nonsynonymous amino acids (annotation table ). the vth1 gene is located on watson strand of chromosome ix (supplementary figure 3, section a). it encodes for putative membrane glycoprotein with 1,549 amino acids. and it may function in vacuolar protein sorting (sgd, 2008). ethanol can disrupt the vacuolar membrane and release the proteases into cytoplasm which results in inactivation of intracellular enzymes (van uden, 1989). teixeira et al. (2009) demonstrated that many of the genes required for ethanol tolerance in yeast are related to intracellular trafficking, including vacuolar protein targeting, endosome transport, and transport mediated by the endosomal sorting complexes. they described that the target point to overcome stress imposed by lipophilic agents such as ethanol, are the membrane transporters. ethanol stress alters vacuole morphology from segregated structures to a single, large organelle (meaden et al., 1999). chandler et al. (2004) found that gene expression profiles of ethanol stressed cells are different in the later stages of ethanol stress. and the major induced genes (yro2, ald4, arg4, lap4, pcl5, ssu1, ygl117w) at this stage are associated with energy utilization, general stress response and vacuole function. fujita et al. (2006) found hundred and thirty-seven mutants as ethanol sensitive mutant with a considerable number of vacuole function-related genes being necessary for growth in the presence of ethanol stress. the outcomes of different research studies of genome104 wide screens for ethanol tolerance commonly agree on genes associated with vacuole function and amino acid biosynthesis as important factors for ethanol tolerance (stanley et al., 2010). the desired phenotype, tolerance to high concentration of ethanol, might be related to mutations that have been observed in vth1 gene. the polymorphism observed in the vth1 gene may have resulted in molecular modification (via ems mutagenesis and pre-treatment to stress condition) that had positive effects on vacuole morphology, alteration in vacuole-based functions such as maintaining intracellular ph and ion homoeostasis, vacuole protein sorting and transportation in the cell (stanley et al. 2010). the m7 sequence showed high similarity (99%) to cox6 gene (yhr051w). one mutation was observed in cox6 gene. the snp was found between the mutant gene and the wild type allele in the alignment between the m7 fragment and the cox6 gene sequence (supplementary figure 4, section c). snp was in coding region (table 1; supplementary figure 4, section b). the snp did not change the amino acid sequence and produced synonymous amino acid (annotation table). the cox6 gene is located on watson strand of chromosome viii and encodes for subunit iv of cytochrome c oxidase (ec 1.9.3.1; supplementary figure 4 section a). the protein contained 148 amino acids and is the terminal member of the mitochondrial inner membrane electron transport chain and its expression is regulated by oxygen concentrations (sgd, 2008). mitochondrion, the organelle that is found in all eukaryotic cells is required for cellular resa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 3. aflp fingerprints of genomic dna between mutant 1 (lane 1) and the wild type strain (lane2). amplification products are shown from primers ecori/ac and msei/g. there was 20 μg dna per lane. the 100 bp promega dna ladder was used as a size standard (lane 3). arrowed are 10 polymorphic bands detected and eluted from this gel for dna sequencing. piration. the additional mitochondria rolls include participation in the biosynthesis of organic acids, amino acids, and phospholipids, the degradation of fatty acids and the storage of metal ions (scheffler, 1999). majority of the yeast genes encoding mitochondrial proteins are subject to glucose repression (derisi et al., 1997), and repression under hypoxic conditions (ter linde and yde, 2002) which fermentation of alcoholic beverages is a good example because the concentration of glucose is high while the concentration of oxygen is low. several studies suggested the presence of mitochondria during the fermentation of alcoholic beverages. for example, data from a genome-wide studies indicated that genes encoding mitochondrial proteins were at higher rate of expression during the brewing of sake (japanese rice wine), one of the alcoholic beverages (wu et al., 2006; kitagaki and shirnoi, 2007). teixeira et al. (2009) indicated a high proportion of the mitochondrionrelated genes were also required for ethanol stress resistance. they were mostly involved in mitochondrial protein synthesis, respiration, and mitochondrial dna maintenance. scheffler, (1999) and kitagaki and shirnoi, (2007) pointed out that it is biologically reasonable for the presence of mitochondria during alcohol fermentation. the reason is that yeast cells require to rapidly transfer from anaerobic to aerobic metabolism even in anaerobic conditions. for example when glucose is depleted and oxygen becomes available yeast cells use mitochondria to utilize the produced alcohol. various studies commonly agree that the first obstacle that yeast cells under ethanol stress initially struggle with is maintaining energy production which leads to an increment in expression of genes associated with energy-generating activities such as glycolysis and mitochondrial function and decrease in expression rates of many genes associated with energy demanding processes, such as growth (stanley et al., 2010). the desired phenotype, tolerance to high concentration of ethanol, might be related to mutations that have been observed in cox6 gene. the polymorphism observed in the cox6 gene may have resulted in molecular modifications (via ems mutagenesis and pre-treatment to stress condition) that improved the energy-generating activities, protein synthesis, respiration, and mitochondrial dna maintenance in mutants (stanley et al., 2010). conclusions from the molecular point of view, the polymorphisms that resulted from ems mutagenesis might have stimulated the activation of specific molecular stress response mechanisms in mentioned genes which resulted in higher levels of resistance to stress conditions, high concentration of ethanol. interestingly mutant showed better growth than the wild type under the lower available carbon stress conditions caused by media derived from cellulosic biomass (hemmati, lightfoot and anderson, unpublished). in future genome sequencing followed by gene knockouts and/or complementing the mutation(s) might identify the significant polymorphisms underlying the altered growth during ethanol stress. from the fermentation point of view and future investigation of kinetics of ethanol production, it would be very valuable to evaluate the mutants’ activity under the enhanced enzymatic fermentation provided by thomas (2009). it might provide more insight to stress tolerance among mutants. acknowledgements the authors would like to express the sincere appreciation to dr. david clark for his contribution, valuable suggestions and constructive advices throughout the research project. dr. navinder saini is thanked for introducing silver staining to dr. lightfoot’s team at siuc (southern illinois university carbondale) genomic research laboratory. grant funding was from cfar. references aguilera a and t benitez (1985) role of mitochondria in ethanol tolerance of saccharomyces cerevisiae. 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he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 107 supplementary figure 1. section b: can1 gene coding region is marked by start codon atg and stop codon tag in larger and bold fonts. the region that c2 segment matches with can1 gene is highlighted in gray. section c: the gray region from section a is aligned with c2 segment and polymorphisms are marked by larger and bold fonts. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 108 supplementary figure 1. section d: can1 gene coding region is marked by start codon atg and stop codon tag in larger and bold fonts. the region that m4 segment matches with can1 is highlighted in gray. section e: the gray region from section a is aligned with m4 segment and polymorphisms are marked by larger and bold fonts. supplementary figure 1, section c: the gray region from section a is aligned with c2 segment and polymorphisms are marked by larger and bold fonts. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 109 supplementary figure 1. section e: the gray region from section a is aligned with m4 segment and polymorphisms are marked by larger and bold fonts. supplementary figure 1. section f: can1 gene coding region is marked by start codon atg and stop codon tag in larger and bold font. the region that m5 segment matches with can1 is highlighted in gray. supplementary figure 1. section g: the gray region from section a is aligned with m5 segment and polymorphisms are marked by larger and bold font. supplementary figure 2, section a: ideogram of the sls1 gene on chromosome xii and a snapshot of predicted structural information about the protein/cdna sequence. a legend on the right-hand side of the image indicates what data that section of the image is displaying. ss: secondary structure, d: disordered regions, tm: transmembrane regions, cc: coiled coil regions and sp: signal peptide. cited from saccharomyces genome database, sgd, and yeast resource center, yrc. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 110 supplementary figure 2. section c: the gray region from section a is aligned with m2 segment and polymorphisms are marked by larger and bold fonts. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 111 supplementary figure 2, section b: sls1 gene coding region is marked by start codon atg and stop codon taa in larger and bold fonts. the region that m2 segment matches with sls1 is highlighted in gray. supplementary figure 3, section a: ideogram of the vth1 gene on chromosome ix and a snapshot of predicted structural information about the protein/cdna sequence. a legend on the right-hand side of the image indicates what data that section of the image is displaying. ss: secondary structure, d: disordered regions, tm: transmembrane regions, cc: coiled coil regions and sp: signal peptide. cited from saccharomyces genome database, sgd, and yeast resource center, yrc. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 112 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 113 supplementary figure 3. section b: vth1 gene coding region is marked by start codon atg in larger and bold fonts. the region that m3 segment matches with vth1 is highlighted in gray. supplementary figure 3. section c: the gray region from section a is aligned with m3 segment and polymorphisms are marked by larger and bold fonts. supplementary figure 4. section a: ideogram of the cox6 gene on chromosome viii and a snapshot of predicted structural information about the protein/cdna sequence. the approximate locations of point mutations, base substitutions, are marked by pink arrows. a legend on the right-hand side of the image indicates what data that section of the image is displaying. ss: secondary structure, d: disordered regions, tm: transmembrane regions, cc: coiled coil regions and sp: signal peptide. cited from saccharomyces genome database, sgd, and yeast resource center, yrc. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 114 supplementary figure 4. section b: cox6 gene coding region is marked by start codon atg and taa in larger and bold fonts. the region that m7 segment matches with cox6 is highlighted in gray. section c: the gray region from section a is aligned with m7 segment and polymorphisms are marked by larger and bold fonts. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 115 supplementary figure 4. section c: the gray region from section a is aligned with m7 segment and polymorphisms are marked by larger and bold fonts. atlas journal of biology 2016, pp. 295–307 doi: 10.5147/ajb.2016.0143 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) evolutionarily degenerate biological structures: terminology through time, and the question of terminological consensus philip j. senter department of biological sciences, fayetteville state university, 1200 murchison road, fayetteville, nc 28301, usa. received: september 17, 2016 / accepted: november 20, 2016 __________________________________________________ * corresponding author: psenter@uncfsu.edu 295 abstract the existence of evolutionarily degenerate biological structures (edbs) is a major concept in biology. biologists have often used the terms “vestige,” “rudiment,” and their adjective forms “vestigial” and “rudimentary” for edbs since the nineteenth century. some authors have advocated stricter usage of the terms than others have. for example, some have advocated restriction of the term “rudimentary” to embryonic structures, whereas others have also applied it to postembryonic edbs. likewise, some have restricted the term “vestigial” to putatively functionless structures, whereas others have applied it to structures that retain some function. here, i sought to determine whether a consensus has been reached in the usage of such terms for edbs. a sample of 200 articles in primary scientific literature from the twentieth and twentyfirst centuries shows that through both centuries it has been more common to call edbs “rudimentary” than to restrict the term “rudimentary” to embryonic structures, and it has been more common to attribute function or possible function to structures called “vestigial” than to restrict the term “vestigial” to putatively functionless structures. the consensus in both centuries has been less-strict usage of such terms; such usage is, in several ways, more logical than strict usage. keywords: vestigial structures, rudimentary structures, evolution, scientific terminology. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction biologists have long recognized the existence of biological structures that have become drastically reduced and/or have lost salient functions during the course of evolution (hereafter called evolutionarily degenerate biological structures or edbs). through the last three centuries, different authors have preferred different terms for such structures, but various forms of the terms “vestige” and “rudiment” have been the most popular. some authors have suggested strict definitions for such terms (brues, 1903; lull, 1920; hall, 2003), while others ignore strict definitions and use the terms more loosely (e.g. miralles et al., 2012; nweeia et al., 2012; woon and stringer, 2012). this study was undertaken to determine whether a consensus has developed in primary scientific literature of the twentieth and twenty-first centuries as to the proper level of strictness for terms for edbs. such terms are often spelled the same in english as they are in other languages. below, therefore, for the sake of clarity, words in languages other than english are italicized. biologists used the noun “vestige,” its adjective form “vestigial,” and their cognates (hereafter collectively called v-terms), as well as the noun “rudiment,” its adjective form “rudimentary,” and their cognates (hereafter collectively called r-terms) for diminutive biological structures, even before such structures were widely recognized as evolutionary degenerate. in a frenchlanguage article, saint-hilaire (1798) called the tiny clavicles of the ostrich rudiments and called the flightless wings of the cassowary vestiges. cuvier (1799) used the french terms rudiment and vestige for numerous diminutive animal organs in a treatise on animal anatomy. both authors used the terms rudiments a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) and vestiges synonymously and without implying evolutionary change. several nineteenth-century biologists also used similar terms without evolutionary implications, implying only that the structures were relatively tiny in comparison to their homologs in related taxa. examples include the use of the german words spur (equivalent to “vestige”) and rudiment by mayer (1825) for parts of the diminutive hindlimb skeletons of snakes and reduced-limbed lizards; the use of the danish word rudiment by eschricht and reinhardt (1861) for the pelves and hindlimbs of whales; and the use of r-terms by strothers (1881) for whale hindlimbs. the earliest published reference to apparently-degenerate structures as evidence of biological evolution is in erasmus darwin’s 1791 book botanic garden. darwin noted “apparently useless or incomplete appendages to plants and animals, which seem to shew they have gradually undergone changes from their original state” (p. 8). as examples he listed stamens without anthers, styles without stigmas, the halteres of flies, the side toes of pigs, and nipples on male mammals. he used no v-term or r-term for such structures. in his french-language book philosophie zoologique, lamarck (1809) was the earliest author to use such a term to express the opinion that such structures were the evolutionary remnants of more fully-expressed structures in the organisms’ ancestors. lamarck called the blind eyes of mole rats and olms vestiges. he used these and other examples to argue that biological evolution occurs and that an organ that is useless in an animal’s environment degenerates through the generations. robert chambers’ (1844) vestiges of the natural history of creation also cited such structures as evidence for biological evolution. despite the book’s title, chambers used r-terms for such structures. examples that he cited include ostrich wings, snake hindlimbs, and the human coccyx. in the category “rudimentary organs” he also included structures that appear and then vanish in the embryo (e.g. teeth in baleen whales), and—echoing erasmus darwin—structures that are present in a sex for which they are useless (e.g. male nipples). he considered rudimentary organs to be degenerate forms of more fully-developed ancestral organs in some cases and precursors to more fully-developed descendant organs in other cases. lamarck and chambers explicitly cited fossil succession as evidence of biological evolution. richard owen subsequently did likewise, and to emphasize the point he cited specific examples of fossil species with morphology intermediate between older fossil species and modern species (owen, 1846, 1849). he used the term “rudiments” for diminutive biological structures with larger homologs in related species (owen, 1849, 1866), and implied that such diminutive structures are evolutionarily degenerate (owen, 1849). charles darwin (1860, 1871) largely followed chambers’ terminology. he used r-terms for putatively degenerate structures, for structures that are present in a sex for which they are useless, and for structures that appear and then vanish in the embryo. however, he coined the term “nascent organs” for precursors to more fully-developed descendant organs, rather than calling them “rudiments.” in the second half of the nineteenth century it became commonplace for biologists to explicitly opine that apparentlydegenerate structures were indeed derived from ancestrally more fully-expressed structures. for edbs führbinger (1870) used the german terms rudiment and spur, marsh (1879) used “rudiments” and “remnants,” cope (1864, 1892, 1894) and rothschild (1900) used r-terms, and some authors interchangeably used r-terms and v-terms (morgan, 1891; bernard, 1893; wiedersheim, 1895; wortmann, 1898). by the twentieth century the theory of biological evolution was accepted by most biologists, and the use of r-terms and v-terms for post-embryonic structures was understood to imply evolutionary degeneration. however, some authors insisted that r-terms be restricted to embryonic structures and not applied to edbs, to which only v-terms should be applied (brues, 1903; lull, 1920; hall, 2003). another call for terminological strictness came from biologists who included functionlessness in the definition of “vestigial,” implying or stating outright that v-terms ought not be used for a structure with a known function (bellairs, 1950; kinsky, 1971; scadding, 1981, 1982). such terminological strictness began in the twentieth century. in contrast, some eighteenth-century and early twentieth-century authors explicitly opined that an edbs could maintain a minor function even after having lost a major one (darwin, 1860, 1871; brues, 1903; waddington, 1937; stickel and stickel, 1946). in the primary scientific literature of the twenty-first century some authors advocate or employ terminological strictness of the two kinds mentioned above (hall, 2003; buckland-nicks et al., 2011), whereas others do not. because terminological consensus is important for communication, it would be useful to determine whether a consensus has developed regarding the degree of strictness in the use of terms for edbs. if a consensus has developed, then it would be advisable for future authors to use terminology as per the consensus so as to communicate with maximum effectiveness. methods i compiled a sample of 200 publications that mention edbs, 100 apiece from the twentieth and twenty-first centuries. the twenty-first century was therefore sampled more densely, which is appropriate because of its higher publication rate. for the search i used online search engines such as jstor (www.jstor. org), science direct (www.sciencedirect.com), and journal finder (library.uncfsu.edu/journal-finder), as well as the help of nine graduate students who were instructed in the use of those three search engines. students using journal finder were instructed to search within eight journals with frequent reference to edbs (am j bot, ann bot, evolution, evolution and development, journal of experimental biology. journal of morphology, j zool, and proc natl acad sci usa), which together provided 83 of the 200 articles. a publication was included in the sample only if it satisfied the following criteria: (1) it is an example of primary scientific literature (an article in a peer-reviewed journal), (2) it uses v-terms and/or r-terms in reference to edbs, (3) it is not written from an explicitly anti-evolution perspective. the third criterion was used because of potential conflict between the second crite296 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) rion and publications written from an anti-evolution perspective. once the sample of 200 articles was compiled, i examined the use of r-terms and v-terms to determine which articles were relevant to the question of prevailing level of strictness in the use of such terms for edbs. an article was deemed relevant to r-term strictness level if it included explicit or implicit advocacy of restriction of r-terms to embryonic structures (by outright insistence on such restriction or by unambiguously employing such strict usage), or if it took the opposite stance by applying r-terms to edbs. an article was deemed relevant to v-term strictness level if it included explicit or implied advocacy of restriction of vterms to putatively functionless structures (by outright insistence on such restriction or by characterizing specific edbs as functionless), or if it took the opposite stance by attributing a known or possible function to edbs to which v-terms were applied. results the results are detailed in table 1 and summarized in table 2. of the 200 articles, 64 were deemed relevant to strictness level for r-terms: 39 from the twentieth century and 25 from the twenty-first century. of these 64 articles, 59 (92%) applied r-terms to edbs, while five (8%) advocated or applied restriction of r-terms to embryonic structures. among the 39 relevant 297 table 1. usage of forms of the terms “vestige” and “rudiment” in reference to evolutionarily degenerate biological structures (edbs) in 200 twentiethand twenty-first century publications in primary scientific literature. ea = explicit advocacy of strict usage. f = function (known or possible) admitted for the edbs in question, or for edbs in general. l = edbs in general, or the edbs in question, characterized as functionless. r = form(s) of the term “rudiment” used for edbs. su = strict usage without explicit advocacy. v = form(s) of the term “vestige” used for edbs. publication term used for edbs level of strictness for r-terms level of strictness for v-terms brues, 1903 v ea f matthew, 1908 r, v bechtel, 1921 v osborn, 1921 r, v camp, 1923 r essex, 1927 v sewertzoff, 1931 r chubb, 1932 v dawson, 1936 v munro, 1937 v waddigton, 1937 r, v f fisher, 1940 v colbert, 1941 v pavan, 1945 r stickel and stickel, 1946 v f stokely, 1947a v stokely, 1947b r, v bellair,s 1950 r l bellairs and underwood, 1951 r, v f colbert and mook, 1951 v hosokawa, 1951 r knobloch, 1951 v l woods and inger, 1957 r boke, 1959 r, v satchell, 1959 v gans, 1960 v emerson, 1961 v l mlynarski and madej, 1961 r f stephenson, 1961 r, v neville, 1963 v radinsky, 1963 v f gasc,1966 v f list, 1966 r, v a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 298 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. continued. publication term used for edbs level of strictness for r-terms level of strictness for v-terms gasc, 1968 r frick and taylor, 1968 r, v mcdowell, 1969 v kinsky, 1971 r, v l patton and taylor, 1971 r, v heye,r 1972 v patton and taylor, 1973 r, v taylor and webb, 1976 r, v l tidemann, 1976 r greer, 1977 v l carpenter et al., 1978 v f lande, 1978 r, v f webb and taylor, 1980 r, v brygoo, 1981 v scadding, 1981 v l slobodchikoff and wismann, 1981 v naylor, 1982 v f scadding, 1982 v l wilson, 1982 r, v greer, 1985 r f greer and cogger, 1985 v land, 1985 v thomason, 1985 v van der merwe, 1985 v f boucher, 1986 v stephens, 1986 v brandoni and brooks, 1987 v greer and mys, 1987 v hancox, 1988 v ludwig and gibbs, 1989 v morton and thurston, 1988 v carle and whiton, 1990 r grimaldi, 1990 r, v heffner and heffner, 1990 r, v gillespie, 1991 v mayer and charlesworth, 1991 r, v l renous et al., 1991 v jouin et al., 1992 v l mcfadden et al., 1994 v f perle et al., 1994 v triemer and lewandowski, 1994 v černý and čižinauskas, 1995 v su fong et al., 1995 r, v l deckel, 1996 v f elbrächter and schnepf, 1996 v gilson and mcfadden, 1996 v f call and dilcher, 1997 v f crespi and vanderkist, 1997 v tague, 1997 r, v turc and lecour, 1997 v bhatnagar and meisami, 1998 r a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 299 table 1. continued. publication term used for edbs level of strictness for r-terms level of strictness for v-terms doving and trotier, 1981 v doweld, 1998 r, v murali et al., 1998 v f argiriadi et al., 1999 v f corley et al., 1999 v cohn and tickle, 1999 r f douglas, 1999 v f klasing, 1999 v rodríguez-riaño et al., 1999 v weston et al., 1999 v f scholtz, 2000 v takeda, 2000 v ea gibert et al., 2000 v sato et al., 2000 v f tchernov et al., 2000 r yan et al., 2000 v walker-larsen and harder, 2001 v l beardsley and olmstead, 2002 v bejder and hall, 2002 v f grimaldi et al., 2002 v kearney, 2002 v f narbona et al., 2002 v peterkova et al., 2002 r, v roxburgh and penshow, 2002 v rudall et al., 2002 v sekiguchi et al., 2002 v f strittmatter et al., 2002 r, v tague, 2002 r, v ashman, 2003 v emig, 2003 v grimaldi, 2003 v hall, 2003 v ea f liman and inman, 2003 v zhang and webb, 2003 v eastman and lannoo, 2004 v engel and grimaldi, 2004 v kearney and stuart, 2004 r maslakova et al., 2004 v pol and norell, 2004 v simões-lopes and gutstein, 2004 v f streltsov et al., 2004 v whiting et al., 2004 r beutel and weide, 2005 v golonka et al., 2005 v gotoh et al., 2005 v kearney et al., 2005 v l miura, 2005 v f a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 300 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. continued. publication term used for edbs level of strictness for r-terms level of strictness for v-terms müller et al., 2005 v napoleão et al., 2005 v narbona et al., 2005 v regoes et al., 2005 v f ronse de craene, 2005 r, v witter et al., 2005 v espinasa and jeffery, 2006 v f franz-odendaal and hall, 2006 v f garnier et al., 2006 v l gomez and shaw, 2006 v grant, 2006 v f kohlsdorf and wagner, 2006 v ostrovsky et al., 2006 v prince and johnson, 2006 v f rehorek and smith, 2006 v su rodríguez-riaño et al., 2006 r, v f sidell and o'brien, 2006 v bowsher et al., 2007 v maxwell and larsson, 2007 v l mehta and wainwright, 2007 v tamatsu et al., 2007 v watabe et al., 2007 v brandley et al., 2008 v gobin et al., 2008 v l hunt et al., 2008 v jonz and nurse, 2008 v f mcgowan et al., 2008 v sherman et al., 2008 r, v f witton and naish, 2008 v yoshizawa and jeffery, 2008 v bateman and fleming, 2009 v crottini et al., 2009 r jerez and tarazona, 2009 v ostrovsky and rodríguez, 2009 v renvoisé et al., 2009 v sauer and hausdorf, 2009 v f tekleva and krassilov, 2009 r, v wilkens and purschke, 2009 r, v burnham et al., 2010 v campbell et al., 2010 v senter, 2010a r, v f senter, 2010b r, v f zubidat et al., 2010 v f barfod et al., 2011 v buckland-nicks et al., 2011 r, v l chan-ard et al., 2011 r, v gomes rodrigues et al., 2011 v moch and senter, 2011 r, v f ortega-chávez and stauffer, 2011 v a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 301 table 1. continued. publication term used for edbs level of strictness for r-terms level of strictness for v-terms siler and brown, 2011 v xu et al., 2011 r, v l yu et al., 2011 v l arkhipkin et al., 2012 r, v f bensimon-brito et al., 2012 v ea crole and soley, 2012 r f daver et al., 2012 v hartstone-rose et al., 2012 v labonne et al., 2012 v f miralles et al., 2012 r f nweeia et al., 2012 r, v l olympska, 2012 v woon and stringer, 2012 r, v godefroit et al., 2013 v gotoh et al., 2013 v huang et al., 2013 v hutson and hutson, 2013 v longo et al., 2013 v f sato et al., 2014 v f xu et al., 2014 v table 2. . number of articles using forms of the terms “vestige” and “rudiment” in specific ways in reference to evolutionarily degenerate biological structures in 200 twentiethand twenty-first century publications in primary scientific literature. see table 1 caption for abbreviations. 1901-2000 2001-14 1901-2014 n 100 100 200 v 87 94 181 r 36 23 59 v&r 22 17 39 ea 2 2 4 su 1 0 1 f 19 23 42 l 10 9 19 twentieth-century articles, 36 (92%) applied r-terms to edbs, while three (8%) advocated or applied restriction of r-terms to embryonic structures. among the 25 relevant twenty-firstcentury articles, 23 (92%) applied r-terms to edbs, while two (8%) advocated or applied restriction of r-terms to embryonic structures. the ratio of less-strict to more-strict usage of r-terms is therefore equal between the two centuries, and the prevailing usage in both centuries has been to allow application of r-terms to edbs. of the 200 articles, 61 were deemed relevant to strictness level for v-terms: 29 from the twentieth century and 32 from the twenty-first century. of these 61 articles, 42 (69%) attributed a function or possible function to edbs to which v-terms are applied, while 19 (31%) advocated restriction of v-terms to putatively functionless structures. among the 29 relevant twentiethcentury articles, 19 (65.5%) attributed a function or possible function to edbs to which v-terms are applied, while 10 (34.5%) advocated restriction of v-terms to putatively functionless structures. among the 32 relevant twenty-first-century articles, 23 (72%) attributed a function or possible function to edbs to which v-terms are applied, while nine (28%) advocated restriction of v-terms to putatively functionless structures. in both centuries, therefore, the prevailing usage of v-terms applies them to structures with a known or suspected function. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 302 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) discussion although terminological strictness can be conducive to communication in some cases, the consensus found here indicates that this is not the case with v-terms and r-terms for edbs. this is none too surprising, because less-strict usage of both sets of terms is more logical than stricter usage in several respects. for example, strict application of v-terms only to functionless structures is illogical because it is impossible to prove that a structure is functionless. a structure with no known function may have a function that is yet to be discovered (scadding, 1981), and a structure that has lost a major function may retain a minor one (darwin, 1860; hall, 2003). strict usage would therefore ultimately render v-terms inapplicable to any structure. it is more logical to apply the term “vestigial” to a structure that is demonstrably a vestige (a remnant of an ancestrally greater structure) than to define v-terms so strictly as to render them obsolete. a term is pointless if its definition is so strict as to prohibit its usage. in addition, rendering v-terms obsolete would provide ammunition to anti-evolution authors who employ the strict usage of v-terms to cast doubt upon biological evolution by claiming that vestigial structures do not exist because functionlessness cannot be demonstrated (bergman and howe, 1990; sarfati, 2002). it would be incongruous for evolutionary biologists to deliberately provide support for the anti-evolution movement. less-strict usage of r-terms for edbs is logical from an evodevo perspective. the evolutionary process that produces edbs often includes the arresting of the structure’s development at an early stage (brues, 1903; bejder and hall, 2002; espinasa and jeffery, 2006; rehorek and smith, 2006), and biologists have long used r-terms for structures in early developmental stages. because edbs can therefore be considered to be persistently rudimentary, r-terms are appropriate for edbs. a recent call for terminological strictness recommended that a given edbs be called a “rudiment” in the embryo and a “vestige” in the adult (hall, 2003), but such terminological differentiation is unnecessary, because a structure with immature morphology is morphologically still a rudiment, even if it is in a mature body. to insist that a persistent rudiment not be called a rudiment merely because it persisted as one, is no more logical than to insist that a town not be called a town because it has never grown into a city. also, biologists frequently use the term “rudimentation” for the evolutionary process that produces edbs (berger dell’mour, 1985; tague, 2002; maxwell and larrson, 2007). it is linguistically logical to call the product of rudimentation a rudiment. stricter usage is not only illogical but is also in opposition to the consensus. as shown here, prevailing usage in primary scientific literature applies r-terms to edbs and v-terms to structures that retain a function. the few calls for greater terminological strictness have therefore been met with an implicit veto by the rest of the scientific community. it is therefore recommended here that r-terms not be restricted to embryonic structures and that v-terms not be restricted to putatively functionless structures. acknowledgments i would like to thank the following individuals for contributing to the search for articles that formed the sample used here: zenis ambrocio, julia b. andrade, katanya k. foust, jasmine e. gaston, ryshonda p. lewis, rachel m. liniewski, bobby a. ragin, khanna l. robinson, and shane g. stanley. i would also like to thank matthew wedel and two anonymous reviewers for constructive comments that resulted in improvements to this paper. references argiriadi ma, c morisseau, bd hammock, and dw christianson (1999) detoxification of environmental mutagens and carcinogens: structure, mechanism, and evolution of liver epoxide hydrolase. proc natl acad sci usa 96: 10637-10642. arkhipkin ai, va bizikov, and d fuchs (2012) vestigial phragmocone in the gladius 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fontana et al., 2007; larson-meyer et al., 2006; redman et al., 2007; martin et al., 2007). cr also has beneficial effect on cognitive performance in ghrko-al and normal-al mice. however, cr significantly increased its levels in both phenotypes. although these findings do not support the hypothesis that cr would increase the capacity of ros defense mechanisms in ghrko mice by increasing antioxidant enzymes levels, they do agree with some of the reported effects of cr on their expression. we suspect that gh resistance and cr may affect aging by different mechanisms and if cr delays aging in ghrko animals it is not due to changes in the activity of antioxidant enzymes. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 137 humans, mediated by increased insulin sensitivity and reduced inflammatory activity, leading to higher synaptic plasticity and stimulation of neurofacilitatory pathways in the brain (witte et al (2009). in animals, cr delay the occurrence of age-associated disease such as diabetes, cancer and cardiovascular disease and enhanc insulin sensitivity (colman et al., 2009; wang et al., 2009). however, the biological mechanism by which caloric restriction may exert its antiaging action is not yet understood. mechanisms that are thought to contribute to cr effects include retardation of growth, reduction of body fat, reduced insulin and glucose levels, reduced reproductive capacities, delayed neuroendocrine changes, improved dna repair capacities, reduced body temperature and metabolic rate, amelioration of oxidative stress damage, and hormesis (sohal and weindruch, 1996; mattison et al., 1998; masoro, 1998). the free radical theory of aging proposes that oxidative stress is a causal factor in aging process. the imbalance between free radical production and cellular antioxidant defense leads to the accumulation of oxidative damage to the cellular macromolecules. cellular endogenous antioxidant defense includes superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and others. the importance of antioxidant enzymes in defying oxidative stress is evidenced by the observation that overexpression of antioxidant enzymes extends lifespan in drosophila (orr and sohal, 1994; parks et al., 1998) and that treatment of c. elegans with synthetic sod/cat mimetics extended its lifespan (melov et al., 2006). studies in drosophila, c. elegans, and mutant mice provide evidence that insulin-like signaling contributes to the aging process. mutations in the daf-2 insulin receptor-like gene or the downstream age-1 gene extend the life span of c. elegans twoto threefold (kenyon et al 1993). ames and snell dwarf mice, deficient in gh (growth hormone), prl (prolactin) and tsh (thyroid stimulating hormone), live much longer than their normal siblings (brown-borg et al., 1996; flurkey et al., 2001). cr was shown to cause significant extension of lifespan in long-lived ames dwarf mice (bartke et al., 1999) and in long-lived daf-2 mutant c. elegans (lakowski and hekimi, 1998). moreover, the activity of two important antioxidant enzymes, cat and cu/ znsod in the liver, kidney, and hypothalamus of ames dwarf mice is higher than that in their normal siblings (brown-borg et al., 1999; hauck and bartke, 2000). gh receptor/gh binding protein knockout mice (ghrko) are gh-resistant and igf-1 deficient and were also reported to live significantly longer than their normal counterparts (tatar et al., 2003). studies conducted in our lab had shown that in ghrko mice, cr did not increase the overall, median or average life span of animals and it was suggested that somatotropic signaling played a key role in mediating the effects of cr to enhance longevity (bonkowski et al., 2006). in the current study we examined the effects of cr on antioxidant enzymes, namely catalase (cat) , superoxide dismutase (sod1) and glutathione peroxidase (gpx1) activity and on lipid peroxidation (lp) as an oxidative damage marker in livers and kidneys of these ghrko mice. materials and methods animals ghrko female mice and normal (n) littermate controls were produced in a closed colony derived from animals provided by dr. j. kopchick and maintained at southern illinois university by mating knockout (−/−) males with heterozygous (+/−) female carriers of the disrupted ghr/gh-binding protein gene. animals were housed on a 12-h light, 12-h dark cycle at 22 ± 2 °c and were fed (lab diet formula 5008, ralston purina corp., st. louis, mo) and watered ad libitum, except as noted below. all animal procedures were approved by the laboratory animal care and user committee at southern illinois university school of medicine. ghrko mice and n siblings were fed either ad libitum (al) or submitted to 30% cr protocol (eight animals per phenotype per diet) as described previously (bonkowski et al., 2006). samples at 9 months of age, animals were anesthetized by isoflurane, bled by cardiac puncture, and decapitated. to avoid stressing animals, the time between taking the animal from the cage, anesthetizing, bleeding, and decapitation was minimized (≈1 min). livers and kidneys were rapidly removed, quickly frozen on dry ice, and stored at −80°c until processed. trunk blood was collected in tubes containing edta. after collection, blood was centrifuged at 6000 × g for 15 min at 4°c, the plasma was collected and stored at −80°c. total proteins were obtained from whole tissue homogenates. approximately 100 mg liver/kidney samples were homogenized in 500 μl ice-cold homogenizing buffer [20 mm tris (ph 7.5), 150 mm nacl, 1% triton 100, with protease inhibitors cocktail and phosphatase inhibitors cocktails (sigma-aldrich corp.)] and spun at 14,000 xg for 45 min. the supernatant was removed and stored at −80°c. protein concentrations were determined using the bicinchoninic acid assay (pierce corp., rockford, il) according to the manufacturer’s instructions. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 0 10 20 30 40 0 5 10 15 20 25 30 35 n al n cr ko al ko cr age (weeks) b od y w ei gh t (g ) figure 1. weekly body weight changes for female normal and ghr-ko mice that were fed al or subjected to 30% cr for 9 months starting at 2 months of age. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 138 glucose concentrations were determined using a onetouch ultra glucose meter (lifescan, milpitas, ca). insulin was assessed by elisa (linco research, inc. st. charles, mo), and corticosterone was determined using ria kits from icn. catalase, cu/zn sod, and gpx activity catalase activity was determined by uv method (spectronic genesys 5 spectrophotometer: milton roy, rochester, ny, usa), as previously described (aebi, 1974). one unit of the enzyme was defined as μmol h2o2 disproportionated/min/mg protein. total cu/zn sod activity was determined by adaptation of the method described by sun et al., (1994) . one arbitrary unit (au) of sod activity was defined as the amount of cellular protein causing 50% inhibition of the rate of nitroblue tetrazolium reduction. catalytic activity of the samples was determined by comparison to a standard curve of commercial cu/zn sod (sigma, st. louis, mo, usa). gpx1 activity was determined as described by (tappel, 1978 ). one unit of activity was defined as the amount of enzyme required to oxidize 1 μmol of reduced nicotinamide adenine dinucleotide phosphate to nadp per minute per milligram of protein. cellular thiobarbituric acid-reactive substances (tbars) were measured (as reaction equivalent to malondialdehyde) as described by hashimoto et al. (1999). tbars formation was expressed as nanomoles of mda per milligram of protein. statistical analysis data are expressed as the mean ± se. the statistical evaluation was performed using two-factor anova (phenotype and diet), followed by fisher’s protected least significant difference test as apost hoc test. a t test was also used to evaluate the effect of diet within phenotypes and phenotype within diet.p < 0.05 was considered significant. all statistical analyses were performed using statview 5.0 software (sas institute, inc., cary, nc). results and discussion despite the fact that the effects of cr and insulin/insulin-like signaling disruption share many similarities, there is evidence that they have their distinct actions. it was previously reported n al n cr ko al ko cr 0 10 20 30 a b c d c a t ac tiv ity (u /m g pr ot ei n) n al n cr ko al ko cr 10 15 20 25 a ab b c s o d 1 ac tiv ity (u /m g pr ot ei n) a b n al n cr ko al ko cr 150 175 200 225 250 a a b c g p x a ct iv ity (u /m g pr ot ei n) c n al n cr ko al ko cr 4 5 6 7 8 9 10 11 12 a a a b lp n m m d a /m g pr ot ei n d figure 2. hepatic antioxidant enzyme activity (a) catalase, (b) cu/znsod, (c) gpx1, and (d) lipid peroxidation (lp) in female ghr-ko (ko) and normal (n) mice subjected to caloric restriction (cr) or fed ad libitum (al). data reported as mean ± se. groups that do not share a common superscript are significantly different (p<0.05). that ames dwarf mice have extended life as compared to wild type animals (brown-borg et al 1996). furthermore, cr extended the lifespan of long-lived ames dwarves (bartke et al., 2001). studies on murine models suggested that cr significantly increases the activity of several antioxidant enzymes including catalase, superoxide dismutase, and glutathione peroxidase (koizumi et al 1987, rao et al 1990, gomi et al 1998). ames dwarf mice, having mutations for less pituitary secretions, exhibited enhanced activity of antioxidant enzymes in the liver and kidney than age matched normal controls which showed that increased activity of these enzymes have protective effect against oxidative damage and consequently increases the life span (brown-borg et al., 1999, 2000 and hauck and bartke, 2000). as mentioned earlier, cr did not increase the overall, median or average life span of ghrko animals and it is suggested that somatotropic signaling is important in mediating the effects of cr on life span of animals (bonkowski et al., 2006). in the current study we sought to elucidate the effects of cr on the activity of antioxidant enzymes in the liver and kidney of these ghrko animals. catalase is an antioxidant enzyme found in all aerobic cells that catalyzes the decomposition of hydrogen peroxide to oxygen and water. a study by perez et al. (2009) has shown that transgenic mice overexpressing sod1, catalase or and manganese superoxide dismutase (mnsod) did not extend the life span of animals. in our study, ghrko animals had higher catalase activity in the liver and cr reduced it in both normal and ghrko animals (figure 2a). in contrast, in the kidney catalase activity was lower in ghrko animals with no effect of diet (figure 3a). these tissue specific differences might suggest different mechanisms of somatotropic signaling and cr on antioxidant capacity. it also questions the notion that catalase mediates the longevity induced by cr. cuznsod (sod) is the major superoxide dismutase isozyme found in cells. mice deficient in sod1 showed 30% reduction in life span (elchuri et al., 2005; perez et al., 2009) however, overexpression of sod did not affect the life span in mice (huang et al, 2000). sod1 in the kidney was shown to be protective against oxidative damage. injury to diabetic rodent kidneys has been reduced by overexpression of the antioxidant sod (craven et al, 2001). moreover, sod activity was found to be reduced in diabetic nephropathy (fujita et al, 2009). nishikawa et al demonstrated the importance of ros generation in mediating hyperglycemia-induced cellular damage (nishikawa 139 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a n al n cr ko al ko cr 4.0 4.5 5.0 5.5 6.0 a a b a c a t ac tiv ity (u /m g pr ot ei n) n al n cr ko al ko cr 20 30 40 50 60 70 80 90 a a b b s o d 1 ac tiv ity (u /m g pr ot ei n) b n al n cr ko al ko cr 30 40 50 60 70 80 a b b a g p x a ct iv ity (u /m g pr ot ei n) c n al n cr ko al ko cr 5 10 15 20 a a a b lp (n m m d a /m g p ro te in ) d figure 3. renal antioxidant enzyme activity (a) catalase, (b) ) cu/znsod, (c) gpx1, and (d) lipid peroxidation (lp) in female ghr-ko (ko) and normal (n) mice subjected to caloric restriction (cr) or fed ad libitum (al). data reported as mean ± se. groups that do not share a common superscript are significantly different (p<0.05). 140 et al, 2000). in the current study, ghrko animals had higher sod1 activity in the liver and cr reduced it in these animals (figure 2b). in the kidney, ghrko animals have significantly lower sod activity with no effect of diet (figure 3b). it was shown that increasing sod concentrations in mammalian cells produce a paradoxical pro-oxidant action by which protection against oxidative stress is lost and injury was even exacerbated, with increased lipid peroxidation (reviewed in mccord and edeas, 2005). reduced sod activity in ghrko kidney might provide a balanced protection against ros injury. these results also suggest different effects of gh/igf-1 and cr on antioxidant capacity. studies in gpx1-deficient mice have revealed many useful rules of gpx1 in regulating acute oxidative stress. gpx1 knockout mice appear to be phenotypically normal, however they are highly susceptible to injury induced by paraquat (a superoxide generator), cerebral ischemia-reperfusion (stroke), and cold-induced head trauma (de haan et al, 2003). however, gpx1 and sod2 deficiency in mice does not induce lifespan reduction despite increased oxidative damage (zhang et al, 2009). in our study, in contrast to catalase and sod1, gpx1 activity was lower in ghrko liver but higher in the kidney (figures 2c and 3c). it was previously shown that ghrko and ames dwarf mice have less severe glomerulonephritis as compared to normal animals (ikeno et al, 2003). ghrko kidneys appear to be protected against oxidative damage as indicated by increased gpx1 activity. however, cr did increase gpx1 activity in normal animals but decreased it in ghrkos. this adds to the finding that cr did not further increase the lifespan of ghrko animals (bonkowski et al, 2006). this study 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zhang y, y ikeno, w qi, a chaudhuri, y li, a bokov, sr thorpe, jw baynes, c epstein, a richardson, h van remmen (2009) mice deficient in both mn superoxide dismutase and glutathione peroxidase-1 have increased oxidative damage and a greater incidence of pathology but no reduction in longevity. j gerontol a biol sci med sci 64 (12): 1212-1220. 141 atlas journal of biology 1 (3): 41-46, 2011 doi: 10.5147/ajb.2011.0046 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) study of some lichens of qatar roda f. al-thani* and hadia a. al-meri department of biological and environmental sciences, college of arts and sciences, qatar university, qatar received: january 26, 2011 / accepted: april 9, 2011 introduction qatar is a peninsula and a small country located at longitude 51º 15’ e and latitude 25º 30’ n on the eastern banks of saudi arabia with the kingdom of bahrain beyond the gulf on its northwestern boundaries. the mainland of qatar is characterized by dominance of a stony desert (which constitute >85% of qatar) with isolated scattered depressions (rodat) with few trees and shrubs characteristic of arid zones. the climate is harsh in summer and mildly cool in winter being the period of seasonal rains. it is very hot and humid between june and august, and pleasant between november and february. the average annual rainfall is 81 mm, average maximum temperature is 31ºc, average minimum temperature is 22ºc, absolute maximum temperature is 50º c, absolute minimum temperature is 1ºc, average morning relative humidity is 71 %, and average afternoon relative humidity is 43%. lichens live on various surfaces (soil, trees, rocks and walls) __________________________________________________ * corresponding author: ralthani@qu.edu.qa and in various environments: some survive in the dry arid conditions of deserts and others on frozen soil of the pole regions. lichens grow very slowly, sometimes only a few cm in a whole year. humans use lichens in food preparation, as dyes for textiles and rugs (william, 2000). lichen metabolites exert a wide variety of biological actions including antibiotic, antimycobacterial, antiviral, anti-inflammatory, analgesic, antipyretic, antiproliferative and cytotoxic effects (melgarejo et al., 2008). even though these manifold activities of lichen metabolites have now been recognized, their therapeutic potential has not been fully explored and thus remains pharmaceutically unexploited (müller, 2002). the utility of lichens is due to the production of a range of secondary compounds by them (boustie and grube, 2005). some lichens are very sensitive to air pollution and some of them accumulate toxic materials (e.g. so2) (bačkor et al., 2003). accordingly, lichens are suited as biological indicators for monitoring environmental quality. pollution can be assessed by chemical analysis of the lichen thalli. lichens are unique organisms formed by an association of green algae or cyanobacteria and fungi of the ascomycetes or basidiomycetes. there are three forms of lichens: crustose, foliose, fructicose. up to date mainly crustose lichens are common on rocky outcrops and only one each of the foliose and fructicose lichens have been encountered in qatar. encounter of lichens is mentioned in various reports (babikir and kürschner, 1992; abdel bari, 1997a,b; abulfatih et al., 2001) but none of these publications covered a detailed study of lichens of qatar. meanwhile, there have been studies of lichens in neighboring 41 short communication abstract the desert regions of north and central qatar were surveyed for lichens. twelve species were reported. the most common lichens are of the crustose type and all fungi of the lichens collected are ascomycete. keywords: lichens, crustose, qatar. 42 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) arabian gulf countries. most literature records are from saudi arabia. bokhary et al. (1993) and abu-zinada et al (1986) listed about 100 species. brown (1998) provided a provisional list of species occurring in kuwait about 40 distinct species have been found on the northern side of kuwait bay. mandeel and aptroot (2004) recorded only seven species, all crustose, from bahrein kingdom. brown et al. (2002) recorded 36 species of lichens from the sultaanate of oman. hellyer and aspinall (2005) have been found that lichens as a prominent feature of coastal rocks on some islands in united arab emirates and rocky environments including several species of the genus caloplaca, lecania subcaesia, buellia subalbula, verrucaria sp. and ramalina maciformis. schultz (1998) reported a total of 36 lichen taxa were found in the south of the yemen arab repuplic. field collection of lichens (deposited at the herbarium of biological and environmental science department, qatar university) commenced in 1994 and the first publication on the record of fructicose lichen was in 1997 (abdel bari, 1997a). in 2004 a study on lichens in qatar was undertaken as a student project (lichens of qatar). this raised further interest in the group particularly as qatar is now undergoing major constructions all over and fear of loss of biodiversity is a great concern. materials and methods during numerous field trips, stone fragments with lichens were randomly collected from different sites (mostly from rocky hills). these were labeled with details and kept safe. selected samples are cleaned, sprayed with water and photographed. photos include general plates and close up of morphological appearance. identification and classification was carried out by 3 methods: morphology: based on the identification of lichens by their colors, types of growth, types of substrates and presence or absence of the fruiting body. anatomy: the lichen thallus typically consists of three basic layers: cortex, (algal and fungi) layer and medulla. these are not always present. longitudinal or transversal sections in the thalli of the lichens were used to identify the composition of the lichen thallus layers. chemical analysis: lichens can be easily distinguished with a simple spot test of koh, hypochlorite or p-phenyldiamine solutions. the color reactions occur because lichens produce acids which react with chemical solutions changing the color of the lichen thallus. results identification keys of reported species the most common lichens in qatar are of the crustose type (fig. 1-2) and all fungi of the lichens collected are ascomycete. lichens were collected from north and central qatar. as to date a total of 12 species were recorded. these fall in 11 genera of 10 families (table 1, fig. 3-4). all species are new records for the country. macroscopic and microscopic characters acarospora sulphurata (arnold) arnold asci multispored; ascospores colourless, 2-4 x 2-3 μm, non-septate; apothecia immersed, appearing as irregular cracks, yellowish brown; prothallus indistinct. collema tenax (sw.) ach. em. degel. thallus rosette-like and thick that is variable in size. it is usually dark olive green or black in color adpressed to the substratum. apothecia are small gloeoheppia turgid (ach.) gyeln. squamules convex, markedly swollen even when dry, adpressed and appearing almost areolate from above. lecidella euphorea (firke) hertel apothecia lecideine, with a black margin and plane to convex disc. buellia subalbula (nyl.) müll. arg. thallus white; k-; ascospores 14-17 x 7 μm ramalina maciformis (delise) bory fruticose lichen and characterized by greenish-yellow and an erect thallus with thick yellwish epithecium. the ascocarp disc is pale green and is surrounded by a corticate exciple. the asci contain 8 colourless spores which are 2or sometimes 4-septate. the spores are ellipsoid or slightly curved caloplaca brouardii (b. de lesd.) zahlbr thallus forming rosettes, placodioid, with radiating lobes at the margins but becoming areolate centrally; apothecia absent; numerous scale-like isidia (phyllidia) developing from the surface of the lobes caloplaca aurantia (pers.) j. steiner thallus has many colour forms: typically, it has dull orange-coloured margins, thallus and apothecia k+ (purple). xanthoria parietina (l.) th. fr thallus forming rosettes, often exceeding 2 μcm diam; lobes flat-tened against the substratum; soralia absent; orange apothecia common. diploschistes albescens lett greyish-white to greyish pruina thallus, 1-3 mm thick. ascospores brown, muriform, 20-40 x 9-17 μm with 5-6 transverse septa, 12-14 μm wide. 43 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. most common lichen species in qatar. fig. 1. above: general view of the stony desert in mainland qatar; growth of crustose lichens on rocks on surfaces facing the sun (r.h.s). below: clay loam soils with crustose lichens. species family koh chemical hypochlorite tests p-phenyldiamine type of substratum acarospora sulphurata (arnold) arnold acarosporaceae kcprocks collema tenax (sw.) ach.em.degel. collemataceae kcprocks gloeoheppia turgid (ach.) gyeln. gloeoheppiaceae kcprocks lecidella euphorea (flӧrke) hertel lecanoraceae kcprocks lecidea sp. lecideaceae kcprocks buellia subalbula (nyl.) müll.arg. physciaceae kcprocks ramalina maciformis (delise) bory ramalinaceae kcprocks caloplaca brouardii (b. de lesd.) zahlbr caloplaca aurantia (pers.) j. steiner xanthoria parietina (l.) th.fr teloschistaceae k+( violet) k+(violet) k+(violet) c+(dark read) c+(dark read) c+(dark read) ppprocks diploschistes albescens lett. thelotremataceae kcpon compacted sand in rock crevices. verrucaria sp. verrucariaceae kcprocks 44 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 2. dense growth of different species of crustose lichens in stony deserts. fig. 3. caloplaca aurantia, a common arrange-colored lichen on stony deserts in qatar. 45 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 4. common lichens in qatar. lecidella euphoria ramalina maciformis buellia subalbula xanthoria parietina discussion qatar is well-known for its active economy and its lichen flora is therefore likely to lose much ground before it has ever been explored. this is the more unfortunate as the lichen flora is only poorly known. lichens biodiversity in qatar is low except at some investigated area at north and central qatar which most of lichens were isolated. this area is still without any pollution or construction. all species recorded during this study have been found in various countries of arabian peninsula. lichens are among the most valuable biomonitors of atmospheric pollution (nimis et al., 2002). the diversity of epiphytic lichens is commonly used as a sensitive indicator of the biological effects of air pollutants. mapping of lichen diversity is becoming routine in several countries since it is quick and inexpensive and provides results on which predictions for human health can be based (cislaghi and nimis, 1997). field collection of lichens must continue to determine species richness and abundance and their distribution. thus a study of lichens from areas of major petro-chemical industrial cities may prove useful in the detection of the nature of the pollutants and the extent of air pollution from major industrial cities in qatar. references abdel-bari em (1997a) record of a fruiticose lichen ramalina farinacea (l.) ach. for qatar. qatar university science journal 17(2): 265-270. abdel-bari em (1997b) addition to the flora of qatar. qatar university science journal 17(2): 303-312. abulfatih ha, em abdelbari, a alsubaey, and ym ibrahim (2001) vegetation of qatar. scientific and applied research center (sarc): university of qatar, doha qatar, p. 360. abu-zinada ah, dl hawksworth, and ha bokhary (1986) the lichens of saudi arabia with a key to the species reported. arab gulf journal for scientific research sp. pub. 2: 1-54. babikir aa, and h kürschner (1992) vegetation patterns within a coastal saline of ne-qatar. arab gulf journal for scientific research 10: 61-75. bačkor m, k paulíková, a geralská, and r davidson (2003) monitoring of air pollution in košice (eastern slovakia) using lichens. polish journal of environmental studies 12 (2):141-150. brown g (1998) notes on the lichen flora of kuwait. nova hedwigia 67: 267-273. brown g, m schultz, and md robinson (2002) saxicolous and terricolous lichens from the foothills of northern oman. nova hedwigia 75: 177-188. 46 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) bokhary ha, s parvez, and ah abu-zinada (1993) lichen flora from high altitude areas of saudi arabia. nova hedwigia 56: 491-496. boustie j and m grube (2005) lichens-a promising source of bioactive secondary metabolites. plant genetic resources 3: 273-278. cislaghi c and pl nimis (1997) lichens, air pollution and lung cancer. nature 387: 463-464. hellyer p and s aspinall (2005) the emirates a natural history. support of the environment agency, abu dhabi and dolphin energy limited. trident press limited pp. 427. mandeel q and a aptroot (2004) lichens of bahrain. willdenowia 34: 539-542. melgarejo m, o sterner, j vila-castro, and p mollinedo (2008) more investigations in potent activity and relationship structure of the lichen antibiotic (+)using acid its derivate dibenzoylusnic acid. revista boliviana de quimica 25 (1): 24-29. müller k (2002) pharmaceutically relevant metabolites from lichens. applied microbiology and biotechnology 56: 9-16. nimis pl, c scheidegger, and pa wolseley (2002) monitoring with lichens – monitoring lichens. kluwer academic publishers, the netherlands. pp. 1-4. schultz m (1998) short communications, studies on lichens from southern yemen (arabian peninsula). the lichenologist 30: 293-297. william p (2000) lichens. the natural history museum, london, uk. pp. 112. atlas journal of biology 2 (1): 88–93, 2012 doi: 10.5147/ajb.2012.0063 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) review of the rpt3 genes encoding part of the 26s proteasome associated with loci underlying disease resistance in soybean shivani malik, sukesh bhaumik and david a. lightfoot* department of biochemistry and molecular biology, southern illinois university, carbondale il 62910, usa received: february 24, 2012 / accepted: march 28, 2012 __________________________________________________ * corresponding author: ga4082@siu.edu 88 abstract the 26s proteasomal complex is a multifunctional proteolytic machinery of the cell. the proteasome plays role in myriad of cellular functions, which have been further diversified by its separable proteolytic and non-proteolytic sub-complexes. protein quality control and turnover, cell cycle regulation, gene regulation and dna repair are among the key processes controlled by the proteasome. disease resistance in plants invokes changes in all the processes controlled by the 26s proteasome. in this review, the potential contribution of genes encoding the proteasome to disease resistance in soybean (glycine max l. merr.) was examined. keywords: proteasome; rpt3; scn; sds; qtl; soybean; glycine max. introduction the 26s proteasome is a versatile, non-lysosomal protein degradation complex of the cell (glickman et al., 1998; voges et al., 1999; coux, 2002). it consists of two sub-complexes: the 20s catalytic particle (cp) and the 19s regulatory particle (rp). the 19s rp in turn consists of a “base” and a “lid”. the 20s cp has a hollow cylinder like structure consisting of a stack of two alpha and two beta rings in the α7-β7-β7α7 order. three of the seven β subunits are catalytically active and possess chymotrypsin-like (β5), trypsin-like (β2) and caspase-like activities (β1) (dick et al., 1998; groll and clausen, 2003). the base of 19s rp consists of a ring of six atpases (rpt1-6) and three nonatpases (rpn1, rpn2 and rpn13) (glickman et al., 1998; voges et al., 1999; coux, 2002; dick et al., 1998; groll and clausen, 2003; horwitz et al., 2007). the 19s base possesses molecular chaperonin activity that enables it to fold and unfold proteins (braun et al., 1999). a chain of four or more polyubiquitin moieties marks the protein destined to be degraded (chau et al., 1989; finley et al., 1994; wang et al., 2006). the ubiquitin receptor (rpn10) in the 19s lid binds to this polyubiquitinated protein (deveraux et al., 1994; elsasser et al., 2004) which leads to its unfolding by the 19s base by atp hydrolysis. the unfolded protein is fed into the 20s cp where the beta subunits proteolyze it (larsen and finley, 1994; navon and goldberg, 2001; verma et al., 2004; guterman and glickman, 2004; hanna et al., 2006; seong et al., 2007a,b; shreiner et al., 2008; husnjak et al., 2008). through this mechanism, the proteasome regulates review article a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 89 abundance and localization of a large number of transcription factors influencing several cellular processes (hilt and wolf, 1996; he et al., 1998). it also degrades oncoproteins, cell cycle dependent cyclins, cyclin dependent kinase inhibitors, thus influencing multitude of cellular processes directly or indirectly. in addition to this proteolytic role, the proteasome has been implicated to regulate transcriptional activation in a proteolyticindependent manner (collins and tansey, 2006; bhaumic and malik, 2008; ferry et al., 2009; lassot et al., 2007). it serves to recruit transcriptional co-activators, chromatin remodeling complexes and enables assembly of the pre-initiation complex at gene promoters to activate transcription. thus, the proteasome serves as a central hub orchestrating several regulatory mechanisms (figure 1). this makes it a good candidate for analysis of its contribution, if any, to some of pathological states of plants (in this study soybean). analysis in soybean soybean [glycine max (l.) merr.] is the world’s most important legume crop grown for its protein and oil content (messina et al., 1997). nematode attack (by heterodera glycines i.) by far has the most damaging effect on soybean crop leading to huge economic losses. soybean cyst nematode (scn) has been counteracted by non-host crop rotation practices, use of nematicides and development of resistant cultivars. use of resistant varieties is a key strategy in scn management. although, about 130 sources of scn resistance have been identified within the glycine sp., and more than 30 resistance loci identified, not much is clearly known about the underlying scn resistance genes (afzal et al., 2008). scn resistance is controlled by several genes at multiple quantitative trait loci (qtl) (concibido et al., 2004) and identification of genes contributing to these loci will enable better management of scn. fine mapping analysis of scn resistance loci has revealed two major loci controlling resistance to scn hg type 0 (race 3) in soybean cv. ‘forrest’ (meksem et al., 2001). one is the rhg1 locus on linkage group g and other is rhg4 on linkage group a2. major loci were also found on linkage groups b1 and d2 in many plant introductions (pis; afzal et al., 2009; yue et al., 2001; webb et al., 1995). proteomic studies have shown that among the proteins increased in scn resistant rhg1 near isogenic lines (nils) was a proteasomal component (afzal et al., 2009; figure 1). it is interesting to note that proteasome forms an important hub in the scn a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. a part of the predicted interactome for soybean showing six proteins from the 2d gel analysis of (32). the multi catalytic endopeptidase shown in blue is the beta-3 subunit of the 20 s proteasome core protein. the proteins encoded by the rpt3 genes of the 19s regulatory particle are represented by a single pink spot (arrowed). the 14 proteins of the 20s particle and 9 proteins of the 19 s regulatory particle interact to form the 26s proteasome. also shown in color were proteins changed in abundance by scn infestation of roots; the thaumatin like protein, cytosolic heat shock protein and triose phosphate isomerase. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 90 resistant protein interactome web with at least 21 interacting partners. perhaps variations in the proteasome contribute to the scn qtl. soybean behaves like a diploidized-tetraploid and homology searches using blast showed there were two or four copies of each of the six atpases subunits. of these, rpt3 had two copies on chromosomes 11 (linkage group b1) and 18 (linkage group g). it is intriguing that the copy of same atpase was conserved on two chromosomal locations. on chromosome 11, rpt3 maps to a position of 32,631,398-32,634,285. this location overlaps with satt415 (position: 32,627,233 -32,627,528), a marker in the region mapped to scn qtl (figure 2a). this suggests that rpt3 gene might be associated with scn qtl. the other copy of rpt3 lies on linkage group g (chromosome 18) at position 4,388,793-4,389,751. satt688 (3,264,2263,264,398) and satt130 (4,619,181-4,619,599) are within 2 mbp of rpt3 on chromosome 18 (figure 2b). both satt688 and satt130 are associated with scn qtl. scn qtl associated with satt688 were 29-1, 29-4, 29-8 while satt130 is linked to scn figure 2. ideograms of 20 cm (8.24 mbp) regions containing the rpt3 genes in soybean. panel a: ideogram of a region of chromosome 11 with two markers satt415 and sat_364 close to rpt3 gene, marked by arrows. rpt3 is located at position 32,631,39832,634,285 which includes marker satt415.panel b: ideogram of a region of chromosome 18 with two markers satt130 and satt688 close to rpt3 gene, marked by arrows. rpt3 is located at position 4,388,793-4,389,751. qtl 17-4, 18-5 and 19-4. on linkage group g there was also a qtl for resistance to sudden death syndrome that overlaps the rps3 genes and the scn qtl an interesting feature of the rpt3 gene on both the chromosomes was a close conservation of the flanking genes (figure 3). conservation of two copies of rpt3 with a set of few common genes suggests that this genetic unit may be playing important physiological roles in soybean. serine theronine protein kiases, e2 ubiquitin lyases, harpin induced proteins, kip1 like proteins and dead box helicases were all clustered with the rpt3 genes at both loci. the proteasomal component was increased during pathogen infestations probably to degrade the oxidized or damaged proteins or as a defense against oxidative stress posed by scn feeding (giulivi et al., 1994; lee et al., 2006). mapping of proteasomal genes close to scn qtl underscores the contribution of proteasome in scn resistant reaction. thus, it might serve as an important gene in contributing to resistance to scn. the proteasome has an interesting architecture, which couples to its exquisite regulation. it has a base of six atpases, which sits atop the 20s cylinder. upon atp binding, the c-terminal hydrophobic tyrosine-x-motif of the base atpases docks into the α subunits of the 20s core. atp hydrolysis by the base then serves to open the gate to proteolytic 20s core to feed the proteins into the 20s barrel (smith et al., 2005). previous work (afzal et al., 2009) and mapping of both the 20s and 19s components to scn qtl indicates that a part of its role is scn resistance is through its proteolytic activity. the 26s proteasome recognizes proteins to be degraded by a polyubiquitin chain of four or more ubiquitin moieties. the ubiquitin chain on a cellular protein is formed by isopeptide bond formation between c-terminus of ubiquitin and €-amino lysine side chain of the target protein or another ubiquitin molecule (pickart and eddins, 2004; pickart and fushman, 2004; wang et al., 2006) by the sequential action of e1 activating, e2 conjugating and e3 ligating enzymes. nature of the lysine linkage is a key factor determining the fate of the protein. k-48 linked poly ubiquitin chains are targeted for degradation while k-68 mostly serves in signaling pathways (wang et al., 2006; chau et al., 1989; finley et al., 1994). in addition, the 19s base atpases can exist as an apis (aaa atpases independent of 20s) complex separate from the 20s core (sun et al., 2002). this complex plays a crucial role in regulation of transcriptional activation in yeast and humans (lassot et al., 2007; gonzalez et al., 2002; lee et al., 2005; bhat et al., 2008; malik et al., 2009; truax et al., 2010). thus, 19s atpases might also function to regulate transcription of other genes contributing to scn reaction. the 19s atpases have been shown to play key roles in histone covalent modification in yeast and mammals (lee et al., 2005; ezhkova and tansey, 2004; laribee et al., 2007; kinyamu et al., 2008; koues et al., 2008, 2009). in yeast, inactivation of rpt6/sug1 leads to decreased dimethylated histone h3 lysine 4 (h3k4 di-me) and acetylated histone h3 (lee et al., 2005). h3k4 methylation is associated primarily with gene activation but also contributes to gene silencing of mating type locus. acetylation of h3 is a strong gene activating signal serving to remodel promoter chromatin. these modifications also serve has a binding sites for effector proteins which perform their designated functions (strahl and allis, 2000; sims and reinberg, 2006) allowing the proteasome to exercise widespread control over various pathways. the 19s base also has chaperonin like activity and might also be serving to play some role in scn resistance consistent with increased abundance of chaperonins in scn resistant nil roots (afzal et al., 2009). 91 figure 3. genes flanking rpt3 in the regions within 200kbp on chromosomes 11 and 18. compare the common genes flanking rpt3 genes on both the chromosomes. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) concluding remarks the proteasome with its far ranging functions is an attractive candidate gene for regulating diseased states in plants. bioinformatics analyses based on protein abundances, protein interactions and the location of genes encoding those proteins has inferred the proteasome may be contributing to scn resistance in soybean. this link however, needs to be confirmed using other approaches. these approaches in soybean include candidate gene tilling, ecotilling and complementation analysis, as has been performed for soybean cv ‘forrest’ (liu et al., 2010). 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of biology 2016, pp. 274–291 doi: 10.5147/ajb.2016.0141 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) nodulation and symbiotic nitrogen fixation in the biofuel legume tree pongamia pinnata phoebe nemenzo-calica1,2, arief indrasumunar1,2, paul scott1,2, peter dart2, and peter m. gresshoff1,2* 1 centre for integrative legume research; 2 school of agriculture and food sciences, the university of queensland, st lucia, brisbane qld 4072, australia received: august 15, 2016 / accepted: september 9, 2016 __________________________________________________ * corresponding author: p.gresshoff@uq.edu.au 274 abstract the legume tree pongamia pinnata (also called millettia pinnata) is a non-food crop that can grow on marginal land not destined for the cultivation of food crops. it is an important candidate for the production of biofuel (bio-oil, biodiesel and aviation biofuel) from its oil-rich seeds. an important trait is its ability to grow in marginal, nitrogen-limited soils. this growth ability is attributed to the nitrogen-fixation activity of root nodules. cutting both cotyledons in halves had little effect on shoot dry weight, nodule number and weight per plant at 8 weeks. these parameters were reduced when both cotyledons were removed; plants were visibly yellow. surprisingly when no nutrients were supplied and with both cotyledons intact, plants grew as well as those with nutrients. to isolate rhizobial inoculants from soil, samples were collected from meandu mine, a coal-mining site near kingaroy, queensland (australia), where pongamia is currently grown to rehabilitate the soil. pongamia nodules were also collected from mt. coot-tha botanic gardens (brisbane), where pongamia was introduced decades ago. established ‘baiting technique’ and ‘one-drop-one-nodule technique’ were applied to isolate potential rhizobia from the soil samples and nodules. all isolates were screened and characterised for symbiotic effectiveness and nitrogenase activity. a total of 21 putative rhizobial samples were isolated – five from soil samples and 16 from pongamia nodules. out of these iso this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. lates, seven were slow-growers while 14 were fast-growers. these isolates were coded as pr-uq. among the isolates, the fast-growers pr-uq-03 and pr-uq-05 were considered as the superior strains compared to the slow-growers pr-uq-01 and pr-uq-04, which were more effective than the rest of the isolates. the new isolates enhanced nodule number, shoot length and total plant dry weight and resulted in better plant growth than the available bradyrhizobium japonicum strains cb1809, usda110 and cb564. acetylene reduction assay (short term) and nitrogen difference analysis (long term) further confirmed that pongamia fixed more nitrogen upon inoculation with these rhizobia. based on 16s rdna sequence analysis, pr-uq-01 and pr-uq-04 are closely related to bradyrhizobium elkanii and bradyrhizobium pachyrizi, while pr-uq-03 and pr-uq-05 are both related to rhizobium mesoamericanum. cotyledons supply the seedling with nutrients for a considerable period of early seedling growth. the newly isolated, fast-growing rhizobia pr-uq-03 and pruq-05 and the slow-growing pr-uq-01 and pr-uq-04 are related to rhizobium and bradyrhizobium species and aid in the nodulation, nitrogen fixation and seedling/sapling growth of pongamia pinnata. keywords: biofuel, bradyrhizobium, nodulation, nitrogen fixation, rhizobium, 16s rdna. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 275 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) introduction pongamia pinnata, a fast growing, outcrossing, papillionaceous tree legume with high oil content seeds found in northern australia, papua new guinea, indonesia and india, is a potential feedstock for biofuel production (biswas et al., 2013; gresshoff et al., 2015; scott et al., 2008). it is salinity and drought tolerant (especially when established beyond the seedling stage), and grows well in low fertility soils (kazakoff et al., 2011). this legume tree produces seeds, which contain about 40% (w/w) oil, predominantly 50% of which is the mono-unsaturated oleic acid (c18:1) that can be used for biodiesel production (after transesterification with methanol or ethanol; kazakoff et al. 2011). the oil is low in saturated palmitic (c16:0) and stearic (c18:0) acids, and is non-edible. following oil extraction the resultant seedcake finds utility as supplemental animal feed for poultry, cattle and sheep, though only in small portions (10-20% of total feed; (biswas and gresshoff, 2014; chandrasekaran et al., 1989; konwar, 1987a, 1984, 1987b, c; natanam et al., 1989a; natanam et al., 1989b; natanam et al., 1989c; natanam, 1989; ravi et al., 2000; singh et al., 2006). multiple pod components (including the oil, seed cake, and pod walls) can be used for energy production via co-combustion, or fermentation. pongamia oil is made up predominantly of triglycerides, which can be converted by transesterification to biodiesel (fatty acid methyl esters; fame) or by hydrogenation to aviation a1 jet fuel (kazakoff et al., 2011; klein-marcuschamer et al., 2013). the potential of pongamia as a biofuel feedstock is now acknowledged (dwivedi et al., 2011; kazakoff et al., 2012; kesari and rangan, 2010; klein-marcuschamer et al., 2013; murphy et al., 2012; naik et al., 2008; samuel et al., 2013). according to the us department of energy (international energy outlook 2009), the current demand for oil from fossil fuels is around 85 million barrels per day (approximately 13.5 billion litres). in 2030, it is expected that oil demand will be around 106 million barrels per day (approximately 16.9 billion litres). pongamia and other biofuel feedstocks can contribute substantially to the future energy demands of the domestic and industrial economies. pongamia is a strong candidate as a biofuel because it meets two criteria as an effective, second-generation biofuel, namely (1) it must be a non-food crop that can grow on marginal land not used for the cultivation of food crops, and (2) the use of vegetable oils from plants must provide an environmentally acceptable fuel, the production of which is greenhouse gas neutral, with reductions in current diesel engine emissions. moreover, the composition of seed oil and the properties of fatty acid methyl esters (fames) of pongamia meet both north american and european industry standards (scott et al., 2008). pongamia is yet to undergo any directed domestication that has accompanied the development of other modern annual and perennial crops. some selection for yield and tree architecture may have occurred in india over the thousands of years of ‘village’ agriculture. there is no published record of breeding programs for pongamia. to initiate a directed domestication program, desirable traits need to be defined in concert with functional genomics studies to identify and characterise the relevant traits. the suggested domestication traits include nitrogen fixation efficiency, which is deemed as highly important, as it relates to the environmental and economic costs of nitrogen fertiliser supplementation. other suggested traits include repeated annual cropping, crop uniformity, seed mass per tree (yield), extractable seed oil content, oil composition and stability, growth vigour at seedling and adult stage, erect growth and architecture, canopy density, seed abscission, resistance to insects, nematodes, fungi and bacteria, flowering time, water-use efficiency, hardiness to cold, acid soils, drought and salinity. many of these traits will be multigenically controlled and breeding will be an on-going task for the emerging pongamia biofuel industry. pongamia nodulates well with a wide range of strains including bradyrhizobium and rhizobium such as bradyrhizobium sp. strain cb564 and rhizobia sp. strain ngr234 (pueppke and broughton, 1999; rasul et al., 2012; scott et al., 2008), bradyrhizobium japonicum cb1809 and usda110 (both slow growing rhizobia and widely used commercial inoculants for soybean) as well as fast growing rhizobia (arpiwi et al., 2012; samuel et al., 2013). some reports indicate that trees, including pongamia, are mainly nodulated by fast-growing rhizobia than by slow-growing rhizobia (moreira et al., 2006). however, the geographic and soil type distribution and taxonomy of rhizobia strains capable of nodulating pongamia and their effectiveness in symbiotic nitrogen fixation are little understood. an indirect technique to isolate rhizobia from soil is to use an uninoculated host plant as ‘bait’ in sample soils and then later on isolate putative inoculant from the surface-sterilised nodule. this baiting technique, which uses nodules as effective traps for selective enrichment, allows isolation of rhizobia classified in different cross-inoculation groups (gault and schwinghamer, 1993). in a study by rasul et al. (2012), 29 rhizobia strains were isolated by planting sterilised pongamia seedlings into soils collected from andhra pradesh, maharashtra and karnataka in southern india. the ability of these strains to fix nitrogen was not assessed and there was no superior isolate identified in this study, although evaluation of nodulation and molecular analysis were conducted on these rhizobia. another baiting experiment was conducted in western australia in which 40 strains were isolated from soil samples (arpiwi et al. 2012). in this study, the superior rhizobia strain was determined as bradyrhizobium yuanmingense. all rhizobia strains from both of these studies were able to form ‘creamy or white opaque’ colonies on growth media with congo red, which is a characteristic of all rhizobia. on the basis of phenotypic, phylogenetic distinctiveness and molecular data, a novel species of the genus rhizobium, rhizobium pongamiae was isolated from nodules of pongamia in north guwahati, assam, india (kesari et al., 2013). it is a gramnegative, non-motile, fast-growing, rod-shaped bacterium, which grew optimal at 280c, ph 7.0 and in medium supplemented with 2% nacl. it exhibits higher tolerance to the prevailing adverse conditions, for example, salt stress, elevated temperatures and alkalinity and was found to fix nitrogen using the acetylene reduction assay. pcr detected a nifh gene, encoding the iron component of nitrogenase (kesari et al., 2013). in the symbiotic relationship with host legumes, rhizobia can promote the host plants’ growth by providing available nitrogen, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) which would normally be restricted to them. therefore, isolation, characterisation and selection of suitable rhizobia strains must be conducted to promote the growth of pongamia and increase its potential yields. this study aimed to identify effective rhizobia that can nodulate and fix nitrogen with pongamia in queensland, australia. materials and methods cotyledon nutrient carry-over to determine the role of cotyledons in nodulation and avoid the carry-over of nutrients from cotyledons, 40 pongamia seedlings growing in sterile vermiculite at 20 days old were divided into five treatments: (1) inoculated with cb1809 but no nutrient solution added; (2) uninoculated and no nutrient solution added; (3) inoculated with cb1809 with nutrient solution (b&d n-free solution); (4) uninoculated but with nutrient solution; and, (5) uninoculated but with nitrogen (2 mm ammonium nitrate solution) and nutrient solution. two replicates per treatment were used. their cotyledons were either cut, removed, or left intact as soon as primary leaves uncurl; all of the seedlings for each treatment were coded as: 0cot = both cotyledons were removed; 1cot = one cotyledon removed; 2cot = both cotyledons intact; 1/2 cot = both cotyledons cut into halves. after eight weeks, the plants were uprooted and nodulation was assessed. isolation of rhizobia the soil samples were characterised by ph and nutrient component analysis. to get the ph of the soil samples, samples were air-dried and homogenised to <1 mm diameter. six grams of each sample were weighed and put inside a 50-ml falcon tube. two replicates were made for each sample. then, 30 ml of deionised water was added into each tube (1:5 soil solution ratio). the soil samples were mixed for an hour on an end-overend shaker at room temperature. the ph was then measured using the ph meter by directly dipping the ph electrode into each tube. for total carbon and nitrogen level analysis, dried samples were subjected to combustion and analysed using a leco truspec analyser. for total nutrient analysis, samples were extracted and analysed using a varian vista pro icpoes instrument. baiting technique was employed to isolate the rhizobia from soil samples from the meandu mine (six different sites). soil samples were filled into sterile pots. pongamia seeds were sizesorted and weighed to select the best seeds, surface-sterilised with 5% commercial bleach for one minute and rinsed with 70% ethanol twice followed by washing seven times sterile distilled water, after imbibition and sown directly into the soil. plants were maintained in the glasshouse at 27-300c daytime and 17200c night time temperature during summer season and watered with 100-ml b&d nutrient solution (stored at room temperature) once and 100-ml sterile distilled water afterwards as needed. plants were protected from cross-contamination by covering the surface with white sterile beads with a pipe inserted for watering purposes. the plants were uprooted after 10 weeks. larger, pink nodules that were close to the crown or primary roots were selected for the isolation of rhizobia in the laboratory. data were gathered for shoot length, number of nodes, number of leaflets, number of nodules, root, shoot, nodule and total plant dry weights, nodule fresh weight (preserved), soil type and properties, soil analysis and nodule morphology. to isolate rhizobia from the pongamia nodules collected from mt. coot-tha (brisbane, queensland) and from the nodules produced from baited plants in soil samples, the one-drop-onenodule technique was employed, in which five nodules (large and pink inside) per replicate plant were selected, placed inside sterile tubes and then surface-sterilised with 95% ethanol for 5 seconds and 3% commercial bleach for 5 minutes. each nodule was crushed into a drop of sterile distilled water in an empty sterile petri dish using sterile forceps. the nodule extract was then streaked three-ways into the yeast extract mannitol agar with congo red (yema+cr). plates were incubated for 6-10 days at 280c. well-isolated colonies that appeared on plates after 3-5 days of incubation were sub-streaked into another yeast extract mannitol agar plate and were designated as “fast-growers”, while colonies that grew within 6-10 days of incubation were designated as “slow-growers”. all isolates were purified by serial sub-streaking into yema+cr until pure cultures (judged by consistent colony morphology) were achieved. nodulation and symbiotic effectiveness under glasshouse conditions the rhizobia that produced the highest number of ‘active’ nodules in pongamia were determined based on the assessment of nodulation and symbiotic effectiveness tests. pure cultures of the isolates and the three available rhizobia strains in the laboratory known to nodulate soybeans (i.e., cb1809, usda110 and cb564) were inoculated into ymb for 48 hours with shaking. the culture suspensions were then inoculated into surface-sterilised pongamia sown in sterile vermiculite in 15 cm pots which were thinned out later into one plant per pot. five replicate plants were done for each treatment. plants were maintained under glasshouse conditions, watered with n-free nutrient solution (b&d nutrient solution) and sterile distilled water and were harvested after 12 weeks. surfaces of the pots were covered with sterile white beads, in which a pipe was used as opening for watering purposes. the holes of the pipes were covered with sterile aluminium foil to prevent cross-contamination. the data gathered include: shoot length, number of nodes, number of leaflets, number of nodules, root, shoot, nodule and total plant dry weights, nodule fresh weight (preserved), and nodule morphology. all data were statistically analysed and the symbiotic effectiveness was computed using the formula (bergersen, 1988): symbiotic effectiveness = dry weight (inoculated) x 100 dry weight (uninoculated) 276 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) time course of nodulation surface-sterilised seeds were sown into sterile vermiculite and inoculated with the best rhizobia isolates. an uninoculated control was also included. five replicates were used per treatment and the plants were uprooted at 4 to 8 weeks for data collection: nodule number, nodule fresh weight, shoot and root dry weights, total n level in dried shoots and roots, area of zone of infection using a dissecting microscope attached to nis-elements software program for image analysis and leghemoglobin concentration using spectrophotometry (bio-rad smartspec™ 3000 spectrophotometer, usa, 2014). assessment of symbiotic nitrogen fixation plants inoculated with the best isolates were subjected to acetylene reduction assay (ara) to assess the nitrogen fixation activity in the nodulated roots and compared to soybeans. the root system was carefully shaken to remove the vermiculite and were cut and quickly transferred into sealed bottle containers with suba-seals (sigma-aldrich, australia). the 10% of total gas volume was removed and replaced with 10% acetylene. at 0 and 60 minutes, the gas samples were obtained by a syringe and were directly assayed to gas chromatography (injected into a shimadzu gc-17a gas chromatograph (fid detector, 2 m self-packed porapak n 60 column) set with a column oven temperature as 90 °c, detector and injection chamber at 110 °c and gas pressure 90 kpa.). the short assay time was used to avoid root cutting associated drop of measurable ara. meanwhile, the nitrogen difference method was also employed to estimate the amount of fixed nitrogen in all dried plants inoculated with rhiziobia and the reference treatments. characterisation of rhizobia the isolates were morphologically characterised based on their colony size in diameter, colour, shape, margin, elevation and texture. growth curves in liquid culture of all isolates were also determined. one millilitre of pure culture was inoculated to 99 ml of yeast extract mannitol broth and incubated with shaking at 28°c. at 0, 18, 24, 45, 48, 68 and 72-hour incubation, 0.1 ml of the culture suspension was diluted to 10-6 to 10-8 using sterile dilution blanks and plated on ym agar at 28°c. viable count was determined after 5-10 days of incubation. growth curves were plotted based on the viable count for each incubation time. the antibiotic resistance of each isolate was also determined. isolates were grown in ym broth (ymb) with shaking until the titre of about 108 cells/ml was achieved. a dilution of 10-6 was made and then plated into yema+cr containing different antibiotics. after 6-12 days at 28°c, viable count of the isolates was determined implicating their antibiotic resistance or sensitivity, while their morphological characteristics on the media with antibiotics were also noted. the isolates were also grown in various growth conditions to determine their tolerance in different temperature (22°c, 37°c and 45°c), ph (4.0, 7.0 and 9.0), and salinity (0.1%, 0.5% and 1% (w/v) nacl). identification of the rhizobia strains rhizobia were grown in ymb in flasks and then transferred into 1.5 ml centrifuge tubes. tubes were centrifuged at 16,000 g for 10 min (using an eppendorf centrifuge 5415d, f45-24-11, germany). the resulting supernatant was decanted. the rhizobia were resuspended in 467 µl of 10 mmol/l tris-hcl, ph 8 and 1 mmol/l edta. then 30 µl of 10% sds and 3 µl of 20 mg/ml proteinase k was added into the tubes. tubes were incubated for 1 hour at 37°c. phenol:chloroform :isoamylalcohol (25:24:1; 500 µl) was added. tubes were centrifuged in an eppendorf centrifuge (5415 d) at 16,000 g for 2 min. sodium acetate (3 m, 0.1 ml, ph 5.2) and 0.6 ml of isopropanol were added to precipitate the dna. tubes were incubated at least 30 minutes on ice. the tubes were centrifuged in an eppendorf centrifuge (5415 d) for 15 minutes at 16,000 g. the dna pellet was washed in 500 µl of 70% ethanol. the tubes were centrifuged again for 15 minutes. the dna was dried and resuspended in 100 µl of sterile milliq water. a total of 100 µl of pcr cocktail was used with the following recipe: 200 µm each of datp, dctp, dgtp and dttp; 0.4 µm of each universal primer 27f: gagtttgatcctggctcag (dorsch and stackebrandt, 1992) and 1492r: tacggttaccttgttacgactt (goodfellow and stackebrandt, 1991); 1x dna polymerase and 50-500 ng of template dna and covered with 50 µl of sterile mineral oil. the pcr profile used was initial denaturation at 94°c for 2 min; denaturation at 94°c for 1 min at 40 cycles; annealing at 52°c for 30 seconds; extension at 72°c for 30 seconds and final extension for 2 min. five µl of each of the pcr products were loaded to the wells of a 1% agarose gel with 2 µl loading dye along with a 1 kb molecular ladder. the resulting molecular bands were isolated from the gel and sent to agrf (brisbane) for dna sequencing and identification. the sequences were aligned using muscle 3.8.31 and refined using gblocks 0.91b. the phylogeny was run using phyml 3.1/3.0 alrt and rendered using treedyn 198.3 (dereeper et al., 2008). results cotyledon nutrient carry-over all of the uninoculated treatments did not produce nodules. for the treatments without n-free nutrient solution added, either inoculated with cb1809 or not, the data between replicates had higher variation while replicates for treatments watered with nutrient solution had closer values. treatment with ammonium nitrate and with both cotyledons cut in halves had the longest average shoot length of 52 cm, while the same treatment but with intact cotyledons had the most number of nodes and leaflets, with mean values of about 8 and 16, respectively. see supplemental table s1. figure 1 shows the nodulation assessment of all the treatments which revealed that the highest number of nodules was observed in the inoculated treatments added with nutrient solution and with both cotyledons intact. both inoculated treatments were able to produce several nodules but higher number was 277 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) observed in the treatments with nutrient solution. there were also no huge differences in the plant structure, nodulation and growth conditions among treatment plants with intact cotyledons, one cotyledon removed and both cotyledons cut into halves. see supplemental figure s1. treatment plants with ammonium nitrate had the best growth and health conditions as evidenced by their dark green shoots. uninoculated plants without both cotyledons were able to survive after 16 weeks and showed differences from the rest of the treatments, as they had the poorest plant growth and symbiosis parameters, such as the lowest number of nodes, leaflets, shoot height, nodule number, fresh and dry nodule weight, and shoot and root dry weights. the appearance of visible difference in growth conditions among the inoculated, uninoculated, with or without nutrient solution and with nitrogen treatment plants had been obvious earlier in plants with both cotyledons halved. growth variations were noted in just 3-4 weeks after inoculation while variations for other plants (either both cotyledons intact, one removed or both cotyledons removed), were observed at later stages of around 7-8 weeks after inoculation or at harvesting. isolation of rhizobia a total of 21 potential rhizobia were successfully isolated from the different soil samples and pongamia nodules collected in queensland, australia (see supplemental table s2). meanwhile, supplemental table s3 shows the results of soil analysis for each soil sample. soil 1 and 3 were sandy while the rest were loamy. soil 1 (shale/sandstone) had the highest n level of 0.062 %wt, while soil 2 (farmhouse control site) had the lowest n level of 0.497 %wt. only soil 1 had a neutral ph while the 278 figure 1. nodule number vs the five different treatments: inoculated with cb1809 but no nutrient solution added; uninoculated and no nutrient solution added; inoculated with cb1809 with nutrient solution (b&d n-free solution); uninoculated but with nutrient solution; and, uninoculated but with nitrogen (2 mm ammonium nitrate solution) and nutrient solution. figure 2. nodulation of pongamia pinnata plants. uninoculated plants (a) were observed to grow poorly having yellowish leaves and stunted growth without nodules (b). inoculated plants (c) using the isolates from baiting techniques grew better with nodulated roots (d). rest were slightly acidic. the mineral levels in soil 1 were mostly higher than the rest of the soil samples, while soil 4 and 6 had average levels. nodulation and symbiotic effectiveness uninoculated plants (control) grew poorly compared with inoculated plants which had higher shoot and root dry weights (see supplemental table s4). no nodules were found on the uninoculated plants which confirmed their ‘no contamination’ status. inoculated plants showed the following: (i) pr-uq-01 had the highest plant dry weight with 2nd largest nodule number; (ii) pruq-03 had highest shoot length, 2nd highest shoot dry weight among the nodule isolates and 2nd highest n content; (iii) pruq-04 had the largest nodule number among the soil isolates (figure 2) and (iv) pr-uq-05 had the highest nodule number among the nodule isolates. these four strains were therefore selected for further studies. time course of nodulation all control plants had no nodules from week 4 to 8 as shown in supplemental table s5. total plant dry weight increased from 1.23 g at week 4 up to 2.11 g in week 8. the total n level in shoots and roots increased from 5.12% wt at week 4 to 6.22% wt at week 7 but decreased to 5.89% wt on week 8. pruq-03 plants nodulated at week 4 with 38 nodules per plant and continue to produce nodules until week 8 with 80 nodules per plant. the total plant dry weight increased from week 4 at 1.859 g to week 6 at 2.406 g but almost without any changes at weeks 7 and 8. leghemoglobin concentration at week 4 was 0.132 mg/ml and increased to 0.640 mg/ml in week 8 (see figure 3). the area of zone of infection of the biggest active nodule was 1.58 mm2 at week 4 and continued to increase its a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 279 figure 3. leghemoglobin content (mg/ml) versus the absorbance readings at 600 nm of the standard haemoglobin, pr-uq-03 nodules and pr-uq-05 nodules from week 4 to week 8. area up to 3.89 mm2 at week 8. the total n level in shoots and roots was 6.56% wt at week 4 and no significant changes until week 8. on the other hand, pr-uq-05 plants produced 56 nodules per plant at week 4 which continued to increase until week 8 producing 78 nodules per plant. the total plant dry weight also doubled from 1.139 g in week 4 up to 2.283 g at week 8. leghemoglobin concentration increased from week 4 at 0.235 mg/ml to week 8 at 0.663 mg/ml. the area of zone of infection of its biggest nodule started at 1.86 mm2 at week 4 and increased to 3.08 mm2 at week 8. the total n level in shoots and roots was 4.30% wt at week 4 and continued to increase at week 8 measuring 5.12% wt. the inoculation of pr-uq-05 showed significant increase in nodule number, total plant dry weight, leghemoglobin concentration, area of zone of infection and total n level in shoots and roots from week 4 to week 8 while inoculation with pr-uq-03 increased nodule number, leghemoglobin concentration and area of zone of infection from week to week 8 and no significant increase for total plant dry weight and total n level in shoots and roots over the weeks. assessment of nitrogenase activity all nodulated pongamia and soybean (as control) plants inoculated with the best isolates were observed to have nitrogenase activities in their nodules as implicated in the increased acetylene-dependent ethylene production from 0 to 60 minutes sampling time incubated at room temperature as shown in table 1. in contrast, the non-nodulated, uninoculated controls had zero to almost negligible values. pongamia plants were observed to have higher nitrogen fixing (i.e., acetylene reduction) activity compared with soybeans when inoculated with pr-uq-05, but show no significant difference when inoculated with pr-uq-03. the n difference method further confirmed that the new in0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 ab so rb an ce r ea di ng s at 6 00 nm leghemoglobin concentration (mg/ml) standards pr-uq-03 pr-uq-05 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 280 table 1. volume of ethylene (ml) produced by pongamia and soybeans inoculated with pr-uq-03 vs pr-uq-05 with uninoculated control after 60-minute incubation at room temperature. values shown were the average of three replicates used per treatment. oculant isolates pr-uq-03 and pr-uq-05 aid in the nitrogen fixation of pongamia as seen in the increase of n level content in the shoots and roots of pongamia seedlings in a 12-week growth period (see table 2). inoculated plants had higher n level content in shoots and root compared with uninoculated control and cb564. consistently, pongamia plants inoculated with pr-uq-05 had the highest nodule number, nodule fresh weight and nitrogen fixation activity. characterisation of the ‘best’ rhizobial inoculant the selected rhizobia pr-uq-01 and pr-uq-03 were able to utilise mannitol as the only carbon source. pr-uq-03 grew within 5 days of incubation, thus considered a “fast grower”, while pr-uq-01 appeared within 6-10 days of incubation making it a “slow grower”. suppplemental table s2 summarises the characteristics of all the 21 isolates on yema+cr and with six different types of antibiotics (10 mg/l in media). the rhizobia pr-uq-03 and pr-uq-05 were selected as “best” among the 21 isolates due to their symbiotic performance and being the fast-growers. both strains were grown into yeast extract mannitol agar with congo red with varying incubation temperatures (22°c, 37°c, and 45°c); different ph (4, 7 and 9) and on different nacl concentration (0.1%, 0.5% and 1%). both pr-uq-03 and pr-uq-05 can grow on ph 7.0 media only. also both strains were able to survive at 22°c incubation, but did not grow at 37°c and 45°c. both pr-uq-03 and pr-uq-05 were able to grow on 0.1% and 0.5% nacl supplementation treatments shoot nitrogen content (wt %) root nitrogen content (wt %) total nitrogen content (wt %) n fixed* (wt%) total plant dry weight (grams) n fixed/g dry wt uninoculated control 1.24+0.05 c 1.18+0.06 c 2.42+0.10 b 0 b 1.991 0 cb564 2.87+0.30 ab 2.2+0.26 ab 5.07+0.53 a 2.65 a 2.157 1.229 pr-uq-03 3.55+0.16 a 2.59+0.18 a 6.15+0.32 a 3.73 a 2.452 1.521 pr-uq-05 3.46+0.47 a 2.56+0.42 a 6.02+0.88 a 3.6 a 2.777 1.296 * values followed by the same letter are not significantly different at 5% significance level. shown are the mean values and se. legend: yellow highlights – high values; blue highlights – low values. *n fixed (wt%/g dw) = n yield by inoculated plant (wt%) – n yield by uninoculatedplants (wt%). table 2. the nitrogen difference method to estimate fixed nitrogen of pongamia pinnata inoculated with the isolates compared with cb564 and uninoculated control for 12-week growth period under glasshouse conditions. of the medium. both rhizobia isolates did not grow on 1% nacl supplementation. the size of the colonies also observed to vary among strains grown on the different media. at ph7.0 and 0.1% nacl supplemented media incubated at 28°c, colonies of pr-uq-05 were bigger in size (1-2 mm diameter) compared to colonies on 0.5% nacl media (at 28°c) and on normal media at 22°c incubation. the number of colonies of pr-uq-05 at 22°c was also decreased by 25%. the cold incubation affects the growth of pr-uq-05 but did not totally inhibit the rhizobia growth. pruq-03 colonies were lesser in number but definitely bigger in size (3 mm diameter) except those that grew on 0.5% nacl (1-2 mm diam) compared with pr-uq-05. no growth was observed in all control plates (inoculated with sterile distilled water only). supplemental table s6 shows the characteristics of the selected rhizobia pr-uq-03 and pr-uq-05. identification and molecular characterisation of the best rhizobia dna was extracted successfully from the four selected rhizobia strains: pr-uq-01, pr-uq-03, pr-ur-04 and pr-uq-05 and was subjected to pcr using primers 27f and 1492r. results showed that 16s rdna of the four isolates was amplified which was further used for dna sequencing. see supplemental figure s2. the predicted molecular band (1,250 bp) from the pcr gel was extracted and purified. another gel electrophoresis using the purified pcr products was done to confirm one single treatments volume of ethylene (ml)/plant number of nodules/plant nodule fresh weight (g)/plant pr-uq-03 – soybeans 0.21+0.01 ns 14+2 c 0.01+0 c pr-uq-03 – pongamia 0.30+0 ns 65+2.03 b 0.50+0.01 b pr-uq-05 – soybeans 0.06+0.03 ns 17+7.36 c 0.02+0.01 c pr-uq-05 pongamia 2.76+1.18 ns 86+3.06 a 0.868+0.03 a * values followed by the same letter are not significantly different at 5% significance level. ns= not significantly different(standard error). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 281 band amplified. all four rhizobia strains produced only a single molecular band; thus, the pcr products were purified. restriction enzyme digestion using the three enzymes-acli, dpniii and haeiii, was done to confirm that the four rhizobia strains belong to different genera. results showed that the pattern of band fragments differed among the four strains implicating that they belong to different genera. purified pcr products were then sequenced at agrf (brisbane, australia) and the resulting sequences were subjected to blast and phylogenetic analysis. blast results: (e values=0) revealed that pr-uq-01 is bradyrhizobium elkanii, pr-uq-03 is rhizobium mesoamericanum, pruq-04 is bradyrhizobium elkanii and b. pachyrizi and pr-uq-05 is rhizobium mesoamericanum (all at 99% certainty). comparison of sequences using blast alignment between pr-uq-01 and 04 as well as pr-uq-03 and 05 revealed that these two sets of strains were highly similar (>99%), although the restriction nuclease digestion pattern and symbiotic tests results differ among them. figure 4 shows the phylogenetic analysis of the four selected isolates compared with other rhizobia strains which revealed that both pr-uq-01 and pr-uq-04 were closely related to bradyrhizobium elkanii and b. pachyrizi with bootstrap value of 94 while pr-uq-03 and pr-uq-05 were both closely related to rhizobium mesoamericanum with a boostrap value of 89. figure 4. phylogenetic relationship among the four rhizobial isolates nodulating pongamia (dereeper et al., 2008). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 282 discussion the basis of all the experimental setups in this study was derived from the cotyledon experiment which was performed to determine the role of cotyledon in nodulation and growth of pongamia seedlings and to avoid the carry-over of nutrient from the cotyledon to the plants which might affect the results of the succeeding experiments. cotyledon experiment had shown that all of the uninoculated treatments did not produce nodules which implied good glasshouse maintenance. the addition of nutrient solution lessens the variation between replicate plants in each treatment. both inoculated treatments were able to produce several nodules but higher number was observed in the treatments with nutrient solution. hence, the addition of nutrient solution at the described rate (weak in normal horticulture) improved the nodulation of pongamia. the appearance of visible difference in growth conditions among the inoculated, uninoculated, with or without nutrient solution and with nitrogen treatment plants had been obvious earlier in plants with both cotyledons halved. growth variations were noted in just 3-4 weeks after inoculation while variations for other plants (either both cotyledons intact, one removed or both cotyledons removed), were observed at later stages of around 7-8 weeks after inoculation or at harvesting. this implied that the cotyledons affect the nodulation and growth of pongamia. by cutting both cotyledons into halves, the nutrient carry-over will be controlled and thus the variation of result can be now attributed to the different treatments and not to nutrients in cotyledon. therefore, the cotyledon factor will now be eliminated in future studies by simply cutting the cotyledons into halves. moreover, the pongamia cotyledon can also provide enough nutrients for the plants to grow even up to 16 weeks with or without nutrient solution added. this study was able to successfully isolate a total of 21 potential rhizobia from the different soil samples in a mining site in kingaroy, queensland where pongamia had been grown to rehabilitate the area while the pongamia nodules used in the isolation were collected from botanical gardens in mt. coot-tha, queensland where pongamia were introduced several years ago. out of the six soil samples located in different areas in the mining sites, only two produced nodules in the baited pongamia seedlings which can be attributed to the presence of average level of nutrients (not too high or low), ph and loamy soil characteristics which can support rhizobia growth. the baiting experiment using these soil samples from mining sites further revealed that rhizobia that can nodulate pongamia are present in the soil with poor nutrient conditions and where pongamia are not previously grown. to determine the best strains, the 21 isolates from the baiting experiment and isolation from pongamia nodule were screened for strain symbiotic effectiveness by inoculating into pongamia under uniform growth conditions. four strains pr-uq-01, pruq-03, pr-uq-04 and pr-uq-05 showed promising results in the screening test but the fast growers pr-uq-03 and pr-uq-05 were selected and were used as inoculants for the other experiments beause pr-uq-03 had highest shoot length, 2nd highest shoot dry weight among the nodule isolates and 2nd highest n content while pr-uq-05 had the highest nodule number among the nodule isolates. in the time course of nodulation experiment, the inoculation of pr-uq-05 showed significant increase in nodule number, total plant dry weight, leghemoglobin concentration, area of zone of infection and total n level in shoots and roots from week 4 to week 8 while inoculation with pr-uq-03 increased nodule number, leghemoglobin concentration and area of zone of infection from week 4 to week 8 and no significant increase for total plant dry weight and total n level in shoots and roots over the weeks. this implied that both strains improved nodulation in pongamia. this results is further supported by the acetylene reduction assay and n difference method which revealed that nodulated pongamia inoculated with pr-uq-03 and pr-uq-05 were observed to have nitrogenase activities in their nodules. the nitrogen fixation present in the nodules of the inoculated plants contributed to the better growth conditions and nodulation of these plants. pongamia plants were observed to have higher nitrogen fixing (i.e., acetylene reduction) activity compared with soybeans when inoculated with pr-uq-05, but show no significant difference when inoculated with pr-uq-03. this means that pr-uq-03 and pr-uq-05 have low symbiotic effectiveness with soybeans as soybeans are known to be strain specific legumes while pongamia on the other hand, is known to be promiscuous legume and can be nodulated with any species of rhizobia. the n difference method further confirmed that the new inoculant isolates pr-uq-03 and pr-uq-05 aid in the nitrogen fixation of pongamia as seen in the increase of n level content in the shoots and roots of pongamia seedlings in a 12-week growth period. pr-uq-03 and pr-uq-05 were both isolated from pongamia nodules. pr-uq-03 had 2-3 mm diam colonies which were white/creamy, high convex and slimy while pr-uq-05 had 3-4 mm diam colonies with white but prolong incubation observed to produced red center, high convex and very slimy. both utilised mannitol as the only carbon source, “fast growers”, had optimum growth at ph 7.0, temperature of 28°c although it can survive at 22°c, and in yeast extract mannitol agar with 0.1% to 0.5% nacl. both strains were resistant to amp, spec and tet. 16s rdna amplification of both strains yielded 1250 bp using primers 27f and 1492r. blast results: (e values=0) revealed that both pr-uq-03 and pr-uq-05 are related to rhizobium mesoamericanum (all at 99% certainty). comparison of sequences using blast pr-uq-03 and 05 revealed that these strains were highly similar (>99%), although the restriction nuclease digestion pattern and symbiotic tests results differ between them. phylogenetic analysis revealed that both pr-uq-03 and pr-uq-05 were both closely related to rhizobium mesoamericanum with a boostrap value of 89. conclusions and future work pongamia plants can fix nitrogen from their efficient symbiosis with either fast or slow-growing rhizobia with nodules starting to form within 2-3 weeks after inoculation and continuing throughout the tested 10-12 weeks growth period. cutting both cotyledons in halves had little effect on shoot dry weight, nodule number and weight per plant at 8 weeks, but were reduced when both cotyledons were removed and plants were visibly yellow. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 283 by cutting both cotyledons into halves, the nutrient carry-over will be controlled and thus the variation of result can be now attributed to the different treatments and not to nutrients in cotyledon. therefore, the cotyledon factor will now be eliminated in future studies by simply cutting the cotyledons into halves. surprisingly when no nutrients were supplied and with both cotyledons intact, plants grew as well as those with nutrients. cotyledons supply the seedling with nutrients for a considerable period of early growth (8 to 16 weeks after germination), while symbiotic nitrogen fixation supports pongamia on its later growth and eventual reproduction (flowering and seed formation). the fast growers, rhizobium-related strains pr-uq-03 and pr-uq-05, isolated from pongamia nodules are likely the best rhizobia strains among the 21 isolates with confirmed nodulation and symbiotic effectiveness in pongamia and with an established active nitrogenase activity. further study is still recommended to test the survival of these rhizobia strains in the field to be used in the improvement of plant performance in soil where native rhizobia nodulating pongamia are sparse or absent. abbreviations pr-uq: pongamia rhizobia-university of queensland cb: csiro brisbane usda: united states department of agriculture cot: cotyledon b&d: broughton and dilworth yema: yeast extract mannitol agar ymb: yeast extract mannitol broth cr: congo red ara: acetylene reduction assay competing interests the author declares that they have no competing interests. authors contributions pnc conducted experiments, evaluated results and wrote the manuscript. ai, ps, pd and pmg contributed to the conception, interpretation and supervision of the 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performance of lambs. animal feed science and technology 126: 157-167. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 285 nemenzo-calica et al., 2016 – supplementary data table s1. effect of cotyledon in nodulation of pongamia pinnata seedlings under glasshouse conditions for 16 weeks growth period. shown are the mean values and standard errors for the two replicates used in each treatment. legend: 0cot = both cotyledons were removed; 1cot = one cotyledon removed; 2cot = both cotyledons intact; 1/2 cot = both cotyledons cut into halves. treatments shoot length (cm) # of nodes # of leaflets nodule number nodule fresh weight (g) shoot dry weight (g) root dry weight (g) nodule dry weight (g) total plant dry weight (g) + cb1809 and –nutrients 2cot 41+2 6+1 10+1 123+78 1.00+0.4 2.70+0.8 1.72+0.3 0.15 4.57+1.1 1cot 25+2 6+1 6+1 94+29 0.41+0.2 1.23+0.1 0.93+0.1 0.06 2.22+0.2 1/2cot 29+2 7+2 11+2 93+20 0.97+0.4 1.57+0.1 1.27+0.2 0.13 2.97+0.3 0cot 16+1 3 3 34+10 0.15 0.39 0.19 0.02 0.60 -cb1809 and –nutrients 2cot 37+1 5+1 7+1 0 0 2.75 1.70+0.7 0 4.45+0.7 1cot 26+4 3+1 3+1 0 0 1.13+0.3 0.58+0.2 0 1.72+0.4 1/2cot 35+3 6+1 8+2 0 0 2.13+0.2 1.51+0.1 0 3.64+0.3 0cot 25 5+1 6+2 0 0 0.94 0.50 0 1.44 + cb1809 and +nutrients 2cot 42+3 8 15 142+9 0.87+0.1 2.13+0.1 0.62 0.21 2.96+0.2 1cot 39+2 5+1 10+1 116+4 0.89+0.1 1.56+0.1 0.50 0.18 2.24 1/2cot 37+2 5+1 11+1 133+13 0.73 1.25+0.1 1.25+0.1 0.17+0.5 2.68+0.8 0cot 31 3+1 7+1 83+3 0.45 0.73 0.26 0.01 1.04+0.1 -cb1809 and +nutrients 2cot 41+1 6+1 14+1 0 0 2.79 0.94+0.1 0 3.73+0.1 1cot 37+2 6+1 9+2 0 0 1.13+0.3 0.48+0.1 0 1.62+0.2 1/2cot 37+4 5+1 8+2 0 0 1.22+0.3 0.65+0.2 0 1.87+0.5 0cot 35+1 4+1 10+2 0 0 0.71+0.2 0.26+0.1 0 0.97+0.2 -cb1809, +nutrients and +n 2cot 43 8+1 16+1 0 0 2.66+0.1 1.00 0 3.65+0.1 1cot 35+3 7 10 0 0 2.86+0.5 0.96+0.3 0 3.81+0.8 1/2cot 52+2 8 15 0 0 1.70 1.13+0.1 0 2.82+0.1 0cot 38+1 6+1 12+2 0 0 1.37+0.2 0.53 0 1.90+0.2 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) code origin morphological/physiological characteristics pr-uq-01 meandu mine soil 6 slow-grower; resistant to amp, spec, tet & kan; 1-1.5 mm diam; whitish-pinkish; flat; circular; slimy pr-uq-02 pongamia nodule (mt. coot-tha) fast-grower; resistant to amp and tet; 1mm diam; white; circular; low convex; slimy pr-uq-03 pongamia nodule (mt. coot-tha) fast-grower; resistant to amp, spec & tet; 2-4 mm diam; white; high convex; very slimy pr-uq-04 meandu mine soil 4 slow-grower; resistant to amp, spec & tet; 1-1.5 mm diam; whitish-pinkish; flat; circular; slimy pr-uq-05 pongamia nodule (mt. coot-tha) fast-grower; resistant to amp, spec & tet; 2-4 mm diam; white; high convex; very slimy pr-uq-06 meandu mine soil 4 fast-grower; resistant to amp, spec & tet; 1 mm diam; whitepinkish; low convex; circular; slimy pr-uq-07 meandu mine soil 6 fast-grower; resistant to amp, spec & tet; 2.5-3 mm diam; pinkish-reddish; flat to low convex; irregular shape; very slimy pr-uq-08 meandu mine soil 6 slow-grower; resistant to amp & tet; 0.5 mm diam; whitepinkish; flat to low convex; circular; slimy pr-uq-09 pongamia nodule (mt. coot-tha) fast-grower; resistant to rif and amp; 3-4 mm diam; white; high convex; circular; very slimy pr-uq-10 pongamia nodule (mt. coot-tha) fast-grower; susceptible to all tested antibiotics; 3-4 mm diam; semi-transluscent; flat; circular; slimy pr-uq-11 pongamia nodule (mt. coot-tha) fast-grower; resistant to rif, amp, str, spec, tet and kan; 2 mm diam; white; flat; circular; slimy pr-uq-12 pongamia nodule (mt. coot-tha) fast-grower; resistant to rif, amp, str, spec, tet and kan; 3-4 mm diam; reddish; low convex; circular; slimy pr-uq-13 pongamia nodule (mt. coot-tha) fast-grower; resistant to rif, amp, str, spec and tet; 1 mm diam; white; flat to low convex; circular; slimy pr-uq-14 pongamia nodule (mt. coot-tha) fast-grower; resistant to rif and amp; 3-4 mm diam; white; high convex; circular; very slimy pr-uq-15 pongamia nodule (mt. coot-tha) fast-grower; resistant to rif, amp, str, spec and kan; 2mm diam; white; low convex; circular; slimy pr-uq-16 pongamia nodule (mt. coot-tha) fast-grower; resistant to rif, amp, str, spec, tet and kan; 3-4 mm diam; opalescent; flat; circular to irregular; slimy pr-uq-17 pongamia nodule (mt. coot-tha) fast-grower; resistant to rif, amp, str and kan; 3-4 mm diam; reddish; irregula; flat; slimy pr-uq-18 pongamia nodule (mt. coot-tha) slow-grower; resistant to rif, amp, str, spec and tet; 1 mm diam; white; circular; flat/low convex; slimy pr-uq-19 pongamia nodule (mt. coot-tha) slow-grower; resistant to rif, amp, str, spec and tet; 1 mm diam; white; circular; flat/low convex; slimy pr-uq-20 pongamia nodule (mt. coot-tha) slow-grower; resistant to rif, amp, str, spec and tet; 1 mm diam; white; circular; flat/low convex; slimy pr-uq-21 pongamia nodule (mt. coot-tha) slow-grower; resistant to rif, amp, str, spec and tet; 1 mm diam; white; circular; flat/low convex; slimy table s2. putative rhizobia isolated from different soil samples and pongamia nodules. 286 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 287 table s3. the physical property, ph, n level and mineral content of the soil samples. parameters mm soil 1 mm soil 2 mm soil 3 mm soil 4 mm soil 5 mm soil 6 source kingaroy 2 east shale/ sandstone farmhouse control site d3 southwest control site d5 southwest spoil southwest topsoil kingaroy 2 east topsoil physical property sandy loamy sandy/ loamy loamy loamy loamy ph 7.01 5.07 5.15 5.24 4.97 5.18 n level wt% 0.062 0.497 0.238 0.262 0.179 0.101 phosphorus [olsen]* (ppm) 3 46 37 9 14 10 potassium [am. acet.] (meq/100g) 0.21 0.43 0.46 0.23 0.39 0.30 calcium [am. acet.] (meq/100g) 0.92 5.92 3.66 1.67 1.35 3.69 magnesium [am. acet.] (meq/100g) 4.59 1.62 1.17 3.50 1.56 1.91 sulphur [mcp]* (ppm) 376 30 13 74 24 15 boron [cacl2]* (ppm) 0.3 0.7 0.4 0.4 0.5 0.5 copper [dtpa] (ppm) 1.1 0.7 0.3 1.1 0.6 0.2 iron [dtpa] (ppm) 33 71 54 69 124 66 manganese [dtpa] (ppm) 8.7 25.9 6.2 13.3 6.9 4.8 zinc [dtpa] (ppm) 3.7 7.1 1.8 4.3 0.8 0.4 sodium [am. acet.] (meq/100g) 2.0 <0.1 0.1 1.2 0.4 0.2 aluminum [kcl] (meq/100g) 0.15 0.44 0.39 0.26 1.05 0.76 al base saturation (%) 1.9 5.2 6.7 3.8 22.2 11.1 aluminum (ppm) 14 40 35 23 95 68 sodium (ppm) 449 <18.4 25 285 87 37 calcium (ppm) 184 1184 732 334 270 738 magnesium (ppm) 551 194 140 420 187 229 potassium (ppm) 82 168 179 90 152 117 legend: yellow highlights – high values; blue highlights – low values. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 288 treatments shoot length (cm) # of nodes # of leaves # of nodules dry weights (grams) shoot root nodule total plant control 28+2 5+1 8+1 0 1.26+0.2 0.73+0.1 0 2.00+0.2 cb1809 42+8 8 15 142+9 2.13+0.2 0.62 0.21 2.75+0.2 usda110 40+4 5+1 9+1 81+13 2.12+0.4 0.70+0.1 0.16 2.98+0.3 cb564 32+3 6+1 13+2 63+24 1.35+0.3 0.81+0.2 0.10 2.16+0.5 pr-uq-01 47+1 6 13+1 148+18 2.65+0.2 0.80+0.1 0.28+0.1 3.73+0.4 pr-uq-02 33+2 7+1 14+2 71+7 1.47+0.1 0.99+0.2 0.16 2.45+0.3 pr-uq-03 42+6 6+1 13+2 84+19 2.02+0.5 0.94+0.2 0.16 2.96+0.7 pr-uq-04 43+3 5 13+2 152+19 2.10+0.1 0.67 0.21 2.78+0.1 pr-uq-05 36+2 5+1 11+2 143+29 1.33+0.1 0.76+0.1 0.15 2.09+0.2 pr-uq-06 44+2 6+1 15+2 99+17 2.07+0.3 0.70+0.1 0.27 3.05+0.3 pr-uq-07 41+1 6+1 13 118+12 2.12+0.2 0.78+0.1 0.22 2.98+0.1 pr-uq-08 42+3 6 14+2 127+15 2.03+0.2 0.91+0.1 0.24 3.18+0.3 pr-uq-09 34+2 6+1 15+2 61+26 1.56+0.2 1.13+0.1 0.15+0.1 2.69+0.2 pr-uq-10 38+4 7+1 10+2 48+20 1.54+0.3 0.98+0.1 0.09 2.51+0.4 pr-uq-11 36+2 8+1 16+2 115+9 1.70+0.3 1.08+0.1 0.15 2.77+0.3 pr-uq-12 28+2 5+1 11+2 35+15 0.98+0.1 0.76 0.05 1.74+0.2 pr-uq-13 39+1 7+1 15+1 50+8 2.05+0.2 1.19 0.13 3.24+0.2 pr-uq-14 39+2 8+1 16+2 66+6 1.97+0.2 1.05 0.14 3.02+0.2 pr-uq-15 41+3 7 16 81+16 1.79+0.2 0.82+0.1 0.16+0.1 2.61+0.2 pr-uq-16 29+2 6+1 10+1 25+12 0.81+0.2 0.46+0.2 0.05 1.27+0.4 pr-uq-17 34+4 6 10+1 25+15 0.83+0.2 0.23 0.04 1.06+0.2 pr-uq-18 30+3 6+1 13+1 19+12 0.93+0.2 0.35+0.1 0.03 1.28+0.3 pr-uq-19 31+3 7 11+1 44+9 1.18+0.2 0.71+0.1 0.09 1.89+0.4 pr-uq-20 29+4 6+1 11+1 54+6 0.85+0.2 0.44+0.1 0.07 1.29+0.3 pr-uq-21 30+4 6+1 10+1 32+11 0.83+0.1 0.37+0.1 0.04 1.20+0.2 shown are the mean values (with se) for all the five replicates used in each treatment. legend: yellow highlights – high values; blue highlights – low values. table s4. strain symbiotic effectiveness of the 21 isolates compared with usda110, cb1809, cb564 and uninoculated control with pongamia pinnata at 12-week growth period under glasshouse conditions. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 289 tr ea tm en ts n o d u le # n o d u le fr es h w t (g ) r o o t d w ( g) sh o o t d w ( g) to ta l pl an t d w ( g) a bs o r ba n c e (6 00 nm ) le g h em o g lo bi n (m g/ m l) r o o t n le v el (w t % ) sh o o t n le v el (w t % ) to ta l n le ve l (w t % ) a re a of z on e of in fe ct io n of l ar ge st n od ul e (m m 2 ) c o n tr o l w ee k 4 0 0 0. 48 8 0. 74 2 1. 23 0 0 2. 13 2. 99 5. 12 0 c o n tr o l w ee k 5 0 0 0. 60 5 1. 15 9 1. 76 4 0 0 2. 40 3. 51 5. 91 0 c o n tr o l w ee k 6 0 0 0. 70 3 1. 21 9 1. 92 2 0 0 2. 23 3. 71 5. 94 0 c o n tr o l w ee k 7 0 0 1. 01 3 0. 99 7 2. 01 0 0 0 2. 63 3. 59 6. 22 0 c o n tr o l w ee k 8 0 0 0. 97 4 1. 13 8 2. 11 2 0 0 2. 59 3. 30 5. 89 0 pr -u q -0 3 w ee k 4 38 0. 28 9 0. 96 0 0. 89 9 1. 85 9 0. 06 4 0. 13 2 2. 80 3. 76 6. 56 1. 58 pr -u q -0 3 w ee k 5 53 0. 40 6 1. 13 0 0. 17 2 2. 38 2 0. 17 2 0. 44 2 3. 09 3. 76 6. 85 2. 07 pr -u q -0 3 w ee k 6 63 0. 49 7 1. 48 6 0. 92 2. 40 6 0. 20 5 0. 53 7 2. 96 3. 51 6. 47 2. 63 pr -u q -0 3 w ee k 7 80 0. 60 5 1. 02 8 1. 12 0 2. 14 8 0. 22 2 0. 58 6 2. 57 3. 51 6. 06 2. 81 pr -u q -0 3 w ee k 8 80 0. 72 2 0. 96 6 1. 12 8 2. 09 4 0. 24 1 0. 64 0 2. 48 3. 31 5. 79 3. 89 pr -u q -0 5 w ee k 4 56 0. 51 5 0. 47 1 0. 66 8 1. 13 9 0. 10 0 0. 23 5 2. 11 2. 19 4. 30 1. 86 pr -u q -0 5 w ee k 5 59 0. 54 6 0. 66 0. 95 6 1. 62 2 0. 13 2 0. 32 7 2. 43 3. 15 5. 58 2. 14 pr -u q -0 5 w ee k 6 73 0. 57 9 0. 86 2 0. 90 1 1. 76 4 0. 20 7 0. 54 3 2. 78 3. 29 6. 07 3. 11 pr -u q -0 5 w ee k 7 72 0. 74 4 0. 71 5 0. 55 8 1. 27 3 0. 23 1 0. 61 2 2. 16 2. 82 4. 99 2. 55 pr -u q -0 5 w ee k 8 78 0. 77 3 1. 00 4 1. 27 8 2. 28 3 0. 24 9 0. 66 3 2. 16 2. 96 5. 12 3. 08 ta bl e s5 . t im e co ur se o f n od ul at io n fo r p on ga m ia in oc ul at ed w ith th e be st rh iz ob ia p ru q -0 3 an d pr -u q -0 5 co m pa re d w ith u ni no cu la te d co nt ro l f ro m w ee k 4 to w ee k 8 gr ow th p er io ds u nd er g la ss ho us e co nd iti on s. va lu es a re a ve ra ge fo r fiv e re pl ic at es . a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 290 parameters pr-uq-03 pr-uq-05 origin pongamia nodule, botanical gardens, mt. coot-tha, brisbane, australia pongamia nodule, botanical gardens, mt. coot-tha, brisbane, australia colony morphology 2-3 mm diam; white; high convex; slimy 3-4 mm diam; white but later observed to have red center in longer incubation; high convex; excessively slimy nodulation positive positive ph 7 7 nacl tolerance/salinity 0.1 – 0.5 % 0.1 – 0.5 % temperature 22-28°c 22-28°c carbon utilisation mannitol mannitol antibiotic resistance resistant to amp, spec & tet resistant to amp, spec & tet turbidity in ym broth >72 hours <48 hours incubation 3 days <3 days table s6. characteristics of pr-uq-03 and pr-uq-05 based on various parameters. figure s1. pongamia seedlings at different treatments. all pongamia plants with both cotyledons removed (0cot) had consistent lowest shoot length, shoot dry weight, nodule number and nodule dry weight (yellow). uninoculated plants without nutrients but both cotyledons intact (2cot) grew as well as those with nutrients implicating that cotyledons supply the seedling with nutrients for a considerable period of early seedling growth. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 291 figure s2. amplified 16s rdna (1,250 bp) of the four best rhizobia isolates: lane 1 = negative control; lanes 2 and 3 = pr-uq-01; lanes 4 and 5 = pr-uq-03; lanes 6 and 7 = pr-uq-04; and, lanes 8 and 9 = pr-uq-05. (blue arrows indicate the predicted pcr product). atlas journal of biology 3 (1): 206–211, 2014 doi: 10.5147/ajb.2014.0134 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) snp-e: a new method for multiple sequence alignments analysis and accurate single nucleotide polymorphism evaluation melody n. hemmati-sholeh1, larry a. sholeh2, and david a. lightfoot1,* 1 genomics core facility; department of plant soil and agricultural systems, and the illinois soybean center, southern illinois university at carbondale, carbondale, il 62901, usa; 2 department of electrical and computer engineering, southern illinois university at carbondale, carbondale, il 62901, usa received: june 19, 2014 / accepted: september 5, 2014 __________________________________________________ * corresponding author: ga4082@siu.edu 206 abstract identification of single nucleotide polymorphisms (snps) and insertion-deletion mutations are important for discovering the connection between the genetic mutations and complex diseases. the objective of this study was to develop a sensitive and accurate computational method for snp detection among multiple sequence alignments (msas) to be run on microsoft office suitetm and windowstm. the snp-evaluator, was designed to simulate the process of human eye visual change-identification. analysis of three 82-kbp genomic loci derived from sanger sequencing and the corresponding snps from 31 genomes from illuminatm sequencing of soybean (glycine max l. merr.) demonstrated that the snp-e was an effective method for medium-scale genomic research. keywords: single nucleotide polymorphism (snp); sanger; illuminatm; cultivar; variation. introuction identification of single nucleotide polymorphisms (snps) in multiple sequence alignments (msa) involves looking across msa and identifying base discrepancies (wegrzyn et al., 2009). snps and mutations among highly polymorphic regions are often associated with useful traits such as resistance to disease (ruben et al., 2006; srour et al., 2012) and identification of them is important to discover the link between the genetic mutations and complex diseases (zhang et al., 2005). the genomes of crop plants, animals and humans contain regions of diversity much less than 98% identity among individuals interspersed in more conserved region. one such region is the rhg1/rfs2 locus of soybean (glycine max l. merr.) which appeared to span over 150kbp and encompassed more than 20 genes. various methods have been developed (chang, 2009) and already available for snp detection and calling single-nucleotide polymorphisms from next generation sequencing data (depristo et al., 2011; kobold et al., 2009; li et al., 2009; nijveen et al., 2013) however, most of these methods require expensive highdepth sequencing to perform satisfactory (xu et al., 2012) or they are java-based programs such as seq-snping (chang et al., 2009) or they require linux command line skills to run and a separate program to visualize the results (nijveen et al., 2013) or requires phred commands in case of pinesap. so, there was a need for a method to efficiently and accurately call, analyze and identify snps in msas for medium scale loci, ~ 100 kbp. the method needed to be run on a popular and user friendly system such as microsoft office suitetm to prevent the need for complex and costly operating system such as unix. snp-evaluator was created and demonstrated that can accurately align relatively diverse sequences and allow researchers to identify this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 207 snps of interest for further analyses. snp-e, has the ability to search among msa and recognize base polymorphisms. it is capable of making a confident identification (base-call) because visual confirmation is not a reliable option for sequences from next generation sequencer with higher number of sequences at the cost of higher error rates and patchy sequence coverage. also, this method needed to give the young researchers who are still enhancing their skills in different programming languages and would like to work with medium-scale genomic sequences, more flexibility for next generation data analysis. furthermore, by applying this method, researchers have the ability to associate a specific alignment nucleotide identification number to each nucleotide (nucleotide’s id), customize data analysis and apply functions and formulas on data (nucleotides). moreover, this method functions as a visible tool due to its table format that can provide snp-e the capability to simulate the process of human eye visual change-identification. materials and methods the genomic dna region used here was the 82kbp isolated and embedded in a bac, b73p06, which encompassed 10 genes and one highly polymorphic region of about 59 kbp (743 snps from 1,500 bp to 60,500 bp). a multiple sequence alignment between rhg1/rfs2 locus on chromosome 18, of three sequences from soybean cultivars; ‘forrest’ bac b73p06 (hemmati and lightfoot, 2011), asgrow 3244 and williams 82 (srour et al., 2012) was performed through ncbi-msa. the out-put of ncbi-msa in fasta format by flat query anchored with dots for identities was used as raw data. exceltm applications was used as a sufficient interface to communicate data, snps, and snps-detection. snp-e design phases designing snp-e consisted of 3 major phases. first phase was data migration of msa-fasta-format to column-alignmentformat (caf), second phase was data conversion of columnalignment-format (caf) to vertical-alignment-format (vaf) and the third phase was single nucleotide polymorphism evaluation, snp-e. data migration of msa-fasta-format to column alignment format (caf) to convert this raw data to excel applicable format, data migration of fasta format sequence of multiple sequence alignments from a horizontal alignment text format to excel vertical alignment format was needed. a cascade conversion process was applied to convert entire msa-fasta text format to excel format, column. text to column function was applied to entire msa-fasta. up to this stage a 3×4 of msa-excel format had been created. each ncbi-msa-fasta converted to msa-excel format consisted of column a indicating sequence id, column b indicating sequence starting numbers, column c containing 60 nucleotides of each sequence from each row with no assigned identification number to each nucleotide and finally, column d indicating sequence ending numbers (figure 1). data conversion of column alignment format (caf) to vertical alignment format (vaf) the greatest challenge at this stage was extraction of each sequence form the multiple sequence alignments while conserving alignments in excel format, vaf. in order to overcome this challenge, data filtering was applied to call for alreadyaligned-sequences; forrest, asgrow and williams 82 (w82) by calling their sequence id, query (forrest), 31218 (asgrow 3244) and 31219 (w82) accordingly. the purpose of filtering was to isolate each sequence from the fasta format alignment. then, each isolated sequence (asgrow 3244, w82 and forrest) was transferred to emeditor to be converted to a single vertical sequence in a column by applying a separation method such as line break or end-of-line (eol). next, each vertical isolated sequence (vis) was imported from emeditor to excel in a single column. and the final outcome of this process was a vertical alignment format (vaf). sequence numbering method (snm) and challenges the purpose of this step was to assign an ascending number (id) to each nucleotide from each sequence located at separate columns in excel. gaps were regions where the greatest challenge in snm occurred. to synchronize snm with sequence alignment gaps sag, a combination of logic functions i.e. “if”, were applied, in order to stop counting nucleotide numbering at gap regions and release counting at nucleotides regions. single nucleotide polymorphism evaluation, snp-e in order to identify snps among aligned sequences, a combination of logic functions i.e. “if” and “or” were applied. all snps were flagged in a separate column as the result of logic function, “0” for “false” and “1” for “true”. filtering on trues revealed snps for further analysis. the algorithm flowchart of snp-e was pictured in figure 2. the step by step guideline is also accessible through http://pbgc.siu.edu/docs/illustration. docx link. integration of 31 genomes snps the available snp motifs of 31 genomes by lam et al. (2010) which identified over 400 more potential snp positions in this region (lam et al., 2010) was compared to the results of snp-e from three analyzed sequences mentioned above (master-snps). the comparison demonstrated that the snp motif s could be added by reference to the bp position of williams 82. for those regions that misalignments had occurred, they were clear from the snp motifs disagreement and could be logically nudged to the nearest likely correct positions. once aligned the snps were de-convoluted to single columns each corresponding to a single genotype (hauge et al., 2006; lam et al., 2010). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 208 fi gu re 1 . t he fi rs t s te p of c on ve rt in g m sa -f a st a te xt f ro m at to e xc el fo rm at . a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 2. snp-e algorithm flow chart. 209 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 210 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) results and discussion snp-e results automated snp-e recorded 800 snps from polymorphic region (figure 3-panel a) of the alignment among forrest, asgrow 3244 and williams 82 genomic sequences (figure 3-panel b). of those, 263 were total nucleotide insertions. no, manual annotation was needed for snp evaluation at any stage of process. the results have been stored in snp-e-master-excel-file. results of integration of 31 genomes snps the results indicated that the master-snps file (snp-e-master-excel-file) was homogenous ascending sequentially with the 31-snps-genome-consensus alignments. the msa between snps results from snp-e-master-excel-file, and 31-columnsnps-sequence resulted in one aligned-master-snps and 31-column-snps-consensus-sequences. the final results showed that 78% snps aligned automatically for each of the 31-snpsgenomes and no manual annotation was involved. therefore, sanger sequence of the bacs and next gen sequence of genome inferred that there were more than 1,400 snps in the 80 kbp region introgressed from peking. 27.97 % of the indels aligned automatically for each of the 31-snps-genomes and no manual annotation was needed. sanger sequence has more strength in detecting indels than nextgen sequencing, so this was to be expected (chang et al., 2009; depristo et al., 2011; kobold et al., 2009; li et al., 2009; nijveen et al., 2013). the methods to compare sequences have to account for the sequence quality of the different available methods. abbreviations snp-e, single nucleotide polymorphism evaluation msas, multiple sequence alignments ncbi, national center for biotechnology information caf, column alignment format vaf, vertical alignment format eol, end of line vis, vertical isolated sequence snm, sequence numbering method sag, sequence alignment gap figure 3. examples of alignments in snp-e. panel a presents a small highly polymorphic region of the automated final alignment of forrest, asgrow 3244 and williams 82 genomic sequences. panel b presents a view of a typical region among the total number of snps. panel c presents a view of a typical region among the total number of indels. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) acknowledgements the physical map location of b73p06 was supported by the nsf under grant no. 9872635 and 0487654. any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the nsf. references chang h, l chuang, y cheng, c ho, c wen, c yang (2009) seq-snping: multiple-alignment tool for snp discovery, snp id identification, and rflp genotyping. omics 13:253-260. depristo m, e banks, kv garimella, j maguire, c hartl, a philippakis, g del angel, m rivas, mea hanna (2011) a framework for variation discovery and genotyping using next-generation dna sequencing data. nature genetics 43:491–498. hauge b, m wang, j parsons, l parnell (2006) methods of introgressing nucleic acid molecules associated with soybean cyst nematode resistance into soybean. us patent 7:154,021. hemmati m, d lightfoot (2011) glycine max cultivar forrest clone bac 73p06 genomic sequence. genbank: hq0089381. kobold td, k chen, t wylie, d larson, md mclellan, e mardis, g weinstock, r wilson, l ding (2009) varscan: variant detection in massively parallel sequencing of individual and pooled samples. bioinformatics 25:2283–2285. lam h, x xu, x liu, w chen, g yang, f wong, m li, w he, n qin, bea wang (2010) resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection. nature genetics 42:1053-1059. li h, b handsaker, a wysoker, t fennell, j ruan, n homer, g marth, g abecasis, r durbin (2009) the sequence alignment/map format and samtools. bioinformatics 25:2078 – 2079. nijveen h, m van kaauwen, d esselink, b hoegen, b vosman (2013) qualitysnpng: a user-friendly snp detection and visualization tool. nucleic acids research advance access:1-4. ruben e, a jamai, j afzal, v njiti, k triwitayakorn, m iqbal, s yaegashi, r bashir, s kazi, p arelli , c town, h ishihara, k meksem, d lightfoot (2006) genomic analysis of the rhg1/rfs2 locus: candidate genes that underlie soybean resistance to the cyst nematode. mol genet genom 276:503-516. srour a, a afzal, n saini, l blahut-beatty, n hemmati, d simmonds, h el shemy, c town, h sharma, d lightfoot (2012) the receptor like kinase transgene from the rhg1/rfs2 locus caused pleiotropic resistances to soybean cyst nematode and sudden death syndrome. bmc genomics 13:368. wegrzyn j, j lee, j liechty, d neale (2009) sequence analysis, pinesap—sequence alignment and snp identification pipeline. bioinformatics application note 25:2609-2610. xu f, w wang, p wang, m jun li, p chung sham, j wang (2012) a fast and accurate snp detection algorithm for next-generation sequencing data. nature communications 3:1258. zhang j, d wheeler, i yakub, s wei, r sood, w rowe, p liu, r gibbs, k buetow (2005) snpdetector: a software tool for sensitive and accurate snp detection. plos computational biology 1:395-404. 211 atlas journal of biology 2 (2): 116–124, 2012 doi: 10.5147/ajb.2012.0093 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a new strain of bacteria degrading tnt and 2,4/2,6-dnt from explosives contaminated soil jinsoo kim1, yong-kju yu2, fei yan3, john bang4, taek you5, and sang-seob lee1* 1 department of life science, kyonggi university, suwon, korea 442-760; 2 department of biological engineering, kyonggi university, suwon, korea 442-760; 3 department of chemistry, north carolina central university, nc, usa; 4 department of environmental, earth, and geospatial sciences, north carolina central university, durham, nc 27707, usa; 5 department of biological sciences, campbell university, buies creek, nc 27506, usa received: may 17, 2012 / accepted: june 17, 2012 __________________________________________________ * corresponding author: sslee@kyonggi.ac.kr 116 abstract the 2,4,6-trinitrotoluene (tnt), 2,4-dinitrotoluene (2,4-dnt), and toluene derived from a nitroaromatic compound(nac) cause high toxicity and mutagenicity to environment. one of best methods to remove their toxicity from contaminated soil is known biological remediation with soil bacteria. total of 235 strains of explosives-removing soil bacteria were isolated from the shooting gallery at a military base in korea. they were identified as acinetobacter, agrobacterium, alcaligenes, flavobacterium, klebsiella, pseudomonas, serratia, and citrobacter species. after further screened for the better removal efficiencies, kt22 identified as serratia sp., kd4 identified as klebsiella sp., and kd6 identified also as klebsiella sp. showed the highest removal efficiency for tnt, 2,4dnt, and 2,6-dnt, respectively. the optimal removal conditions were shown as follows: cell concentration 1 g/l, ph 7, and temperature 25-30°c. in a luria-bertani (lb) medium containing 100 mg/l of tnt, kt22 strain could remove tnt over 99% and showed a good biomass growth after 6 h of incubation. furthermore, when the kt22 strain was mixed with a bacillus sp., the mixed culture showed the improved tnt removal efficiency. conclusively, this new strain could be most effective to remove nac toxicity with rhizosphere remediation system on contaminated soil. introduction 2,4,6-trinitrotoluene (tnt) is a nitroaromatic compound (nac) commonly used in explosives, and 2,4-dinitrotoluene (2,4-dnt) and 2,6-dinitrotoluene (2,6-dnt) are two of the six dnt isomers that are used primarily as chemical intermediates in the production of toluene diamines and diisocyanates, dyes, explosives, and propellants. two products derived from tnt are 2,4-dinitrotoluene (dnt) and toluene (spanggord et al., 1991; duque et al., 1993). these toxic compounds are often the main contaminants of soil and groundwater at their manufacturing, processing, and disposal facilities (kalafut et al., 1998). especially, tnt has been widely used during and even after wwii, resulting in widespread soil contamination (scheibner et al., 1997), and was also used throughout the entire korean peninsula during the korean war. the high toxicity and mutagenicity of tnt and some of its metabolites have led to a harmful effect in their fates in the environment. numerous cases of munitions workers who have developed liver damage and anemia owing to the tnt exposure have been documented (voegtlin et al., 1919; hamilton, 1921; bridge et al., 1942; sax, 1963). in addition, tnt has been shown to have toxic effects in rats and mice (dale, 1921; channon et al., 1994), fish, algae, and oyster larvae (smock et al., 1976; won et al., 1976). the international agency for research on cancer (iarc) has also determined that 2,4and 2,6-dnt are possibly carcinogenic to humans. in addition, 2,6-dnt showed hepatocarcinogenic effects in fischer-344 rats, and exposure to a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 117 high levels of dnts in animals caused lowered numbers of sperm and reduced fertility (popp, 1983; richart, 1984). the most practical approach toward the remediation of explosives is currently soil incineration, but it can be a costly, energy-intensive process that destroys much of the soil, leaving ash as the primary residue with estimates approaching $800/ ton (funk et al., 1993). chemical treatment, on the other hand, requires the exercise of rigid controls to avoid the discharge of unreacted materials and is not practical in many situations. it is believed that biological remediation of explosives is the most economical and reliable method of response to this problem at a cost that ranges $30-$150/yd3 (montemagno and irvine, 1990; preslan et al., 1993). composting, one of the biological remediation methods applied to explosives, has been proven to be effective and is compatible with the incineration method (williams et al., 1992). bio-treatment with bacteria has focused on less expensive method of bioremediation, because many different species of bacteria potentially reduce nitro groups on the aromatic ring in soil with no requirement for additional nutrients. recently researchers have tried to hybrid more than two techniques to overcome weak points of each technique, and improve efficiency in the remediation fields. for instance, phytoremediation, which is a combined technique with bioremediation using soil bacteria and plant, was applied to remediate the contaminated soil. for nac group is fairly toxic and combined one is applied to various environmental conditions, bacteria which could degrade tnt under aerobic and/or anaerobic condition are required to grow well with other organisms. to develop an enhanced rhizosphere remediation system with nac removing soil bacteria, we were isolated and identified them in this study. then, they were further screened for high removal efficiencies toward tnt and 2,4/2,6-dnt, using batch tests. effects of pure vs. mixed culture with common soil microbes on the removal efficiencies were also evaluated. materials and methods isolation, cultivation, and identification of explosives-removing soil bacteria all the samples for the microbial isolation were obtained from the shooting gallery at a military base in kyonggi-province, korea. samples were obtained with the bottom soil, and the ph and temperature were 5.6, 9.5-10.50c, respectively. four different types of media, i.e., nutrient broth, medium 1, medium 2, and king’s b medium, were used as the growth medium for culturing tnt and 2,4/2,6-dnt removing bacteria. the composition of each medium was as follows per liter of deionized water. nutrient broth: bacto beef extract 3.0g, bacto peptone 5.0g, tnt 0.1 g; medium 1: k2hpo4 7.0 g, kh2po4 3.0 g, mgso4 0.1 g, nacl 0.1 g, nh4cl 0.25 g, peptone 0.5 g, yeast extract 0.1 g, succinate 5.0 g, and tnt 0.1 g; medium 2: mgso4 0.1 g, k2hpo4 3.5 g, kh2po4 1.5 g, tnt 0.1 g, yeast extract 1.0 g, and trace element solution 1.0 ml, consisting of ethylenediaminetetraacetic acid (edta) 0.25 g, feso4•7h2o 0.1 g, and trace element solution sl-6 1.0 ml; king’s b medium: peptone 20.0 g, k2hpo4 1.5 g, and mgso4•7h2o 1.5 g. agar at 1.5% (w/v) was added for the solid medium. the explosive stock solution was prepared by dissolving 0.5 g tnt in 50 ml n,n-dimethylformamide to have a final concentration of 10,000 mg/l. the final tnt concentration of test media was adjusted to 100 mg/l using stock solution. in case of aerobic cultivation, 20 ml cultivation solution was added in a 100 ml flask, inoculated, and then cultivated. facultative anaerobic cultivation was performed using test tubes full of the medium. cultures were incubated at 28±20c. for the identification of isolates, gram staining and biochemical tests were performed. cell size, motility, and morphology were determined microscopically (x1,000) under the light microscope (olympus bh-2, japan). chracteristics of the results were compared with the properties described in the bergey’s manual of systematic bacteriology (krieg et al., 1994). for genetic characteristics, the 16s rrna gene was amplified by pcr with 27f/ 1492r primers (universal primers for bacteria) in 35 amplification cycles at 940c for 45 sec, 550c for 60 sec, and 720c for 60 sec. for purification of pcr products, unincorporated pcr primers and dntps from pcr products were removed by using the montage pcr clean up kit (millipore co., usa). the purified pcr products were sequenced by using 27f/ 1492r primers. sequencing was performed by using big dye terminator cycle sequencing kit v.3.1 (applied biosystems, usa). sequencing products were resolved on an applied biosystems model 3730xl automated dna sequencing system (applied biosystems, usa) at the macrogen, inc., seoul, korea. the nearly complete sequence of the 16s rrna gene (1427nt) was compiled with seqman software (dnastar inc.). the 16s rrna gene sequences of the related taxa were obtained from genbank. screening of highly efficient explosives-removing strains each of those 235 isolated strains of soil bacteria was screened for the tnt and 2,4/2,6-dnt removal efficiencies, using a luria-bertani (lb) medium containing 100 mg/l of tnt or 2,4/2,6-dnt, under aerobic conditions. after 12 and 18 h of cultivation, concentrations of tnt and 2,4/2,6-dnt were measured on a gas chromatograph (hp-6890, usa) equipped with the electron capture detector (gc-ecd) and rtx-tnt column. ecd has been widely used for the determination of nacs (walsh, 2001). nitrogen was used as a carrier gas at the flow rate of 60 ml/min. injector and detector temperatures were 250 and 300°c, respectively. the column temperature was programed as follows: held for 1 min at 80°c; temperature increased to 180°c at the ratio of 10°c/min; temperature increased to 300°c at the ratio of 30°c/min; and held for 3 min at 300°c. batch experiments for high explosives removal efficiencies in order to determine the optimum culture conditions for the explosives-removing bacteria, the batch culture was performed under various conditions. for the effect of explosive concentration on bacterial growth rate, the bacterial cell concentration was set at 1.0 g wcw (wet cell weight)/l. tnt and 2,4/2,6dnt concentrations in lb medium and basal mineral medium a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 118 were modified to 10, 100, and 200 mg/l, respectively. for the optimum cell concentration, tnt and 2,4/2,6-dnt concentrations in lb medium and basal mineral medium were set at 100 mg/l but the bacterial cell concentrations were modified to 0.5, 1.0, and 1.5 g wcw/l. for the optimum ph and temperature, ph was modified to 6, 7, 8, and 9 and temperature to 15, 20, 25, and 30°c. in addition, to evaluate the possibility of even higher removal efficiencies for tnt and 2,4/2,6-dnt, the mixed cultures consisting of isolates mixed with either bacillus strains, purple non-sulfur bacterial strains, or pseudomonas strains, were compared with single cultures in terms of the tnt removal efficiency. results and discussion bacterial isolation and identification in total, 235 strains of soil bacteria were isolated from the military shooting gallery and they were identified as follows, according to the classification keys of bergey’s manual of systematic bacteriology (krieg et al., 1994): acinetobacter (51 strains), agrobacterium (2), alcaligenes (2), azorhizobium (15), citrobacter (14), flavobacterium (27), hafnia (9), klebsiella (8), klyvera (16), pantoea (5), proteus (18), pseudomonas (36), serratia (22), and yersinia (10). among 235 isolates, 168 strains could remove tnt and were identified as pseudomonas (24), flavobacterium (13), citrobacter (14), proteus (12), yersina (6), hafnia (4), klyvera (15), pantoea (4), klebsiella (4), serratia (21), azorhizobium (9), and acinetobacter (42). fifty one strains removed 2,4-dnt and were identified as pseudomonas (11), flavobacterium (13), proteus (4), agrobacterium (2), yersina (4), hafnia (3), klyvera (1), pantoea (1), klebsiella (4), serratia (1), azorhizobium (1), and acinetobacter (6). sixteen strains removed 2,6-dnt and were identified as acinetobacter (3), alcaligenes (2), azorhizobium (5), flavobacterium (1), hafnia (2), proteus (2), and pseudomonas (1) (table 1). the other 77 strains were not shown explosives-removal efficiency, but could survive. the strains were tolerant to explosives as bacteriostatics. for the screening of strains with high explosives removal efficiencies, 147 isolates were tested for tnt and 2,4/2,6-dnt removal. results showed that 113 strains removed tnt at the average removal efficiency of 84.4%, 27 strains removed 2,4dnt at the average efficiency of 89.8%, and 7 strains removed 2,6-dnt at the average efficiency of 72.6%. among these isolated tested, kt22 strain showed the highest removal efficiency for tnt (100%, after 6 h), kd4 strain for 2,4-dnt (100%, after 18 h), and kd6 strain for 2,6-dnt (97.7%, after 18 h), respectively (table 2). high efficiency bacteria kt22 was identified as serratia sp., and kd4 and kd6 were identified as klebsiella sp. (figure 1, table 3). on the basis of the 16s rrna gene sequences revealed that strain kt22 showed the high sequence similarity to serratia marcescens (100%), strain kd4 and kd6 showed the high sequence similarity to klebsiella oxytoca (99%) as blast searching results. (a) (b) (c) figure 1. micrographs of the selected bacterial strains (x 1,000). (a) kt22, serratia sp., tnt removing strain; (b) kd4, pseudomonas sp., 2,4-dnt removing strain; (c) kd6, pseudomonas sp., 2,6-dnt removing strain. 119 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ta bl e 1. m or ph ol og ic al a nd b io ch em ic al c ha ra ct er ist ic s of is ol at es f ro m th e m ili ta ry s ho ot in g ga lle ry . iso late s* 51 2 2 15 14 27 9 8 16 5 18 36 22 10 ide ntif ica tion ac ine tob act er ag rob act eri um alc alig ene s az orh izo biu m cit rob act er fla vob act eri um ha fnia kle bsi ella kly ver a pan toe a pro teu s pse udo mo nas ser rat ia ye rsin ia mo rph olo gic al ch ara cte rist ics gra m s tain ing sha pe rod rod / c occ i rod rod rod rod rod rod rod rod rod rod rod rod siz e 0.8 x 1 .52.5 0.5 -0. 8 x 1.0 -1. 5 1.0 x 1 .52.0 0.8 -1. 0 x 1.5 -2. 0 1.0 x 1 .52.0 0.8 -1. 0 x 1.5 -2. 0 0.8 -1. 0 x 1.5 -2. 0 0.8 -1. 0 x 1.5 -2. 5 0.8 -1. 0 x 1.0 -1. 5 0.8 -1. 0 x 1.5 -2. 5 0.8 -1. 0 x 1.5 -2. 0 0.5 -0. 8 x 1.5 -2. 0 0.5 -0. 8 x 1.2 -2. 0 0.8 -1. 0 x 1.0 -1. 5 re plic atio n b b b b b b b b b b b b b b mo tilit y + + + + end osp ore fo rm ing bio che mi cal c har act eri stic s ca tala se + + + + + + + + + + + + + + ox ida se + + + + + nit rat e r edu cta se + + + + + + + + + + ca sei n + v sta rch hy dro lys is + ure a + + + + + ge lati n h ydr oly sis + v + mr v v + + + + + + + + vp (+) + + v + ind ole ga s fr om glu cos e v + v + 120 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 2. results of screening tests for selected isolates. table 3. morphological and biochemical characteristics of the selected isolates (kt22, kd4, and kd6) with the highest explosives removal efficiencies. explosive strain no. initial conc. (mg/l) final conc. (mg/l) r.e. (%) strain no. initial conc. (mg/l) final conc. (mg/l) r.e. (%) tnt kt22 107.7 0.0 100.0 kt60 107.7 41.7 61.3 kt23 107.7 4.2 96.1 kt88 144.5 71.5 50.5 kt 92 107.7 11.2 89.6 kt147 107.7 59.8 44.5 kt76 107.7 19.7 81.7 kt43 107.7 72.4 32.8 kt104 107.7 32.0 70.3 kt110 107.7 95.2 11.6 2,4-dnt kd4 68.1 0.0 100.0 kd27 125.0 46.0 63.0 kd9 125.0 0.5 99.6 kd14 125.0 84.5 32.4 kd28 125.0 0.6 99.5 kd3 68.1 49.1 27.9 kd23 125.0 17.7 85.8 kd13 125.0 104.2 16.6 2,6-dnt kd6 103.9 2.4 97.7 kd53 71.4 30.9 56.7 kd59 103.9 4.3 95.9 kd55 103.9 64.0 38.4 kd64 103.9 12.7 87.8 kd62 103.9 79.8 23.2 kd58 103.9 30.0 71.1 kd63 103.9 97.3 6.4 kd54 103.9 36.3 65.1 kd52 71.4 75.3 r.e.: removal efficiency. strain kt22 kd4 kd6 shape rod rod rod size (μm) 0.8 x 2.0 0.8 x 2.0-2.5 0.8 x 1.5 gram reproduction b b b motility + + + endospore forming catalase + + + oxidase + + nitrate reductase + + + casein starch + urea + gelatin mr (+) vp + indole gas from glucose b: binary replication, v: various results, +: positive reaction, -: negative reaction, and weak reaction is given in the parentheses. batch culture three bacterial strains (kt22, kd4, and kd6) which showed the highest explosives removal efficiencies were selected for the batch experiments in order to determine optimal culture conditions. effects of various factors, including cell concentrations, explosives concentrations, medium composition, temperature, ph, and pure vs. mixed cultures, on explosives removal efficiencies were evaluated. determination of optimal cell concentration in order to determine the optimum cell concentration for the removal of tnt, different cell concentrations (0.5, 1.0, 1.5, and 2.0 g wcw/l) of kt22 strain and the stanier’s basal mineral medium were used. as shown in table 4, the removal efficiency for tnt used as sole carbon source was 38.8, 51.2, 49.5, and 52.5% at the cell concentration of 0.5, 1.0, 1.5, and 2.0 g wcw/l, respectively, after 6 h of incubation. therefore, the optimum cell concentration was determined as 1 g/l because the higher amount of biomass at 2.0 g/l did not show significantly higher tnt removal efficiency. effects of explosives concentrations on bacterial growth rates using the cell concentration of 1 g wcw/l, effects of explosives concentrations on bacterial growth rates were evaluated. three different concentrations (10, 100, 200 mg/l) of tnt and a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 121 2,4/2,6-dnt in the basal mineral medium were used. results showed the higher bacterial growth rate and tnt removal efficiency at the lower tnt concentration (figure 2a and table 5). likewise, 2,4-dnt showed similar effects for kd4 strain (figure 2b). however, in case of 2,6-dnt, kd6 strain showed similar growth rates regardless of 2,6-dnt concentrations (figure 2c). effect of medium composition on explosives removal efficiency to determine the effect of medium composition on the explosive removal efficiency, lb medium and stanier’s basal mineral medium were compared, using kt22, kd4, and kd6 strains. the tnt removal efficiency for kt22 was 99.7% in lb medium and 51.2% in stanier’s basal medium, and the 2,4/2,6-dnt removal efficiency for kd4 and kd6 was 99.1% and 92.7% in lb medium and 63.0% and 34.5% in stanier’s basal medium, respectively. therefore, each selected strain showed higher removal efficiency for the corresponding explosive in lb medium which is a rich medium containing additional carbon and nitrogen sources such as peptone and yeast extract, compared to stanier’s basal mineral medium which contained explosives as sole carbon sources. these results are also in good agreements with previously published studies on the effects of supplemental nitrogen sources and carbon sources on the biodegradation of tnt (won et al., 1974; boopathy et al., 1997; park et al., 2003). these studies showed that in the presence of other carbon sources except aspirate, pseudomonas putida u-t202 could degrade tnt over 92% and showed a good biomass growth after 30 h of incubation (park et al., 2003; won et al., 1974) also reported that the tnt oxidation required the addition of glucose or nitrogenous substances to achieve accelerated transformation and in a medium supplemented with 0.5% yeast extract, 100 mg/l of tnt was completely transformed to intermediates such as monoaminodinitrotoluene (madnt), diaminomononitrotoluene, or azoxy compound. cell conc. (g (wcw)/l) 0 h (mg/l) 6 h (mg/l) r.e. (%) 0.5 84.7 51.9 38.8 1.0 84.7 41.5 51.2 1.5 84.7 42.8 49.5 2.0 84.7 40.2 52.5 wcw: wet cell weight, r.e.: removal efficiency. table 4. effect of cell concentrations of kt22 strain on tnt removal efficiency. explosive conc. (mg/l) tnt 2,4-dnt 2,6-dnt 0 h (mg/l) 6 h (mg/l) r.e. (%) 0 h (mg/l) 12 h (mg/l) r.e.(%) 0 h (mg/l) 18 h (mg/l) r.e. (%) 10 9.6 5.1 46.9 9.1 0.0 100.0 12.5 8.1 35.5 100 76.5 37.9 50.3 89.4 33.1 63.0 103.9 68.0 34.5 200 149.4 121.2 18.9 166.7 74.7 55.2 192.0 172.7 10.1 r.e.: removal efficiency. table 5. effects of explosives concentrations on removal efficiencies. determination of optimum temperature and ph for the ranges of temperature (15-30°c) and ph (5-8) tested in this study, the explosive removal efficiencies for those three selected strains were the highest at around 25°c and at ph 7, as shown in figures 3 and 4. explosive removal efficiency for mixed culture to further investigate the effect of the presence of pure culture (containing one type of explosive-removing strain) vs. mixed culture (containing more than one type of explosive-removing strains) on the explosive removal efficiency, kt22 strain which showed a high tnt removal efficiency was used as a representative tnt-removing strain. for the mixed culture, kt22 strain was mixed with bacillus or pseudomonas strain, both commonly occurring soil bacteria, or with purple non-sulfur bacterial strain capable of degrading some recalcitrant organic compounds. as shown in figure 5, tnt removal was the highest for the mixed culture consisting of kt22 and bacillus sp., with the removal efficiency of 76.6% after 6 h, compared to 51.2% when kt22 strain used alone. this study carries several significant scientific and industrial application merits. firstly, serratia sp. has been rarely reported for its efficiency in tnt biodegradation. moreover, three bacterial strains (serratia sp. kt22, klebsiella sp. kd4, and klebsiella sp. kd6) showed higher removal efficiencies on the explosives than the efficiencies reported in other previous studies. finally, serratia sp. kt 22 showed synergistic removal efficiency in a mixed culture with bacillus sp. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 2. effects of explosives concentrations on bacterial growth rates. (a) effect of tnt concentrations on growth of kt22; (b) effect of 2,4-dnt concentrations on growth of kd4; (c) effect of 2,6-dnt concentrations on growth of kd6. (a) (b) (c) (a) (b) (c) figure 3. effect of temperature on explosive removal efficiency. 122 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) despite the significant level of advancement made in the environmental remediation techniques by using chemical, physical and biological media, numerous side effects induced by chemicals used or the underlying challenges in identifying the bacteria with high removal efficiency always have been limiting factors. the new strategic approaches preferred by may municipal and governmental agencies around the world, however, are dominantly bacteria-using bioremediation techniques. therefore, the bacterial strains with higher bioremediation potency shown here could further facilitate explosive cleaning up processes in real life applications in addition to the scientific values mentioned somewhere else in this report. acknowledgments this study was supported by a grant 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platyfish (poecillidae: xiphophorus maculatus) tyler e. frankel and jack s. frankel department of biology, howard university, washington, dc 20059, usa received: march 24, 2017 / accepted: april 14, 2017 __________________________________________________ * corresponding author: jfrankel@howard.edu 371 abstract the ability of individuals within a population to survive and thrive is highly dependent upon the maintenance of genetic variation and phenotypic diversity, thereby ensuring adaptation to dynamic environments. a fundamental method of maintaining such variation is through a negative assortative mating strategy, in which individuals would be expected to reproductively select members of the opposite sex that exhibit dissimilar phenotypes. employing three uniform body color morphs, red, yellow and blue, of the platyfish (xiphophorus maculatus), this study was designed to investigate whether x. maculatus females would preferentially be attracted to males exhibiting an alternative color, thereby enabling an examination of the effect of male body coloration on mate choice by adult females. mate choice was determined based on the initial preference of each female, as well as the amount of time females spent associating with each male. initial preferences were analyzed using a binomial distribution test, and overall preference data using wilcoxon signed rank tests. red females initially selected for dissimilar colored males, and spent a significantly larger amount of time associating with blue and yellow males, as did yellow females with red and blue males. blue females initially selected and spent a significantly larger amount of time associating with red males but, interestingly, showed no selective preference between blue and yellow males. in these experimental trials, the overall strong mate selection exhibited by female platyfish for males of dissimilar coloration is suggestive of a negative assortative mating strategy and provides evidence for the maintenance of color polymorphism in nature populations. keywords: xiphophorus maculatus, platyfish, negative assortative mating, courting behavior, mate choice. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction population heterogeneity and phenotypic diversity are major factors enhancing the long-term survival of a species and are closely linked to the ability of that species to respond to environmental change (bazin et al., 2006). one of the evolutionary mechanisms by which populations can maintain such genetic heterogeneity is through mate choice via assortative mating strategies (pryke and griffith, 2007). individuals exhibiting negative assortative mating (i.e. disassortative mating) are expected to select mates that exhibit dissimilar phenotypes (workman, 1964; jiang et al., 2013). this selection process not only prevents the loss of newly developed genotypes due to genetic drift, but also encourages the re-establishment of genotypes that have decreased in frequency due to brief, sudden selective pressures (rapid environmental shifts, predation, etc.), potentially preventing the development of a homogeneous population. conversely, a positive assortative mating strategy would cause individuals to choose mates exhibiting phenotypes similar to their own. this strategy could prevent the incorporation of potentially deleterious alleles into populations during times when environmental conditions are stable (whitlock and agrawal, 2009; agrawal and whitlock, 2012; arbuthnott and rundle, 2012). diversity in coloration has been shown to affect species recognition and mate selection in poeciliid fishes (endler, 1983). indeed, much of the research examining sexual selection in fish has utilized members of the poeciliidae, including guppies (poecilia reticulata), swordtails (xiphophorus helleri), and platyfish (xiphophorus maculatus), all of which exhibit a wide range of color variation due to both artificial and natural selection (basolo, 2006; porter and frankel, 2014). such variation in coloration patterns of certain central american poeciliids have been shown to have effects on predation and species recognition (endler, 1983). this also supports the theory of coloration aiding in sexual selection preferences in p. reticulata, specifically a fea tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) male’s natural affinity for orange and other coloration patterns (houde, 1997). additional research using pygmy swordtails (xiphophorus pygmaeus) has shown that females exhibit preference for males exhibiting a blue coloration and an aversion to yellow males when given a choice between the two (kingston, 2003). as a member of the poeciliidae, platyfish are ideally suited for studies involving mate choice due to several factors, including (1) their relatively uniform body morphology and size among males and females (kallman, 1975; basolo, 2006); (2) the use of an ovoviviparous reproductive strategy which allows for easy sexing of males and females through the presence or absence of a gonopodium (rosenthal and de leon, 2006); and (3) the historically well-documented and easily identifiable courtship and mating behaviors that are exhibited by both sexes (noble, 1938; schlosberg et al., 1949; rosen and tucker, 1961). wild platyfish populations are highly polymorphic for color patterns (borowsky and kallman, 1976; basolo, 2006) and, as a result, it is often difficult to isolate mate preferences due to coloration alone using wild individuals. as the popularity of x. maculatus in the aquarium trade has grown, new color variants have been developed through artificial selection. while more vivid than those typically found in these populations, careful selection of individuals that display similar color patterns, coupled with the retention of natural mating behaviors, makes them ideally suited for studies involving coloration and mate choice. this study was designed to investigate whether assortative mating for alternative body coloration is observed amongst female x. maculatus. it is hypothesized that females will both preferentially select males of alternate coloration and spend significantly more time associating with those males as compared to males exhibiting the female’s coloration. materials and methods to investigate female preference for males of dissimilar body coloration based on three distinct color morphs, a series of experimental trials was conducted employing similar constructs as described for studies on pygmy swordtails (kingston, 2003). in the work presented here, three phenotypes of x. maculatus were chosen, red, yellow, and blue color morphs. these phenotypes were selected for their uniformity in overall coloration from individual to individual within these color variants. healthy, sexually mature (>140 d post-birth) individuals of each color variety were obtained from a local retail distributor (g&g aquatics, lorton, virginia, usa). because previous research using the congeneric x. variatus has shown that body size and length can impact female selection (maclaren et al., 2011), male size was standardized in this study based on total length (mm) and weight (g). males deviating beyond one standard deviation in total body length and/or body mass from the general population were not utilized for this study. males and females that displayed differences in melanin pigmentation or marking expression were also excluded at this time. selected males and females were separated by coloration and housed in single sex, 75.7 l stock tanks. because females have been shown to copy the mate choice of other individuals in studies involving other poeciliids (dugatkin, 1992), the walls of all stock tanks were covered to limit any visual interactions between individuals of various colorations and sexes. this also served to reduce stress from environmental stimuli. fish were fed a commercial flake diet twice a day until satiated and maintained under a 14 hr : 10 hr (light : dark) photoperiod at a temperature range of 26 ± 10c. water quality (ammonia, nitrite, and nitrate) was monitored weekly, and the ph was maintained between 7.3 and 7.5. experiments were conducted utilizing a standard (56.7 l) capacity aquarium, with clear plexiglas® partitions placed equidistant from the center of the tank and sealed with aquarium silicone to create two isolated holding areas for the male subjects. these partitions allowed the female in each experiment to visually interact with males in both compartments, but prevented chemical and physical interactions. the back and side panels of the tank were covered to prevent interference from any external stimuli. a black curtain was hung three feet from the front of the tank to further prevent behavioral modification during the trials as a result of external stimuli (fig. 1). for each trial, two males from the labeled tanks were randomly selected from the experimental male populations. to account for any side bias, one male exhibiting the female’s color morph was placed randomly into one of the side chambers of the experimental tank (fig. 1). another male exhibiting either red, yellow, or blue coloration was placed in the opposite chamber. to prevent an order effect, the order in which the various colorations were introduced to the females was randomized. a single female was then placed into a 10 cm x 10 cm x 25 cm plexiglas® holding chamber located in the center of the tank. after an acclimation period of 2 minutes (during which the female was allowed to observe but not interact with the two males), the holding chamber was slowly and gently removed, and the female was allowed to associate with both males. courting behaviors and male associations exhibited by the female were recorded using a web camera (logitech g920) and ispy recording software (version 6.6.7.0) for an 8 minute period. using these methods, 28 females were tested for each color combination. to test for side bias, females were also tested with two males of her own coloration on both sides a and b of the experimental tank. mate preference trials were conducted under an approved 372 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. diagram of behavioral recording setup. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 373 howard university iacuc protocol (iacuc-gsas-11-03). to ensure consistency and prevent observer bias, we utilized a single-blind experimental design. to determine female preference, we recorded the total amount of time (s) each female spent associating with either male, and time was only recorded when both the female and male were actively engaged. we defined association based on the descriptions of platyfish courtship provided by schlosberg et al. (1949), which include behaviors such as posing, sigmoidal flexing, and arching. the amount of time female x. maculatus spend associating with males has been shown to be a reliable indicator of mate selection and final reproductive outcomes in previous studies involving various xiphophorus species (cummings and mollaghan, 2006; walling et al., 2010). all data analyses were performed using sas 9.3 (sas institute, cary, nc, usa). we compared male preference displayed by females of each coloration by subtracting the amount of time spent by each female associating with the alternately colored male, from the amount of time she spent associating with the same colored male, and compared these values to a null expected value of zero using the wilcoxon signed-rank test (p = 0.05). results and discussion a total of 252 initial preference and association trials were performed over the course of this study. females of all color morphs did not show any side bias throughout the course of the study, as indicated by their equal association times when tested against two males of their own coloration. when tested against red and blue males, red females spent significantly more time associating with blue males than with red males (p = 0.0001) (fig. 2). red females also spent significantly more time associating with yellow males compared to red males (p = 0.0028) (fig. 2). yellow females spent significantly more time associating with alternately colored blue males (p = .0065) and red males (p = 0.0004) compared to the yellow males used in the trials (fig. 3). blue females significantly preferred associating with red males over blue males (p = 0.0032), but showed no significant difference in association time when tested against blue and yellow males (p = 0.7040) (fig. 4). overall, the results of this study show a distinct association preference of red and yellow female platyfish toward males of dissimilar coloration. because courtship and display behaviors entail an increased risk of predation in the wild, mate assessment by females must be performed using cues that are readily apparent and virtually instantly assessable (sullivan, 1994). based on the results of this study, platyfish appear to generally utilize a negative assortative mating strategy when selecting for males based on body coloration, although the total time each female spent associating with each male varied from individual to individual in all three color morph populations. this, along with the findings of other studies showing varying degrees of affinity for male ornamentation by females (houde, 1997; morris et al., 2003), suggests that while a tendency exists for the selection of dissimilar males, there is variation in the existence and strength of female preference. red and yellow females showed a high affinity towards both alternate color morphs when making an initial selection, and continued to show a significant preference for dissimilarly colored males over time. this finding was repeated when blue females were exposed to red males, but not when blue females were exposed to yellow males. while there have been previous studies examining the effects of morphological characteristics on mate choice in platyfish, this is the first to examine assortative mating in x. maculatus based on body figure 2. bars represent average time ± se red females spent associating with red or yellow males and red or blue males during association trials. asterisks indicate significant differences (p < 0.05). figure 3. bars represent average time ± se blue females spent associating with blue or red males and blue or yellow males during association trials. asterisks indicate significant differences (p < 0.05). figure 4. bars represent average time ± se yellow females spent associating with yellow or blue males and yellow or red males during association trials. asterisks indicate significant differences (p < 0.05). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 374 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) coloration. negative assortative mating has been postulated to be a rare occurrence compared to other mating strategies (jiang et al., 2013). however, our results provide evidence for the use of a negative assortative mating strategy based on intraspecific advertised body coloration in female platyfish. this result is consistent with the findings of previous studies examining the effects of various dissimilar phenotypes on mate choice in a range of taxa, including coloration in the siamese fighting fish betta splendens (clotfelter et al., 2006), mouth-opening direction in the cichlid perissodus microlepis (takahashi and hori, 2008), plumage coloration in the pigeon columba livia (johnston and johnson, 1989), striping coloration in the white-throated sparrow zonotrichia albicollis (houtman and falls 1994), and body size in the sand lizard lacerta agilis (olsson, 1993). we did not see evidence of a negative assortative mating strategy when blue females were allowed to select between blue and yellow males over time (fig. 4). this lack of preference in a specific coloration of females for a specific coloration of males is an intriguing finding, albeit difficult to explain. the aversion to gold males has been observed in other studies examining mate choice in the pygmy swordtail (kingston, 2003) and various neotropical cichlid fish (elmer et al., 2009). additionally, researchers examining mate selection in poeciliids have noted an increased risk of predation associated with the display of conspicuous male ornamentation (endler, 1987; rosenthal et al., 2001; godin and mcdonough, 2003), and a larger cost to immune system function associated with increased carotenoid pigment expression in gold males (hill, 1999). thus, it is possible that the lack of preference by blue females when asked to choose between blue and yellow males is due to the higher risk of predation associated with yellow coloration, or due to the negative impacts of yellow coloration on immune function. the fact that red females showed a preference for yellow males indicates the relative strength of assortative mating, and a possible instance in which the observed female polymorphic preference may have played an active role in altering overall preference for the population. we recognize that the phenotypes utilized in this study are the result of artificial selection, and would be rare, or likely not found, in natural populations. however, the clear association in our results between alternative coloration and female mate preference illustrates how the relatively subtle, but highly varied, coloration polymorphism (basolo, 2006) may be maintained in feral populations of platyfish. further research is required to determine if these preferences for coloration exist within wild populations of this species. while multiple theories exist regarding the evolution of female choice for alternative phenotypes, the exact mechanisms for the evolution of this preference provides impetus for future research. most explanations for why negative assortative mating and mate choice exists focus on prevention of inbreeding and maintaining genetic diversity. in poeciliids, traits such as orange coloration (hughes et al., 2013), fin spotting (culumber and rosenthal, 2013), sword length (basolo, 1990), and body size (maclaren and fontaine, 2011) are known to influence female choice and maintain phenotypic diversity in wild populations. that said, our results are inconsistent with previous findings in guppies, in which females were attracted to increasing degrees of one coloration in males (houde, 1997; grether et al., 2001; rodd et al., 2002), suggesting that patterns of sexual selection may be species specific even among members of the same family of fishes. we contend that the platyfish is a powerful model species for studying mating preferences based on coloration due to their homogeneous body morphology (mass, body length, etc.), readily observable and well documented courting behaviors, and the existing body of knowledge concerning genetic linkages and color polymorphisms (basolo, 2006; culumber, 2014). most importantly, the recent sequencing of the platyfish genome has opened the door for the discovery of genetic underpinnings for the evolution of life history traits such as reproductive behaviors and mate selection (schartl et al., 2013). we conclude that the female platyfish utilized in this study follow a negative assortative mating strategy when selecting for male mates based on body coloration. these findings are significant due to their implications involving the maintenance of genotypic variation and should be tested in wild platyfish with different color phenotypes. while this study has examined female preference based on individual male coloration, the presence or 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soybean seedlings in greenhouse assays hemlata sharma1,2 and david a lightfoot2,3* 1 dept of plant breeding & genetics, rajasthan college of agriculture, mpuat, udaipur, india; 2 department of plant, soil and agricultural systems, southern illinois university, carbondale, il, 62901, usa; 3 the illinois soybean center, southern illinois university, carbondale, il, 62901, usa received: june 30, 2013 / accepted: march 18, 2014 __________________________________________________ * corresponding author: ga4082@siu.edu 175 abstract cercospora sojina (hara), an air-borne pathogen, infects soybean [glycine max (l.) merr.] leaves causing frog-eye leaf spot (fls). three major genes (rcs1-3) underlie resistance to the major races of fls but two were not yet mapped. in addition quantitative trait loci provide partial resistance to many strains. fls race 2 was an isolate first collected in the 1950’s when damaging fls first arose. ‘essex’ was partially resistant while ‘forrest’ was partially susceptible to mixed races of fls. the objective here was to identify quantitative trait loci underlying resistance to fls race 2 in the greenhouse using recombinant inbred lines (rils) derived from the cross of essex by forrest. c. sojina race 2 (atcc 44531) was used to induce leaf symptoms on one hundred f5:14 rils derived from the cross of essex by forrest. the leaf symptoms were measured at 21 days after manual infestation by wounding (dai) and again at 42 dai to show resistance to reinfestation of new leaves from the primary lesions without wounding. bags over leaves were not used to better simulate field conditions. however, there was no significant correlation between fls severity at 21 and 42 dai (r =0.08 and p= 0.005). at 21 dai there was a strongly significant qtl near satt319 on lg c2 (chromosome 7; lod 3.8; r2 52%) where the essex allele reduced leaf symptoms by 0.7 units. at 42 dai there was a strongly significant qtl near satt632 on lg a2 (chromosome 8; r2 was 15%; lod was 3.6) where the essex allele reduced leaf symptoms by 0.4 units. neither locus mapped introduction frogeye leaf spot (fls), caused by the fungus cercospora sojina (hara), has been an important soybean disease, that has caused both seed yield losses and seed quality deterioration (wrather et al., 1996; 2003). yield reductions in the range of 10 to 60% due to fls have been reported. the use of resistant cultivars was the most efficient and cost effective means of controlling this disease (mian et al., 2009). however, the rise this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. to the location of rcs3. by anova thirteen additional minor loci were detected on lgs a1, b1, f, g, h, i, j, k, l, m and o. at two loci (lg b1 and o) the forrest allele appeared to reduce fls at both 21 and 42 dai. eight loci may have reduced fls at 21 dai (0.006 < p < 0.049; 4% < r2 > 9%) of which 5 had beneficial alleles from forrest. seven loci may have reduced fls at 42 dai (0.001 < p < 0.04; 4% < r2 < 15%) of which 4 had beneficial alleles from forrest. therefore, quantitative resistance to race 2 of fls was inferred to have major loci contributions from essex and minor loci contributions from both forrest and essex. resistance was dependent on plant age. breeding and selection for fls will be complex and may be more efficient with the markers, germplasm and models of inheritance reported here. keywords: cercospora; tolerance; qtl; resistance; soybean; frog-eye leaf spot. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 176 of dozens of new c. sojina races (yorinori, 1992; mian et al., 2009) has reduced the effectiveness of the three major genes deployed to date. a new approach to the search for sources of resistance may involve loci underlying partial resistance and their incorporation into breeding programs (gravina et al., 2004; mian et al., 2008; 2009). three single genes conditioning resistance to c. sojina were; rcs1 from `lincoln’ that conferred resistance to race 1 (athow and probst, 1952); rcs2 for resistance to race 2 identified from cultivar `kent’ (athow et al., 1962); and rcs3 from ‘davis’ that was found to condition resistance to race 5, among others (boerma and phillips, 1983; phillips and boerma, 1982; yorinori, 1992). dominant genes for resistance to race 5 that were not alleles of rcs1-3 were found in ‘peking’, ‘ransom’, ‘stonewall’, and ‘lee’ (pace et al., 1993; baker et al., 1999). on its release (smith and camper, 1973) ‘essex’ was reported to show partial resistance to both frogeye leaf spot (caused by c. sojina k.), and purple seed stain disease [caused by c. kikuchii (mastsumoto & tomoyasu) m.w. gardner]. essex had, among its immediate parents, lee, a potential source of rcs3 like loci. ‘forrest’ had, among its immediate ancestors, peking the source of an rcs3 like locus. forrest also had, on either side of its phylogeny, lee and its sibling line ‘d49-2491’, both potential sources of rcs3 like loci (hartwig and epps, 1973; lightfoot et al., 2005). both essex and forrest shared cultivars ‘cns’ and ‘s100’ as ancestors, potential sources of fls resistance genes. essex was reported resistant to fls race 2 in the field but susceptible to fls race 5 (phillips and boerma, 1981). further, race 2 of c. sojina appeared (athow et al., 1962) in the late-1950s, in the carolinas and mid-west of the usa. therefore, it is likely essex, forrest and their parents were unintentionally selected for quantitative resistance to fls race 2 in the field. however, whilst it was reported that essex had field resistance to fls it was not reported whether forrest was resistant (hartwig and epps, 1973) and it was later shown not to carry rcs3 (missaoui et al., 2007). therefore, the objective of this study was to identify major loci underlying resistance and/or polygenes underlying qtl for partial resistance or tolerance to the foliar symptoms of fls race 2 under controlled conditions in the greenhouse using seedlings of an advanced ril population. materials and methods materials the experimental material in the publicly released population ef94 included ninety four f5:14 rils (hnetkovsky et al.,1996; lightfoot et al., 2005) derived from the cross of essex (smith and camper, 1973) x forrest (hartwig and epps, 1973) and the two parents. in addition the 6 rils removed from ef94, rils 95-100 were returned to the population tested. the susceptible check used was ‘blackhawk’ and the resistant check used was ‘kent’. the strain of race 2 was provided by dr. j. phillips through the american type culture collection (atcc) as strain number 44531. it was last deposited in 2008. the strain was received desiccated and was grown on v8 media plates (kent et al., 2008) at 22 ºc in the dark. siuc retains a duplicate, frozen, desiccated isolate for distribution on request. alternately the strain can be obtained from the atcc. infestations with c. sojina plates used for inocula were washed with distilled water and 10 µl of this was used for spore count on a hemo-cytometer under a microscope. spore counts of 104 spores per cm3 were used. the one hundred rils were planted in 15 cm square pots the greenhouse at the southern illinois university horticulture research center in carbondale, il. parents and non-inoculated control plants were included in the experiments. all plants were sown in sterilized 1:1 (v/v) of sand and soil and grown to the v3 stage (about 21 days; fehr and caviness, 1977). the first fully expanded leaves of 3 plants per genotype were selected to be infested by c. sojina. three more plants per genotype were selected for non-inoculated controls (natural infestation). across both repeats of the experiment 6 inoculated plants and 6 noninoculated plants were scored. plants were arrayed in randomized complete blocks. the plants to be infested were inoculated by rubbing the youngest full expanded leaflets with 600 mesh carborundumtm paper. leaflets were sprayed to runoff with the spore solution. infested leaves were not bagged as in mian et al (2008) so symptoms would develop more slowly and reflect field infestations to a greater degree. experiments were conducted from october to december in 2008 and repeated in full from october to december in 2011. plants were grown with a 14 h photoperiod and 10 h of darkness using sunlight supplemented by growlights providing 1,500–2,000 mol photons m-2 s-1. greenhouse air temperature ranged from 20±2ºc at night to 27±2ºc during the day. humidity ranged from 75-85% (v/v) as judged by indicator cards. in total 6 plants per genotype contributed to the trait means. the mean disease severity (ds) of the non-inoculated plants was subtracted from the infested plant mean for each genotype. fls ds was rated at 21 days after inoculation (dai; 42 days after germination), and 42 dai. the ds rating was determined on the basis of the degree of leaf damage of the whole canopy (chlorosis/necrosis) on each plant, and was rated on a scale of 0 to 9 (0 was 0%/0% ; 1 was 1–10%/1–5%; 2 was 10–20%/6– 10%; 3 was 20–40%/10–20%; 4 was 40–60%/20–40%; 5 was >60%/>40% of leaf surface chlorosis/necrosis, respectively; 6 was up to 33% premature defoliation; 7 was up to 66% premature defoliation; 8 was more than 66% premature defoliation; and 9 was premature plant death). means, plus and minus the standard error of the means (sems), were reported. the ds scores for cultivar blackhawk (susceptible check) was 5 at 21 dai and 5 at 42 dai. the ds scores for kent (resistant check) was 0 at 21 dai and 0 at 42 dai. trait analyses since the assay was conducted under controlled conditions in the greenhouse the broad sense heritability (h2 ) of ds was calculated from variance components as: h2 = σ2g/( σ2g + σ2e) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 177 where σ2g = genotypic variance and σ2e = error variance. pearson’s correlation coefficient analysis was performed on the mean trait data in order to test for correlations among traits. ril’s and parent trait mean comparisons were made by lsd. mapping quantitative trait loci a linkage map was created using mapmaker–exp 3.0 (lander et al., 1987); the (ri-self) genetic model; cm in haldane units and a two point analysis. the ril population was used to map a total of 413 markers, including 238 satellite markers and 177 other dna markers (kassem et al., 2006; yesudas et al., 2010). the log10 of the odds ratio (lod) for grouping markers was set at 3.0 and the maximum distance was 50cm. conflicts were resolved in favor of the highest lod score after checking the raw data for errors. marker orders within groups were determined by putting them in increasing order of sequence number in the soybean genome (schmutz et al., 2010). the map and disease scores were analyzed with anova (sas, cary, nc) by single point analyses. markers were listed if they were significant at 0.001< p <0.05 at either 21 or 42 dai. markers that were unlinked, or in marker sparse areas of the map, were considered to be independent tests (kassem et al., 2006; shultz et al., 2007; yesudas et al., 2010). composite interval analysis used winqtl cartographer (version 2.0; basten et al., 2001) for qtl mapping and estimation of locus effects as described previously (kassem et al., 2006; shultz et al., 2007; yesudas et al., 2010). results trait heritability the broad sense heritability for fls ds in the rils was 83.9 % at 21 dai and 64.8 % at 42 dai. the genotype x environment (gxe) interaction was not significant since the greenhouse conditions were closely controlled. that was used as the justification to use the mean data (njiti et al. 2001). the correlation between 21 dai and 42 dai mean ds across the population was r = 0.08 suggesting the traits were largely independent. trait distributions ds from the replication and repeats at either 21 dai or 42 dai showed similar severity and were highly correlated so data were pooled and those means used for further analyses (fig. 1). at 21 dai the distribution of mean ds was nearly normal and neither significantly skewed or kurtotic (fig. 1). the mean ds distribution ranged from 0 to 5. the mean ds+sem for essex was 1.85+0.02 and significantly different (p < 0.001) from forrest was 3.8+0.02 as judged by lsd. the 20 most resistant and 13 most susceptible lines were segregation events that were significantly (p < 0.05) better than essex or worse than forrest (hereafter transgressive segregants). at 42 dai the distribution of mean ds was positively skewed (p < 0.05) towards resistance. the distribution was continuous and had a significant negative kurtosis that reflected a flattened distribution (fig. 1). the mean ds distribution ranged from 0 to 5, similar to that seen for bigenic resistance to scn (lightfoot, 2008). the mean ds+sem for essex was 1.65+0.02 and significantly different (p < 0.001) from forrest that was 2.45+0.02. the 19 most resistant and 3 most susceptible lines were significant (p < 0.05) transgressive segregants. major qtl detection at 21 dai there was a major qtl on near satt319 on lg c2 (chromosome 7; lod was 3.8; r2 was 52%) where the essex allele reduced fls leaf symptoms by 1.3 ds units (table 1; fig. 2). at 42 dai there was a major qtl near satt632 on lg a2 (chromosome 8; r2 was 15%; lod was 3.6) where the essex allele reduced fls leaf symptoms by 0.46 ds units. the two loci represented major gene effects and underlay a large portion of the variation in trait distributions (fig. 1). notably, both loci derived beneficial alleles from essex, the most resistant parent. figure 1. frequency distributions of mean ds among rils from the exf cross in greenhouse seedling assays at 21 dai (a) and 42 dai (b). the ranges into which essex (e) and forrest (f) mean scores fell are arrowed. a b essex forrest essex forrest a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 178 minor qtl detection in addition to the two major loci there were thirteen minor loci that may be associated with resistance to fls race 2 detected by anova (table 2) at 0.001< p <0.05. some of these loci had beneficial alleles from forrest. at two loci the forrest allele appeared to reduce fls at both 21 and 42 dai. the first locus was associated with satt444 on lg b1 (chromosome 11) at 85.9 cm. the locus explained 6% of the variation in fls at 21 dai (p=0.017) with the forrest allele reducing ds by up to 0.59 units. a locus in the same region explained 12% of the variation in fls at 42 dai (p=0.008) with the forrest allele reducing ds by up to 0.66 units. the second locus was only weakly associated with variation in fls near satt446 on lg l (chromosome 19; p<0.049). six loci were solely associated with reduced fls at 21 dai (0.006 < p > 0.045; 4% < r2 > 9%) of which 5 had beneficial alleles from forrest. the first locus was associated with scar cfr2 on lg f (chromosome 13) at 1.1 cm. the locus explained 9% of the variation in fls at 21 dai (p=0.006) with the forrest allele reducing ds by up to 0.75 units. the second locus was associated with scar cgg116 on lg g (chromosome 18) at 10.1 cm. the locus explained 6% of the variation in fls at 21 dai (p=0.006) with the essex allele reducing ds by up to 0.66 units. the third, fifth and sixth loci listed in table 2 were only weakly associated with variation in fls the fourth locus was associated with satt249 on lg j (chromosome 16) at 12.3 cm. the locus explained 8% of the variation in fls at 21 dai (p=0.009) with the essex allele reducing ds by up to 0.77 units. five loci were only associated with reduced fls at 42 dai (0.001 < p > 0.042 ; 4% < r2 > 15%) of which 4 had beneficial alleles from forrest. the first locus was associated with satt276 on lg a1 (chromosome 5) at 17.2 cm. the locus explained 13% of the variation in fls at 42 dai (p=0.007) with the forrest allele reducing ds by up to 0.64 units. the second locus was associated with satt589 on lg a2 (chromosome 8) at 34.0 cm. the locus explained 11% of the variation in fls at 42 dai (p=0.01) with the forrest allele reducing ds by up to 0.64 units. the third locus was associated with satt440 on lg i (chromosome 20) at 112.7 cm. the locus explained 15% of the variation in fls at 42 dai (p=0.001) with the essex allele reducing ds by up to 0.95 units. the fourth locus was associated with both satt555 and sat_116 on lg k (chromosome 9) at 42.7 and 52.3 cm respectively. the locus explained 11% of the variation in fls at 42 dai (p=0.009) with the forrest allele reducing ds by up to 0.67 units. the fifth locus was minor in effect (p = 0.045) and was associated with satt259 on lg o (chromosome 10) at 39.8 cm. discussion the broad sense heritability for fls ds was high at 21 dai and moderate at 42 dai. the earlier date may be higher in heritability because the trait was the direct effect of inoculation. in comparison the later fls ds score was the product of a more natural inoculation and indirect infestation of new leaves from diseased leaves. the correlation between 21 dai and 42 dai mean ds suggested the traits were largely independent and that the later ds score was the product of different resistance response mechanisms. infection after wounding measured at 21 dai eliminates or reduces the effectiveness of resistances derived from barriers like the cuticle, anti fungal waxes, and even cell wall integrity. trait distributions differed in normality, skewness and kurtosis, again suggesting the traits were underlain by different response mechanisms. the most resistant transgressive segregants for both ds scores were ranked in order ef 7, 28, 2, 8, 61, 86. these lines would be useful parents for crosses designed to select loci underlying quantitative resistance to fls (gravina et al., 2004; mian et al., 2009). quantitative resistance to c. sojina race 2 was shown to be underlain by two major qtl in the seedlings of ef94. the two loci were effective at different stages of seedling development, suggesting they were conditional qtl. the locations of the qtl suggested the loci are not allelic to rcs3 (lg j). the locations of rcs1 and rcs2 had not been determined by 2011, so the loci detected might be allelic to these qualitative resistance loci. however, in view of the lee ancestry of essex, the loci are not allelic to rcs1-3 (pace et al., 1993; baker et al., 1999). therefore, the loci represent new quantitative resistance loci possibly underlying a more broad and durable type of resistance found with other diseases (hnetkovsky et al., 1996). the loci mapped to locations containing several other resistance loci for scn, sds, insect herbivory and seed yield (yuan et al., 2002; kassem et al., 2006; yesudas et al., 2010). since essex provided the beneficial allele at the qtl on lg c2 the resistance alleles to fls, sds and seed yield were in coupling. however, on lg a2 resistance alleles to fls and insect herbivory were linked in repulsion to scn resistance. therefore, the major loci detected here may be useful for the genetic improvement of resistance to c. sojina by selection of new recombination events to break repulsion and create qtl stacks to increase resistance to c. sojina. the minor loci detected here were on eleven linkage groups (a1, b1, f, g, h, i, j, k, l, m and o). minor loci might represent type ii errors. alternately the loci might be in sparse areas of the map. a third possibility is the loci were major genes but with alleles that were not very diverged in essex and forrest. a fourth a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) trait qtl l.g. marker/ interval peak cm position lod additive r2 essex allelic mean + sem (n) forrest allelic mean + sem (n) fls 21 dai 1 c2 satt319-satt079 120 3.8 -0.58 0.52 2.44±0.01 (44) 3.80±0.01 (45) fls 42 dai 2 a2 satt632-a2d8 50 3.6 -0.92 0.15 1.93±0.01 (47) 2.36±0.01 (38) table 1. detection of qtl underlying resistance to cercospora sojina at 21 dai and at 42 dai by cim. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 179 ta bl e 2. m in or lo ci p ot en tia lly c on tr ib ut in g to r es ist an ce to f ls d et ec te d by a n o va f ro m s in gl e m ar ke rs f ro m s pa rs e ar ea s of th e ef 94 m ap a ss oc ia te d w ith tr ai ts. s ig ni fic an t a ss oc ia tio ns a re in b ol d an d r2 v al ue s ar e sh ow n. fl s 2 1 da i fl s 4 2 da i m ar ke r lg cm ch r p r2 e + se m f + se m p r2 e + se m f + se m *s at t2 76 a1 17 .2 5 0. 44 7 2. 97 + 0. 24 2. 93 + 0. 19 0. 00 7 0. 13 2. 55 + 0. 25 1. 81 + 0. 21 *s at t5 89 a2 34 .0 8 0. 26 5 3. 01 + 0. 23 2. 80 + 0. 20 0. 01 0 0. 11 2. 45 + 0. 20 1. 81 + 0. 17 *s at t4 44 b1 85 .9 11 0. 01 7 0. 06 3. 16 + 0. 17 2. 52 + 0. 24 0. 00 8 0. 12 2. 52 + 0. 21 1. 86 + 0. 18 *c fr 2 f 1. 1 13 0. 00 6 0. 09 3. 21 + 0. 22 2. 46 + 0. 19 0. 47 2 2. 19 + 0. 19 2. 17 + 0. 22 *c g g -s ca r g 10 .1 18 0. 01 6 0. 06 2. 50 + 0. 20 3. 16 + 0. 20 0. 06 0 2. 39 + 0. 21 1. 93 + 0. 18 *s at t2 93 h 89 .1 12 0. 01 4 0. 06 3. 22 + 0. 20 2. 55 + 0. 23 0. 11 3 2. 17 + 0. 20 2. 22 + 0. 21 *s at t4 40 i 11 2. 7 20 0. 23 9 2. 88 + 0. 19 2. 63 + 0. 24 0. 00 1 0. 15 1. 87 + 0. 17 2. 82 + 0. 24 *s at t2 49 j 12 .3 16 0. 00 9 0. 08 2. 67 + 0. 20 3. 40 + 0. 20 0. 25 4 2. 26 + 0. 21 2. 30 + 0. 21 *s at t5 55 k 42 .7 9 0. 17 5 3. 02 + 0. 21 2. 72 + 0. 23 0. 01 4 0. 10 2. 43 + 0. 21 1. 90 + 0. 18 *s at _1 16 k 52 .3 9 0. 44 7 2. 75 + 0. 27 3. 00 + 0. 21 0. 00 9 0. 11 2. 53 + 0. 19 1. 86 + 0. 20 *s at t4 46 l 11 .5 19 0. 04 9 0. 04 3. 23 + 0. 22 2. 71 + 0. 19 0. 04 2 0. 05 2. 44 + 0. 22 1. 95 + 0. 17 *b 35 h0 7 m 8. 1 7 0. 02 0 0. 05 2. 58 + 0. 24 3. 17 + 0. 16 0. 27 2 2. 10 + 0. 19 2. 28 + 0. 22 *s at t3 23 m 60 .0 7 0. 04 5 0. 04 3. 06 + 0. 20 2. 57 + 0. 21 0. 13 3 1. 91 + 0. 19 2. 45 + 0. 20 *s at t2 59 o 39 .8 10 0. 08 4 2. 56 + 0. 21 3. 00 + 0. 21 0. 04 5 0. 04 2. 00 + 0. 19 2. 49 + 0. 22 180 figure 2. locations of the major qtl found in the essex by forrest population on linkage groups a2 (42 dai) and c2 (42 dai) for fls ds (black arrows). also shown are qtl for hg type 0 (black stippled arrow); seed yield (light grey solid arrow); and herbivory by japanese beetle (dark grey solid arrow). the size of the arrow reflects the interval significantly associated by qtl cartographer or mapmaker at lod > 2.0 or anova at p < 0.001. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 181 possibility is that loci for resistance to other fls races contribute some quantitative resistance to race 2. the minor loci might be candidates for the non-allelic dominant genes for resistance to fls race 5 or 6 that were found in peking, cns, s100, d49-2491 and lee (pace et al., 1993; baker et al., 1999). conclusions resistance to race 2 of fls was inferred to be a quantitative trait with major loci contributions from essex and minor loci contributions from both forrest and essex. the genes were not in the regions of rcs3 but one or two of them might be allelic to rcs1 or rcs2. however, it is more likely is that race 2 resistance preceded the development of rcs genes and was quantitative (smith and camper, 1973; phillips and boerma, 1981). since all major loci and most minor loci were active at only one stage of seedling development it was inferred that this quantitative resistance to fls was conditional upon wounding state or growth stage. the method recommended by mian et al., (2008) for qualitative resistance was quite different since it does not wound the leaf, wraps the sprayed leaf in a plastic bag for 48h and scores fls only at 14 dai. therefore, the three different methods may not detect the same loci. however, the breeding and selection for fls may be more efficient with the markers, transgressive segregants and models of inheritance reported here. alleles from essex are common among modern us cultivars (lam et al., 2010). alleles from forrest can be selected in derived lines like hartwig and it’s derivatives (anand, 1992). recombination events breaking the repulsion between resistance to fls and resistance to scn would be useful advances for breeding. the resources available for genomics from forrest may assist in the isolation of genes underlying the major quantitative trait loci for resistance to many fls races (lightfoot, 2008). acknowledgements the authors thank dr. p. gibson, o myers jr. and m. schmidt for assistance with germplasm development and maintenance from 1991-2000. we thank the boyscast program and the government of india for the fellowship to hs. references athow kl and ah probst (1952) the inheritance of resistance to frogeye leaf spot of soybeans. phytopathology 42: 660–662. athow kl, ah probst, cp kartzman, and fa laviolette (1962) a newly identified physiological race of cercospora sojina on soybean. phytopathology 52: 712–714. anand sc (1992) registration of ‘hartwig’ soybean. crop sci 32: 1060–1070. baker wa, db weaver, j qiu, and pf pace (1999) genetic analysis of frogeye leaf spot resistance in pi54610 and peking soybean. crop sci 39: 1021–1025. basten cj, bs weir, and z zeng (2001) qtl cartographer version 2.0. raleigh, nc: department of statistics, north 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(2010) genome sequence of the palaeopolyploid soybean. nature 463: 178–183. shultz jl, s kazi, ja afzal, r bashir, and da lightfoot (2007) the development of bac-end sequence-based microsatellite markers and placement in the physical and genetic maps of soybean. theor appl genet 114: 1081–1090. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 182 smith tj and hm camper (1973) registration of essex soybeans. crop sci 13: 495. wrather ja, tr anderson, dm arsyad, j gai, dl ploper, a portapuglia, hh ram, and jt yorinori (1996) soybean disease loss estimates for the top ten producing countries during 1994. plant dis 79: 107–110. wrather ja, sr koenning, and tr anderson (2003) effect of diseases on soybean yields in the united states and ontario (1999 to 2002). plant health progr (online doi 10.1049). yesudas cr, h sharma, and da lightfoot (2010) identification of qtl in soybean underlying resistance to herbivory by japanese beetles (popillia japonica, newman). theor appl genet 121: 353–362 yorinori jt (1992) management of foliar fungal diseases in brazil. p. 185–193. in l.g. copping et al (ed.) pest management in soybean. elsevier applied science, london. yuan j, vn njiti, k meksem, mj iqbal, k triwitayakorn, ma kassem, gt davis, me schmidt, and da lightfoot (2002) quantitative trait loci in two soybean recombinant inbred line populations segregating for yield and disease resistance. crop sci 42: 271-277. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) atlas journal of biology 2017, pp. 355–363 doi: 10.5147/ajb.2017.0149 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) identification of loci underlying seed yield in recombinant inbred and near isogeneic soybean lines derived from flyer by hartwig samreen kazi1,2, jeffry l. shultz1,3, ahmed jawaad afzal1,2,4, yi-chen lee1,2, and david a. lightfoot1,2* 1 plant biotechnology and genomics core-facility, department of plant, soil, and agricultural systems, illinois soybean center, southern illinois university, carbondale, il 6290, usa; 2 molecular biology and medical biochemistry program, southern illinois university, carbondale, il 62901, usa; 3 present address: school of biological sciences, louisiana tech university, ruston la, usa; 4 present address: lums, lahore, pakistan. received: january 6, 2017 / accepted: february 19, 2017 __________________________________________________ * corresponding author: ga4082@siu.edu 355 abstract two major determinants of soybean [glycine max (l.) merr.] seed yield were resistances to the soybean cyst nematode (scn) and sudden death syndrome (sds). two loci were identified rhg1/rfs2 and rhg3/rfs5 (for resistance to scn, heterodera glycines (i.) hg type 1.3(race 14), hg type 0 (race 3) and sds caused by fusarium virguliforme (roy & rupe)). the aim of this study was to identify quantitative trait loci (qtl) underlying seed yield. used were 142 microsatellite markers and the recombinant inbred line population (ril) ‘flyer’ × ‘hartwig’ (f × h; n=92). flyer (f) was high yielding but scn and sds susceptible. hartwig (h) was lower yielding but resistant to all scn hg types and sds. four regions on 3 chromosomes were associated with seed yield. the first region on chromosome 9 (gm9, qyld09.1), identified by the microsatellite marker satt539-satt242 (lod 2.9, 13% variation) derived the beneficial allele from hartwig (f allele 2.76 ± 0.06 mg/ha; h allele 2.98 ± 0.03 mg/ha). the second region on gm9 (qyld09.2) between satt337 and satt326 spanned 1.4 cm (lod of 5.31, 20.2% variation) and the beneficial allele derived from flyer (0.22 mg/ha f allele 2.98 ± 0.03, h allele 2.77 ± 0.04 mg/ha). the third and fourth qtl were identified in genetic linkage groups d2 (qyld19.1) and g (qyld18.1) in regions previously associated with resistance to scn. the region encompassing rhg1/rfs2 on gm18 between the microsatellite marker tmd1 and satt610 spanned 15.5 cm (lod 3.05, 15.8 % variation, f allele 2.37 ± 0.035; h allele 2.91 ± 0.058 mg/ha). the region on linkage group d2 between satt514 and satt488 spanned 32.6 cm (lod 2.57, this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the main goal of plant breeders is to increase crop yield (fehr, 1987). overtime yield improvement has two major inputs; genetic gain and production efficiency (specht et al., 1999). genetic gain expressed as seed yield potential, largely derives from improvement in stress and disease resistances. farmers are responsible for production efficiency by choosing the best varieties and the best production technology. soybean seed yield is a multigenic trait (mansur et al., 1993). genotype by environment (g × e) interactions, genomic duplications and epistasis complicates phenotypic selection for soybean seed yield (lark et al., 1996). further, seed yield at harvest maturity (r8) is a composite trait composed of loci that control plant performance during the growing season (sun et al., 2006; palomeque et al., 2009). hence a wide set of contributing loci are expected. to date the yield potential of lines derived by intercrossing has been difficult to predict without extensive field tests that are expensive and time consuming (maughan et al., 1996). components of yield such as maturity, lodging, growth habit (fehr 1987; specht et al., 1999) and disease resistance in pathogen infested environments (concibido et al., 2004; kas13.3% variation, f allele 2.79 ± 0.049; h allele 3.1 ± 0.043 mg/ha). the qtl detected will allow marker assisted selection to stack seed yield, with pest resistance traits (rhg1/rfs2/ qyld18.1; h/h/h allele) and recombinant loci (rhg5/rfs2/ qyld19.1; h/f/h alleles). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) sem et al., 2006) have major effects on yield potential in many segregating populations. dna molecular markers have been used extensively to identify and screen for qtl that underlie soybean seed yield and yield components (mansur et al., 1994; mansur et al., 1996; orf et al., 1999b; yuan et al., 2002; kassem et al., 2006). the yield qtl identified to date are large genomic regions where at least two distinct alleles have been shown to influence yield. individual yield qtl regions can explain up to 25% of yield variability and can be detected by bulked segregant analysis. some have been stable in different genetic backgrounds and environments (yuan et al., 2002) and some have not (reyna and sneller 2001). some seed yield loci have been shown to be clusters of loci active at different states in plant and seed development (palomeque et al., 2009) molecular markers closely linked to accurately mapped seed yield qtl allow the possibility of early selection of lines by genotype in soybean (palomeque et al., 2009). toward this end, hundreds of seed yield qtl have been mapped in diverse germplasms. several seed yield qtl have been identified in every linkage group of the soybean genome. for example, orf et al. (1999a,b) identified qtl for seed yield in different linkage groups a2, b1, b2, d1a+q, d1b+w, d2, f, j, l, and m of the soybean genome. other authors identified qtl for yield on linkage groups c2, h, m (specht et al., 1999), f (reyna and sneller 2001), j, l, m (mansur et al., 1993, 1996), and n (kassem et al., 2006) of the soybean genome. three qtl for yield were found on linkage groups c1, i, and k using 136 ssr markers and a ril population of essex × forrest (e × f) (yuan et al., 2002). two genomic regions associated with yield were also identified and mapped on linkage group k using a ril population of flyer × hartwig (f × h)(yuan et al., 2002). here we describe further analysis of seed yield loci in the recombinant inbred line (ril) population derived from f × h (yuan et al., 2002; kazi et al., 2008). the population was used because it segregates for growth habit (determinate to semideterminate) and about 100 -114 days in maturity. yield qtl could be examined in the presence of these potentially confounding factors. here, we report seed yield qtl identified in this populations based on a more extensive map than reported previously. materials and methods plant material the genetic material used in this study consisted of the f × h rils (n = 92; yuan et al., 2002; kazi et al., 2008). populations were advanced to the f5:14 from 2003 to 2005 and seed were released in 2007 (kazi et al., 2008). the cross was selected because it showed segregation for seed yield and many pest resistances, including well characterized reactions to sds, and scn. hartwig was lower yielding but consistently resistant to sds in most locations (njiti et al., 1997, 2001; mueller et al., 2003) and strongly resistant to most hg types of scn (anand 1992). flyer was higher yielding but susceptible to most scn hg types and to sds (yuan et al., 2002; kazi et al., 2007, 2008, 2010). methods of field trait measurements field trait measurements were measured from 1997 to 2010 by methods described in yuan et al., (2002). briefly, the f × h ril population along with two parents was grown for increase in non-infested fields. locations were planted with 4 row plots 6.1 m long and row spacing was 0.75 m apart in a randomized complete block design with three replications. weeds were controlled with preand postplant herbicides and hand weeding. rows were examined to remove contaminants or off-types at flowering. only the center two of the four rows were harvested. seed yield was measured with cleaned seed at 13 % (w/v) moisture without discarding broken seed. lodging was score on a 1-5 scale before harvest. days after planting to maturity was measured at r6 and r8. sds analysis the population was planted in sds infested environments as described in yuan et al. (2002) and kazi et al. (2007). briefly at the southern illinois university carbondale agonomy research center (arc), ullin (u) and ridgway (r) disease incidence (di), disease severity (ds) and root infection severity (is) were measured from 1997 to 2000. for disease rating rils were planted as 2 row plots in a randomized complete block design with 3 replications. scn scores scn resistance was determined as described by yuan et al. (2002) with the following modifications. the complete f5:11 f × h ril (n=92) population was tested for reactions for scn hg type 0 (pa-3; race 3). of the hg type 0 resistant lines the reactions to hg types 1.3.6.7 (pa-14, race 14) and hg type 1.2.3.5.6.7 (pa2, race 2) were determined (kazi et al., 2007). five, three and two single-plant replications were used respectively per ril line. cultivar hutcheson was used as the susceptible control while peking, pickett, pi 88788 and pi 90763 were used as the standard differentials to determine hg biotype. dna marker analysis dna was extracted and used for microsatellite amplifications as in yuan et al. (2002) as modified by kazi et al. (2007). briefly, more than 350 barc-satt markers spaced at 5-10 cm intervals (song et al., 2004) were used. in addition, 140 siucbes-ssr primers from the build 2 mtp bes clones (shultz et al., 2006ab, 2007) were used chosen to be spaced at 10,000 kbp intervals from the soybean physical map (shultz et al., 2006ab, 2007). amplification reactions in 10 µl, used 15 ng of genomic dna, 0.75 µl taq polymerase (1 unit), 0.75 mm dntps, and 15 nm of forward and reverse primers. the pcr products were separated on 4 % (w/v) agarose gels by electrophoresis at 92 v for 4 hours. gels were stained with ethidium bromide, dna visualized under uv light and photographed. flyer and hartwig amplified dna samples were included as controls to facilitate manual scoring. 356 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) heritability estimation the heritability (h2) estimates, a ratio of genotypic variation over phenotypic variation of seed yield were calculated using variance components obtained through analysis of variance (anova; sas institute inc. cary, nc), as described in fehr (1987). narrow sense heritability for each trait was calculated. all correlations were calculated using the proc corr function of sas. construction of the genetic linkage map following kazi et al. (2007) a linkage map was created using mapmaker/exp 3.0 (lander et al., 1987). briefly, centimorgans (cm, haldane units), lod 3.0, and maximum distance 50 cm were used. heterogenous scores for lines were excluded. the recombinant inbred line (ri-selfing genetic model) was used with error detection. conflicts among the positions of linked markers in f × h were resolved in favor of experimental evidence when the maps generated at lod 3.0 disagreed with the composite map of song et al. (2004). construction of qtl maps following kazi et al. (2008) three methods were used to detect qtl, single point analysis; interval mapping and composite interval mapping. a. single point analysis was accomplished by line mean comparisons by anova, with mean separation by lsd as described by njiti et al. (1998). a significant difference (p < 0.005) was considered to be a preliminary indication of an association between a marker and a qtl for the trait in question. a value of p ≤ 0.0005 was suggested by an approximate bonferroni correction. however, at genomic regions where gaps between adjacent markers were greater than 10 cm in the map associations 0.005>p>0.0005 were accepted as a potentially significant association. precedents (hnetkovsky et al., 1996; chang et al., 1997; njiti et al., 1997; kassem et al., 2006) have shown these criteria to be valid in later maps (meksem et al., 2001a; njiti et al., 2002; yuan et al., 2002; triwitaykorn et al., 2005; ruben et al., 2006). b. interval maps of qtl were first analyzed by mapmaker/ qtl 1.1 using the f2 -backcross genetic model for trait segregation (kazi et al., 2008). putative qtl were inferred when lod scores exceeded 2.0, equivalent (but not equal) to a single marker p < 0.005 in one-way anova. the position of the qtl was inferred from the lod peaks at individual loci detected by maximum likelihood tests at positions every 2 cm between adjacent linked markers. c. composite interval maps of qtl (cim) used winqtl cartographer (version 2.5; jansen and stam 1994; basten et al., 2001). following kazi et al. (2008) a walk speed of 2 cm and the forward regression method were selected. qtl were inferred when lod score peaks exceeded 2.0. to confirm linkage, experiment-wise threshold level was calculated from 1,000 permutations of each genotype marker against the phenotype in the population. linkage was reported as significant if the two statistics for a marker were significant. the use of lower lod values has been justified previously (hnetkovsky et al., 1996; prabhu et al., 1999; kazi et al., 2007, 2008, 2010). results seed yield trait distributions grand seed yield (mg ha-1) across four environments and 2 years among the 92 recombinant inbred lines from flyer × hartwig showed no significant departure from normality (p>0.0001; figure 1). the frequency distribution of mean yield was skewed (-0.54) toward low yield limiting parent hartwig. the distribution was continuous, uni-modal and a significant kurtosis (0.82) that reflected a peaked distribution. there was no line whose yield was higher than flyer and 18 lines had a mean lower than hartwig. nine of the extreme lines were significant transgressive segregants (standard deviation 0.29; p < 0.0001). the mean seed yield at nashville in 1998 (n98) showed unimodal and relatively intermediate distribution (figure 2a). the distribution is skewed towards low yield like hartwig. fifteen lines with a mean yield higher than flyer were significant transgressive segregants (at standard deviation 0.31; p<0.0001). twelve lines had a n98 mean lower than hartwig and were significant transgressive segregants. as shown in figure 2b, the frequency distribution of the seed yield at ridgway in 1998 (r98; mg ha-1) showed a bimodal distribution. there was evidence for peaks at 3.51 and 4.01. the distribution was skewed towards low yield. a negative kurtosis (-0.08) reflected a platykurtic distribution as these curves have fewer extreme scores than found in a normal distribution. four lines with a mean yield higher than flyer (figure 1) were three were significant transgressive segregants (at standard deviation 0.50; p < 0.0001). nineteen lines had a mean lower than 357 figure 1. frequency distributions of the mean seed yield (mg ha-1) across four field locations among 92 recombinant inbred lines from a cross between cultivars flyer and hartwig. the mean yield score for the parents were arrowed. the least significant difference between parent and line means was 0.015 (p < 0.05). the number of scn resistant rils in each yield class is shown by light colored boxes. the highest yielding rils were also scn resistant. ril 95 was resistant to both races 3 and 14, whereas ril 78 was only resistant to race 3. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 358 hartwig and nine were significantly transgressive. the frequency distribution at harrisburg in 1999 (h99; mg ha-1) showed unimodal distribution as there was peak below 2.78 (figure 2c). a significant positive kurtosis (1.20) reflected a leptokurtic (‘lepto’ means slender or narrow) distribution and skewed towards lower yield (left). here, hartwig yielded well too. there were five lines that have higher mean yield than flyer. (p<0.0001). as shown in figure 2d, the frequency distribution of the seed yield at nashville in 1999 (n99; mg ha-1) showed an approximately normal (p = 0.001) and continuous distribution, although there is evidence for a peak below 2.28. its distribution was skewed towards low yield hartwig. a significant kurtosis (0.15) reflected a mesokurtic distribution. thirteen lines with a mean yield higher than flyer (figure 1) were statistically significant transgressive segregants (at standard deviation 0.31; p < 0.0001). eighteen lines had a mean lower than hartwig and nine were significantly transgressive segregating lines. trait heritability and correlations the heritability of mean seed yield on a line mean basis among the recombinant inbred lines was 57% across four environments. among locations heritability ranged from 72 to 89%. all of the scn resistant progeny had higher yields than the hartwig, scn resistant parent. however, none yielded better and only two as well as the scn susceptible parent flyer (figure 1). there were a significant correlations between mean yield and resistance to scn race 3(p = 0.005, r2 = 31%); scn race 14 (p = 0.005, r2 = 24%); mean sds in other field locations infested with f. virguliforme (p = 0.001, r2 = 33% for dx) and (p = 0.006, r2 = 19% for is at r8). there was no significant association between yield and growth habit (p = 0.06, r2 = 4.0%) or yield and lodging (p = 0.055, r2 = 9%) in the locations tested for yield. yield means were 2.92 + 0.03 and 2.79 + 0.07 mg ha-1 for indeterminate (n = 72) and determinate (n = 22) lines, respectively. significant genomic regions for yield there were two regions detected on linkage group k (figure 3) associated with mean seed yield across four environments and two consecutive years by cim (table 1; figure 3). the first region on linkage group k, identified by the microsatellite marker satt539-satt242, was significantly associated (p=0.0005, r2=20.5%). the interval identified by im had a peak lod score of 2.9 and explained about 13% of total variation of soybean yield. however, the interval was not associated with seed yield at every location. the interval was significantly associated (p=0.0001, r2=34.5%) with yield at harrisburg, il in 1999 (h99) and at nashville in 1999 (p=0.01, r2=6%). it was not associated with yield at ridgway or at nashville in 1998 (p=0.056, r2=2.4%). neither satt539 nor satt242 was associated with soybean yield at ridgway in 1998 by im or anova (not shown). the region derived beneficial allele for seed yield from hartwig (the flyer allelic mean was 2.76 ± 0.06 mg ha-1; the hartwig allelic mean was 2.98 ± 0.03 mg ha-1). the second region of linkage group k associated with mean seed yield (figure 3) was the interval between satt337 and satt326. the interval contained a mean seed yield qtl that spanned 1.4cm had a peak lod of 5.31 and explained about 25.2% (0.4 mg ha-1) of total variation in soybean yield (figure 3). the region derived the beneficial allele from flyer (table 1). however, at individual locations the interval was no associated with mean seed yield. in addition, by anova the associated locus was only significantly associated with seed yield in n98 and the mean of all locations (table 1). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) h f a b c d h f f h h f figure 2. frequency distributions of the mean yield (mg ha-1) at four field locations (nashville 98, ridgway 98, harrisburg 99 and nashville 99) among 92 recombinant inbred lines from the cross f × h. the mean yield for each parent flyer, hartwig and lines mean were arrowed. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 359 population, marker yield mean±sem (mg ha-1) (alleles from) and map position location p value r2 lod qtl var flyer hartwig satt337 lg k (gm9) n98 0.0042 10 5.9 27 3.20 ± 0.06 2.69 ± 0.08 47.4 cm n99 0.0001 27 2.2 26 2.53 ± 0.05 2.36 ± 0.04 mean 0.0006 14 2.7 14 2.98 ± 0.03 2.77 ± 0.05 satt326 lg k (gm9) n98 0.0001 26 5.4 26 3.20 ± 0.06 2.69 ± 0.08 49.5 cm n99 0.0082 9 1.6 8 2.52 ± 0.06 2.33 ± 0.04 mean 0.0004 15 3 15 2.98 ± 0.04 2.76 ± 0.05 satt539 lg k (gm9) r98 0.049 5 1.8 5 3.39 ± 0.09 3.62 ± 0.07 2.0 cm h99 0.0006 15 2.6 13 2.45 ± 0.07 2.74 ± 0.04 n99 0.012 8 1.5 8 2.35 ± 0.05 2.54 ± 0.05 mean 0.0008 14 2.5 13 2.77 ± 0.06 2.99 ± 0.03 tmd1 lg g (gm18) ‡ n98 0.0047 11 2.5 10 2.88± 0.07 3.38± 0.05 r98 0.9552 3 0.5 2 3.50± 0.04 3.70± 0.07 h99 0.0029 10 2.8 11 2.54 ± 0.07 2.99± 0.06 n99 0.0001 16 4.1 17 2.33± 0.06 2.79± 0.07 mean 0.0007 9.7 3.1 27.4 2.32 ± 0.03 2.64 ± 0.05 satt514_lgd2 (gm17) 85.7 cm mean 0.0006 8.0 2.52 12.1 2.77±0.048 3.0±0.041 †mean involves all location used for evaluation within each population. ‡ also significantly associated with scn resistance (within the intron of the receptor like kinase at rhg1). table 1. examples of markers associated with seed yield at harvest among recombinant inbred lines in two soybean populations. essex × forrest was evaluated in carbondale, il (c96) and ridgway, il (r96) in 1996 and in desoto, il (d97) in 1997. flyer × hartwig was evaluated in nashville, il (n98 and n99) in 1998 and 1999; ridgway, il (r98) in 1998 and harrisburg, il (h99) in 1999. clustered yield qtl were found on lg k (gm9) and g (gm18) that could be fine mapped in nils. loci on c1 (gm4) and i(gm20) proved difficult to isolate in nils and so might be inaccurately mapped or blends of conditional qtl. marker location p value r2 (%) loda qtl var.b yield means ± sem (mg ha -1) with alleles from flyer hartwig a. anova anova im im im im satt337 (k) satt326 (k) nashville 98 mean yield nashville98 yieldmn98 mean yield 0.0001 0.002 0.0001 0.006 0.001 12.4 4.6 15.4 10 6.2 2.06 2.2 2.14 2.6 2.50 25.2 20.2 14.5 7.7 5.5 3.19±0.05 2.98±0.03 3.14±0.07 3.38±0.05 2.97±0.04 2.69± 0.07 2.77±0.04 3.35±0.05 3.08±0.07 2.75±0.04 b. cim cim cim cim satt337satt326 (k) nashville 98 ridgway 98 mean yield 5.31 2.48 2.55 25.2 13.6 13.8 2.98±0.03 2.77±0.04 satt539satt242 (k) nashville 98 ridgway 98 mean 98 harrisburg99 nashville 99 mean 99 mean yield 0.056 0.0001 0.0001 0.01 0.001 0.0005 2.4 7.12 34.5 6.0 5.1 6.5 1.04 3.76 2.81 1.3 2.65 2.9 5.6 13.1 14.5 9.2 13.6 13.0 3.01±0.08 3.13±0.08 2.44±0.03 2.34±0.05 2.41±0.05 2.76±0.05 2.94± 0.07 3.34±0.05 2.747±0.05 2.52±0.52 2.61±0.03 2.98±0.03 a. lod was the probability of the presence of a locus. b. var. was the amount of variability in seed yield explained by the marker loci. table 2. regions detected by cim (a.) and anova or im (b.) that were associated with mean yield (mg ha-1) across various locations in the flyer by hartwig (ril) population on linkage group k. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 360 figure 3. genetic map of seed yield qtl on linkage group d2, g and k (chromosomes 17, 18 and 12 respectively) in the f × h ril population. estimated map distances are shown (cm). qtl are presented to the right of the linkage group as arrows. discontinuous black arrows are qtl underlying seed yield. the qtl underlying resistance to sds are black stippled arrows. the qtl underlying resistance to scn race 14 are grey stippled arrows. the qtl underlying resistance to scn race 3 are black solid arrows. figure 4. parents and ancestors of hartwig and flyer. sds and scn resistance is indicated along with approximate year of release and percent of heterozygosis in a single plant estimated by gbs where known (blue number; li et al., 2014). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 361 two additional qtl were identified in genetic linkage groups d2 and g of the f × h population. one region on linkage group g (chromosomes 17 and 18: figure 3) identified by the microsatellite marker tmd1 was significantly (p=0.0005, r2=9.7 %) associated with mean yield over two years (1998, 1999) in the population. the adjacent marker was satt610. the interval between these two markers spanned a genetic distance of about 15.5 cm contained a yield qtl (figure 3) and had a peak loglikelihood (lod) of 3.05 and explained about 15.8 % of total variation in mean yield (table 2). this region was also significantly associated with resistance to scn hg types 0 and 1.3.5 (kazi et al., 2007, 2009) and is the likely location for rhg1. the region on linkage group d2 (figure 3) identified by the microsatellite marker satt514 was significantly (p=0.0006, r2=7.4%) associated with seed yield (across environment grand mean yield; 1998-1999) in the population at four locations. the adjacent marker was satt488. the interval between these two markers spanned a genetic distance of about 32.6 cm. the yield qtl had peak log-likelihood (lod) of 2.57 and explained about 13.3% of total variation (table 2). discussion the f × h population was very important for the analysis of seed yield qtl and other agronomic traits because it segregates for many traits (kazi et al., 2007). a qtl for dap (days after planting) maturity was found to be located on linkage group c2 by satt277 that gave a highly significant peak-lod of 6.45 and underlies segregation across maturity groups 4 and 5. previously, this locus has been found significantly associated with lodging and plant height in minsoy × archer (orf et al., 1999a), seed weight and seed yield in noir1 × archer (orf et al., 1999a) and plant height/maturity date in noir1 × archer (orf et al., 1999b). for quantitative traits, genotype and environment interactions (g × e) are commonly observed (kearsey and farquar 1998). specific environmental conditions have effects on quantitative trait values like yield and lodging (brim 1973). environment seems to be an important factor in the number of qtl detected. for instance, in f × h, there was no yield qtl that associates with all environments, suggesting that these qtl might be environment specific (palomeque et al., 2009). the qtl that was identified based on average data from two years may be the most significant qtl for breeders, because these may represent the genes that will lead to yield stability irrespective of the environment. qtl detected in one population could be detected at similar positions in different populations (lin et al., 1995). most populations had a reasonably good distribution of markers across the major linkage groups, like in e × f (meksem et al., 1999) but inevitably gaps were present that prevented detection of qtl across populations. also, different qtl could be identified in different populations due to genetic background effects. this is probably the reason why a large effect is detected in one population but not in others (brummer et al., 1997). as all populations were not mapped with the same markers, it is difficult to provide reliable estimates of the number of qtl that are common across various populations. however, common qtl are detected. in this study, f × h ril verified putative qtl for yield on linkage group k. the genetic marker satt337 was significantly associated with yield in both f × h and e × f population (yuan et al., 2002; table 3). this indicates that the qtl associated with this marker is consistent. here the susceptible parent, flyer, in f × h provided the beneficial allele. as shown in figure 4, these parents share a significant amount of their genome with essex and forrest i.e. flyer originated from the cross essex × l24. on the other hand, hartwig was derived from a cross between forrest3 × pi437654. two new qtl for yield were also detected marker or interval location p value r2 (%) loda qtl var.b (%) yield means ± sem (mg ha-1) with alleles from flyer hartwig a. anova anova im im im im tmd1 (g/18) satt610 (g/18) mean yield mean yield 0.0007 0.01 9.7 9.0 .07 2.57 27.4 11.8 2.32±0.03 2.72±0.09 .64±0.05 .96±0.052 satt514 (d2/17) mean yield 0.0006 8.0 2.52 12.1 2.77±0.048 3.0±0.041 b. cim cim cim cim tmd1satt610 (15.5cm) (g/18) mean yield 3.05 15.8 2.37±0.035 2.91±0.058 satt514satt488 (32.6cm) (d2/17) mean yield 2.57 13.3 2.79±0.049 3.1±0.043 a. lod was the probability of the presence of a locus. b. var. was the amount of variability in seed yield explained by the marker loci. table 3. regions on linkage groups g (18) and d2 (17) detected by anova and im (a.) or cim (b.) that were associated with mean yield (mg ha-1) and disease resistance in the flyer by hartwig (ril) population. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 362 in linkage group g (tmd1) and d2 (satt514) but they were not responsible for yield drag associated with scn resistance. 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cell model in vitro john bang1, susan yeyeodu2, naila gilyazova2, sam witherspoon2, and gordon ibeanu2,3* 1 department of environmental, earth and geospatial sciences, north carolina central university, 2105 mary m. townes science building, 1801 fayetteville st., durham, nc 27707, usa; 2 biomanufacturing research institute and technology enterprise, north carolina central university, 302 east lawson st., durham, nc 27707, usa; 3 department of pharmaceutical sciences, north carolina central university, 302 east lawson st., durham, nc 27707, usa received: july 11, 2011 / accepted: august 19, 2011 __________________________________________________ * corresponding author: gibeanu@nccu.edu introduction recent developments in medicine and industry involve increasingly the production and use of nanomaterials. while the intended uses of these materials are to benefit human health and the environment, it is not yet clear whether their existence and use has harmful consequences. carbon nanotubes (cnts) have come to the forefront in the past decades as one of the most promising nanomaterials. although other species have been reported, there are basically two types of cnts which differ in the number of helical layers they contain. these are designated as single-walled carbon nanotubes (swcnts) and multi-walled carbon nanotubes (mwcnts). cnts possess certain properties which are highly desirable in several fields and applications, including their small size, electrical conductivity, strength and flexibility (baughman et al., 2002; malarkey and parpura, 2007; saito et al., 2009). however, cnts have recently been rated environmentally toxic (kahru and dubourguier, 2010). early in vivo studies with non-carbon based nanoparticles demonstrated that either ingestion or inhalation of these particles could result in their accumulation in the brain (el-ansary and al-daihan, 2009). while inhaled cnts are toxic to pulmonary, 70 abstract as the use of nanomaterials continues to flourish in industrial and biomedical applications so does the concern that these materials may have unanticipated and undesirable effects on human health and the environment. inhaled or ingested nanoparticles have been detected in the brain, among other organs, raising the question as to their effect on neuronal viability. the effects of multi-walled carbon nanotubes (mwcnts) on the viability of neuroscreen-1 (ns-1) cells, a neuronal cell model, were studied. resazurin reduction and in situ hoechst and propidium iodide (pi) double staining indicated mwcnts reduce cell viability. caspase 3/7 activity on par with etoposide-induced apoptosis was not observed until 72 hours and was only 20-30% higher than controls. however, increased levels of annexin v in the outer leaflet of the cell plasma membranes after 24 hours and a minimum two-fold increase in relative mitochondrial depolarization after 48 hours both suggested mwcnts promoted apoptosis in this neuronal cell model. keywords: neuroscreen-1, carbon nanotubes, hypodiploid, neuronal viability, pheochromocytoma, pathway finder. 71 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) circulatory and immune systems (madl and pinkerton, 2009) their effect on the central nervous system is not known. therefore, further studies in vivo are needed to determine the effects of cnt exposure in the central and peripheral nervous systems. in vitro studies have provided another approach to the study of cnt toxicity where conditions can be more easily manipulated. reports using primary or immortal cell lines of the cns glia and neurons have yielded mixed results. for example, the bv2 microglia cell line was unaffected by mwcnts (kateb et al., 2007), while the dna content of primary microglia in mixed neuro-glia or glia-enriched cultures were reduced by exposure to swcnts (belyanskaya et al., 2009). the pc12 pheochromocytoma line widely used as a neuronal cell model (greene and tischler, 1976) shows various signs of stress and/or apoptosis when exposed to relatively low concentrations of cadmium telluride quantum dots (lovric et al., 2005) or mn, ag or cu nanoparticles (hussain et al., 2006; rossi et al., 2006). however, this same cell line showed no signs of toxicity after exposure to mwcnts (xu et al., 2009). furthermore, in primary cortical neurons, mwcnts protected against toxicity induced by the nonionic detergent pf127 (bardi et al., 2009). we report here the extent and nature of mwcnt toxicity using the ns-1 cell line, a rapidly growing, highly adherent line derived from pc12 cells. specifically, we examined mwcnt effects on cell metabolism, plasma membrane translocation and permeability, dna integrity, caspase 3/7 activity and mitochondrial membrane depolarization. we chose to study mwcnts rather than swcnts for several reasons. first, mwcnts are currently more widely available at significantly lower cost than swcnts. second, swcnt synthesis is favored at a higher metal to carbon ratio (lam et al., 2006), a process that increases the level of metal impurities and the likelihood of toxicity. third, swcnts have proven more toxic in in vitro studies (lovric et al., 2005; magrez et al., 2006). finally, humans and other organisms are exposed to mwcnts generated by fuel combustion (lam et al., 2006). materials and methods carbon nanotubes mwcnts were either purchased from nanomaterials store (fremont, ca) or received as a gift from jie liu (duke university, durham, nc). both materials, designated cnt-ca and cnt-d, were synthesized by chemical vapor deposition with fe or co. cnt-ca was >99% pure (diameter=10-20 nm, length=10-30 microns). cnt-d contained <0.1% fe as judged by inductively coupled plasma (icp) spectroscopy. dynamic light scattering estimated particle hydrodiameter at 85-115 nm (p<0.5). mwcnts were solubilized by sonication and vortexing in treatment media (rpmi 1640 containing 100 µg/ml penicillin/streptomycin). the remaining insoluble particles were removed by filtration through a 0.45 micron polyethersulfone (pes) filter (pall corporation, port washington, ny). the concentration of soluble mwcnts was 190 ppm (cnt-ca) and 295 ppm (cnt-d) as determined by total organic carbon (toc) analyzer (shimadzu, japan). tissue culture ns-1 cells purchased from cellomics inc. (pittsburgh, pa) were maintained in culture media consisting of rpmi 1640, 10% fetal bovine serum (fbs), 2 mm glutamine and 100 µg/ml penicillin/ streptomycin (pen/strep) at 37oc in 5% co2, 90% humidity and plated at 1x104 cells per well in 96-well or 5x105 cells per well in 6 well microtiter plates for assays. viability assay (redox system) cells were plated overnight at in 96 well plates and then treated with mwcnts. at indicated times resazurin (sigma, st. louis, mo) in dulbecco’s phosphate buffered saline without calcium and magnesium (pbs) was added to the wells (final concentration 0.1 mg/ml) and incubated at 37oc for 2 hours. the reduction of resazurin to resorufin was measured fluorometrically (λexc560 nm/λem590nm) using a pherastar plus plate reader (bmg labtech, cary, nc). data were processed using mars data analysis (bmg labtech) software. viability assay (in situ) following cnt or control treatment for 48 h, cells were double stained with a mixture of hoechst 33342 (invitrogen, carlsbad, ca) and propidium iodide (pi) (sigma, st louis, mo). the dyes were added to a final concentration of 2 µg/ml and 5 µg/ ml, respectively. cells were incubated for 30 min at room temperature (rt) and imaged on a bd pathway findertm 855 high content screening (hcs) imaging platform using a 20x lwd objective. a 3x3 montage image was collected for each well. each treatment condition was performed in quadruplicate (average of 1000 total nuclei per well). the resulting images were segmented for nuclei using bd attovisiontm software version 6.1. apoptosis/necrosis assay (annexin v/pi double stain) cells were plated overnight, rinsed with serum free medium (sfm) and treated with cnts or 10 µm etoposide (calbiochem, darmstadt, germany) at 37°c for the indicated times. after rinsing with sfm, cells were harvested and washed in cold pbs and resuspended to a density of 1x106 cells/ml in annexin-binding buffer (10 mm hepes, 140 mm nacl and 2.5 mm cacl2, ph 7.4). a 5 µl aliquot of annexin v-fitc and pi (5 µg/ml final concentration) were added to 100 µl samples. samples were incubated at rt for 15 min and diluted with 400 µl annexin binding buffer, gently mixed and transferred to ice. samples were analyzed on a bd facsariatm cytometer (becton dickinson, mountain view, ca) using facsdivatm software version 6.1 using λexc488 nm/λem518nm. mitochondrial membrane potential (jc-1) ns-1 cells were plated overnight at 37°c, washed twice with pbs and incubated with cnt preparations at 37°c for 48 h prior to analysis. cells were harvested, washed and loaded for 1 h at 37oc with 6 µg/ml jc-1 (invitrogen, carlsbad, ca) and data a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) were collected on a bd facsariatm cytometer using facsdivatm software version 6. valinomycin treated cells (1 µm) were used to produce gates separating cells with polarized versus depolarized (valinomycin-treated) mitochondria. to enhance spectral separation, the red “j-aggregate” fluorescence generated by polarized mitochondria was monitored at 610 nm and the green jc-1 monomer was detected at 530 nm. fluorescence compensation was applied to enhance resolution of cells with decreased mitochondrial membrane potential. cell cycle analysis (pi) adherent cells were harvested, washed in pbs and suspended at1x106 to 1x107 cells in 500 µl pbs to a uniform single cell suspension. cells were transferred to ice and fixed in 4.5 ml cold 70% ethanol for at least two hours. cells were rinsed briefly in pbs and suspended in 1 ml pi staining solution containing 0.1% v/v triton x-100 (sigma, st. louis, mo), 0.2 mg/ml dnase-free rnase a (sigma) and 20 µg/ml pi for 30 min at rt. samples were analyzed on a bd facsariatm cytometer using facsdivatm a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) software version 6.1. the hypodiploid population of cells was identified as apoptotic. apoptosis assay (caspase 3/7) caspase 3/7-like activity was assayed using the apo-one homogeneous caspase-3/7 assay kit (promega, madison, wi) with 10 µm etoposide as a positive control (hishita et al., 2001). fluorescence (λexc485 nm/λem535nm) was measured using a pherastar plus microplate reader. statistical analysis data with error bars are presented as means ± standard deviation (sd) of at least three separate experiments done in triplicates. anova and turkey’s multiple comparison tests were performed in graphpad prism 5.0 (graphpad software inc., san diego, ca). values of p less than (<) 0.05 were considered statistically significant. 72 fig. 1. viability of ns1 cells exposed to cnts. ns1 cells were exposed to sfm or carbon nanotubes for 24 h (a) or 48 h (b) and assayed for metabolic activity. alternately, cells were double stained with hoechst 33342 and pi as in situ measure of viability (c). percentage of viable cells was calculated as the ratio of (hoechst nuclei – pi nuclei)/hoechst nuclei. results represent means ± sd of three experiments in triplicates. *, *** indicates p<0.01, and p<0.001 compared to sfm. results mwcnts reduce ns1 neuronal cell viability in order to investigate the neurotoxicity of mwcnts, we selected ns-1 cells, a subclone of the pc12, a pheochromocytoma line used as in vitro neuronal model (greene and tischler, 1976). ns-1 metabolic activity was assayed using resazurin conversion to resorufin as a biochemical measure of cell viability (magnani and bettini, 2000). mwcnts from two sources inhibited ns-1 viability as compared with cells grown in either culture medium or sfm (fig 1). figure 1a shows treatment of cells with mwcnts resulted in a decrease of more than 40% in resazurin conversion capability after 24 h. the toxic effects of cnts were even more pronounced by 48 h when the extent of metabolic inhibition was approximately 70% (fig. 1b). only the highest concentrations of solubilized mwcnts (190 ppm for cnt-ca and 295 ppm for cnt-d) showed toxic effects; when diluted, the mwcnts had no effect on cells (data not shown). cell viability was also measured using an in situ assay of plasma membrane integrity (fig. 1c). automated measurements compared total cell nuclei (stained with hoechst 33342) and nuclei from cells with compromised plasma membranes (stained with pi). less than half the cells treated with mwcnts had intact plasma membranes by 48 h. in both viability assays, cnt-ca was slightly but consistently more toxic than cnt-d. in order to test the limits of mwcnt toxicity, we conducted the majority of our subsequent studies with cnt-ca. mwcnts promote gradual phosphatidylserine translocation there are two well described paths to cell death: apoptosis, which is a natural and controlled process, and necrosis, which is unordered and more destructive. since mwcnts reduced ns-1 cell viability, we tested whether mwcnts stimulated apoptosis, necrosis or both using a double-stain flow cytometric assay. one of the earliest events in apoptosis is the translocation of phosphatidylserine (ps) phospholipids from the cytoplasmic face to the extracellular face of the plasma membrane. the protein annexin v (anxv) has a high affinity for ps in the presence of calcium and, when conjugated with a fluorescent probe such as fitc, can be used to assay for newly exposed ps. when simultaneously stained with pi, a dye specific for staining the nucleus in cells with compromised membrane, test cells can be identified as belonging to one of four categories: normal (anxv and pi negative), early apoptotic (anxv positive, pi negative), late apoptotic (anxv and pi positive) and necrotic (anxv negative, pi positive). ns-1 cells plated in cell growth medium were allowed to attach and recover following overnight incubations in a humidified 37°c incubator. the growth medium was replaced with the cntca solution after a brief pbs rinse. the cells were harvested at 6, 10, 24, 48 or 72 h, and 1x106 cells double stained with anxv-fitc and pi, and analyzed by flow cytometry to determine the condition of the cells. a topoisomerase ii inhibitor, etoposide (10 µm), was used as a positive control for loss of plasma membrane asymmetry. fig 2a shows the scatter plots cytograms a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 73 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 2. analysis of the effects of cnts on cell state by flow cytometry. ns1 cells were treated with sfm, cnt-ca or etoposide, double-stained with annexin v-fitc and pi and analyzed by flow cytometry. panel 2a represents the scatter plots at 10 h (a-c) and 24 h (d-f) exposures to sfm (a, d), cnt (b, e) and etoposide (c, f). the line graphs in panel 2b represent the time course of generation of early apoptotic (a), or necrotic (b) cell populations in cells treated with sfm (○), cnt-ca (*), or etoposide (■). 2a 2b generated at 10 and 24 h after treatment with sfm, cnt or etoposide. each result is shown as a panel partitioned into four sections with cells represented as dots distributed in the four staining categories, an(-) pi (+), an(+) pi(+), an(+) pi(-), and an(-) pi(-). in comparing the data from treated cells harvested at 10 h (fig. 2a, panels a-c), only etoposide produced a notable increase in the population of early apoptotic cells, an(+) pi(-), when compared with sfm and cnt treated cells, as shown in the lower right quadrant in panels a-c. by 24 h (fig. 2a, panels d-f), cnt treated cells showed an increase in the population of anxv reactive (ps translocated) cells, both with and without a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 3. cnts depolarize mitochondrial membrane. ns1 cells were exposed to cell gm (a), sfm (b), cnt-d (c), or cnt-ca (d) for 48 h or valinomycin for 2 h (e), harvested and loaded with jc-1 dye for 1 h. cells were analyzed by flow cytometry using gates to separate and quantify fluorescence of green monomer and red aggregates in cell populations. fig. 4. cnts induce caspase 3/7 activity. ns1 cells were exposed to sfm, cnt-ca or etoposide for 24, 48 or 72 h and assayed for caspase 3/7 activity. results represent the means + s.d. of four independent experiments tested in triplicates. *** indicates p<0.001 compared with sfm. table 1. percentage of hypodiploid (apoptotic) cells produced by cnt and control treatments. 74 treatment 48 h 72 h gm 6.27 6.29 sfm 16.39 22.18 cnt-ca 27.64 46.04 etoposide 46.68 38.48 increased membrane permeability (fig. 2a, panel e, upper and lower right quadrants), relative to the control (sfm) cells (panel d) and on par with etoposide treated cells (panel f). however, data collected over the full time course of the experiment indicated a more complex pattern. cell death can occur by two distinct methods, apoptosis and necrosis. although anxv-fitc and pi double staining provides an efficient means to identify cells which have undergone apoptosis, the identification of cells that died through necrosis by this method is less well defined for single observations. however, when samples are analyzed over a period of time it is possible to identify population of cells which have undergone necrosis. therefore to clarify whether cnt induces necrosis in addition to apoptosis, we analyzed the effects of the treatments by plotting the data obtained at 6, 10, 24, 48 and 72 h for the apoptotic an(+) pi(-) and potentially necrotic an(-) pi(+) cells. figure 2b displays the time course of generation of these two abnormal cell states following sfm, cnt, and etoposide treatment. we note first that etoposide-induced ps translocation preceding loss of membrane integrity (early apoptosis) is maximal at 10 h (fig. 2b-a). in contrast, neither sfm nor cnt treated cells display any sign of increased anxv reactivity at 10 h. second, there was little difference in the percentage of necrotic cells among the treatment groups for the first 24 h (fig. 2b-b), and since the percentage of necrotic cells is relatively low and generally decreased with time, the data suggest that neither etoposide nor mwcnts induce neuronal cell death via necrosis. mwcnts induce mitochondrial membrane depolarization since mwcnts appear to induce neuronal apoptosis, albeit at a slow (48-72 h) rate, we explored their effects on three key cellular processes associated with apoptosis: mitochondrial membrane depolarization, dna fragmentation, and induction of caspase activity. we examined cnt effects on mitochondrial membrane potential as a measure of intrinsic apoptosis using jc-1, a dye which is selectively permeable to mitochondria and emits an altered wavelength as it aggregates in the interior of normally polarized mitochondria. ns-1 cells seeded in 10 cm2 culture plates with complete growth medium (gm) were incubated overnight at 37°c in a humidified incubator. the cells were washed with pbs and treated with treated with 10 ml of mwcnts, sfm or gm for 48 h. cells treated for 2 h with 1 µm valinomycin, an antibiotic that negatively modulates mitochondrial oxidative phosphorylation through the formation of potassium ion permeable pores, served as depolarized control (fig 3). facs analysis of the treated cells showed that both cnt-d and cnt-ca decreased the number of polarized cells at 74.7% and 67.6% respectively (fig 3, panels c, d) compared with 87.3% for sfm control (fig 3, panel b). cnt-ca was slightly more destructive to the mitochondrial membrane at 32.4% (fig. 3, panel d) than cnt-d at 25.3% (fig. 3, panel c), relative to sfm at 12.7%. over 93% of the population of cells grown in gm exhibited intact mitochondrial membrane while cells treated with the ionophore valinomycin resulted in greater than 43% mitochondrial depolarization. mwcnts produce an increased hypodiploid population of cells using pi dna stain and flow cytometry, we measured the formation of cells with a lower pi signal than that of normal diploid cells (hypodiploid cells). the percentage of hypodiploid cells measured at 48 h and 72 h following exposure of cells to cnt-ca and etoposide with gm and sfm as controls are shown in table 1. cnt exposure for 48 h increased the percentage of apoptotic cells by four-fold compared with gm. by 72 h the difference in cells treated with cnt-ca compared to those maintained in gm was more than seven-fold. cells in sfm also began to show signs of apoptosis at the two time points evaluated. however, at 72 h cnt-induced apoptosis was more than twice that induced by serum withdrawal. also, cnt produced a greater percentage of hypodiploid cells by 72 h compared to etoposide under serum-free conditions. mwcnts induce delayed caspase 3/7 activity the two major pathways known to induce apoptosis, the receptor-mediated extrinsic pathway and the mitochondrial-activated intrinsic pathway, both involve the activation of the executioner caspase 3 protein. caspase 7, another apoptotic executioner protein, shares substrate specificity with caspase 3 and its activity is also measured by this assay. therefore, we looked for signs of caspase 3/7 activity in ns-1 cells treated with cnts compared to sfm and etoposide (fig 4). cnt-ca induced marginal but significant caspase 3/7 activity over sfm but less that etoposide after 24 h of incubation. by 48 h there was a slight overall decrease in the levels of caspase 3/7 compared to those observed at 24 h. however, by 72 h cnt-treated cells continued to produce a sustained level of activated caspase 3/7 while no change was observed in sfm cultures cells. compared to the 24 h time point, the pronounced decrease in caspase 3/7 activity in etoposide treated cells is likely due to compound toxicity resulting in low numbers of viable cells after 72 h under etoposide tension. these results imply that caspase 3/7 is activated in ns-1 cells by exposure to cnt and participates in cell damage, apoptosis and ultimately, death of neuronal cells. discussion the wide use of nanoscale materials in products as diverse as foods, cosmetics, drugs and medical devices raise unique safety concerns for industry and regulators. reports on the toxicity profile of these materials are limited. however, it is becoming evident that their effects on cells systems are very dynamic and are dependent on cell types and the physicochemical characteristics of the material. in this study we report the effects of mwcnts on ns-1, a neuronal cell model derived from pc12. our findings show that only the highest concentrations of solubilized mwcnts (190 ppm cnt-ca and 295 ppm cnt-d) compromised the viability of ns-1 cells. in comparison with the toxic effects of non-carbon nanoparticles on pc12 cells, mwcnts are relatively well-tolerated. much lower concentrations (5 ppm /5 µg/ml) of mwcnts had no effect on pc12 viability (xu et al., 2009) even though cadmium telluride quantum dots (lovric et al., 2005), and mn, ag or cu nanoparticles are cytotoxic at just 10 ppm (hussain et al., 2006; rossi et al., 2006). like pc12, the human neuroblastoma line sh-sy5y also tolerated mwcnts at low concentrations, i.e. 5-10 ppm [19]. nevertheless, we found that at sufficiently high concentrations, mwcnts reduced ns1 viability as measured metabolically by cellular enzyme conversion of resazurin to resorufin and structurally using the comparison of total nuclear staining by hoechst 33342 with nuclear staining of cells with damaged plasma membranes by pi. cnt-induced cell death was apoptotic and not necrotic. ns-1 cells were treated with cnts compared to serum-free or etoposide controls for various lengths of time and double-stained with a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 75 ps reactive anxv-fitc and pi. while necrotic cell populations remained at or below levels measured at the initial 6 h time point, early and late apoptotic cell populations increased with length of cnt exposure. etoposide optimally induced early stages of apoptosis by 10 h; this timeframe is consistent with that reported by denecker et al. (2000). in contrast, induction of apoptosis by cnts was not easily observed until 24 h of exposure. although cnt-induced apoptosis occurred more slowly, by 72 hours cnt toxicity was more extensive than that of etoposide. similar timeframes for cnt and etoposide toxicity were also seen in assays measuring the loss of dna integrity and the modest increase in caspase 3/7 activity. our observation that sensitivity to etoposide decreases by 72 h is likely a function of its mechanism of action as a dna topoisomerase ii inhibitor; only the subset of cells in s and g2 phases are affected and in the absence of serum, cells enter into quiescent g0 and are unsusceptible to etoposide. thus, our experimental design does not allow for comparison of cnts and etoposide at time points beyond 24-48 h. as to the more important question of overall cnt toxicity in the brain, recent in vitro studies of microglia, astrocytes and complex primary cultures have suggested neurons may be more resilient than their accessory cells. although we are unaware of any data comparing cnt effects on neurons and accessory cells, long and coworkers (long et al., 2007) compared the effects of tio2 nanoparticles on neurons and microglia. immortalized rat n27 mesencephalic neurons tolerated up to 120 ppm tio2 nanoparticles for three days without a significant reduction in hoechst live cell staining. in contrast, mouse bv2 microglia was found to be highly sensitive to tio2 nanoparticles at only 2.5 ppm. in addition, complex primary cultures of rat striatum (containing glia and neurons) incurred a 14% loss of neural cells after 6 h in 5 ppm tio2 nanoparticles. similarly, belyanskaya et al. [8] tested the effects of single walled (sw) cnts at 30 ppm on primary cultures of embryonic chicken spinal cord and found an overall decrease in dna content after 6 days. using cell type specific antibodies they determined that the loss of dna was due to glia, not neural, cell death. since complex primary cultures contain several cell types, it is not clear yet whether microglia or other neuroglia type cells are responsible for neural cell damage. for example, astrocytes may also play a role in neuronal toxicity. when primary rat neurons were co-cultured with purified primary astrocytes or microglia, neurons were protected from agents that induce reactive oxygen and nitrogen species (tanaka et al., 1999). on the other hand, toxic agents such as the alzheimer’s a β peptide stimulate primary rat astrocytes to produce ros and pro-inflammatory cytokines (johnstone et al., 1999). along these lines, sayes and co-workers have shown that neuronal human astrocytes (nha) are damaged by as little as 0.24 ppm nano-c60 fullerene, another carbon nanoparticle (sayes et al., 2005). if astrocyte distress promotes neuronal toxicity as does microglia distress, cnts may be indirectly toxic to neurons because of cnt damage to supporting glia, not because of their effect on the neurons themselves. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 76 conclusion the widespread application of nanotechnology and the potential impact of mwcnts on the environment and human health, particularly in occupational exposure settings, have led us to initiate an investigation of the effect of this material in a cultured neuron model. we find that mwcnt toxicity occurs at a slower rate than etoposide and requires relatively long exposure to induce cell death. cell death occurs via apoptosis with serine translocation by 24 h, mitochondrial membrane depolarization by 48 h, dna fragmentation by 48-72 h and modest caspase 3 activation by 72 h. further research is ongoing to determine the molecular mechanism of delayed mwcnt toxicity in cultured neurons. acknowledgments this work was supported in part by a grant from the goldenleaf foundation and funds from the state of north carolina. the authors wish to thank dr. susan peacock, j.d., ph.d. for assistance with the review of this manuscript. the authors appreciate dr. appala raju badireddy and dr. mark wiesner, center for environmental implications of nanotechnology, duke university, durham, for their technical support in carbon nanotube characterization. references bardi g, p tognini, g ciofani, v raffa, m costa, and t pizzorusso (2009) pluronic-coated carbon nanotubes do not induce degeneration of cortical neurons in vivo and in vitro, nanomedicine 5: 96-104. baughman rh, aa zakhidov, and wa de heer (2002) carbon nanotubes--the route toward applications. science 297: 787-792. belyanskaya l, s weigel, c hirsch, u tobler, hf krug, and p wick (2009) effects of carbon nanotubes on primary neurons and glial cells, neurotoxicology 30: 702-711. denecker g, h dooms, g van loo, d vercammen, j grooten, w fiers, w declercq, and p vandenabeele (2000) phosphatidyl serine exposure during apoptosis precedes release of cytochrome c and decrease in mitochondrial transmembrane potential. febs lett 465: 47-52. el-ansary a and s al-daihan (2009) on the toxicity of therapeutically used nanoparticles: an overview. j toxicol 754-810. greene la, and as tischler (1976) establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, proc natl acad sci u s a 73: 2424-2428. hishita t, s tada-oikawa, k tohyama, y miura, t nishihara, y tohyama, y. yoshida, t uchiyama, and s kawanishi (2001) caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line p39. cancer res 61: 2878-2884. hussain sm, ak javorina, am schrand, hm duhart, sf ali, and jj schlager (2006) the interaction of manganese nanoparticles with pc-12 cells induces dopamine depletion. toxicol sci 92: 456-463. johnstone m, aj gearing, and km miller (1999) a central role for astrocytes in the inflammatory response to beta-amyloid; 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2 laboratory of plant biotechnology and molecular biology, faculty of sciences, meknes, morocco; 3 faculty of sciences, kenitra, morocco. received: november 18, 2013 / accepted: october 31, 2014 __________________________________________________ * corresponding author: achbani105@gmail.com 218 abstract pseudomonas marginalis is an important postharvest pathogen causing soft rot in a wide variety of harvested fruits and vegetables. isolated strains from rotten onion bulbs based on morphological characteristics, were tested for pathogenecity on tobacco. pathogenic strains underwent a biochemical test which detected the presence of p. marginalis. symptoms were reproduced by inoculating this species to onion leaves and bulbs. based on arn16s sequencing, reported associated species (pantoea agglomerans, pseudomonas fluorescens, klebsiella oxytoca) were also confirmed. this study reports for the first time the presence of p. marginalis bacterium in morocco causing the soft rot of onion bulb, in association with pantoea agglomerans, pseudomonas fluorescens, and klebsiella oxytoca. keywords: pseudomonas marginalis, soft rot, onion, morocco. introduction in morocco, the onion crop (allium cepa l.) has a high economical importance, representing 11% of nationally produced vegetable crops (anonyme, 2011). however this crop is exposed to many physiological and phytosanitary constraints (conn et al., 2012), among which, the onion bulb soft rot represents the most serious post harvest disease. for onion storage two methods used in france, storage in a traditional dryer and temporary storage under plastic tunnels, also storage methods affect the soft rot incidence (gourc et al., 2007). the pseudomonas and pectobacterium bacterial genus are considered as the main bacteria that caused the damages in usa during storage (agrios, 2005). by means of pectinolytic enzyme products (pectin lyase and pectate lyase) (hayashi et al., 1997, liao et al., 1997), p. marginalis causes the post-harvest soft rot (conn et al., 2012; scortichini, 1994) of many harvested crops including: onion (kim et al., 2002 ; wright et al 1992 ; dallaire, 2009), tomato (ibe and grogan, 1983), salad (blancard et al., 2003), potato (elumalai and mahadevan, 1995), broccoli (charron et al., 2002) and carrot (godfrey and marshall., 2002). p. marginalis is a foliar as well as a post-harvest disease (conn et al., 2012; scortichini, 1994). it’s a bacterium present in europe, india, south american, usa, japan, new zealand and austria, etc. development of the soft rot disease caused by p. marginalis is optimal at low temperatures (between 5 and 25°c), the bacteria develop at 0°c and it can induce the soft rot at 5°c on onion crop (kim et al., 2002). in morocco, no evidence of such bacteria causing soft rot on onion or another crop. the main objective of this work was to isolate and identify the this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 219 causal agent of the bacterial soft rot on onion bulbs in morocco using biochemical, molecular and pathogenicity tests. materials and methods isolation onion bulbs showing soft rot symptoms (fig. 1) were sampled in march 2012 from vegetables storage areas at elhajjeb (n 33°41.45, w 5°22 00) (meknes-tafilalt, morocco). onion bulbs showing water-soaking or yellowish-brown rot were used for isolation. these were washed with tap water and cut longitudinally. the diseased scale tissues were cut into 5 mm cubes by using sterilized surgical blade. three pieces of onion scale were ground in 1 ml of distilled water using a mortar and pestle. the suspension was streaked onto lpga (yeast extract, 5g; peptone, 5g; glucose, 10g; agar, 18g; and distilled water, 1 l) and king b (proteose peptone, 20 g; k2hpo4•3h2o, 2.5 g ; mgso4•7h2o, 6 g ; glycerol, 15 ml ; agar, 15 g; and distilled water, 1 l) mediums. six bacterial isolates were identified by biochemical and physiological tests including gram strain; lopat (levan production, oxidase reaction, potato soft rot, arginine dishydrolase [adh] and tobacco hypersensitivity) test, hydrolysis of tween80, mobility test, indole, mannitol, catalase, hugh & leifson (h.l), the bacteria development in low and high temperature (4 and 40°c), the salt tolerance (7 and 10% of nacl) and api 20 gallery. all tests were repeated at last twice (schaad et al., 2001). pathogenicity test cultures of bacterial isolates obtained from onion bulbs, labeled 2078-6-1, 2078-6-2, 2078-6-3, 2078-6-4, 2078-6-5, were used in a greenhouse pathogenicity experiment. in vivo, seven weeks old tomato plants grown in greenhouse and sixteen weeks old onion plants grow in nursery were inoculated. a suspension of tested bacterial isolate (1×108 cfu/ml) in sterile distilled water from 24-h cultures was used as the inoculums; plants in one experimental variant were inoculated by injection of approximately 0.2 ml of inoculums into leaves of onion and tomato plants. the plants were incubated in a greenhouse at 20°c to 25°c and 60–70% relative humidity. sterile water was used as control (kudela et al., 2010). in vitro, onion bulbs and leaves, and tomato leaves were inoculated by a suspension (108 cfu/ml) of 24-h bacterial culture; the vegetable material was incubated in moist chamber at a temperature of 30°c, after five days of incubation symptoms were observed. pcr amplification to complete the biochemical results, a molecular identification by sequencing was performed. the extraction was made using genelute mammalian genomic kit (anonyme, 2010). quantification of dna was performed using spectrophotometry. amplification was performed with primers fd1 (cagagtttgatcctggctcag) and rp2 (agagtttgatcctggctcag) at a pcr kit (invitrogen). the pcr was carried out in a total volume of 25 µl of the following reaction mixture: 2.5µl 10x buffer, 2µl dntp (10mm), 0.125µl of each primer (100 µm), 0.75 µl mgcl2 (50 mm), 0.2µl taq (5u/µl) and 5µl dna. the pcr was performed using the following protocol: initial denaturation at 96°c for 4 min, followed by 35 cycles of denaturation at 96°c for 0.1min, annealing at 52°c for 0.4min, and a extension at 72°c for 2min, followed by an additional extension at 72°c for 4 min. electrophoresis was performed in 1.5% agarose gel. purification of pcr products was carried using the enzyme exo-sat according to the following schedule: 37°c for 15min following by 80°c for 15min. 16s dna sequencing and sequence analysis selected pcr fragments, amplified from the isolates tested for pathogenicity, were sequenced in both strands, with the pa and ph’ primers, using the bigdye terminator cycle sequencing ready reaction fs kit. so the sequencing of amplification product was carried out in a total volume of 10µl of the following reaction mixture: 1µl bigdye, 3µl sequencing buffer x5, xµl primer (3.2-5pmol), (0.75-1.5) µl dna matrice, (2.5-3.25) µl h2o (miliq). the sequencing was performed using the following protocol: initial denaturation at 96°c for 1 min, followed by 25 cycles of denaturation at 96°c for 10s, annealing at 50°c for 5s, and a extension at 60°c for 4min, followed by an additional extension at 72°c for 4 min. reading of sequencing results was done using ncbi-blast software (altschul et al., 1997). results and discussion bacterial colonies which were consistently isolated from infected samples were fluorescent on king’s medium b. all six (2078-6-1, 2078-6-2, 2078-6-3, 2078-6-4, 2078-6-5 and 2078-6-6) strains caused hypersensitive reaction (hr) on tobacco leaves, indicating that they were pathogenic. they were gram-negative. according to the lopat tests, the isolates were negative for levan production and positive for oxidase test, pectinase test (fig. 2) and arginine dihydrolase. based on the lopat and gram tests (schaad et al., 2001) and kim et al. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. onion soft rot. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 220 (2002) results, representative isolates of the causal agent of soft rot of onion bulbs in morocco was identified as p. marginalis, they also showed that strains can grow in a concentration of 7 and 10% of salt at 24°c with ph7. these results were different from those of membre and burlot (1994) who showed that, a lesser concentration of 2.5% of nacl reduced p. marginalis growth and inhibited pectinolytic enzyme production. the conflicting results may be due to strains natures and isolation origins. all of our strains were catalase positive and capable of hydrolyzing esculin, but not tween80 and gelatin. none of the strains were able to produce indole and reduce nitrate. however, all strains showed ability to utilize arginine, lysine (ldc), ortnithine table 1. p. marginalis strains (2078-6-1, 2078-6-2, 2078-6-3, 2078-6-4, 2078-6-5,) behavior on gallery api20, classic biochemical tests and growth at different temperatures. (onpg : determination of enzyme beta-galactosidase presence, adh : transformation of arginine by dishydrolase argnine, ldc : transformation of lysine by decarboxylase lysine, odc : transformation of ornithine by decarboxylase ornithine, cit : utilization of citrate as alone source of carbone, h2s : production of hydrogen sulfate from thiosulfate, ure : liberation of ammoniac from urea by urease, tda : formation of indolepyruvique acid from tryptophan by desaminase tryptophan, ind : formation of indole from tryptophan, vp : formation of acetone from sodium piruvate, gel : liquefaction of gelatin, glu : glucose, man : mannitol, ino : inositol, sor : sorbitol, , rha : rhamnose, sac : sucrose, mel : melibiose, amy : amygdaline, ara : arabinose (formation of acid by utilization of carbon hydrate tests ) ((-): negative; (+): positive; nd: no determinate). (-): negative; (+): positive; nd: indeterminate strains 2078-6-1 2078-6-2 2078-6-3 2078-6-4 2078-6-5 2078-6-6 levan (+) (+) (+) (+) (+) (+) oxidase (+) (+) (+) (+) (+) (+) pectinase (+) (+) (+) (+) (+) (+) adh (+) (+) (+) (+) (+) (+) h. tabac (+) (+) (+) (+) (+) (+) growth at 41°c (+) (+) (+) (+) (+) (+) growth at4°c (+) (+) (+) (+) (+) (+) h.l (-) (-) (-) (-) (-) (-) catalase (+) (+) (+) (+) (+) (+) hydrolysis of tween 80 (-) (-) (-) (-) (-) (-) mobility (+) (+) (+) (+) (+) (+) esculine (+) (+) (+) (+) (+) (+) onpg (+) (+) (+) (+) (+) (+) ldc (+) (+) (+) (+) (+) (+) odc (+) (+) (+) (+) (+) (+) cit (-) (-) (-) (-) (-) (-) h2s (+) (+) (+) (+) (+) (+) ure (+) (+) (+) (+) (+) (+) tda nd nd nd nd nd nd ind (-) (-) (-) (-) (-) (-) vp (-) (-) (-) (-) (-) (-) gel (-) (-) (-) (-) (-) (-) glu (+) (+) (+) (+) (+) (+) man (+) (+) (+) (+) (+) (+) ino (-) (-) (-) (-) (-) (-) sor (+) (+) (+) (+) (+) (+) rha (+) (+) (+) (+) (+) (+) sac (+) (+) (+) (+) (+) (+) mel (+) (+) (+) (+) (+) (+) amy (+) (+) (+) (+) (+) (+) ara (+) (+) (+) (+) (+) (+) no2 (-) (-) (-) (-) (-) (-) n2 (-) (-) (-) (-) (-) (-) 221 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) the sequence of isolates strains (2078-6-1, 2078-6-2, 2078-6-3, 2078-6-4, 2078-6-5, 2078-6-6) sequence identity/similarity genbank accession numbers gcctaggaatctgcctggtagtggggga taacgtccggaaacggacgctaataccgc atacgtcctacgggagaaagcaggggac cttcgggccttgcgctatcagatgagccta ggtcggattagctagttggtggggtaatg gctcaccaaggcgacgatccgtaactggt ctgagaggatgatcagtcacactggaact gagacacggtccagactcctacgggagg cagcagtggggaatattggacaatgggc gaaagcctgatccagccatgccgcgtgtg tgaagaaggtcttcggattgtaaagcactt taagttgggaggaagggccattacctaat acgtgatggttttgacgttaccgacagaat aagcaccggctaactctgtgccagcagcc gcggtaatacagagggtgcaagcgttaat cggaattactgggcgtaaagcgcgcgta ggtggtttgttaagttggatgtgaaatccc cgggctcaacctgggaactgcattcaaaa ctgactgactagagtatggtagagggtg gtggaatttcctgtgtagcggtgaaatgc gtagatataggaaggaacaccagtggcg aaggcgaccacctggactgatactgacac tgaggtgcgaaagcgtggggagcaaac aggattagataccctggtagtccacgccg taaacgatgtcaactagccgttgggagcc ttgagctcttagtggcgcagctaacgcatt aagttgaccgcctggggagtacggccgc aaggttaaaactcaaatgaattgacgggg gcccgcacaagcggtggagcatgtggtt taattcgaagcaacgcgaagaaccttacca ggccttgacatccaatgaactttccagaga tggattggtgccttcgggaacattgagac aggtgctgcatggctgtcgtcagctcgtg tcgtgagatgttgggttaagtcccgtaac gagcgcaacccttgtccttagttaccagca cgtgatggtgggcactctaaggagactg ccggtgacaaaccggaggaaggtgggg atgacgtcaagtcatcatggcccttacggc ctgggctacacacgtgctacaatggtcgg tacagagggttgccaagccgcgaggtg gagctaatcccagaaaaccgatcgtagtc ctgatcgcagtctgcaactcgactgc he586394.1 table 2. table 2. the sequence identity/similarity. (the sequences of the isolates strain amplified by rna 16s, present a 99% similarity with the lmg 2214 pseudomonas marginalis several strain (1204b/1208b with a score equal to 2165 bits (2400)). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) (odc), thiosulfate (h2s), urea (ure), glucose (glu), mannitol (man), sorbitol (sor), rhamnose (rha), sucrose (sac), melibiose (mel), amygdaline (amy), arabinose (ara), but they did not use citrate (cit), tryptophan (ind), sodium piruvate (vp), gelatin (gel), inositol (ino) (table 1). the strains can grow at 4°c; low temperatures during this season (2011-2012) may have favored soft rot p. marginalis bacteria development. these results correlate with those of kim et al. (2002), who showed that the optimal temperature development is 0°c. also we have shown the development strains at 40°c. pathogenicity test the 2078-6-1, 2078-6-2, 2078-6-3, 2078-6-4, 2078-6-5 and 2078-6-6 strains gave a positive reaction in vitro (fig. 3) and in planta. in vitro tests showed that there was development of soft rot after five days of incubation on onion bulbs by p. marginalis as indicated by chlorosis of infected area and necrosis development at onion and tomato leaves. in planta tests necrosis development was observed in onion and tomato leaves, after five days of incubation and after 14 days onion leaves died. dallaire (2009) and (ibe and grogan, 1983) show that p. marginalis is a cause of onion and tomato decay. molecular results result of dna quantification showed that the quantity of dna was sufficient for pcr (20.18 ng/µl). a band with 1550 bp size was observed in agarose gel (fig. 4). analysis of the 16srdna sequence, by blast-ncbi, revealed that, the strains isolates (2078-6-1, 078-6-2, 2078-6-3, 2078-6-4, 2078-6-5and 2078-6-6) were identical (100% identity over 1208 nucleotides) and that they were most closely related to 16s rdna sequences from several strains of p. marginalis (lmg 2214) (table 2). we also found the presence of associated bacteria namely: pantoea agglomerans, pseudomonas fluorescens, and klebsiella oxytoca. el-hendawy (2004) showed that serratia marcescens and klebsiella oxytoca are associated bacteria with p. marginalis in onion crop. figure 3. in vitro symptoms induced by artificial incubation. (a) yellowing of onion leaves. (b) necrosis in tomato leaves. (c) soft rot in onion bulbs, after five days of inoculation. figure 4. electrophoritic profile of p. marginalis stains: (1) 2078-6-1. (2) 2078-6-2. (3) 2078-6-3. (4) 2078-6-4. (5) 2078-6-5. (6) 2078-6-6. (t+) p. marginalis reference strain. (t-) negative control (h2o). figure 2. pectinase test. symptoms induced by artificial inoculation of isolates of p. marginalis. 222 to our knowledge, this is the first report of bacterial bulbs soft rot of onion in morocco. because the soft rot of onion is the most danger disease during storage, this study is considered as a basic of others works that will target the p. marginalis ecologic and behaviors, to solve the soft rot problem by applying of a biological control. a development of a molecular method is very important for a rapid detection of p. marginalis in soft rot bulbs. references agrios gn (2005) plant pathology. fifth edition, elsevier academic press, 922p. altschul sf, madden tl, schaffer aa, zhang j, zhang z, w miller, and dj lipman (1997) gapped blast and psi-blast: a new generation of protein database search programs. nucleic acids res (25): 3389–3402. anonyme (2010) genelute™ mammalian genomic dna miniprep kit, user guide, 16p. anonyme (2011) ministère de l’agriculture et de la pêche maritime. web site : http://www.marocagriculture.com. blancard d, h lot, and b maisonneuve (2003) maladie des salades identifier, connaître et maîtriser, inra, paris, 277p. charron cs, ce samsm, and ch canaday (2002) impact of glucosinolate content in broccoli (brassica oleracea) on growth of pseudomonas marginalis, a causal agent of bacterial soft rot. plant dis (86): 629-632. conn ek, js lutton, and sa rosenberger (2012) onion disease guide. plant health, 72p. dallaire c (2009) la pourriture molle de l’oignon : levures ou bactéries? rien de mieux qu’un test de laboratoire! fiche technique, québuc, 4p. el-hendawy hh (2004) association of pectolytic fluorescent pseudomonads with postharvest rots of onion. phytopathol. mediterr (43): 369–376. elumalai pp and a mahadevan (1995) characterization of pectate lyase produced by pseudomonas marginalis and cloning of pectate lyase genes. physiological and molecular. plant pathology (46): 109-119. godfrey sac and jw marshall (2002) identification of cold-tolerant pseudomonas viridiflava and p. marginalis causing severe carrot postharvest bacterial soft rot during refrigerated export from new zealand. plant pathology (51): 155–162. gourc d, d monnier, and jd payet (2007) oignon guide pratique, ile de la réunion, france. fiche technique (www.armeflhor.fr). hayashi k, y inoue, m shiga, s sato, r tankano, k hirayae, t hibi, and s hara (1997) pectinolytic enzymes from pseudomonas marginalis. maff03-01173 45 (7): 1359-1363. ibe sn and rg grogan (1983) effect of controlled oxygen and carbon a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 223 dioxide atmospheres on bacterial growth rate and soft rot of tomato fruits caused by pseudomonas marginalis. plant disease (67): 1005-1008. kim yk , sd lee, cs choi, sb lee, and sy lee (2002) soft rot of onion bulbs caused by pseudomonas marginalis under low temperature storage. plant pathology j 18 (4): 199-203. kudela v, v krejzar, and i pankova (2010) pseudomonas corrugata and pseudomonas marginalis associated with the collapse of tomato plants in rockwool slab hydroponic culture. plant protect. sci (46): 1-11. liao c-h, de mccallus, wf fett, and yg kang (1997) identification of gene loci controlling pectate lyase production and soft-rot pathogenicity in pseudomonas marginalis. can. j. microbiol (43): 425-431. membre jm and pm burlot (1994) effects of temperature, ph, and nacl on growth and pectinolytic activity of pseudomonas marginalis. applied and environmental microbiology 60 (6): 2017-2022. schaad nw, jb jones, and w chun (2001) laboratory guide for identification of plant pathogenic bacteria. 3rd ed. aps press, st. paul, 373p. scortichini m (1994) leaf spot and blight of dieffenbachia amoena caused by pseudomonas marginalis pv. marginalis. plant pathology (43): 941-943. wright pj, rd cronin, and cn hale (1992) afield andstorage rot of onion caused by pseudomonas marginalis. new zealand journal of crop and horticultural science (20): 435-438. atlas journal of biology 2017, pp. 402–406 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) the procedure of seed potato certification in morocco fadoua eljai1, meryem slamini2, ibtissam mzabri2*, ibtihal bekkouch2, nour eddine kouddane2, and abdelbasset berrichi2 1 laboratory of management and development of companies and organizations, superior school of technology, university mohammed first, p.o. box 473, oujda 60000, morocco; 2 biology of plants and microorganisms laboratory, faculty of sciences, p.o. box 717, oujda 60000, morocco received: june 16, 2017 / accepted: july 4, 2017 __________________________________________________ * corresponding author: btissammzabri@gmail.com 402 abstract the potato (solanum tuberosum l.) is a geophyte of the solanaceae family and it represents the first crops in morocco. thanks to its high nutritional value, the seed requirements attain around 160000 t of which 70% originate from the previous production, which is the reason behind the limitation of the market’s development. considering the importance of the certified seeds, morocco has signed a program contract to develop this sector. the production of certified potato seedling is a whole pro-cess that begins with the selection of plants and goes on until the marketing of the product where the producer should absolutely respect the cultivation processes and hygiene standards. the establish-ment and the insurance of compliance with these standards is carried out by the national office of sanitary safety and food supplies (onssa). the purpose of the following work is to summarize and represent briefly the certification standards of potato seeds in morocco. keywords: seed, potato, onssa, certification, standards, morocco. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the seed is the first key element in plant production. the use of quality seed represents one of the investments with the highest multiplier effect of the entire agricultural economy. beside the im-portance of seeds in crop production, the seed trade provides many jobs in rural areas where they generate significant revenues globally. the global seed market was estimated at 42 billion usd in 2011 (isf, 2011). this estimation attracted the attention of scientists and required scientific research, investment and the use of advanced technology in the field of seed production. yet, experience shows that the seed industry is failing to develop satisfactorily due to the lack of appropriate legis-lation. concerning the legal and economic globalization, the seed sector has, in the past years, had a multitude of legal rules with the aim of strengthening the food quality and safety through the har-monization of the national legislation on seed trade. a number of international organizations, con-ventions and treaties have been set up to deal with the regulation of the seed trade. since the be-ginning of the 1970’s, the seed sector in morocco has known a sustained growth thanks to a juridical arsenal for the seed industry, which has been complemented by the promulgation in 1997 of law 9-94 on the protection of plant varieties which entered into force on 28 october a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 2002 (onssa, 2015). these national regulations are added to the global organizations of which moroc-co is a member, such as the organization for economic co-operation and development (oecd), the international seed testing association (ista), the international union for the protection of new va-rieties of plants (upov), the international seed federation (isf) and the un food and agriculture organization (fao). these different international organizations constitute a regulatory framework that monitors the interests of all breeders, producers and consumers. the potato (solanum tuberosum l.) is a geophyte of the solanaceae family. recently, it has become a leading product in the seed in-dustry thanks to the cultural and culinary revolution that created the fast food chains. in morocco, potato cultivation tops on the market garden with an area of 60 000 ha, accounting for 23% of the total area of vegetable crops and an annual output of 1.5million tons with average yields between 17 and 18 t / ha (skiredj.,2007). considering the high demand for potatoes, it is practically culti-vated in all regions of morocco with four types of cultivation, namely early cultivation; where plant-ing occurs in september-december, seasonal cultivation in january -february, the culture of back-season in august and the culture of mountain in may (chibane, 1999). the global seed demand is around 160 000 tons, including 20 000 tons for early-growing crops. 30% of these needs are cov-ered by imports and 70% by common plants taken from previous productions. given the importance of seed certification, morocco signed a program contract in 2009 to develop the sector of certified potato seed production, which will cover 30% of certified seed needs in the near future (2020) instead of the current coverage that represents only 2% (aissi, 2014). the following study aims to establish the certification process for potato seeds in morocco by re-ferring to the standards established by the national office for food safety (onssa, 2015). the stages of potato seed certification the process of potato seed certification requires several stages and several generations of multiplication under tight control throughout the production chain (figure 1). the selection of high-performing plants this selection constitutes the first link of this chain, where we distinguish two techniques: i. mass selection: this is the oldest empirical method. it consists of selecting the plants that appear to be the most interesting in a population and using their seeds as seeds for the next crop (burg, 2004). ii. selection by hybridization: it consists in crossing two species or two varieties with each other, which gives plants new characteristics. this technique makes use of a long varietal selection (bouharmont, 1995). iii. entry in the official catalogue iv. when a new variety is created, its breeder requests it to be registered in the official catalogue. it must be submitted to the so-called dhs test: distinction, homogeneity and stability and, on the other hand, to the vat test: agronomic and technological value which is based on the assessment of the agronomic and technological performance of the candidate variety in relation to the reference varieties that are already widely cultivat-ed in the territory. in order to officially register in the catalogue, there are three major steps to be followed: (i) the application for registration should be submitted by the breeder to onssa, this application has to be accompanied by samples of 300 kg of seedlings for varieties whose seed may be marketed in morocco or abroad as well as 50 kg for varieties whose seeds may be propagated in morocco for export, (ii) a receipt for payment of the registration fee paid by the applicant to onssa, and (iii) a certificate signed by the breeder and / or the holder of the variety authorizing the filing of the ap-plication in the event that the applicant is not the breeder. after registration in the official catalog, the seed of the registered variety must undergo several generations of propagation before reaching the farmer. stages of propagation during this phase, the potato seed takes the following names: i. starting plants: corresponding to the original material of the authentic variety, it must be free mainly from degenerative diseases (virus y, virus x, virus a, ...) and quarantine pests and diseases in accordance with current phytosanitary legislation. ii. pre-base plants: seedlings that are usually multiplied from the original material; the number of field generations should not exceed four. iii. basic seedlings (elite and super elite classes): the basic plants come from the multiplication of the pre-base or base plants, provided that the number of basic seedlings must not exceed four and that the number of cumulative pre-base and base generations does not exceed seven. the super elite (se) class corresponds either to the harvest from pre-basic or basic plants, and the number of generation of pre-basic seedlings in the field and basic seedlings is limited to six. for the elite class (e), the crop is either from pre-basic plants or from basic plants and the number of genera-tions of pre-basic plants in the field and basic plants is limited to seven. iv. certified plants (classes a and b): certified plants are originated from the propagation of pre-basic or basic plants, and may also be produced from a first generation of certified plants. de-pending on the level of quality compared to the standards, certified plants can be classified in class a or b. in order to produce different categories of certified potato material, the multiplier must have the following: (1) an authentic genetic material free from disease and nematodes, (2) accessible land with a min-imum area of 1/2 hectare (0.5 ha) for basic seedlings and one hectare (1 ha) for certified seedlings 403 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 404 figure 1. schematic of seed potato certification as a propagation material. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 405 and an installation that meets the production standards for potatoes. the production declaration, 15 days after the installation of the production program, should be addressed to the certification body. taking in consideration the importance of control in the certified potato seed production scheme, the certification body exercises control at all stages, namely: field control, laboratory control, control during storage, control during packaging and control to marketing. in order to do so, an application for inspection must be sent to the certification body, and should be accompanied by (i) receipt of the payment of the inspection fee, (ii) a document that indicates the location of the parcels to be con-trolled (iii) documents justifying the origin of the plants, (iv) analyses of the soil of the seed multipli-cation plots. 1. field control this depends on the type of plant material. for the starting and pre-base material, the breeder or maintainer are the ones responsible for control, while the basic super elite, elite and a or b certified seedlings should be subject to at least 3 periodic surveys. the first visit is made before the crop is put in place and consists of (i) verifying the origin of the plants by checking the labels of the invoices and delivery notes. (ii) sampling soil for nematological tests in a representative pattern that takes into account the field’s architecture, variability and ag-gregation of nematodes (for a surface area of 0.5 to 1 ha, sampling should be between 10 and 50 samples). (iii) verification of the isolation and rotation, the land for production must have at least a three years rotation for the pre-base and base plants and at least two years for the certified plants (class a and b). thus, table 1. standards for minimum insulation distances. category prebase base se and e certified a and b cultivation of solanaceous, cruciferous and stone fruit trees prebase 1 rang vide 20 m 40 m 500 m base se and e 20 m 1 rang vide 20 m 300 m certified a and b 40 m 20 m 1 empty row 200 m plots must respect the prescribed isolation distances (table 1) in order to avoid the transmission of degenerative diseases (viral diseases). the second visit is carried out one month after the full lifting. the purpose of this visit is (i) to examine the general conditions of the plots (presence of weeds, abnormalities of the vegetative, apparatus: deformations, foliar discolorations, etc.), (ii) to assess the rate of contamination by the pests via the diagnosis of 20 m2 (5m * 4m) bands are chosen according to a well-defined sampling scheme that takes into consideration the size and geometric shape of the production plot (the minimum number of controlled bands is 5), (iii) to check the quality of the purification by tearing off the feet of different appearance and not con-forming to the variety, feet suffering from diseases. the third visit is carried out when the varietal characters are visible, it allows (i) the assessment of the rate of contamination by viral, bacterial and plant fungal diseases, (ii) the checking of the varietal purity based on the shape of the leaves, the color of the flowers, the coloration of the collar, the ribs and the hair of the leaves, etc., (iii) the estima-tion of the yield using the formula: yield = stand weight / stand x stand / ha, (iv) set the date of top killing to be carried out within the timeframe set by onssa for seed types and climatic conditions (table 2). table 2 shows the standards set for top killing. 2. laboratory control after harvest, tubers are examined in the laboratory; the main analyzes which are viral, nematolog-ical, bacteriological and mycological. the sample used for these tests should be presented with a minimum of 220 tubers for basic plants and 110 tubers for certified plants. categories classification by date of removal at the deadline during the first 15 days after the deadline between 16 and 20 days after the deadline more than 20 days after the deadline plants de base se e certified plants a b se e a b a a a b b b b b refusal refusal refusal refusal table 2. deadline for topkilling. 406 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 3. control during storage its purpose is to check the storage conditions (temperature, humidity, isolation of varieties), the hy-giene conditions of the storage rooms and the damage that can be caused by the postharvest dis-eases. 4. conditioning control during this phase, the tubers go through several stages, namely (i) sorting of tubers with abnormali-ties, (ii) tuber drying, (iii) sizing of the tubers depending on the type (round varieties 30-65 mm and 28-55 mm for oblong varieties, (iv) treatment of the tubers against crop plant diseases, (v) packag-ing tubers in new bags of 25 or 50 kg that will be sealed afterwards. 5. marketing control the aim of this step is to verify and control the compliance with the packaging stages mentioned above: origin, size, weight of the bags, conditions and shelf life and the health status of the tubers. stage of certification and labeling: during this final stage, the certification body proceeds to the labeling and the sealing of the bags that respect the prescribed stages of production, preservation and packaging. a certified seed bag should carry two labels: one on the bag and the other in the bag, their colors shall vary according to the type of seed and they must bear all the necessary in-formation on the lot in question (lot number, variety, class, caliber, treatment product, etc.). conclusion the seed potato certification is the culmination of a monitoring process that enables the official ser-vice (onssa) to ensure that the submitted seedlings meet the standards established by the inspection body. the use of certified seed is the most efficient means of spreading and disseminating genetic progress, since it ensures high productivity, adaptation to various biotic and abiotic stresses and quality that meets the market requirements. references aissi n (2014) le maroc ne profite pas de sa pomme de terre. [internet]. accessed at: http://www.leconomiste.com/article/947441-lemaroc-ne-profite-pas-de-sa-pomme-de-terre. bouharmont j (1995) création variétale et amélioration des plantes. bases physiologiques et agro-nomiques de l’amélioration de la production végétale, hatier. paris. agronomie moderne 313-337. onssa (2015) office national de sécurité sanitaire et des produits alimentaires. accessed at http://www.onssa.gov.ma/fr/index.php burg h (2004) la production des semences à petite échelle. digigrafi editor. p. 107. chibane a (1999) techniques de production de la pomme de terre au maroc. bull mens liaison d’information du pntta 52: 1–4. isf statistics (2011) seed. accessed at http://www.worldseed.org/resources/seed-statistics. skiredj a (2007) legume fruit maroc. accessed at: http://www.legumefruit-maroc.com/pomme-terre.php. atlas journal of biology 3 (1): 183–205, 2014 doi: 10.5147/ajb.2014.0133 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) transcriptome profiling of the shoot and root tips of s562l, a soybean gmclavata1a mutant saeid mirzaei1,2, jacqueline batley1, brett j ferguson1, and peter m gresshoff1* 1 centre of excellence for integrative legume research, school of agriculture and food sciences, the university of queensland, st lucia, brisbane qld 4072, australia; 2 present address: department of biotechnology, institute of science, high technology and environmental sciences, graduate university of advanced technology, kerman, iran received: march 20, 2014 / accepted: april 6, 2014 __________________________________________________ * corresponding author: p.gresshoff@uq.edu.au 183 abstract plant shoot apical meristems (sam) and root apical meristems (ram) contain stem cells that form overall-plant architecture. mechanisms acting in these regions keep a balance between the stem cell population and differentiation. these mechanisms are well-studied in arabidopsis, but little is known in the legume soybean (glycine max (l.) merr.). in arabidopsis, the leucine-rich repeat (lrr) receptor kinase clavata1 (clv1) is a crucial regulator of this process in the sam. in soybean, the receptor most similar to atclv1 is gmnark, which is involved in nodulation control. in contrast, the homeologous partner of gmnark in soybean, called gmclv1a, appears to have no function in ‘autoregulation of nodulation’ (aon) a role in regulating shoot architecture in the sam. here, the transcriptome of the shoot and root tip areas of a chemically induced and tilling-selected gmclv1a missense mutant, s562l, and its wild type, cultivar forrest, were analysed to identify genes which are affected by impaired function of gmclv1a. among the differentially expressed genes identified, many were categorised as having a role in receptor kinase activity, transcription or defense/ stress-response. molecular categories over-represented in the shoot tip of the mutant include those involved in hormone biosynthesis/activity and secondary metabolism, signalling, photosynthesis, and transport. functional categories including those involved in polyamine metabolism, nucleotide metabolism, rna regulation, protein targeting and protein degradation were under-represented in the shoot tip of the mutant. in the root tip, categories associated with signal introduction soybean (glycine max (l.) merr.), garden pea (pisum sativum), common bean (phaseolus vulgaris) and alfalfa/lucerne (medicago sativa) are some of the important crops belonging to the legume family, which are second to the grasses in providing food for the world’s population. one-third of all dietary protein and one-third of processed vegetable oil for human consumption are provided by grain legumes (gepts et al., 2005; graham and vance, 2003). nitrogen is the most required nutrient of plants and enters into many biological molecules such as amino acids, proteins and nucleic acids. although dinitrogen gas (n2) forms a main part of the earth’s atmospheric gas (78.1%), it cannot be used by most of the plants, generating a global need for nitrogen-containing fertiliser. leguminous plants, however, are able to use dinitrogen gas through a symbiotic association with soil bacteria, collecthis is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ling, transport, protein synthesis and metabolism were overrepresented in the mutant, while categories associated with cell wall degradation, stress, rna regulation, protein degradation and targeting were under-represented in the mutant. factors similar to arabidopsis regulatory components are most likely functioning in specialised shoot structures in legumes. furthermore, gmclv1a may have an unexpected role in the regulation of flavonoid biosynthesis in soybean. keywords: glycine max, legume, plant development, ram, receptor kinase, rnaseq, sam, symbiosis. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 184 tively named rhizobia (ferguson et al., 2003). apical meristems including those of the shoot (sam) and root (ram) are responsible for the aerial and underground organs of the plant, respectively (stahl and simon, 2010; traas and hamant, 2009). sam and ram are specialised regions containing stem cells, which allow plants to grow continuously throughout their life through the control of a pool of stem cells. similar to animal stem cells, plant stem cells are capable of generating diverse tissues and renewing the stem cell population (sharma et al., 2003). therefore, the major function of the sam is to keep a dynamic balance between maintenance of the pluripotent stem cell population and the formation of new organs (leaves and flowers) and enables plants to grow and reproduce (fletcher, 2002). some pathways and regulatory mechanisms in this process have been identified through studies using the model plant arabidopsis, a crucifer. of particular significance is the role of the clavata (clv) signalling network to regulate the size of the stem cell reservoir in the sam. mutations in genes (like clv1, clv2 and clv3) acting in the clv network lead to the proliferation of undifferentiated cells in the sam and the development of an abnormal apical meristem (clark et al., 1993; 1997). within this gene network, clv1 encodes a leucine-rich repeat receptor kinase (lrr-rk) and plays a critical role in this pathway. atclv1-like genes in soybean are gmnark and gmclv1a. gmnark regulates nodulation through a mechanism called autoregulation of nodulation (aon) (searle et al., 2003) and gmclv1a appears to have a function in the sam (mirzaei et al., 2014; submitted). however, to date, little is known about the ‘clv’ network of soybean. recently, through tilling, an emsinduced missense mutation in a putative s-glycosylation site (s562l) in gmclv1a was isolated (batley et al., 2014; submitted). the mutant shows an alternative function from gmnark and behaves as a loss-of-function allele. gmclv1a lacks any measurable effect on nodulation despite sharing over 90% dna sequence with gmnark. the s562l mutation leads to severe nodal identity alterations in the basal parts of the emergent plant such as branching, as well as flower and pod abnormalities (mirzaei et al., 2014; submitted). several methods are available to study gene expression, such as qrt-pcr, microarrays and high-throughput rna sequencing (rna-seq) (ozsolak et al., 2009). qrt-pcr is utilised for studying a small number of genes and samples, microarrays are used for large scale gene expression studies (e.g., whole transcriptome), and rna-seq also evaluates the whole transcriptome, but with less danger of confusing data caused by cross-hybridisation of related genes. in recent years, rna-seq has been widely used for transcript profiling and gene discoveries in plant species including legumes, such as soybean (hayashi et al., 2012; libault et al., 2010; reid et al., 2012). in this study, we compared the transcriptome of the shoot and root tip of the s562l, gmclv1a mutant, and its wild type parent (cultivar forrest) using rna-seq. the clc genomics workbench program was subsequently used to map the rna-seq data to the soybean reference genome and determine the relative transcript abundance. the results provide further evidence to aid the understanding of meristem maintenance in soybean. materials and methods plant growth conditions soybean (glycine max (l. merr.) wild type cv. forrest and the ems-induced and tilling-selected missense mutant s562l were used for this experiment. for rna-seq, seeds were surface-sterilised by immersion in 70% ethanol for 30 s, then rinsed 5 times with sterile water, and were put between filter paper in sterile petri dish and kept in a growth chamber at 25°c in dark conditions. tissue harvest shoot tips (1 mm) and root tips (2 mm) were harvested after 48 hours using a sterile scalpel and immediately frozen in liquid nitrogen. shoot tips of plants 48-hours old were collected under the dissecting microscope after opening the cotyledon and removing the emerging leaves. rna sample preparation and library construction for rna-seq, total rna was extracted from dissected shoot and root tips using the qiagen rneasy minikit with on-column dnase digestion according to the manufacturer’s instructions (qiagen, maryland, usa). the australian genome research facility (agrf) subsequently conducted cdna library construction and rna sequencing. cdna libraries for plant transcriptome sequencing were constructed using the illumina truseq rna kit according to illumina protocols and rna sequencing was performed using the illumina hiseq 2000 platform, with four multiplexed samples run on one flowcell lane generating 100 bp single-end reads. for qrt-pcr experiment, rna was converted to cdna in a 20-µl reaction mixture containing 0.5 mm deoxynucleoside triphosphates (dntps), 1 µl of 50 µm oligo(dt) primers, 40 unit of rnaseout (invitrogen), 0.5 µg of dna-free rna, 1x firststrand buffer (invitrogen), 5 mm dithiothreitol (dtt) and 100 units of superscript iii reverse transcriptase (invitrogen) at 500c for 60 min. finally, cdna was confirmed using gmcons6 primers (libault et al., 2008) (glyma12g05510; f box protein family) and pcr. quantitative real time pcr primers used for quantitative real-time pcr were designed using the online primer design program, primer 3 version 0.4.0 (available at http://frodo.wi.mit.edu). sequences from the soybean genome (phytozome version 8.0; the united states department of energy joint genome institute and centre for integrative genomics; available at http://www.phytozome.net) were used for primer design. the sequences for forward and reverse primers for each gene are shown in table 3. to ensure that the primers were specific and produced only a single band, normal pcr was run using forrest cdna. all primer pairs were found to amplify a single product of the correct size. the relative transcript abundance was detected using sybr a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 185 green pcr master mix (applied biosystems) on an abi 7900ht cycler (applied biosystems) in a 384-well plate. the 384-well plates were set up using an eppendorf epmotion 5075 robotic system and contained no template (water) control and reverse transcription negative (rt-) controls to verify genomic dna contamination of the samples. all reactions were carried out in duplicate of one biological replicate. the qrt-pcr conditions used were as follows: initial denaturation of 95˚c for 10 min, then 45 cycles of 95˚c for 15 sec and 60˚c for 1 min followed by a dissociation stage of 95˚c for 2 mins to assess the specificity of the pcr. the expression level of the genes was normalised to the mrna expression level of soybean gmcons6 (libault et al., 2008) amplified by forward primer 5’-aaaggtgaaattgcctcttcc-3’ and reverse primer 5’-cccaaagatctgccaaatgta-3’. pcr efficiency for each sample was calculated using the linregpcr 7.5 program (ramakers et al., 2003). bioinformatics and data analysis of sequencing output the read quality score was determined using the fastx tool kit. shoot and root read sequence data were mapped separately against the soybean genome (available at phytozome; http:// www.phytozome.net/) using the clc genomics workbench with the rna-seq function and the mapping setting: minimum length fraction 0.9, and minimum similarity fraction 0.8. relative transcript abundance was yielded in “read per kilobase” of exon model per million mapped reads (rpkm) values. this value only uses the mapped reads and relative size of transcripts to determine expression level. differentially expressed genes were identified by comparing expression values between samples and using kal’s test (kal et al., 1999) which considers proportions, rather than raw data. genes were determined to be differentially expressed using thresholds of fold change >=2 with kal’s z test p-value <0.05. results transcriptome sequencing (rna-seq) outputs to help understand how gmclv1a affects the gene expression network in soybean, transcript profiles of the shoot and root tip of s562l gmclv1a mutant and wild type (forrest) were compared at germination. one hundred (100) bp single-end sequence reads generated using an illumina hi-seq 2000 platform had a good quality (phred quality score ≥ 32; figure 1). results of mapping reads against the soybean reference genome (available at phytozome; http://www.phytozome.net/) are summarised in table 1. overall a total of 84-87% of the reads from each sample uniquely mapped to the soybean genome, whereas ~4% were non-specifically mapped. transcriptomes of wild type forrest and mutant s562l shoot tips by having at least one read match, a total of 40,229 genes were expressed in the wild type shoot tip compared with 41,172 genes in the s562l shoot tip. comparison of wild type and s562l shoot tip genes expression value (rpkm) indicated that 631 genes had a differential transcript abundance (kal’s z test; p ≤ 0.05). around 71% of differentially expressed genes (448 genes) had significantly higher expression in the s562l shoot tip relative to the wild type, and 29% of differentially expressed genes (183 genes) genes had less expression. of these, 277 (~62%) of the more-highly expressed genes had a fold change of two or greater, whereas 62 (~34%) lower-expressed genes had a fold change of two or greater. a subset of these differentially expressed genes, which were predicted to encode either protein kinases, transcription factors, protein binding, defense, stress response or catalytic activity, is presented in table s1 [see additional file]. transcriptomes of wild type forrest and mutant s562l root tips a total of 40,460 genes were expressed in the wild type root tip compared with 40,714 genes in the s562l root tip. comparison of gene expression values in the wild type and s562l root tips identified 1,204 genes that had differential transcript abundance (p ≤ 0.05). around 64.5% of differentially expressed genes (777 genes) increased in expression in the s562l root tip relative to wild type and 35.5% of differentially expressed genes (427 genes) decreased. four hundred and forty six (446: ~58%) of these more-highly expressed genes had a fold change of two or greater and 140 (~33%) lower-expressed genes had a fold change of two or greater. a subset of these differentially expressed genes, with activities such as protein kinase and signalling activity, transcription factor activity, protein binding, defence and stress response, and catalytic activity are presented in table s2 [see additional file]. table 1. mapping rna sequencing reads to the glycine max genome. wild type shoot tip s562l shoot tip wild type root tip s562l root tip total reads 39,284,610 44,789,650 34,169,345 33,856,295 uniquely mapped reads 34,063,809 38,886,356 28,999,578 28,343,248 87% 87% 85% 84% non-specifically mapped reads 1,639,245 1,879,153 1,450,399 1,400,816 4% 4% 4% 4% un-mapped reads 3,581,556 4,024,141 3,719,369 4,112,231 9% 9% 11% 12% a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 186 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. phred quality scores of rna sequencing reads. the percentage of base calling accuracy for each base position of the read. the x axis shows read position and the y axis on the left shows the phred score and on the right shows the percentage of base call accuracy. 187 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 2. qrt-pcr analysis of six differentially expressed genes (based on rna-seq) in the shoot samples. this analysis was done in the same shoot samples used for high-throughput rna sequencing. fst: forrest shoot tip, s562lst: s562l shoot tip. 188 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) confirming the expression of some differentially-expressed genes at the shoot tip from differentially-expressed genes, ten candidate genes (eight had over-expression and two had reduced-expression in the shoot or root tip of s562l) were selected based on homology to arabidopsis genes that could influence the clv pathway or were acting in developmental pathways (table 2). the expression of the selected genes was confirmed by qrt-pcr. qrtpcr was carried out on the exact rna samples that were used for deep sequencing. the result showed that the expression of all selected genes was consistent with the deep sequencing results (figures 2 and 3). soybean functional categories regulated in the shoot tip functional categories of genes that were differentially expressed in the s562l shoot tip compared to the wild type shoot tip were analysed using mapman (thimm et al., 2004) and pageman, an integrated program in mapman. this analysis, applying the wilcoxon test with benjamini-hochberg correction, revealed 141 functional pathways (bins and sub-bins) to be statistically different from the other pathways (p<0.05; table s3; see additional file). these pathways are distributed into 22 bins out of 37 bins. categories over-represented in the shoot tip of s562l included photosynthesis, cell wall, secondary metabolism, hormone metabolism, redox regulation, signalling and transport bins and sub-bins. under-represented functional categories included polyamine metabolism, nucleotide metabolism, rna, dna and protein bins and sub-bins. soybean functional categories regulated in the root tip a comparison of the rna-seq root tip outputs of s562l and its wild type found a total of 71 biological functional pathways to be statistically different from all other bins (p<0.05; table s4; see additional file). over-represented categories included photosynthesis, lipid metabolism, amino acid metabolism, secondary metabolism hormone metabolism, redox regulation, nucleotide metabolism, dna, signalling, development and transport and under-represented functional categories included fermentation, cell wall, biosynthesis, stress, rna, protein and cell biology. figure 3. qrt-pcr analysis of four differentially expressed genes (based on rna-seq) in the root samples. this analysis was done in the same root samples used for high-throughput rna sequencing. frt: forrest root tip, s562lrt: s562l root tip. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) discussion rna-seq analysis provided a broad view of the gene expression in the s562l shoot and root tip regions compared with the wild type. our data revealed genes involved in signalling, transcription, metabolism and defense and stress response are over-represented in the s562l shoot and root tip. moreover, genes that belong to the receptor protein kinase and transcription factor families, which are important in signalling and plant development, were also shown to have higher transcript abundance in the shoot and root tip of s562l compared to the wild type. wus encodes a homeodomain transcription factor and is expressed in the organising centre at the sam (schoof et al., 2000). wus regulates the meristem size through cytokinin signaling via repression of several type-a arabidopsis response regulators (arrs) (arr5, arr6, arr7 and arr15) as well as activation of clv3 transcription by binding to its regulatory region (leibfried et al., 2005; yadave et al., 2011). our rna-seq data and qrt-pcr revealed that glyma06g01940 (putative orthologue of at4g35550; wuschel related homeobox 13) was transcribed higher in the s562l shoot tip, which is reminiscent of the finding in arabidopsis where the wus expression domain expanded in the clv sam (schoof et al., 2000). this indicates that gmclv1a of soybean is acting through a similar component as the clv network in arabidopsis. receptor kinases are key elements in ligand-receptor systems to communicate signals in multicellular organism (i.e., plants) and are involved in diverse pathways in growth and development (searle et al., 2003; dievart et al., 2004; shiu et al., 2004). in figure 4. predicted model of the regulatory network of factors acting in the legume sam. in the model, it is proposed that gmclv1a, clv2 and klv perceive a ligand probably similar to clv3 in arabidopsis and then negatively regulate a wus-related protein. there is possibility that aon genes such as nark also interact with gmclv1a in some aspect of plant development. gmclv1a also negatively regulates flavonoids biosynthesis. arrows indicate positive regulation and barred lines indicate negative regulation. ‘?’ implies ‘unknown’. table 2. expression fold changes and putative annotation of genes selected for qrt-pcr. 189 gene id putative annotation proportion fold change shoot samples glyma08g11620 chalcone and stilbene synthase 39.30 glyma10g44170 homogentisate phytyltransferase 1 9.73 glyma06g01940 wuschel related homeobox 13 5.38 glyma04g29250 response regulator 9 8.19 glyma16g33870 succinyl-coa ligase -18.38 glyma05g36310 acc oxidase 1 -5.29 root samples glyma04g00930 gln phosphoribosyl pyrophosphate amidotransferase 1 7.99 glyma06g05300 ccch-type zinc finger family protein 7.15 glyma08g11620 chalcone and stilbene synthase 6.91 glyma07g35630 nac-like, activated by ap3/pi 4.58 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) arabidopsis thaliana, the clv family [19], erecta family (torii et al., 1996; uchida et al., 2013), bam family (deyoung et al., 2006; deyoung and clark, 2008), rpk2 (kinoshita et al., 2010), rpk1 (receptor like protein kinase1) and acr4 (arabidopsis crinkly4) (de smet et al., 2008; 2009) are all receptor kinases and function in regulating cell division and differentiation in shoot and root meristems. lrr rks, differentially regulated in the s562l shoot tip and root tip, are presented in table s1 and table s2 [see additional file]. none of these lrr rks are paralogues of the above mentioned lrr rks, indicating that they represent new candidates that function in signalling at the shoot and root tip and may play a similar role — controlling cell division and differentiation — as the above mentioned receptor kinases. transcription factors play critical roles in plant development by regulating positively or negatively the expression level of relevant genes. analysis of differentially expressed genes revealed their presence in the s562l shoot and root tip (table s1, 2; see additional file). transcripts that are related to ap2 (apetala2) and a group of ap2, ethylene responsive element binding proteins, erebps, were also found to be differentially expressed in the shoot and root tip of s562l (table s1, 2; see additional file). ap2/erebp transcription factors are expressed in different tissues including: flower, leaves, inflorescence stem and root (okamuro et al., 1997) and they are involved in several developmental processes that include: seed development, stem cell identity, floral organ identity, plant growth, nodulation and defense response (agrawal et al., 2011; andriankaja et al., 2007; aoyama et al., 2012; jofuku et al., 1994; krishnaswamy et al., 2011; yant et al., 2010). ap2-related transcripts are under-represented in the shoot while they are overand under-represented in the root tip of s562l (table s1, 2; see additional file). this suggests that the defect in gmclv1a function, which is involved in plant development of soybean, might associate with overand under-represented transcripts of this group of transcription factors which function in developmental processes such as stem cell and thus nodal identity. some transcripts correspond to the nac (nam, ataf1/2 and cuc2) domain containing proteins, are also over-represented in the shoot and root tip of s562l (table s1, 2). nac domain containing proteins are another group of transcription factors that are involved in a wide range of biological processes, including embryogenesis, flower development, sam development, wood formation and shoot branching (aida et al., 1997; hu et al., 2010; mao et al., 2007; ohtani et al., 2011; xie et al., 2000). interestingly, glyma02g26480 is a putative orthologue of ataf1 (at1g01720) was over-represented in the shoot tip of s562l. ataf1 is a homologue of the nam (no apical meristem) gene in petunia (souer et al., 1996). in petunia, nam mutants fail to develop a sam (souer et al., 1996). this indicates that the over-expression of this gene might be due to impaired gmclv1a function in the s562l mutant. however, it has been shown that ataf1 is involved in stress responses (hu et al., 2010; wang et al., 2009). furthermore, glyma13g35550, over-represented in the root tip data set, is also an ataf1-like gene. there are genes highly expressed in the root and shoot tip of s562l that correspond to those of the wrky transcription factors family (table s1, 2; see additional file). wrky transcription factors act as activators or repressors and control many plant biological processes including germination, senescence, biotic and abiotic responses and development. moreover, wrky factors have a key role in the innate immune system of plants (rushton et al., 2010). flavonoids play a pivotal role in plant biology. they protect plants against uv irradiation, attract pollinators and symbionts, and contribute to plant hormone signalling (dixon and pasinetti, 2010; stracke et al., 2007). myb transcription factors also act in various plant biological processes and impact development, biotic and abiotic stresses and metabolism (dubos et al., 2010). there is a member of the myb transcription factors among the differentially regulated genes in the s562l shoot tip, which is involved in flavonol biosynthesis. glyma16g02570, a putative orthologue of myb111, was over-represented in the gene id forward primer (5’-3’) reverse primer (5’-3’) glyma08g11620 tccacccccatcatcatatc ttgcgccttacgaatctctt glyma10g44170 ttcacgacacaaaagggaaac agcatgagcttcaaaaccaa glyma06g01940 tcaaacgctggtggtattattg gacagatggtggcatagacaga glyma04g29250 ctcagagaatgtcccagcaag tttcaacaaatgtggcctcag glyma16g33870 tgacatatgaagcggttttcc tctgaaggcagtcaacgaagt glyma05g36310 accttccaagaacaatgccat tccaatggggttatagaaggtg glyma04g00930 ggtgtacccgggtgaagttat acctcccgaaaacaacagagt glyma06g05300 aacaactccgcctcgtagtaac attttaacattccgcgttgagt glyma07g35630 cctcctggctttaggtttcac gcaattcccaaggatcaaact gene id is according to the phytozome database (http://www.phytozome.net). table 3. primer sequences used for qrt-pcr. 190 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) transcriptome data base of the s562l shoot tip. in arabidopsis, myb111 controls flavonol biosynthesis and is mainly active in cotyledons (stracke et al., 2007). as flavonol accumulation regulates polar auxin transport (kuhn et al., 2011), it is likely that some phenotypes of s562l, such as increased branching, are the result of flavonol accumulation due to higher expression of glyma16g02570. members of this group were also over-represented in the root tip data of s562l. of interest is glyma03g34110, a putative orthologue for atmyb68. it is specifically expressed in the root and responds to environmental conditions, temperature in particular (feng et al., 2004). there is a possibility that over-expression of this gene causes a stronger phenotype of s562l under cold conditions; however, atmyb68 expression is elevated at high temperature (feng et al., 2004). putative orthologues of arabidopsis chalcone synthase (chs) including glyma08g11620, glyma08g11520, glyma0811650 and glyma02g14450, were found to have higher expression in the s562l shoot and root tips compared to wild type shoot and root tips. chs is a key enzyme in flavonoids biosynthesis (winkel-shirley, 2001). this suggests a correlation between flavonoid biosynthesis and clv signalling which may cause developmental outcomes. overall, the existence of numerous transcription factors among the differentially expressed genes identified in this study is consistent with recent studies which show a wide range of transcription factors are active in the sam and ram of soybean (haerizadeh et al., 2009; 2011). furthermore, over-representation of transcripts for myb and wrky transcription factors which are involved in a wide range of plant processes and mainly function in abiotic and biotic stresses, could be explained by sam and ram immunity systems, which may be affected by impaired function of gmclv1a. a recent study demonstrates that clv3, a main regulator of stem cell homeostasis, can also activate innate immunity (lee et al., 2011). moreover, mathesius et al. (2011) by comparison of root tip and differentiated root, highlighted the importance of stress, defense response and flavonoid metabolism in the root apex. conclusions and future work past studies using the model plant arabidopsis revealed regulatory pathways in the sam and ram that sustain stem cells in both shoot and root meristems and exhibited some similarities between molecules and mechanisms. in legumes, it seems there is a divergence in clv1 function as clv1 orthologues in legumes, except for gmclv1a in soybean, are involved in nodulation control. gmclv1a (a paralogue of gmnark, which is a key component in the regulation of nodule formation), acts in shoot architecture, leaf and pod development (mirzaei et al., 2014; submitted). investigation of the shoot tip transcriptome of s562l indicated that gmclv1a suppresses the expression of glyma06g01940 (wuschel related homeobox 13) reminiscent of clv1 function in arabidopsis. this finding along with the evidence of the function of ljclv2, psclv2 (krusell et al., 2011) and ljklv (lotus japonicus klavier) (miyazawa et al., 2010) in the sam indicates that components similar to arabidopsis regulatory elements are most likely acting in specialised shoot structures in legumes (figure 4). furthermore, it seems that gmclv1a negatively controls the flavonoids biosynthesis through the chalcone synthase and the myb111 transcription factor. as auxin polar transport is regulated by flavonoids (kuhn et al., 2011; falcone et al., 2012), there is a possibility that they also have a function in regulating legume shoot structure. further research is required revealing how pathways regulating flavonoids biosynthesis and pathways regulating plant development are connected together in the legume family. moreover, more studies are required to identify other components (known or unknown in arabidopsis) regulating the sam of legumes. as mutations in ljclv2/psclv2 and ljklv lead to hyper-nodulation as well as stem fasciation (krusell et al., 2011; miyazawa et al., 2010), such lines of research may aid in understanding not only the molecular mechanisms underlying sam regulation in legumes, but also pathways acting in nodulation. abbreviations sam: shoot apical meristems ram: root apical meristems clv: clavata lrr-rk: leucine-rich repeat receptor kinase aon: autoregulation of nodulation rpkm: read per kilobase of exon model per million mapped reads arrs: arabidopsis response regulators rpk1: receptor like protein kinase1 acr4: arabidopsis crinkly4 ap2: apetala2 erebps: ethylene responsive element binding proteins klv: klavier dntps: deoxynucleoside triphosphates dtt: dithiothreitol competing interest the authors declare that they have no competing interests. author contribution sm conducted experiments, evaluated results and wrote the manuscript. jb, bjf and pmg contributed to the conception, interpretation and supervision of the research and editing of the manuscript. acknowledgements we thank the australian research council and the university of queensland for support through the centre of excellence scheme. we also thank a/prof. paul ebert and dr david schlipalius at uq for helping with analysing data using the clc genomics workbench. satomi hayashi, dr dugald reid, and dr stephen kazakoff (all cilr) are thanked for technical discussion. 191 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) references agarwal p, s kapoor, and ak tyagi (2011) transcription factors regulating the progression of monocot and dicot seed development. bioessays 33: 189–202. aida m, t ishida, h fukaki, h fujisawa, and m tasaka (1997) genes involved in organ separation in arabidopsis: an analysis of the cupshaped cotyledon mutant. the plant cell online 9: 841–857. andriankaja a, a boisson-dernier, l frances, l sauviac, a jauneau, dg barker, and f de carvalho-niebel (2007) ap2-erf transcription factors mediate nod factor–dependent mt enod11 activation in root hairs via a novel cis-regulatory motif. the plant cell online 19: 2866–2885. aoyama t, y hiwatashi, m shigyo, r kofuji, m kubo, m ito, and m hasebe (2012) ap2-type transcription factors determine stem cell identity in the moss physcomitrella patens. development 139: 3120– 3129. clark se, mp running, and em meyerowitz (1993) clavata1, a regulator of meristem and flower development in arabidopsis. development 119: 397–418. clark se, williams rw, meyerowitz em: the clavata1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in arabidopsis. cell 1997, 89:575-585. de smet i, v vassileva, b de rybel, mp levesque, w grunewald, d van damme, g van noorden, m naudts, g van isterdael, r de clercq, et al. 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2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) mirzaei et al., 2014 supplementary data gene name proportion fold change p-value putative annotation protein kinase glyma13g09870 6.95 0.02 protein kinase superfamily protein glyma13g09810 6.08 0.015 protein kinase superfamily protein glyma11g00320 4.55 0.041 leucine-rich repeat (lrr) family protein glyma20g27480 3.76 0.0009794 cysteine-rich rlk (receptor-like protein kinase) 29 transcription factor glyma05g31800 271.42 0.004925 wrky dna-binding protein 51 glyma08g15050 41.48 0.002085 wrky dna-binding protein 51 glyma15g00570 9.28 0.0004699 wrky dna-binding protein 40 glyma18g44030 9.02 0.029 wrky dna-binding protein 33 glyma05g36970 5.07 0.016 wrky family transcription factor glyma09g00820 3.66 0.005466 wrky family transcription factor glyma09g41050 3.45 0.001657 wrky dna-binding protein 70 glyma08g23380 3.35 0.041 wrky dna-binding protein 40 glyma19g43420 4.11 0.049 bzip transcription factor family protein glyma03g40730 3.14 0.039 bzip transcription factor family protein glyma11g11790 -2.08 0.032 basic-leucine zipper (bzip) transcription factor family protein glyma05g35050 19.91 0.043 myb domain protein 116 glyma10g00930 18.05 0.014 myb domain protein 15 glyma16g02570 8.04 0.00002565 myb domain protein 111 glyma10g32410 7.53 0.003432 myb domain protein 15 glyma02g00820 5.33 0.025 myb domain protein 15 glyma20g35180 3.08 0.01 myb domain protein 15 glyma16g04740 6.30 0.038 nac domain containing protein 47 glyma02g26480 2.10 0.027 nac (no apical meristem) domain transcriptional regulator superfamily protein glyma08g47240 -13.65 0.029 ap2/b3-like transcriptional factor family protein glyma18g38490 -4.65 0.033 ap2/b3-like transcriptional factor family protein glyma18g43750 -2.28 0.022 integrase-type dna-binding superfamily protein glyma13g31010 -2.05 0.0002355 erf domain protein 12 glyma12g12600 5.20 0.018 ring membrane-anchor 1 table s1. a representative list of the genes that were differentially transcribed in the shoot tip of s562l compared with those of the wild type. light green and light pink represent the most up-regulated and down-regulated gene in each category respectively. grey shows genes that were differentially-expressed in both the shoot and root tip. 194 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table s1. continued. protein binding glyma14g07410 73.64 0.007283 calcium-dependent lipid-binding (calb domain) family protein glyma06g44870 30.02 0.019 ankyrin repeat family protein glyma02g41540 22.40 0.044 calcium-dependent lipid-binding (calb domain) family protein glyma03g16600 6.20 0.0008464 glutathione s-transferase tau 8 glyma11g00320 4.55 0.041 leucine-rich repeat (lrr) family protein glyma16g25080 3.68 0.037 disease resistance protein (tir-nbs-lrr class), putative glyma08g08360 3.24 0.045 polygalacturonase inhibiting protein 1 glyma10g33650 3.13 0.0008941 glutathione s-transferase tau 15 glyma01g26220 2.66 0.0000691 glutathione s-transferase tau 8 glyma15g40190 2.33 0.029 glutathione s-transferase tau 19 glyma06g40710 2.22 0.047 disease resistance protein (tir-nbs-lrr class), putative glyma11g33760 2.18 0.043 calcium-dependent lipid-binding (calb domain) family protein glyma15g03160 -2.06 0.035 alfin-like 5 glyma08g44960 -3.39 0.006228 proteasome activating protein 200 glyma07g04820 -3.75 0.002134 chaperone dnaj-domain superfamily protein defense and stress response glyma09g04520 46.20 9.26e-09 pathogenesis-related protein bet v i family glyma07g37280 33.05 0.033 mlp-like protein 423 glyma15g15600 28.99 0.006306 pathogenesis-related protein bet v i family glyma15g15590 15.76 3.558e-10 mlp-like protein 423 glyma17g03340 10.33 3.268e-11 mlp-like protein 423 glyma07g37270 8.05 1.355e-09 mlp-like protein 423 glyma09g04510 4.41 4.124e-10 mlp-like protein 423 glyma07g37240 4.08 0 mlp-like protein 423 glyma17g03350 3.87 0.000003783 mlp-like protein 423 glyma03g41390 6.36 0.006478 senescence-associated gene 21 glyma20g23090 3.86 0.00249 adenine nucleotide alpha hydrolases-like superfamily protein glyma10g02210 3.01 0.006941 senescence-associated gene 21 glyma04g01130 -2.36 0.000005269 cold-regulated 47 195 catalytic activity glyma11g31310 7.52 0.019 amp-dependent synthetase and ligase family protein glyma09g40580 7.20 0.034 nad(p)-binding rossmann-fold superfamily protein glyma08g37670 6.66 0.013 deoxyxylulose-5-phosphate synthase glyma01g44270 6.39 0.002263 4-coumarate:coa ligase 3 glyma04g11250 5.96 0.047 haloacid dehalogenase-like hydrolase (had) superfamily protein glyma01g43460 5.38 0.033 highly aba-induced pp2c gene 3 glyma04g13880 4.97 0.004299 haloacid dehalogenase-like hydrolase (had) superfamily protein glyma18g45260 4.29 0.00007771 nad(p)-binding rossmann-fold superfamily protein glyma06g05280 3.25 0.013 branched-chain amino acid transaminase 2 glyma03g19810 2.81 0.032 haloacid dehalogenase-like hydrolase (had) superfamily protein glyma08g42070 2.23 0.029 atp-dependent caseinolytic (clp) protease/crotonase family protein glyma13g44950 2.22 0.049 4-coumarate:coa ligase 2 glyma07g36770 -2.95 0.039 enoyl-coa hydratase/isomerase a table s1. continued. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 196 table s2. a representative list of genes that were differentially transcribed in the root tip of s562l compared with that of the wild type. light green and light pink represent the most up-regulated and down-regulated gene in each category respectively. grey indicates genes that were differentially-expressed in both the shoot and root tip. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) gene name proportions fold change p-value putative annotation receptor kinase & signalling glyma20g23890 7.67 0.00000151 act-like protein tyrosine kinase family protein glyma08g23340 4.64 0.018 cbl-interacting protein kinase 5 glyma09g33650 4.57 0 phosphoenolpyruvate carboxykinase 1 glyma08g25600 4.41 0.023 leucine-rich repeat transmembrane protein kinase glyma02g46670 3.24 0.021 protein kinase superfamily protein glyma01g02330 3.04 0.0000316 phosphoenolpyruvate carboxykinase 1 glyma13g38170 2.93 0.022 receptor-like protein kinase-related family protein glyma17g05660 2.69 0.037 protein kinase superfamily protein glyma09g41340 2.64 0.0005498 sos3-interacting protein 1 glyma01g37100 2.45 0.05 calcium-dependent protein kinase 28 glyma12g07770 2.31 0.025 mitogen-activated protein kinase 3 glyma02g02790 2.17 0.004924 disease resistance protein (tir-nbs-lrr class) family glyma16g06940 2.07 0.044 leucine-rich repeat receptor-like protein kinase family protein glyma10g30710 -2.15 0.003837 leucine-rich receptor-like protein kinase family protein glyma06g06550 -2.23 0.013 cbl-interacting protein kinase 25 glyma11g00320 2.48 0.046 leucine-rich repeat (lrr) family protein glyma08g04390 -2.12 0.00277 leucine-rich repeat (lrr) family protein glyma15g26790 -2.72 0.0002545 polygalacturonase inhibiting protein 1 197 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) transcription factor glyma19g40470 6.61 0.028 wrky dna-binding protein 35 glyma01g31920 6.40 0.012 wrky dna-binding protein 33 glyma01g43420 6.11 0.00856 wrky family transcription factor glyma08g02580 5.14 0.004004 wrky family transcription factor glyma03g05220 4.90 0.007609 wrky dna-binding protein 33 glyma05g36970 3.37 0.009669 wrky family transcription factor glyma15g00570 3.33 0.002048 wrky dna-binding protein 40 glyma17g15480 2.96 0.023 ethylene responsive element binding factor 1 glyma10g34760 2.65 0.036 ap2/b3 transcription factor family protein glyma17g15460 2.07 0.007356 ethylene responsive element binding factor 5 glyma14g09320 2.06 0.032 related to ap2 1 glyma15g16260 -2.26 0.025 ethylene-responsive element binding protein glyma17g33060 -2.89 0.001162 integrase-type dna-binding superfamily protein glyma19g27790 -2.89 0.0004498 integrase-type dna-binding superfamily protein glyma03g26450 -4.71 0.02 ethylene-responsive element binding factor 13 glyma19g43420 5.27 0.0000127 bzip transcription factor family protein glyma03g40730 2.46 0.002065 bzip transcription factor family protein glyma06g38410 8.29 0.038 nac (no apical meristem) domain transcriptional regulator superfamily protein glyma07g35630 4.58 0.00009966 nac-like, activated by ap3/pi glyma01g06150 4.20 0.024 nac-like, activated by ap3/pi glyma16g04740 4.06 0.004638 nac domain containing protein 47 glyma13g35550 3.87 0.0002846 nac domain containing protein 3 glyma17g23740 3.42 0.044 nac domain containing protein 83 glyma03g34110 7.83 0.045 myb domain protein 68 glyma09g03690 5.65 0.022 myb domain protein 78 glyma02g00820 3.45 0.02 myb domain protein 15 glyma09g37340 -2.27 0.009459 myb domain protein 30 glyma06g05300 7.16 0.009363 ccch-type zinc finger family protein glyma04g05290 6.90 0.0000799 ccch-type zinc finger family protein glyma12g12600 3.11 1.516e-07 ring membrane-anchor 1 glyma10g29750 3.09 0.004816 ring/u-box superfamily protein glyma03g37740 3.07 0.039 ring/fyve/phd zinc finger superfamily protein glyma05g36110 3.00 0.039 ccch-type zinc finger family protein glyma08g03540 2.97 0.036 ccch-type zinc finger family protein glyma11g36040 2.39 0.03 ring-h2 finger a2a glyma07g24480 8.51 0.007142 gdp dissociation inhibitor family protein / rab gtpase activator family protein glyma01g39260 3.29 0.008994 heat shock factor 4 glyma20g32050 -2.02 0.00006938 gata transcription factor 9 glyma02g06560 5.44 0.032 homeobox protein 40 table s2. continued. 198 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table s2. continued. protein binding glyma10g43670 9.06 0.034 f-box family protein glyma16g32230 7.46 0.028 phloem protein 2-a13 glyma02g04420 4.95 0.028 octicosapeptide/phox/bem1p family protein glyma06g17910 4.56 0.000001083 snf1-related protein kinase regulatory subunit gamma 1 glyma13g44260 4.03 0.019 cystathionine beta-synthase (cbs) protein glyma11g33760 3.33 0.0002613 calcium-dependent lipid-binding (calb domain) family protein glyma01g03150 3.29 0.01 octicosapeptide/phox/bem1p family protein glyma05g28820 2.95 0.04 galactose oxidase/kelch repeat superfamily protein glyma01g42420 2.57 0.031 phospholipase d beta 1 glyma13g09290 2.53 0.008898 rni-like superfamily protein glyma08g10890 2.38 0.018 galactose oxidase/kelch repeat superfamily protein glyma15g01610 2.38 0.002209 galactose oxidase/kelch repeat superfamily protein glyma08g23540 2.26 0.001665 chaperone dnaj-domain superfamily protein glyma10g33650 2.17 0.00002115 glutathione s-transferase tau 15 glyma18g01140 2.15 0.008673 galactose oxidase/kelch repeat superfamily protein glyma04g37140 2.04 0.033 snf1-related protein kinase regulatory subunit gamma 1 glyma08g04390 -2.12 0.00277 leucine-rich repeat (lrr) family protein glyma19g24640 -2.39 0.011 galactose oxidase/kelch repeat superfamily protein glyma09g15600 -2.39 0.05 ccch-type zinc finger protein with arm repeat domain glyma16g06690 -2.81 0.002079 galactose oxidase/kelch repeat superfamily protein glyma08g44960 -2.82 0.0002941 proteasome activating protein 200 defense and stress response glyma02g03680 7.10 0.00001028 mlp-like protein 423 glyma15g15590 5.84 0.021 mlp-like protein 423 glyma07g37280 5.28 0.00628 mlp-like protein 423 glyma17g03330 4.55 0.026 mlp-like protein 423 glyma20g23090 2.34 0.02 adenine nucleotide alpha hydrolases-like superfamily protein glyma06g16810 2.23 0.00000118 scorpion toxin-like knottin superfamily protein glyma15g13870 2.04 0.019 catalytic activity glyma06g45950 31.05 0.00003716 isocitrate lyase glyma06g05280 4.69 1.221e-14 branched-chain amino acid transaminase 2 glyma04g05190 4.34 1.416e-08 branched-chain amino acid transaminase 2 glyma03g40720 3.05 0.004779 gdp-d-mannose 3\',5\'-epimerase glyma06g41520 3.03 0.017 nad(p)-binding rossmann-fold superfamily protein glyma20g28320 2.90 4.441e-16 haloacid dehalogenase-like hydrolase (had) superfamily protein glyma13g01420 2.89 0.04 trehalose phosphatase/synthase 11 glyma06g19590 2.70 0.018 trehalose-phosphatase/synthase 9 glyma18g12660 2.41 0.009343 rhamnose biosynthesis 1 glyma11g31310 2.35 0.003131 amp-dependent synthetase and ligase family protein glyma03g19810 2.11 0.002617 haloacid dehalogenase-like hydrolase (had) superfamily protein glyma09g05590 -2.01 0.009678 haloacid dehalogenase-like hydrolase (had) superfamily protein glyma15g16850 -2.35 0.015 haloacid dehalogenase-like hydrolase (had) superfamily protein 199 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table s3. biological categories that are regulated in the s562l shoot tip in comparison to wild type shoot tip as analysed by mapman (benjamini hochberg-corrected; p < 0.05). bin name elements p-value photosynthesis 1 ps 317 0 1.1 ps.light reaction 220 0 1.1.1 ps.light reaction.photosystem ii 98 0 1.1.1.1 ps.light reaction.photosystem ii.lhc-ii 38 1.46223e-10 1.1.1.2 ps.light reaction.photosystem ii.psii polypeptide subunits 60 1.28332e-06 1.1.2 ps.light reaction.photosystem i 39 1.82784e-07 1.1.2.2 ps.light reaction.photosystem i.psi polypeptide subunits 31 9.16001e-08 1.1.5 ps.light reaction.other electron carrier (ox/red) 33 0.042731782 major cho metabolism 2.1.2 major cho metabolism.synthesis.starch 56 0.019806396 2.1.2.1 major cho metabolism.synthesis.starch.agpase 17 0.011872219 2.1.2.2 major cho metabolism.synthesis.starch.starch synthase 15 0.015657959 2.2 major cho metabolism.degradation 147 0.033712098 2.2.1.3 major cho metabolism.degradation.sucrose.invertases 37 0.028318178 2.2.1.3.2 major cho metabolism.degradation.sucrose.invertases.cell wall 22 0.000328236 2.2.1.3.3 major cho metabolism.degradation.sucrose.invertases.vacuolar 7 0.007550217 2.2.2 major cho metabolism.degradation.starch 58 0.043862952 2.2.2.1 major cho metabolism.degradation.starch.starch cleavage 26 0.021405036 minor cho metabolism 3.1 minor cho metabolism.raffinose family 19 0.014279496 3.1.2 minor cho metabolism.raffinose family.raffinose synthases 12 0.003851643 3.1.2.2 minor cho metabolism.raffinose family.raffinose synthases.putative 12 0.003851643 3.2.2 minor cho metabolism.trehalose.tpp 22 0.033634009 3.4.3 minor cho metabolism.myo-inositol.insp synthases 4 0.040258978 cell wall 10 cell wall 875 9.90407e-06 10.5.3 cell wall.cell wall proteins.lrr 22 0.020416261 10.7 cell wall.modification 153 0.009841828 10.8 cell wall.pectin*esterases 153 0.006763777 10.8.1 cell wall.pectin*esterases.pme 130 0.027926432 lipid metabolism 11.9.3.4 lipid metabolism.lipid degradation.lysophospholipases.phospholipase a2 6 0.021694885 amino acid metabolism 13.1.3.6 amino acid metabolism.synthesis.aspartate family.misc 11 0.015036573 13.1.3.6.1 amino acid metabolism.synthesis.aspartate family.misc.homoserine 11 0.015036573 13.1.3.6.1.1 amino acid metabolism.synthesis.aspartate family.misc.homoserine.aspartate kinase 8 0.015473113 sassimilation 14.2 s-assimilation.apr 3 0.048354292 200 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table s3. continued. secondary metabolism 16 secondary metabolism 761 2.10308e-09 16.1 secondary metabolism.isoprenoids 192 0.009275191 16.1.3 secondary metabolism.isoprenoids.tocopherol biosynthesis 24 0.000171833 16.1.3.2 secondary metabolism.isoprenoids.tocopherol biosynthesis.homogentisate phytyltransferase 12 0.003851643 16.2 secondary metabolism.phenylpropanoids 261 6.13238e-05 16.2.1 secondary metabolism.phenylpropanoids.lignin biosynthesis 118 5.45265e-07 16.2.1.1 secondary metabolism.phenylpropanoids.lignin biosynthesis.pal 8 0.000396601 16.2.1.3 secondary metabolism.phenylpropanoids.lignin biosynthesis.4cl 31 0.011872219 16.2.1.6 secondary metabolism.phenylpropanoids.lignin biosynthesis.ccoaomt 15 0.036064324 16.8 secondary metabolism.flavonoids 155 6.45017e-07 16.8.2 secondary metabolism.flavonoids.chalcones 26 2.09935e-08 16.8.5 secondary metabolism.flavonoids.isoflavonols 22 0.000638941 hormone metabolism 17 hormone metabolism 1302 2.80432e-08 17.5 hormone metabolism.ethylene 430 1.19416e-05 17.5.2 hormone metabolism.ethylene.signal transduction 178 0.000622743 17.6 hormone metabolism.gibberelin 101 0.009275191 17.7 hormone metabolism.jasmonate 72 0.00068077 17.7.1 hormone metabolism.jasmonate.synthesis-degradation 70 0.000507643 17.7.1.2 hormone metabolism.jasmonate.synthesis-degradation.lipoxygenase 45 0.009861992 17.7.1.5 hormone metabolism.jasmonate.synthesis-degradation.12-oxo-pdareductase 12 0.014129403 stress 20 stress 1776 7.21114e-07 20.1 stress.biotic 979 0 20.1.7 stress.biotic.pr-proteins 711 0 20.2 stress.abiotic 775 0.013788265 20.2.1 stress.abiotic.heat 375 5.46072e-10 redox regulation 21 redox.regulation 405 0.005916683 21.4 redox.glutaredoxins 89 0.029935211 polyamine metabolism 22.2 polyamine metabolism.degradation 4 0.021694885 22.2.1 polyamine metabolism.degradation.polyamin oxidase 4 0.021694885 nucleotide metabolism 23 nucleotide metabolism 269 0.007504987 23.1 nucleotide metabolism.synthesis 66 0.000192645 23.1.1 nucleotide metabolism.synthesis.pyrimidine 26 0.009342322 23.1.1.1 nucleotide metabolism.synthesis.pyrimidine.carbamoyl phosphate synthetase 7 0.041272522 23.1.2 nucleotide metabolism.synthesis.purine 28 0.025728984 biodegradation of xenobiotics 24.2 biodegradation of xenobiotics.lactoylglutathione lyase 27 0.048107793 201 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) rna 27 rna 6310 1.69051e-36 27.1 rna.processing 582 4.64734e-33 27.1.1 rna.processing.splicing 101 5.37497e-06 27.1.2 rna.processing.rna helicase 73 3.60128e-12 27.2 rna.transcription 232 0.00022923 27.3 rna.regulation of transcription 5289 9.85113e-13 27.3.25 rna.regulation of transcription.myb domain transcription factor family 667 1.55764e-11 27.3.32 rna.regulation of transcription.wrky domain transcription factor family 174 0 27.3.44 rna.regulation of transcription.chromatin remodeling factors 102 3.61765e-06 27.3.50 rna.regulation of transcription.general transcription 60 8.02228e-05 27.3.52 rna.regulation of transcription.global transcription factor group 29 0.015473113 27.3.54 rna.regulation of transcription.histone acetyltransferases 24 0.013788265 27.3.57 rna.regulation of transcription.jumonji family 32 0.014129403 27.3.63 rna.regulation of transcription.phd finger transcription factor 39 0.009861992 27.3.64 rna.regulation of transcription.phor1 39 1.17597e-06 27.3.67 rna.regulation of transcription.putative transcription regulator 397 1.95682e-11 27.3.69 rna.regulation of transcription.set-domain transcriptional regulator family 77 0.033634009 27.3.99 rna.regulation of transcription. unclassified 621 2.00383e-06 27.4 rna.rna binding 267 1.14682e-12 dna 28 dna 939 4.19444e-18 28.1 dna.synthesis/chromatin structure 567 8.43108e-16 28.1.3 dna.synthesis/chromatin structure. histone 71 4.61178e-08 28.2 dna repair 116 0.000367641 protein 29 protein 6420 2.50503e-26 29.1 protein.aa activation 135 2.80432e-08 29.1.30 protein.aa activation.pseudouridylate synthase 24 0.022243842 29.2 protein.synthesis 976 3.46656e-17 29.2.1 protein.synthesis.ribosomal protein 670 0.001970238 29.2.1.1.1.1 protein.synthesis.ribosomal protein.prokaryotic.chloroplast.30s subunit 31 0.009275191 29.2.1.2 protein.synthesis.ribosomal protein.eukaryotic 428 0.000668567 29.2.1.2.1 protein.synthesis.ribosomal protein.eukaryotic.40s subunit 163 0.006166941 29.2.2 protein.synthesis.misc ribososomal protein 22 0.007251695 29.2.2.50 protein.synthesis.misc ribososomal protein.brix 11 0.007550217 29.2.3 protein.synthesis.initiation 184 7.66744e-16 29.2.4 protein.synthesis.elongation 66 0.004180482 29.2.99 protein.synthesis.misc 12 0.037356552 29.3 protein.targeting 490 1.60382e-08 29.3.1 protein.targeting.nucleus 98 1.60564e-08 29.3.3 protein.targeting.chloroplast 66 0.022901299 29.3.4.2 protein.targeting.secretory pathway.golgi 24 0.019980876 29.4.1 protein.postranslational modification.kinase 550 1.31382e-07 29.4.1.57 protein.postranslational modification.kinase.receptor like cytoplasmatic kinase vii 515 2.97425e-07 29.5 protein.degradation 2751 0.000556946 29.5.11 protein.degradation.ubiquitin 1824 0.000367641 29.5.11.5 protein.degradation.ubiquitin.ubiquitin protease 72 4.92242e-08 29.5.11.20 protein.degradation.ubiquitin.proteasom 151 2.00383e-06 table s3. continued. 202 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table s3. continued. signalling 30 signalling 2836 0 30.1 signalling.in sugar and nutrient physiology 98 0.037356552 30.2 signalling.receptor kinases 1307 0 30.2.11 signalling.receptor kinases.leucine rich repeat xi 365 2.49818e-07 30.2.17 signalling.receptor kinases.duf 26 554 0 30.2.24 signalling.receptor kinases.s-locus glycoprotein like 66 0.000142501 30.2.25 signalling.receptor kinases.wall associated kinase 39 0.028132661 30.2.99 signalling.receptor kinases.misc 219 7.58628e-06 30.3 signalling.calcium 496 1.02576e-07 30.5 signalling.g-proteins 482 0.004180482 cell 31.1 cell.organisation 843 0.008218999 31.2 cell.division 161 0.001000067 development 33.1 development.storage proteins 89 1.26332e-05 transport 34.3 transport.amino acids 180 0.002846344 34.9 transport.metabolite transporters at the mitochondrial membrane 141 0.015657959 34.19 transport.major intrinsic proteins 96 9.49911e-05 34.19.2 transport.major intrinsic proteins.tip 37 0.000233643 34.22 transport.cyclic nucleotide or calcium regulated channels 41 0.003764466 34.99 transport.misc 639 1.5495e-18 203 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table s4. biological categories that are regulated in the s562l root tip in comparison to wild type root tip as analysed by mapman (benjamini hochberg-corrected; p < 0.05). bin name elements p-value photosynthesis 1 ps 317 2.23e-06 1.1 ps.light reaction 220 1.56e-08 1.1.1 ps.light reaction.photosystem ii 98 1.88e-06 1.1.1.1 ps.light reaction.photosystem ii.lhc-ii 38 4.79e-04 1.1.1.2 ps.light reaction.photosystem ii.psii polypeptide subunits 60 0.006735025 1.1.2 ps.light reaction.photosystem i 39 7.10e-04 1.1.2.2 ps.light reaction.photosystem i.psi polypeptide subunits 31 5.16e-05 fermentation 5.2 fermentation. pdc 8 0.01995843 cell wall 10 cell wall 875 0.001131608 10.6 cell wall degradation 254 0.004153439 lipid metabolism 11.9 lipid metabolism.l ipid degradation 271 0.0015716 11.9.2 lipid metabolism.lipid degradation.l ipases 113 0.00294316 11.9.2.1 lipid metabolism.lipid degradation.lipases.triacylglycerol lipase 89 0.038454121 amino acid metabolism 13.2 amino acid metabolism.degradation 156 0.018542453 13.2.3 amino acid metabolism.degradation.aspartate family 45 0.046371554 13.2.4 amino acid metabolism.degradation.branched chain group 31 0.002102358 secondary metabolism 16 secondary metabolism 761 0.034592476 16.2.1 secondary metabolism.phenylpropanoids.lignin biosynthesis 118 0.005204386 16.2.1.1 secondary metabolism.phenylpropanoids.lignin biosynthesis.pal 8 0.014817383 16.2.1.3 secondary metabolism.phenylpropanoids.lignin biosynthesis.4cl 31 0.014331695 16.8 secondary metabolism.flavonoids 155 0.011867805 16.8.2 secondary metabolism.flavonoids.chalcones 26 0.001215041 hormone metabolism 17 hormone metabolism 1302 0.004747464 17.2.2 hormone metabolism.auxin.signal transduction 54 0.021272135 17.5.2 hormone metabolism.ethylene.signal transduction 178 0.04073287 17.7 hormone metabolism.jasmonate 72 0.033698515 stress 20.1 stress.biotic 979 0.025578528 20.1.7.6 stress.biotic.pr-proteins.proteinase inhibitors 34 0.002102358 20.2 stress.abiotic 775 0.002668161 20.2.1 stress.abiotic.heat 375 5.16e-05 20.2.3 stress.abiotic.drought/salt 129 0.033698515 204 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table s4. continued. redox regulation 21.4 redox.glutaredoxins 89 0.033698515 nucleotide metabolism 23.1.2 nucleotide metabolism.synthesis.purine 28 0.034892781 23.1.2.1 nucleotide metabolism.synthesis.purine.amidophosphoribosyltransferase 5 0.021645967 misc 26.2 misc.udp glucosyl and glucoronyl transferases 502 0.011867805 26.1 misc.cytochrome p450 398 0.007547013 26.12 misc.peroxidases 185 2.37e-05 rna 27 rna 6310 7.58e-05 27.3 rna.regulation of transcription 5289 0.014817383 27.3.4 rna.regulation of transcription.arf, auxin response factor family 57 0.002008989 27.3.14 rna.regulation of transcription.ccaat box binding factor family, hap2 21 0.008713324 27.3.30 rna.regulation of transcription.trihelix, triple-helix transcription factor family 64 0.008713324 27.3.32 rna.regulation of transcription.wrky domain transcription factor family 174 4.18e-11 27.3.64 rna.regulation of transcription.phor1 39 1.05e-04 27.3.80 rna.regulation of transcription.zf-hd 43 0.002668161 27.3.99 rna.regulation of transcription.unclassified 621 3.38e-07 dna 28.1.3 dna.synthesis/chromatin structure.histone 71 2.24e-08 protein 29.2 protein.synthesis 976 2.44e-09 29.2.1 protein.synthesis.ribosomal protein 670 0 29.2.1.2 protein.synthesis.ribosomal protein.eukaryotic 428 0 29.2.1.2.1 protein.synthesis.ribosomal protein.eukaryotic.40s subunit 163 1.76e-08 29.2.1.2.2 protein.synthesis.ribosomal protein.eukaryotic.60s subunit 265 3.25e-13 29.2.3 protein.synthesis.initiation 184 0.007187207 29.3 protein.targeting 490 0.00294316 29.3.4 protein.targeting.secretory pathway 237 2.13e-06 29.3.4.2 protein.targeting.secretory pathway.golgi 24 0.001431831 29.5 protein.degradation 2751 0.014770075 29.5.11 protein.degradation.ubiquitin 1824 0.034568824 29.5.11.5 protein.degradation.ubiquitin.ubiquitin protease 72 0.003802264 29.5.11.20 protein.degradation.ubiquitin.proteasom 151 5.16e-05 29.7 protein.glycosylation 58 0.029399721 signalling 30 signalling 2836 0.008713324 30.2 signalling.receptor kinases 1307 2.24e-08 30.2.17 signalling.receptor kinases.duf 26 554 2.26e-12 30.2.24 signalling.receptor kinases.s-locus glycoprotein like 66 0.010926445 30.2.25 signalling.receptor kinases.wall associated kinase 39 0.007347366 30.2.99 signalling.receptor kinases.misc 219 0.018034925 cell 31 cell 1584 6.23e-05 31.4 cell.vesicle transport 336 2.13e-06 development 33.1 development.storage proteins 89 1.06e-04 transport 34.19 transport.major intrinsic proteins 96 0.002528114 205 atlas journal of biology 2017, pp. 392–401 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) characterization of diversity of bradyrhizobia on cowpea in iraq reveals unusual strain characteristics suad a. al-saedi1†, naoufal lakhssassi2†, my abdelmajid kassem3, ibrahim b. razaq1†, and khalid meksem2* 1 agricultural researcher directorate, ministry of science and technology, baghdad, iraq; 2 department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901, usa; 3 plant genomics and biotechnology laboratory, department of biological sciences, fayetteville state university, fayetteville, nc 28301-4298, usa. received: april 23, 2017 / accepted: may 7, 2017 __________________________________________________ * corresponding author: meksem@siu.edu. † these authors contributed equally to this work. 392 abstract rhizobium-legume symbiosis is considered as one of the most well established symbiotic nitrogen fixing system for agronomic studies. association between legumes and rhizobia results in the formation of root nodules where symbiotic nitrogen fixation occurs. the current study aimed to authenticate 110 isolates from 20 sites belonging to 10 governorates in iraq, tested their capacity of nodulation with cowpea and classified them depending on the phenotype and genotype presented by sequence analysis of 16s rrna. to fulfill these goals, many approaches have been implemented such as authentication tests, bromothymol blue reaction, colony size and morphology, antibiotic test, sequencing of 16s rrna and phylogenetic analysis. this study provides an easy way to classify the bradyrhizobia sp. strains by genotype analysis depending on the phenotypes (i.e. motility and colony size) by sample preservation and high quality dna isolation from environmental soil samples followed by 16s rrna sequencing. this molecular technique has demonstrated the usefulness of these methods, easy technologies, and their applications to microbiome analysis and environmental science. interestingly, a group of bradyrhizobia identified in the current study was able to secrete acidic products before switching and starting to secrete alkali products after 1, 2 and 3 days. this is an unusual phenotype observed within rhizobia strains. keywords: 16s rrna, nitrogen, symbiosis, locations, strains. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction crop yields are linked to the use of soil or foliar fertilizers that are applied to supply one or more essential nutrients that support plant growth and production (stewart et al., 2005). many reports estimate that 30 to 50% of crop yields are attributed to natural or synthetic commercial fertilizers (gowariker et al., 2009; stewart et al., 2005). there are 14 essential elements for plant growth and development. nitrogen (n), phosphorus (p) and potassium (k) are the three essential elements needed in large quantity by plants, which are usually known as macroelements (salisbury and ross, 1985). not all soils are rich in npk and thus nutrients must be supplied through fertilizers (dittmar et al., 2009). in fact, these three elements must be added to arable soils to secure the sufficiency level of each for optimum crop yield. however, most farmers worldwide, due to high costs and environmental concerns, limit the use of synthetic fertilizers. although much of the nitrogen is removed when protein-rich grains or hay are harvested, significant amounts can remain in the soil for future crops (bisen et al., 2012). this is especially important when nitrogen fertilizer is not used, such as in crop rotation schemes used in less industrialized countries. nitrogen is the most commonly deficient nutrient in many soils around the world; therefore, it is the most commonly supplied plant nutrient. during the last two decades, many studies focused on the widespread applications of natural nitrogen suppliers such as nitrogen-fixing rhizobia sp. (abaidoo et al., 2000; shahzad et al., 2012; steenkamp et al., 2008; abdulameer, 2010; martyniuk et al., 2013). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) rhizobia sp. is soil bacteria that fix nitrogen (diazotrophs) after establishing inside root nodules of legumes (fabaceae) in a very well-known process called biological nitrogen fixation (bnf). rhizobia require a plant host and cannot independently fix nitrogen. the rhizobium-legume symbiosis is one of the most prominent beneficial plant-microbe interactions (gonzález and gonzalez-lópez, 2013). bnf has received high attention because of the central role it plays in the maintenance of soil fertility (sprent and sprent, 1990; chemining’wa et al., 2011). legumes have the potential to contribute to soil nitrogen and increase yields of subsequent or associated non-legume crops through symbiotic nitrogen fixation (brockwell et al., 1995). cowpea (vigna unguiculata l. walp.) is one of the main products of family farming in semi-arid regions such as iraq and is of considerable importance as a protein source for low-income populations of rural areas. it is well known that cowpea is relatively resistant to salinity and drought stress (eaglesham et al., 1992). cowpea rhizobia were first classified as a heterogeneous group of slow-growing rhizobia that nodulates promiscuous tropical and subtropical legume species known as ‘cowpea cross-inoculation group’ (allen and allen, 1981). furthermore, rhizobia, representing the ‘cowpea group’ cowpea rhizobia (bradyrhizobium spp.), are usually slow-growing bacteria of the genus bradyrhizobium belonging to the order rhizobiales of the alphaproteobacteria class (garrity et al., 2005; jordan, 1982; kuykendall, 2005a,b). bradyrhizobium species are gram-negative bacilli (rod shaped) with a single subpolar or polar flagellum. many changes in rhizobia taxonomy have occurred during the last decade due to an increase of available phenetic and genetic information about this group of bacteria (silva et al., 2012). knowledge of the diversity of rhizobia is of paramount importance as it is a source of genetic resources for selection of strains adapted to different conditions. currently, the system of microbial taxonomy addresses a joint analysis of morphological, physiological and different molecular tools. statistical methods are used to evaluate the differences and similarities among microorganisms, providing quantitative measures of similarities among microorganisms. however, rhizobia taxonomy has been changed in recent years due to the use of molecular tools allowing for the identification of new groups of bacteria capable of nodulation and nitrogen fixation in legumes (chen et al., 2005). in this study, a total of 110 strains isolated from 20 sites belonging to 10 governorates in iraq were tested for their ability to fix nitrogen and to symbiotically interact with the cowpea as a plant host. next, we focused on bacterial nodulation by testing new rhizobia isolates from the 20 sites belonging to the most important cowpea production area in iraq. interestingly, the 60 rhizobia strains were genetically identified and classified by sequencing the 16s rrna gene. the 16s rrna gene is a highly conserved component of the transcriptional machinery of all dna-based life forms and thus is highly suited as a target gene for sequencing dna in samples containing up to thousands of different species. material and methods bacterial isolate one hundred and ten bacteria isolates were obtained from cowpea root nodules in 20 different field sites in 10 governorates of iraq, belonging to the most important cowpea production areas in the country. these are namely: al basrah, dhi-qar, misan, wasit, babil, al anbar, baghdad, salahudein, suleimanyah, and ninevah. nodules were surface sterilized by immersing in 95% v/v ethanol for 10 sec. followed by 30% v/v solution of sodium hypochlorite for 4 min and then rinsed with sterile distilled water. they were then placed in 3% v/v hydrogen peroxide for 1 min, followed by rinsing five times in sterile water. the sterilized nodules were crushed in a large drop of sterile water in a petri dish. the nodule suspension was next streak inoculated on yeast mannitol agar (yma) (somasegran and hoben, 1994) and incubated at 28ºc for 5 days. single colonies were selected and streaked onto yma slant and kept at 4ºc for short-term storage with sub culturing every 4 months. long-term storage was carried out by storing the culture broth in 10% glycerol at –80ºc. authentication tests all 110 bacterial strains were authenticated in order to determine if they were cowpea rhizobia by observing nodule formation on cowpea roots grown in autoclaved sandy soil. sterilized seeds of cowpea cultivars were inoculated with different bacteria strains. non-inoculated controls were used to check for cross-contamination. nodulation assay of the cowpea plants cowpea plants were grown on autoclaved sandy and organic soil (50:50) and watered as needed. sixty rhizobium strains were grown in yma medium for 5 days at 28ºc until reaching the exponential growth phase. bacterial inoculation was done at the time of sowing (10% volume by seed weight). inoculated plants were grown in the greenhouse under 27°c, 70% humidity, and 16-hour artificially supplemented light conditions. bromothymol blue reaction all authenticated cowpea rhizobia cells were streaked onto agar plates containing ym medium with bromothymol blue at 25 µl/ml final concentration. then, the plates were incubated at 28ºc. the authenticity of each strain was detected during the 10 day incubation period with observation for color of the indicator dye on the plates (somasegaran and hoben, 1994). fast growing rhizobia changed color of the indicator dye to yellow while slow growing rhizobia turned the indicator dye to blue. colony size and morphology colony size and morphology were measured by incubating single cells onto the center of agar plates containing ym me393 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 394 dium. the plates were incubated at 28ºc for 10 days with morphology observations of movement, convex, flat, etc. the size of the colonies was also measured at the end of the 10-day incubation period. antibiotic test antibiotic tests were conducted by spreading bacteria on petri dishes containing mueller-hinton agar and testing two different antibiotic discs. the discs were equidistant from the others to avoid overlapping zones of inhibition. the antibiotics discs tested were spectinomycin and streptomycin at 100 µg and 300 µg concentrations, respectively. the plates were incubated at 28ºc. the presence or absence of an inhibition zone was noted, indicating susceptibility, resistance, or intermediate resistance. dna isolation in order to extract bacterial dna, the bacteria isolates were incubated into ym broth in a 15 ml sterile tube and incubated under shaking conditions at 200 rpm, 28ºc for 5 days. cells were harvested by centrifugation at 13.2 x 1000 rpm for 10 min then washed once with 0.85% nacl solution to eliminate extracellular polysaccharides before chromosomal dna isolation. bacterial dna was obtained using wizard genomic dna purification kit from promega. polymerase chain reaction (pcr) amplification of the 16s rrna gene genomic dna from each isolate was amplified using the primer fd1 (5`agagtttgatcctggctcag – 3`) and rd1 (5`aaggaggtgatccagcc – 3`) in order to amplify the 16s rrna as described by weisburg et al. (1991). the selected primers were derived from conserved regions of the 16s rrna gene and amplified nearly 1500 bp of full-length 16s rrna gene (weisburg et al. 1991). pcr amplifications were carried out in total reaction volumes of 25 µl containing 2.0 mmol/l mgcl2, 200 µmol/l of dntps, 1 µmol/l of each primer, 30 ng of genomic dna and 1.5 u of pfu dna polymerase (agilent technologies). the temperature profile was as follows: an initial denaturation step at 95ºc for 3 min, 35 cycles of denaturation at 94ºc for 30 sec; then annealing at 51ºc for 1 min and extension at 72ºc for 2 min with a final extension at 72ºc for 7 min. sequencing of 16s rrna products of amplification belonging to approximately 60 strains were purified with qla quick pcr purification kit (from qiagen) prior sequencing and then sequenced at genewiz fig. 1. geographic distribution of the rhizobia bacterial collection sites in iraq. sixty bacteria isolates were obtained from cowpea root nodules of 20 locations from different field sites in 10 governorates in iraq, belonging to the most important cowpea production area in iraq (basrah, dhi-qar, misan, wasit, babil, al anbar, baghdad, salahudein, suleimanyah, and ninevah). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 395 company, usa, using the primers fd1 and rd1. the 16s rrna sequences were first analyzed with seqman-dna star lasergene software; then, the closely related sequences found were analyzed by culstal w program. phylogenetic analysis and genomic structure multiple sequence alignments were performed using the mega4 software package and the clustal-w algorithm. an unrooted phylogenetic tree was calculated with the neighbourjoining method (saitou and nei, 1987), and tree topology robustness was tested by bootstrap analysis of 5,000 replicates. alignment analysis of the 16s rrna sequenced genes from the different rhizobia strains, in addition to the reference 16s rrna sequences were obtained using megalign 4 software. all parameter values corresponded to default definitions. results authentication test in this study, a total of 110 strains isolated from 20 sites belonging to 10 governorates in iraq (fig. 1) were tested to their ability to form nodules. from the 110 bacteria isolated, we authenticated a total number of 60 isolates that re-nodulated their original host cowpea confirming their symbiotic status (table 1). strain id nodule number with cowpea colony size (mm) antibiotic test spectinomycin streptomycin after 5 days after 10 days s i r s i r s1 50 35 85 + + s2 20 85 85 + + s3 55 85 85 + + s4 12 12 20 + + s5 5 85 85 + + s6 25 85 85 + + s7 4 12 20 + + s8 30 10 15 + + s9 35 25 85 + + s10 7 12 20 + + s11 10 8 15 + + s12 25 20 55 + + s13 2 10 15 + + s14 17 10 20 + + s15 29 9 15 + + s16 7 19 85 + + s17 29 10 22 + + s18 7 10 20 + + s19 2 12 23 + + s20 3 12 24 + + s21 18 9 15 + + s22 6 10 15 + + s23 6 19 24 + + s24 7 18 25 + + s25 5 16 25 + + s26 25 10 20 + + s27 15 75 85 + + s28 10 75 85 + + s29 3 70 85 + + s31 10 12 24 + + s32 6 50 85 + + s33 7 50 85 + + s34 4 5 10 + + s35 6 9 16 + + s36 4 8 10 + + s37 2 10 20 + + s38 13 25 85 + + s39 10 85 85 + + s40 2 10 17 + + s41 2 7 10 + + s43 7 13 20 + + s44 3 8 12 + + s45 3 19 26 + + s46 2 20 27 + + s47 89 85 85 + + s48 21 5 10 + + s49 89 12 20 + + s50 89 10 20 + + s51 78 85 85 + + s52 3 20 39 + + s54 16 85 85 + + s55 3 9 20 + + s56 2 9 15 + + s57 7 10 15 + + s58 4 17 85 + + s59 19 12 20 + + s60 1 55 85 + + s61 5 85 85 + + s62 2 12 20 + + s63 10 18 28 + + table 1. phenotypic data of the sixty cowpea rhizobia. second column present the nodule number, third and fourth column show the colony size after 5 and 10 day of growth, last columns present antibiotic discs tested; spectinomycin and streptomycin at 100 mg and 300 mg concentrations, respectively. s= susceptibility, r= resistance or i= intermediate resistance. red signs present the five cowpea rhizobia found to resist to both antibiotics. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 396 fig. 2. reaction type of the sixty cowpea rhizobia with bromothymol blue. the authenticity of each strain was detected during 10 days incubation period, with observation for color of the indicator dye on the plates. fast growing rhizobia changed color of indicator dye to yellow while slow growing rhizobia turned the indicator dye to blue. fig. 3. colony size of the sixty cowpea rhizobia strains after 5 days (a) and 10 days (b) growth. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 397 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fig. 4. neighbour joining (nj) tree showing the phylogenetic relatedness of 16rna sequences for 43 cowpea rhizobia associated with the colony size (c.s) and cell movement. the phylogenetic tree was generated using clustalw and mega4 softwares, and boostrap values are shown using 5,000 replicates. 398 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) the authentication test showed that 38 (63.3%) cowpea rhizobia presented a nodule number less than 10, 8 (13.3%) strains presented between 10 and 20 nodules, 6 (10%) strains presented between 20 and 30 nodules, 3 (5%) strains presented between 30 and 70 nodules, and 4 super-nodulating (6.66%) strains (s47, s49, s50 and s51) presented between 70 and 90 nodules per plant. surprisingly, the four strains from the last super-nodulating group belong to al-basrah governorate. this result may be related to the physical composition of the soil in this area, which differs from the rest of the 10 governorates in iraq. furthermore, all 60 strains were chosen for genotypic characterization (16s rrna target sequencing) and phenotypic analysis including colony size, morphology, bromothymol blue reaction, and antibiotic resistance test. phenomic analysis of the rhizobia strains the bromothymol blue technique is used in bacteriology as a ph indicator in the agar. it changes to yellow in case of acid production during fermentation of lactose or changes to deep blue in case of alkalinization. lactose-positive bacteria build yellow media. however, bacteria that decarboxylate l-cystine cause an alkaline reaction and build deep blue media. this colorant is used essentially in order to classify rhizobia, especially bradyrhizobium (alkaline reaction) from rhizobium (acidic reaction) strains. the reaction of the 60 cowpea rhizobia colonies on bromothymol blue (btb) agar plates revealed two major types of reactions. in the first type of reaction, cells of 6 cowpea rhizobia were found to secrete acidic product during 10 days incubation. surprisingly, 3 strains were able to secrete alkali product during the first 5 days before switching and starting the secretion of acidic product in the last 5 days during the 10 days incubation (fig. 2). furthermore, most cells were in the second type of btb reaction and were found to secrete alkali product. this second type was divided into 4 types of reactions. seven cowpea rhizobia belong to a group that secreted alkali product during the 10 days incubation. surprisingly, the second, third and fourth groups were able to secrete acidic products during the first days before starting to secrete alkali products after 1 (16 strains), 2 (24 strains) and 3 (4 strains) days. this is an unusual phenotype observed within rhizobia strains (fig. 2). furthermore, the 60-cowpea rhizobia colonies were regrouped in 4 groups depending on their capability to grow (colony size) and capacity to invade the whole petri dish (motility). the first group of the four-regrouped colony contained 13 cowpea rhizobia presenting size of colonies between (10–15) mm, of which 8 bacteria were flat in shape and 5 were convex. the second group containing 15 bacteria showed sizes between 15 and 20 mm, of which 6 were flat in shape and 9 were convex. the third group with 7 bacteria had sizes between 20 and 25 mm, 6 of which were flat in shape and one was convex. finally, the fourth group with 25 bacteria were able to grow more than 25 mm until they filled the whole plate during the 10 days of incubation; 20 of them were flat in shape and 5 were convex. interestingly, all bacteria belonging to groups one, two and three were not motile while all bacteria from group four were motile on agar medium (fig. 3). moreover, all 60 strains were tested in the presence of two kinds of antibiotics: spectinomycin and streptomycin. it has been shown that rhizobia strains can be resistant to spectinomycin or streptomycin (ramírez et al., 1998). however, resistance to both antibiotics can be possible if the strain carries a mutation in its genome (zelazna-kowalaska, 1971). the result given in table 1 showed that 15 cowpea rhizobia were sensitive to spectinomycin and 55 sensitive to streptomycin. however, 25 cowpea rhizobia were resistant only to spectinomycin and 5 resistant only to streptomycin, while 20 cowpea rhizobia had an intermediate resistance to spectinomycin. interestingly, five cowpea rhizobia (s31, s35, s51, s61 and s63) were found to resist both antibiotics (table 1) (national committee for clinical laboratory standards–nccls). sequencing the 16s rrna the 16s rrna gene sequencing is commonly used for identification, classification, and quantification of microbes within complex biological mixtures such as environmental and gut samples (schmidt et al., 1991; ley et al., 2005). the 16s rrna gene is a highly conserved component of the transcriptional machinery of all dna-based life forms and thus is highly suited as a target gene for sequencing dna in samples containing up to thousands of different species (cox et al., 2013). conveniently, the 16s rrna gene consists of a conserved and a variable region. while the conserved region makes universal amplification possible, the variable regions allows discrimination between specific different microorganisms (kolbert and persing, 1999). specific primers have been used in this study to target the conserved regions of 16s in order to target conserve and variable regions within the characterized cowpea rhizobia. phylogenetic analysis of the sequenced 16s rrna from the 60 rhizobia separated the strains into four clades (fig. 4). surprisingly, the phylogenetic tree grouped all the strains based on their ability to grow (colony size) and motility. thus, group i contained strains with lower colony size presenting between 10 and 15 mm, group ii strains’ size was between 15 and 20 mm, and group iii contained strains able to grow more than 25 mm until they invade the entire petri dish. finally the last group iv contained strains with colony sizes varying between 20 and 25 mm. discussion in this study we characterized a population of 60 bradyrhizobium found in cowpea nodules, isolated from 20 sites belonging to the most important cowpea production area in iraq for their ability to form nodules, resist to antibiotics, motility, and study their evolutionary relationships among other bradyrhizobium species. in order to fulfill these goals, several techniques were employed such as authentication test, bromothymol blue reaction, resistance to antibiotics (spectinomycin and streptomycin), phenotypic analysis including colonies size and morphology, and phylogenetic analysis of the sequenced 16s rrna. when high density inoculum of a rhizobial strain are inoculated into media containing an antibiotic, a few cells may exhibit resistance as a result of spontaneous genetic changes or a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 399 mutations (josey et al., 1979; beynon and josey, 1980; turco et al., 1986; kuykendall et al., 1988). resistance of a rhizobial strain to a particular antibiotic is a useful marker (schwinghamer and dudman, 1973; pankhurst, 1977; beynon and josey, 1980; turco et al., 1986). it is important that antibiotic-resistant strains that are selected for inoculation experiments have not lost their infectiveness (i.e. ability to form nodules) or their effectiveness (i.e. ability to fix nitrogen) in the symbiosis with the host plant (beynon and josey, 1980; kremer and peterson, 1982; turco et al., 1986). interestingly, from the 60 strains only five cowpea rhizobia (s31, s35, s51, s61 and s63) were found to resist to both antibiotics. it has been shown that rhizobia strains can be resistant to spectinomycin or streptomycin (karanja and wood, 1988; ramírez et al., 1998). however, resistance to both of them can be possible only if the strain carries a mutation in its genome (schwinghamer, 1964; zelazna-kowalaska, 1971; turco et al., 1986). several studies showed that mutants treated with gamma radiation were able to grow at 50°c (chitchanok et al., 2011) or at temperatures higher than 40ºc (hungria et al., 1993), and four of our strains were able to grow at 37°c and 50°c. these strains were probably mutated by the presence of high radiation level in the soil, since several studies showed that certain bacterial strains are sensitive to nuclear radiation (makarova et al., 2001). four strains, (s47, s49, s50, and s51) presenting 6.66% of the authenticated rhizobium were super-nodulated, presenting between 70 and 90 nodules per plant, and belong to al-basrah governorate. this result may be related to the physical composition of the soil in this area, which differs from the rest of the 10 governorates in iraq. al-basrah is located on the shatt-al-arab waterway, downstream of which is the arabian gulf. the city is penetrated by a complex network of canals and streams vital for irrigation and other agricultural use, which makes it one of the most fertile regions for agriculture, located at the juncture of two famous rivers in iraq, al forat and dijlah. the sediments of al-basrah soils are mainly silt clay in texture and silt clay loam with subordinate amount of sand. al-basrah soils are characterized by their wide spectrum of mineralogical composition, non-clay minerals (calcite, dolomite, quartz, halite, gypsum, and feldspar), clay minerals (kaolinite, illite, montmorillonite, palygorskite, chlorite, and mixed-layred clay minerals), and heavy mineral (opaque, pyroxene, hornblende, chlorite, biotite, epidote, garnet, kyanite, staurolite, celestite, zircon, and tourmaline). the organic matter content has widely ranged in present soils; its amount depends upon the intensity of vegetation cover. the detrital supply of clastic materials controls the concentration of silica and aluminum. plant nutrition controls the concentration of fe and k. climatic conditions and hydrogeological setting controls the accumulation of na, ca and mg in basrah soils. the values of major elements in al-basrah saline soils survive seasonal fluctuations because of precipitation and dissolution alternative processes (al-marsoumi and al-jabbri, 2007). moreover, sequence analysis of 16s rrna has also been used frequently for microbial taxonomy, and it is a powerful and accurate method for determining interand intra-specific relationships (stackebrandt and goebel, 1994; janda and abbott, 2007; wang et al., 2007; kim et al., 2012). on the basis of 16s rrna sequences, six of the strains we isolated from cowpea are closely related to both b. japonicum and rhizobium species (representatives to group iv), which regrouped strains presenting colony sizes between 20 and 25 mm without motility. phylogenetic analysis showed that all the rest of the 54 strains descended from the common ancestral copy in the group iv. those 54 descendants evolved into different strains regrouped in 4 different sub-clades or branches. group i contained strains with lower colony size between 10 and 15 mm, and group ii between 15 and 20 mm without motility, and group iii contained strains able to grow more than 25 mm until they invaded the entire petri dish showing a clear motility in the agar. as shown in different studies, phylogenetic analysis supported their descent from a common ancestral copy (gherbi et al., 2008) and generated a topology consistent with previous legume phylogenies (wojciechowski et al., 2004). this gave rise to the hypothesis that the evolutionarily recent legume–rhizobia symbiosis reuses some of the molecular mechanisms of the more ancient arbuscular mycorrhiza (am) symbiosis (hirsh, 2004). using this molecular technique, we resolved the cowpea bradyrhizobial diversity of 60 nodulating strains regrouping them into four different genotypes (fig. 4). this study provides an easy way to classify the rhizobia strains by genotype analysis depending on the phenotypes observed (i.e. motility and colony size) by sample preservation and high quality dna isolation from environmental samples such as soil samples and 16s rrna target sequencing. this demonstrated the usefulness of these methods, using easy technologies, and their applications to microbiome analysis and environmental science. conflict of interest the authors declare that they have no conflict of interest. references allen on and ek allen (1981) the leguminosae. a source book of characteristics, uses and nodulation. london, uk: mocmillan. al-marsoumi amh and mha al-jabbri (2007) basrah soils; geochemical aspects and physical properties–a review of basrah journal of science(c) 25 (1): 89-103. beynon jl and dp josey (1980) demonstration of heterogeneity 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(w w w. at la spu bl is hi ng .o rg ) verticillium dahliae-eggplant as the pathosystem model to reveal biocontrol potential of three trichoderma spp in greenhouse conditions wafaa mokhtari1*, mohamed achouri1, abdellah remah1, and hassan boubaker2 1 plant protection department, institut agronomique et vétérinaire hassan ii, agadir, morocco; 2 biology department, school of sciences, university ibn zohr, agadir, morocco received: january 17, 2018 / accepted: february 17, 2018 __________________________________________________ * corresponding author: w_mokhtari@yahoo.fr 417 abstract in this study three trichoderma species isolated from moroccan soil (natural and agricultural habitats) were investigated for their biocontrol potential against virulent verticillium dahliae on eggplant in green house conditions based on dipping root approach. evaluation of biocontrol efficacy of trichoderma spp. demonstrated effective potential of trichoderma on reducing verticilium disease on eggplant cultivars. disease assessment was established by measuring disease incidence in root units (di-ru) and in the above ground of eggplant cultivars (diau). di-ru% was recorded at 12.5%, 25.0%, 31.3% and 37.5% in t3, tc, t1 and t2 treatment respectively where di-ru recorded in tm2 controls; eggplants inoculated with verticillium only was equal to 100.0%. whereas, disease incidence outcomes in aerial part was diau = 100.0% in treatments tc and t2, 87.5% in t1 and 96.0% in t3 treatment where tm2 controls were assessed with 100.0%. keywords: trichoderma, biocontrol, verticillium dahliae-eggplant pathosystem. abbreviations: g; gram, cm2; centimeter area, cm; centimeter, di; disease incidence, di-au; disease incidence in above ground units, di-ru; disease incidence in roots units; w/w; weight per weight, npk; nitrogen, phosphor, potassium, g/hl; gram per hectoliter, °c; celsius degree, ml; milliliter. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction verticillium dahliae is considered a highly polyphagous soil borne plant pathogen that attacks more than 400 vegetable species including solanacea and cucurbitaceae cultures and olive trees. its polyphagous characteristic causes the attack of wide host range other than the original hosts what makes determination of host specificity and virulence of verticillium isolates of paramount importance regarding its management (tjamos, 1981). verticillium is considered a chronic economic soil borne pathogen that causes vascular wilt and death in many plants in field and green house. no economic losses measure has been done till now in the cultural infestation with verticilium in morocco. however, it is estimated of billions of dollars annually losses all around the world (pegg and brady, 2002). soil disinfection and genetic resistance are the major management strategies used to control verticilium diseases. however, application of biocontrol agents like trichoderma have become more and more used to control this soil borne pathogen due to negative effect of disinfectant on human health and environment (cook and baker, 1983). eggplant is a highly susceptible cultivar wilt and dies shortly after verticillium dahliae invading and expressing first symptoms in it. in fact, verticillium wilt has been reported to be the most destructive and prevalent diseases of eggplant. therefore, eggplant-verticillium is considered one of the best pathosystem models to evaluate resistance and/or tolerance to verticilium wilt for designing the adequate and required disease management strategy (i.e. grafting, biocontrol application) in crop system. for instance, testing biocontrol potential of trichoderma spp. using alike susceptible host controls would allow to idena tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) tify the effective biocontrol potential among trichoderma isolates especially when selecting antagonistic candidates during biocontrol agent screening (huisman and gerik, 1989; blestos et al., 2003). selecting antagonists for specific biocontrol of verticillium diseases was based on screening potential antagonistic candidates like trichoderma spp. many studies demonstrated that trichoderma spp. can inhibit growth or kill soil borne pathogens and revealed different antagonistic interactions between the antagonist trichoderma and the pathogen in vitro and in vivo (lumsden et al., 1993; monte, 2001; yang et al., 2010). actually, few research work have focused on the biocontrol efficacy of trichoderma spp against verticillium in vivo though important antagonistic potential were involved against verticillium dahliae when in vitro assays were applied. in this study, we attempt to evaluate biocontrol potential of three trichoderma species isolated from moroccan soil (natural and agricultural habitats) in green house conditions based on root dipping technique. material and methods experimental design for biocontrol treatments in greenhouse conditions three species of trichoderma identified at the species level (mokhtari et al., 2017); trichoderma afro-harzianum (t8a4), trichoderma reseei (t9i12) and trichoderma guizouhense (t4) were tested for their biocontrol efficacy against verticillium dahliae-eggplant pathosystem. eggplant cultivars were grown for three weeks on 77 peat trays. seedlings were then transplanted into pots after their inoculation with fungi. eggplant seedlings were transplanted in 3 l pots filled with sterile substrate at 3:1 w/w peat to sand ratio. substrate was fertilized using npk and oligo-elements composition at 250 g/hl. experimental design was organized in four randomized complete blocs with four replicates in each experimental unit. that is, four pots were used in each experimental unit. four treatments were tested; t1 was designated for treatment of cultivars inoculated with trichoderma afro-harzianum (t814) and verticillium dahliae, t2 was designated for treatment of cultivars inoculated with trichoderma guizouhense (t4) and verticillium dahliae and t3 was designated for treatment of cultivars inoculated with trichoderma reesei (t9i12) and verticillium dahliae. controls were respectively; tm1 was designated for healthy cultivars with non inoculated plants (negative controls), tm2 was designated for cultivars inoculated with pathogen only (positive control). verticillium has been known of its virulence and lethality towards woody and other crops. pathogenicity test investigated in tm2 controls allowed the exhibition of verticillium dahliae virulence on susceptible eggplants (schnathorst and sibbett, 1971). tc designated for treatment of cultivars inoculated with trichoderma afro-harzianum extracted from a commercial product to be compared with other trichoderma isolates. 418 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) obtaining verticillium isolate for pathogenic test branches and roots of olive trees diagnosed with wilt symptoms were collected, washed with tap water and disinfected one minute in 10% sodium hypochlorite. six small fragments from wilt branches and roots were washed in distilled di-ionized water for one more minute then inoculated in pda and incubated at 19 °c in the dark for 10 days. to obtain pure culture of verticillium dahliae, mycelium of the fungus was collected from tissues baits with sterilized scalpel and inoculated into fresh pda. verticillium dahliae microsclerotia inoculum and eggplant rootdipping inoculation microsclerotia were used exclusively as inoculum propagules to infest eggplant roots. therefore, we used cellophane layer to produce uniform microsclerotia layer on potato dextrose agar (pda, difco) plate. cellophane plates with verticillium microsclerotia were thereafter flooded with sterile di-ionized water and poured through 45 µm stainless steel sieve to remove spores and hyphae. the contents on the sieve were transferred to sterile glass petri dishes and comminuted with a sterile razor. to prepare microsclerotia suspension, microsclerotia obtained were transferred in beaker filled with distilled di-ionized water. after deep vortex, number of microsclerotia was determined with serial dilution ranged from 10-1 to 10-6 to a final microsclerotia concentration of 104 to 105/ml (atibalentja and eastburn, 1997; shiraishi et al., 2014; dongfang et al., 2014). eggplant roots cuts of 2 to 3 cm diameters long were inoculated in 104 to 105/ ml of microsclerotia of verticillium dahliae (atibalentja and eastburn, 1997; gray et al., 1998). disease evaluation and measurement of leaves surface area (lsa) disease assessment was estimated by measuring disease incidence (di) of verticillium dahliae as reported by campbell and neher (1994). disease incidence (di) was measured as the percentage of number of plant units that are visibly diseased. therefore, di percentage was calculated as shown in the equation (1) (campbell and neher, 1994). disease incidence = [number of infected plant units] / [total number of plants in the experiment] x 100 equation 1 disease incidence was measured in above ground units corresponding to aerial parts of cultivars (di-au) and root units (di-ru). diabove ground units was measured based on visible symptoms detected in the above-ground plant area. therefore, symptoms were identified and measured in symptomatic vegetative tissues in leaves (i.e. typical bronze-yellowing, necrosis) and vascular tissues wilt. di-ru was measured based on visible symptoms and signs in the diseased roots and crown (rots and/or discoloration) of related host plant. in fact, this a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 419 method requires destructive sampling of plant, therefore, applied at the end of each experiment. signs were basically examined based on the presence of pathogens’ components like mycelium and propagules under microscopic observation (campbell and neher, 1994). in addition, plant parameters like plant height (ph) in centimeter (cm) and leaf surface area (lsa) in square centimeters (cm2) were also measured. these three parameters were assessed at the end of each experiment yet with non destructive method. whereas, root dry weight (rdw) and plant dry weight (pdw) in gram (g) were measured at the end of each experiment with destructive sampling method (benson and baker, 1974; campbell and neher, 1994). in order to measure pdw, all plants were excavated from pots, roots were washed under running tap water to discard adhering substrate then exposed to the air at ambient temperature (25-30 °c) until they dry. when pdw was measured, roots were cut at crown level and dried in drying chamber at 60 °c for three to four days to measure rdw. in order to measure leaves surface area (lsa) technique described by breda (2003) was used. since leaves are irregular, lsa was measured based on constant mass ratio calculation (breda, 2003). at the end of each experiment leaves were collected, laid on the surface of a4 papers then their outlines were traced. each leaf drawn on a4 paper was cut at the level of outline and a4 leaf drawing pieces were kept to be weighed. surface area (cm2) and the mass (g) of a whole a4 paper were measured too. to calculate lsa (cm2) of each a4 leaf drawing piece mass ratio was correlated to surface area (cm2) of a4 paper as detailed in equation (2). [surface area of a4 paper (cm2)] [mass of a4 leaf drawing piece (g)]/[mass of a4 paper (g)] euation 2 results and discussion reducing verticillium infestation on eggplant cultivars as mentioned in material and methods in vivo antagonistic assay was tested on eggplant. similarly to previous antagonistic assay in green house, disease responses of verticillium were assessed by measuring disease incidence in above ground units (diau) and root units (di-ru). for eggplant, disease assessment and antagonistic evaluation were performed three months after trichoderma spp. treatments. on the whole, trichoderma spp. treatments exhibit biocontrol efficacy reducing verticillium disease in eggplant cultivars yet could not control effectively verticillium severe symptoms on eggplant. as detailed in figure 1 disease incidence in above ground units = 100.0% in treatments tc and t2, 87.5% in t1 and 96.0% in t3 treatment. it can be inferred from diabove ground unit results that trichoderma spp. treatments each containing t1; t. afroharzianum, t2; t. guizouhense, t. reesei and tc; commercial trichoderma were not able to suppress definitively the pathogen. in fact, different symptoms on the above-ground eggplant cultivars were detected; typical bronze-yellowing and necrosis of leaves veins and wilting as illustrated in figures 2 and 3. moreover, significant decrease was recorded in leaves surfaces area (lsa) of cultivars in different treatments relatively 0% 20% 40% 60% 80% 100% 120% t3 t2 t1 tc tm2 tm1 d is ea se i nc id en ce in % di-above ground unit di-roots unit 100 % 87.5 % 100%96 % 100% 100 % 87.5 % 100%96 % 100% 12.5% 37.5% 31.3% 25% 0.0% figure 1. disease incidence assessment in (%) on verticillium-eggplant pathosystem towards evaluation of effect of three antagonists; trichoderma afroharzianum t1, trichoderma reseei t2 and trichoderma guizouhense t3. tc trichoderma extracted from commercial product used as reference. disease incidence was assessed in above ground units di-au and in roots; di-ru. figure 2 and 3. trichoderma treatments of eggplant artificially infested with verticillium microsclerotia; both figures represent above ground symptomatic eggplant treated with different trichoderma spores suspenssions; t. afro-harzianum in t1 treatment, t. reseei in t2 and t. guizouhense in t3 and tc trichoderma extracted from commercial product compared to healthy eggplants tm1 and infested eggplant tm2 controls. 2 3 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 420 compared to healthy plant tm1. for instance lsa = 19.5 cm2 of eggplant occurred in t3 treatment while lsa = 88.1 cm2 occurred in healthy plant tm1, (p = 0.000). lsa dramatically decreased to 2.7 cm2 in tm2 controls (figure 4). interestingly, there seems to be different asset between disease incidence assessed above-ground (di-au) and in root unit (di-ru). di-ru was recorded at 12.5%, 25.0%, 31.3% and 37.5% in t3, tc, t1 and t2 treatment respectively (figure 1). root dry weight (rdw) recorded in eggplant with different treatments was respectively 0.1 gram (g) in tc, 0.2 g in t2 and t3 and 0.6 g in t1 compared to 1.2 g rdw for healthy eggplant controls tm1. it seems that roots weight was significantly affected by disease responses when infested with verticillium with only rdw = 0.2 g in tm2 controls (for more details see figures 4 and 5). in fact, root dry weight recorded support the overall results obtained in di-ru. it may be reasonable to suppose from di-ru values that trichoderma treatments were able to alleviate verticillium root disease in eggplant. moreover, from plant height (ph) results, it can be deduced that eggplant height decreased at some extent. yet, ph was maintained at somewhat remarkable level in treated eggplants compared to tm2 controls. that is, tm2 infested controls hardly reached their 4.2 centimeters (cm) height whereas eggplant cultivars in t1 treatment containing t. afro-harzianum maintained their height at 16.6 cm (p = 0.000). ph in eggplants in t2, t3 and tc treatment noticeably reached 12.6, 13 to 13.5 centimeters respectively compared to tm1 with ph = 26 cm, (p = 0.000). these results were in line with some of previous work on verticillium biocontrol efficacy test in many crop-systems in green house and field conditions. for instance, zheng et al (2011) tested biocontrol potential and efficacy of 105 antagonists including trichoderma spp. in the v. dahliae-cotton pathosystem in green house conditions. in their work, zheng et al (2011) assessed 33 fungal antagonists including trichoderma spp. with biocontrol potential in vitro which displayed efficacy under green house (zheng et al., 2011). carrero-carron et al. (2016) have demonstrated that two of t. asperellum strains reduce significantly the severity of defoliating verticilium disease on olive plants and promoting growth in infested and non-infested olive plants (carrero-carron et al., 2016). these interesting biocontrol properties and potential of trichoderma spp. on root disease like verticillium investigated in the previous and the present studies may be due to a number of reasons including trichoderma antagonistic traits and inoculation method applied. as mentioned in the literature investigating the naturally occurring highly susceptible eggplant and virulent verticillium interactions allowed us to reveal the congruent potential figure 4. evaluation of biocontrol efficacy of trichoderma spp. on plant development parameters on verticillium-eggplant pathosystem. plant development parameters measured were; leaves surface area (lsa), plant height (ph), root dry weight (rdw) and plant dry weight (pdw). figure 5. roots of eggplant artificially infested with verticllium and treated with different trichoderma spores suspenssions; t. afro-harzianum in t1 treatment, t. reseei in t2, t. guizouhense in t3, and tc trichoderma extracted from commercial product compared to controls; healthy eggplants tm1 and eggplant inoculated with verticillium only tm2. 0 20 40 60 80 100 t3 t2 t1 tc tm2 tm1 ls a (c m 2 ) 88.1 26.4 2.7 0 5 10 15 20 25 30 t3 t2 t1 tc tm2 tm1 ph ( cm ) 4.2 26 16.6 0 0.5 1 1.5 t3 t2 t1 tc tm2tm1 r d w ( g) 0.02 1.2 0.6 0 0.5 1 1.5 t3 t2 t1 tc tm2 tm1 pd w (g ) 0.1 18.1 26.4 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 421 of trichoderma species like t. afro-harzianum in this work and exhibiting the antagonistic potential in reducing virulence of such pathogen. another point, trichoderma has been previously demonstrated to be used as compatibly as efficacious in biocontrol when combined with other bcas. thaloromyces flavus and non pathogenic fusarium oxysporum have been recognized as the most effective fungal biocontrol agents against verticillium dahliae disease. in fact, different research work demonstrated the potential of different antagonists like thalaromyces flavus, fusarium oxysporum and phomopsis sp. to control different verticillium wilt in tomatoes, pistachio, cotton and eggplant (marois et al., 1982; tjamos et al., 2004; zheng et al., 2011; angelopoulou et al., 2014). recently yuan et al (2017) research work on biocontrol efficacy of penicillium simplicissimum with di = 41.4%, acremonium sp with di = 39.2%, leptosphaeria sp. with di = 32.4%, and talaromyces flavus with di = 36.9% against verticillium disease showed effective control of the disease when seed soaked with these fungal antagonists (yuan et al., 2017). therefore, combination of trichoderma spp. with thaloromyces flavus, non-pathogenic fusarium oxysporum or penicillium simplicissimum may be used as an integrated 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solanacearum. microbes environment 30 (1): 1-11. zheng y, qy xue, ll xu, q xu, s lu, c gu, and jh guo (2011) a screening strategy of fungal biocontrol agents towards verticillium wilt of cotton. biological control 56 (3): 209-216. atlas journal of biology 2017, pp. 364–370 doi: 10.5147/ajb.2017.0150 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) effect of drought stress on the growth and development of saffron (crocus sativus. l) in eastern morocco ibtissam mzabri*, manal legsayer, fatimzahhra aliyat, mohammed maldani, nour eddine kouddane, azzouz boukroute, ibtihal bekkouch, and abdelbasset berrichi laboratory of biology of plants and microorganisms, faculty of sciences, b.p. 717, oujda 60000, morocco received: january 19, 2017 / accepted: march 4, 2017 __________________________________________________ * corresponding author: btissammzabri@gmail.com 364 abstract saffron (crocus sativus l.; iridaceae) is the most expensive spice in the world. it has been cultivated in morocco for centu-ries and has represented a traditional staple for culinary, medical and cosmetic uses. the present work is about the study of the effect of drought stress on saffron’s morpho-physiological and biochemical param-eters. an experiment has been carried out on a 4-year-old saffron plantation planted in an open field located in the experimental station of the faculty of sciences of oujda. the experimental treatment included three water regimes (t0: control receiving 100% et0, t1: moderate water deficit receiving 60% et0, t2: pronounced water deficit re-ceiving only 40% et0). the results show that the increase in drought stress levels has slightly influenced the different param-eters of saffron growth. at the foliar level, the effect of stress has resulted in a de-crease in the chlorophyll content, a slight decrease in the psii quantum yield and a proline content accumulation as soluble sugars and total phenols, which resulted in keeping the relative water content (rwc) and the malondialdehyde (mad) content at a level similar to that of the control. in gen-eral, the morphophysiological adaptation traits were observed even at severe level of water stress (40% et0) which resulted in an acceptable decrease in stigmas yield. keywords: saffron, drought stress, growth, photosynthesis, performance, corm diameter. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction by 2050, the demand for water is expected to increase by 55%, not only under the pres-sure of a growing population, but also be-cause the continuous increased consumption. as for the agricultural sector, experts believe that the current levies are not sustainable (ligtvoet et al., 2014). the problem is becoming more severe in the arid and semi-arid areas, which constitute about two-thirds of the earth’s surface (benbrahim et al., 2004). the limited water resources in these areas are subject to competi-tion between agricultural and other uses, and the search for better adaptations of plants lacking water is thus becoming a serious issue. plants grown under drought condition have a lower relative water content and leaf water potential. obviously, exposure wheat and rice plants to a drought stress substantially decreased the leaf water potential, relative wa-ter content and transpiration rate (siddique et al., 2001). severe drought stress also de-creases the rate of photosynthesis (kawamitsu et al., 2000). plants survive under drought stress by using various morphological, bio-chemical and physiological responses. (chaves and oliveira, 2004). one of the most common stress tolerance strategies in plants is the accu-mulation of osmolytes including soluble sugars, proline, sugar alcohols (serraj and sinclair, 2002). overall, they contribute toward osmot-ic adjustment, detoxification of reactive oxy-gen species and stabilization of membranes (farooq et al., 2009). saffron, dried stigma of the crocus sativus flower, is considered among the main terroir products of morocco. in 2015, the saffron plantation in morocco was conducted in a sur-face a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) area of around 1600 ha with an aver-age yield of 3.5t, making morocco the fourth saffron producer in the world. according to the edaphic-climatic requirements, saffron is a rustic plant, and thanks to its morphology and physiology, it is able to withstand severe cli-matic conditions (alizadeh et al., 2009). pro-moting saffron cultivation in arid and semi-arid zones will enhance these areas, which are currently hard to cultivate due to the scarcity of water resources and will contribute to the support of low-input agriculture systems, to improve the incomes of small producers and to limit the rural exodus. the present study aims to elucidate the in-fluence of the drought stress on the agro mor-phological and physiological behavior of saf-fron plants under the natural conditions in the semi-arid climate of eastern morocco and un-der controlled hydric conditions. materials and methods experimental site the experiment was conducted in an open field at the experimental research station of the faculty of sciences of oujda, located at 661 m altitude and 34 ° 39 ‘06-71’’ north and 01 ° 53 ‘58-80’’ west (gps back track bush-nell). plant material the plant material used in this trial corre-sponds to saffron plants planted on 18/10/2011 in an open field. the corms used are from the region of taliouine, the main ar-ea of saffron production in morocco. treatments used to assess the effect of drought stress on the saffron crop, the plants were subjected to three different water regimes for a period of two years (2014-2015). these regimes correspond respectively to 100%, 60% and 40% et0 (reference evapotranspiration), taking into account the rainfall of oujda. the water used for watering has an electrical conductivity of 0.7 ms /cm. experimental design the adopted experimental design is ran-domized complete block, includes 3 blocks with a total of 45 saffron plants, the blocks indicate the repeats and sub blocks represent treatments. measured parameters the eco-physiological response of saffron to the hydric stress was evaluated on the morpho-logical, physiological and biochemical pa-rameters frequently used in the work concern-ing the response of plants to the various abiotic stresses including: stigma yield: the flowers were harvested ear-ly in the morning, then the stigmas are spread on flat receptacles in the shade for a few days, and the weight was obtained by weigh-ing the dry stigmas. number of leaves: counted each month for each plant length of leaves (cm): growth in length of the aerial part (leaves) was evaluated every month with a scale in millimeters (mm) from the leaf base to the top. leaf area (cm²): in view of the morphology of the leaves of saffron, the leaf area is estimat-ed directly using the autocad 2010 soft-ware. the total leaf area is estimated by mul-tiplying the number of leaves by the leaf area unit. number and weight of corms: at the end of the cycle, plants have been dug up, corms rid of topsoil, cleaned and de-tunicates then the number and weight of corms have been de-termined. caliber of corms: calibre of corms was deter-mined by using a caliper. three sizes are dis-tinguished: large caliber: ø> 2.5cm, medium caliber: 1.5cm <ø <2.5cm and small caliber: ø <1.5cm. relative water content (rwc %): relative water content of leafs was determined by the method described by barrs, (1966). after-wards, the following formula was used to esti-mate the water content: tre (%) = [(pf-ps) / pt-ps)] * 100, with trf: relative water content, pt: weight in full turgor (g), ps: dry weight (g). leaf water potential (ψf): this measure rep-resents the strength by which water is retained in the plant. the leaf water potential is meas-ured according to the scholander method (scholander et al., 1965). determination of chlorophyll pigments: total chlorophyll is determined according to the method of tran et al., (1995). total chlo-rophyll concentration is determined by follow-ing formula: [chl (a+b)] = (7.15× od 663 + 18.71 × od 646) ×v/m with: chl (a + b) to-tal chlorophyll (mg.g-1fm), v: volume of total extract (ml) m: mass of fresh material (g) and od: optical density. quantum efficiency measurement (φ psii): chlorophyll fluorescence is measured using the fms portable fluor meter model fms (fms 2 pulse modulated chlorophyll fluorescence monitoring system, hansatech, england). this device automatically records the φpsii, which displays the quantum efficiency of the psii. dosage of proline: leaf proline content was determined according to the method of monneveux and nemmar (monneveux et al., 1986). the content of proline was calculated with reference to a proline standard curve. dosage of soluble sugars: leaf soluble sugar was determined according to the method of yemn and willis (1954) reported by sidari et al., (2008). the content of soluble sugars was calculated with reference to a glucose stand-ard curve. determination of membrane lipid perox-ides: malondialdehyde (mda) is determined according to the method of heath and paker, (1968). the amount of mda is calculated using a molar extinction coefficient of 155 nm-1.cm-1, according to the beer-lambert law: ab-sorbance = є x w x [c] (є: molar extinction coefficient, w: width of the tank (1 cm) [c]: concentration). dosage of total phenol: extracting phenolic compounds was performed according to the method described by ollivier et al., (2004).the results are expressed in microgram of caffeic acid /g of saffron fresh material. with reference to caffeic acid standard curve. 365 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) atatistical analyzes the values of different parameters were expressed as the mean. spss statistical analy-sis software was used for analysis of variance, anova and duncan’s multiple range tests were utilized to separate means in 0.05 confi-dence level. in order to examine the interrela-tions among a variables studied to identify the underlying structure of those variables, an acp analysis was done using the xlstat software. results and discussion effect on yield and its parameters the field monitoring showed that drought stress influenced the flowering parameters. the first flowers were observed in the control fol-lowed by treatment 60% et0. the number of flowers is inversely proportional to the intensity of stress. the highest yield was recorded in the control while treatment 40% showed the low-est yield (-29 %). this can be explained by the reduced number of flower buds in the first year of the stress that would depend on the importance of the stocks stored at the corms level during the vegetative phase. however, the decrease in yield observed between water treatments is not statistically significant, which indicates that saffron allows the production of an acceptable yield even under severe and prolonged water conditions (table 1). effects on the aerial part effect on the morphological parameters the obtained results show that the number of leaves, the leaf length, the leaf area are inversely proportional to the intensity of the stress when the lowest values were observed in the treatment 40% et0 (table 2). these pa-rameters vary according to the months. the lowest values observed in april for all treat-ments. this period coincides with the end of the cycle. the reduction of the leaf area following the reduction of the cellular elongation is one of the plants’ first reactions to water deficit. it contributes to the conservation of water re-sources, which allows the survival of the plant (lebon et al., 2004) and is therefore consid-ered as a reaction or adaptation to the lack of water (blum et al., 1996). effect of the plant’s hydric parameters the hydric status of the leaves shows that the relative water content and the leaf water po-tential -ψfdecreased in proportion to the intensity of the applied hydric stress. the most noticeable action is observed in march and april. the average values shows that the rwc decreased from 74.4% in the control to 69 % for the moderate treatment and to 65 % for the severe treatment. however, there were no significant differences among treatments (table 3). the maintenance of a high ψf in plants could be explained by a strategy of avoid-ance which seems linked, to a complex set of morphological characters (mass and volume of the roots, shape of the leaves, etc.) making it possible to maintain a sufficient tissue hydra-tion for normal metabolic function. the maintenance of a high water content in the leaves under water stress could be explained by a high efficiency of osmotic adjustment, which counteracts the decrease in water potential without any significant reduction in rwc (hsiao et al., 1976). effect on biochemical parameters effect on chlorophyll content and photosynthetic activity the results show that the total chlorophyll content and the quantum performance of the psii decreased respectively with increasing drought stress degree. under severe stress conditions (40% et0), the total chlorophyll content and quantum performance of the psii decreased by 39.75% and 9.3%, respectively. the variation in psii quantum performance is almost stable as a function of water stress (table 4). these results are confirmed by oukarroum (2007), who showed that there is no loss in the psii quantum performance in barley under stress. ykhlef and djekoun, (2000) suggest that the survival of plants while there is a lack of water is partly due to the maintenance of the photosynthetic capacity of leaves. however, the rate of reduction of chlorophyll content is statistically significant during the month of april only. this could be explained by a level of relative water content that is high enough, making it possible to mitigate the effects of the ap-plied hydric stress. 366 table 1. effect of different levels of drought stress on the stigma yield. treatment 100% of et0 60% of et0 40% of et0 yield (g)/ treatment 0,37a 0,34a 0,26a table 2. effect of different levels of drought stress on morphological parameters. significant differences in same column are shown by different letters (a,b,c); p<0.05. month treatment leaf number leaf length (cm) leaf area (cm²) 100% of et0 174 a 22 a 525 a 60% of et0 140 b 21 a 373 b january 40% of et0 124 c 19 a 209 c 100% of et0 231 a 27 a 1225 a 60% of et0 170 b 24 a 658 b february 40% of et0 136 c 22 a 420 c 100% of et0 190 a 28 a 960 a 60% of et0 117 b 25a 412 b march 40% of et0 102 b 21 a 295 b 100% of et0 155 a 26 a 513 a 60% of et0 110 b 24 ab 356 b april 40% of et0 80 b 20 b 250 c average 100% of et0 188 a 26 a 805 a 60% of et0 134 b 23 a 449 b 40% of et0 110 b 20 a 294 c a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 367 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 3. effect of different levels of drought stress on water parameters. differences in same column are shown by different letters (a,b,c); p<0.05.*: *the results of april are not represented in the table because the measures have not yielded the results even we apply a very high pressure. month treatment relative water content (%) leaf water potential (mpa) 100% of et0 74a -6.4a 60% of et0 70a -7.8a january 40% of et0 74a -9.7a 100% of et0 75a -7.8a 60% of et0 74a -7.3a february 40% of et0 68a -9.2a 100% of et0 72a -8.6b 60% of et0 70a -10b march 40% of et0 65a -13a 100% of et0 69a * 60% of et0 63a * april 40% of et0 61ab * 100% of et0 74a -7.6 b average 60% of et0 69 a -8.3 b 40% of et0 65 a -10.6 a month treatment total chlorophyll content (mg/g fm) quantum yield of psii1 leaf proline content (µg/g fm) leaf soluble sugars content (µg/g fm) malondialdehyde content (nmol/g fm) total phenols content (µg/g fm) 100% of et0 0.87a 0.76a 207.9b 2990.4a 0.00104a 238.6b 60% of et0 0.79a 0.71a 474.8ab 3867.5a 0.00102a 349.1a january 40% of et0 0.76a 0.73a 1064.9a 3918.3a 0.00104a 307.6a 100% of et0 1a 0.75a 224.7b 2709.3a 0.00104a 236.3b 60% of et0 0.96a 0.73a 462.5b 4030.1a 0.00125a 361ab february 40% of et0 0.79a 0.72a 1212.3a 4301b 0.00132a 474.3a 100% of et0 0.93a 0.76a 226.8b 2394.3a 0.00144a 409.1b 60% of et0 0.86ab 0.75a 294.2b 2689a 0.00149a 417.5b march 40% of et0 0.78b 0.70a 306.8a 2851.5a 0.00203a 560.1a 100% of et0 0.84a 0.75a 144.7b 870.5b 0.00187b 300.8b 60% of et0 0.73a 0.71ab 216.3ab 940.1ab 0.00202ab 370.7ab april 40% of et0 0.51b 0.66b 250a 1070a 0.00268 a 468.6a 100% of et0 0.91a 0.75a 201b 2241b 0.00134a 296.2b average 60% of et0 0.83a 0.72a 361b 2881ab 0.00144a 374.5ab 40% of et0 0.71a 0.70a 708a 3035a 0.00176a 452.6a significant differences in same column are shown by different letters (a,b,c); p<0.05. 1psii: photosystem ii (or water-plastoquinone oxidoreductase). table 4. effect of different levels of salt stress on biochemical parameters. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 368 effect on the concentration of proline and foliar soluble sugars plants were subjected to a water restriction produced more proline and soluble sugars in their foliage (table 4). this finding was in conformity with the result has been reported on vigna unguiculata (souza.,2004), alfalfa (mefti et al., 2001) and wheat (munns et al., 2006; gaudillère et al., 1990). the accumulation of these solutes allows plants to support lack of water, maintaining their relative leaf water content at a high level and preserving their cellular integrity. furthermore, this accumula-tion could be the result of a high catabolism of the protein pool and in this case, it will not serve as a reliable diagnosis of resistance to drought. however, the last two months before dor-mancy (march & april) marked a drastic de-crease of these osmoregulators in all treat-ments, which resulted in a significant contrac-tion in the relative water content. this decrease could be explained by the cutback in photo-synthetic activity following the senescence of leaves, as it could be due to a translocation of the reserves (proteins, sugars ..) to the storage organs. effect on malondialdehyde content and antioxidant activity the malondialdehyde (mda) content was increased with intensity of stress. overall, there were not significant differences between the treatments. in addition, the stressed plants ac-cumulated higher levels of total phenols, which could explain the maintenance of the mda content at a comparable level of the control, and thus preserving membrane integrity (table 4). this result is confirmed by other authors such as leinhos and bergman; (1995) who have studied the involvement of polyphenols in the plant defense system against various types of stress. also, chakraborty and al., (2002) re-ported that phenol accumulation was im-portant in tea-tolerant cultivars. effect on the underground part the results show that the highest weight (437 g) was recorded in the control versus 359 g (-12. 77%) in the treatment 60% et0 and 367 g (-10.92%) in the treatment 40% et0. similarly, the percentage of diameter catego-ries varies according to the water regime ap-plied. control showed a dominance of large and medium catégories, whereas the stressed plants marked a dominance of small diameter category which exceeded 50 % in the sever treatment. however, number of daughter corms was not significantly influenced by drought stress. the highest value was recorded in the 40% et0 treatment with 116 corms (table 5). the high rate of small diameter corms could be due to small quantity of reserves stored during vegetative phase. several authors worldwide (negbi et al 1989; demaestro et al 1993; de juan et al 2003; koocheki et al., 2007; çavuşoğlu et al., 2009) have shown that large caliber en-hance precocity, flowering density and give large daughter corm for the next season. principal component analysis the obtained results show that, with axis 1 correlates all parameters studied which de-scribe the physiological and agronomic be-havior of the aerial part of saffron plant. on this axis, there is a very close correlation be-tween the vegetative growth parameters (la, ln, lh), water parameters of the plant (rwc, lwp) and the chlorophyll content (chlt, psii). in addition, there is a negative correlation between the parameters involved in osmotic regulation (pc, ssc) and the other parameters studied. treatment corms weight corms number corms diameter big diameter medium diameter small diameter 100% of et0 412.4a 108a 27a 50a 23a 60% of et0 359.3a 93a 11b 44ab 45b 40% of et0 367.8a 116a 10b 37b 53b table 5. effect of different levels of drought stress on the underground part. significant differences in same column are shown by different letters (a,b,c); p<0.05. figure 1. representation of the studied characters in the plane (1-2). principal component analysis. la: leaf area, ln: leaf number, lh: leaf height, sy: stigma yield, rwc: relative water content, lwp: leaf water potential, chlt: total chlorophyll content, psii: quantum efficiency measurement, wc: weight of corms, nc: number of corms, pc: proline content, ssc: soluble sugars content, tpc: total phenol content, mdac: malondialdehyde content. sy ln lh la wc nc rwc lwp chlt psiipc ssc mdac tpc -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 f2 ( 12 ,7 6 % ) f1 (87,24 %) variables (axes f1 and f2 : 100,00 %) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 369 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) in other words the increase in the pro-duction of osmoregulators leads to a decrease in the growth parameters which is confirmed by the decrease of these parameters in the sever treatment. with axis 2, which represents the conversion efficiency of the biomass in re-placement corms, the number (nc) and the weight of the corms (wc) are correlated. on this axis, there is a relation of independence between these two parameters (cos α = 0) (figure 1). conclusion drought stress is one of the most important environmental stresses affecting agricultural productivity worldwide, where plants are ex-periencing a reduced growth and a reduced productivity. our results show that the influence of the hydric stress on the morphological, phys-iological and biochemical parameters on saf-fron is not strongly marked at 60% et0. whereas at 40% of et0 the parameters stud-ied were more or less affected. the most no-ticeable effect was demonstrated by a decrease in the plant growth (decrease in num-ber, length and leaf area) as well as a de-crease in the diameter of daughter corms. in general, morpho-physiological adapta-tion traits in water-deficient conditions have been externalized, resulting in an acceptable yield (-29%) in the case of the severe treat-ments (40% of et0). references ligtvoet, w., hilder-ink, h., bouwman, a., puijenbroek, p., lucas, p., & witmer, m. (2014). towards a world of cities in 2050. an outlook on water-related chal-lenges. background report to the un-habitat global report. bilt-hoven: netherlands environmental as-sessment agency alizadeh a, n sayari, j ahmadian, and a mohamadian (2009) study for zoning the most appropriate time of irrigation of saffron (crocus sativus) in khorasan razavi, north and southern provinces. j water soil 23: 109–118. barrs hd (1968) determination of water deficits in plant tissue. in: kozlowski, t.t. 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plant cell environ 25: 333–341. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 370 sidari m, c santonoceto, u anastasi, g preiti, and a muscolo (2008) variations in four genotypes of lentil under nacl-salinity stress. am j agri & biol 3 (1): 410-416. siddique mrb, a hamid, and ms islam (2001) drought stress effects on water relations of wheat. bot bull acad sinica 41: 35–39. souza rp, ec machado, jab silva, a lagoa, and j silveira (2004) photosynthetic gas exchange, chlo-rophyll fluorescence and some associated metabolic changes in cowpea (vigna unguiculata) during wa-ter stress and recovery. environ exp bot 51: 45-56. ykhlef n, a djekoun, m bensari, and d vignes (2000) déficit hydrique excès de lumière et activité photo-chimique chez le blé dur : analyse de la résistance des psii. sci technol 10 : 87 -92. eljiati-et-al-2022 752752 a tla sj ou rn al of bi ol og yis sn 21 58 -9 15 1. pu bl ish ed by a tla sp ub lis hi ng ,l lc (w ww .a tla spu bl ish in g. or g) atlas journal of biology 2022, pp. 752–758 doi: https://doi.org/10.5147/ajb.vi0.229 effect of different ethephon concentrations on shiss removal in ‘khlass’ and ‘sukkary’date palmvarieties abdelazize eljiati1*, imtiaz ahmed1, naoki terada2, atsushi sanada2, kaihei koshio2 1 yousef bin abdul latif and sons agriculture co. ltd. research and development departnment., al-shihiyah 52758, gassim, saudi arabia; 2 tokyo university of agriculture, dept. of international agricultural development, laboratory of tropical horticultural science. 1-1-1 sakuragaoka, setagaya,tokyo, 156 8502, japan. received: december 4, 2021 / accepted: february 28, 2022 abstract after pollination, aborted date fruits known as tricarpel or shiss, remain on the bunch in most of the varieties and exert a competition on fruits for water and nutrients. in sukkary, they drop down at the end of kimri stage, while remain on ‘khlass’ until the harvest. most of them remain as bisr and little turns into tamr that is not appreciated in the date market. they can only be used as paste that has low price compared to dates. in an attempt to get rid of shiss, we sprayed ethephon at different concentrations on bunches of ‘sukkary’ and ‘khlass’ after fruit-set, at hababook stage. beside the shiss drop, undesirable fruit drop also occurs. we are looking for the optimum ethephon concentration where shiss dropped more than fruits. in ‘khlass’, the ethephon concentration 800 ppm showed the highest shiss drop (81%) together with a fruit drop of 20% that occurs at the same time, while in ‘sukkary’, the concentration of 600 ppm was the best by giving a shiss drop equal to 44 % together 12% fruit drop.we consider that the concentration of 800 ppm at hababook stage is the ideal concentration to generate optimum drop in shiss with reasonable percentage of fruit drop. we, therefore highly recommend a trial with this concentration on ‘sukkary’ as well. keywords: abortion, shiss, date palm, fruit, ethephon. introduction the ethephon or the 2-chloroethylphosphonic acid (cepa) is a systemic plant growth regulator, which in its liquid state at the proper ph does not yield ethylene; however, when the ph is elevated, it breaks down to form ethylene (arteca, 1996). however, at ph higher than 4, it breaks down to ethylene, hydrochloric and phosphatic ions. it stimulates the endogenous ethylene production by releasing ethylene in the plant tissue as the cell cytoplasm has a ph higher than 4 (nicotra, a. 1982). the main role of ethylene is to make changes in fruit texture, softening, colour, and other processes involved in ripening. it is also known as the aging hormone in plants. it is well known that ethephon can promote fruit abscission. ethephon has been performed well as a fruit-thinning agent for many crops (abeles et al., 1992). ebert and bangerth (1982) reported that ethylene inhibited the synthesis and translocation of indole-3-acetic acid (iaa) within the fruits, thus reducing sink strength and ultimately inducing the separation area in the peduncle, which causes fruit drop (roberts et al. 2002.). el hamadi et al. (1983) used ethephon at different concentrations from 200 to 400 ppm after fruit set and deduced that the level of thinning increased by increasing the concentration. mohamed et al (2015) concluded that ethephon at 1000 ppm, ten days after pollination are this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. _____________________________________________ *corresponding author: a.eljiati@gmail.com 753753 a tla sj ou rn al of b io lo gy -i ss n 21 58 -9 15 1. pu bl ish ed by a tla sp ub lis hi ng ,l lc (w ww .a tla spu bl ish in g. or g) suitable for obtaining economic yield with best fruit quality. bakr et al. (2006) tested the effect of ethephon on fruit thinning compared with cytophex at different concentrations and dates of application on ‘samany’ date palm variety. they concluded that, ‘samany’ fruit set was decreased when ethephon was sprayed 18 days after pollination especially at 300 ppm. no study has been done before on the use of ethephon in order to remove aborted date fruits known as tri-carpel or shiss that remain on the bunch and compete on fruits for water and nutrients. that was the objective of this investigation. materials andmethods location of the experiment: this trial was carried out in the experimental farm “naam” of yousef bin abdul latif and sons agriculture co. ltd. (yala) in qassim, saudi arabia. the weather conditions of the farm during the 12 days of the experiment on each of the two date varieties ‘sukkary’ and ‘khlass’ are summarized in the figures 1 and 2 as recorded by the weather station of the farm. for ‘sukkary’, during the 12 days of study (6-18 may 2019), the reference evapotranspiration (et0) figure 1.weather conditions of the experimental farm during the 12 days of study on ‘sukkary’ variety (from 6 to 18 may 2019). 754754 a tla sj ou rn al of bi ol og yis sn 21 58 -9 15 1. pu bl ish ed by a tla sp ub lis hi ng ,l lc (w ww .a tla spu bl ish in g. or g) ranged between 4.3 and 5.8 mm day-1, the cumulative precipitation was 2 mm and the maximum wind speed was 5.5 m/s. the maximum temperature recorded during the day ranged between 35.4 and 41.2 °c (figure 1). for ‘khlass’ variety, during the 12 days of study (2-14 july 2019), the reference evapotranspiration (et0) ranged between 5.4 to 6.7 mm day-1, there was no precipitations during this period, and the maximum wind speed was 3.3 m/s. the maximum temperature recorded during the day ranged between 43 and 45.5 °c (figure 2). the experiment: the spray started first on ‘sukkary’, as it is an early maturing variety, then on ‘khlass’. ‘sukkary’was sprayed on 6 may 2019 and the evaluation was done 12 days later on 18 may 2019. ‘khlass’was sprayed on 2 july 2019 and the evaluation was done 12 days later on 14 july 2019. the concentrations of ethephon applied on ‘sukkary’ were: 1000 ppm, 600 ppm and 400 ppm in addition to the control where only pure water is sprayed. after evaluation of results on ‘sukkary’, we changed the concentrations on ‘khlass’ to respectively 1000 ppm, 800 ppm, 600 ppm in addition to pure water as a control. three trees per variety have been used for this experiment. one bunch per tree per treatment was sprayed.we have in total three bunches per treatment. before the spray, the total number of fruits and shiss were counted. after the spray, the bunches are figure 2.weather conditions of the experimental farm during the 12 days of study on ‘khlass’ variety (from 2 to 14 july 2019). 755755 a tla sj ou rn al of b io lo gy -i ss n 21 58 -9 15 1. pu bl ish ed by a tla sp ub lis hi ng ,l lc (w ww .a tla spu bl ish in g. or g) covered with a bunch bag (a mesh bag) to collect dropped shiss and fruits. fruits and shiss remaining on the bunches are counted 12 days after the spray. dropped fruits and shiss are calculated as follow: number of dropped fruits = ifn –ffn number of dropped shiss = isn –fsn where: ifn : initial fruit number on the bunch before spray ffn : final fruit number on the bunch 12 days after spray isn: initial shiss number on the bunch before spray fsn : final shiss number on the bunch 12 days after spray the percentage of fruits and shiss dropped from the total initial number existing before the spray is calculated as follow: percentage of fruit drop (%) = number of dropped fruits / number of fruits on the bunch before the spray percentage of shiss drop (%) = number of dropped shiss / number of shiss on the bunch before the spray results anddiscussion when bunches of ‘sukkary’ were sprayed with 1,000 ppm of ethephon, 94% of shiss dropped together with 54% of fruits (table 1). at 600 ppm ethephon, 44% of the shiss dropped, against 12% of fruit drop. at the concentration of 400 ppm ethephon, 28 % of the shiss dropped and 17 % of fruits. for the control where pure water is sprayed on the bunches, 4% of the shiss dropped, together with 8% of fruit drop. the concentration of 1,000 ppm ethephon, despite generating a desirable high shiss drop, significantly different from the one generated by the concentration of 600 ppm ethephon, but it did unfortunately the same for fruit drop (table 1).this is why we recorded a high correlation between ethephon concentration from one side and shiss drop (r² = 0.9902) and fruit drop percentages (r² = 0.8164) from the other side (figure 3). therefore, we have to look for an optimal concentration of ethephon that generates a significant increase in shiss drop with a figure 3. correlation between the ethephon concentration and the percentage of shiss (a) and fruit drop (b) generated in ‘sukkary’ date variety. a) b) y = 0.0011x 0.185 r² = 0.9902 0% 20% 40% 60% 80% 100% 0 200 400 600 800 1000sh iss dr op in su kk ar y% ethephon concentration in ppm y = 0.0007x 0.1685 r² = 0.8164 0% 20% 40% 60% 0 200 400 600 800 1000 fr ui td ro p in su kk ar y% ethephon concentration in ppm 756756 a tla sj ou rn al of bi ol og yis sn 21 58 -9 15 1. pu bl ish ed by a tla sp ub lis hi ng ,l lc (w ww .a tla spu bl ish in g. or g) table 1.percentage of drop generated in fruits and shiss of ‘sukkary’, 12 days after spray by ethephon at different concentrations. a) b) ethephon concentration control (purewater)1000 ppm 600 ppm 400 ppm percentage of shiss drop (%) * 94% a 44% b 28% c 4% d percentage of fruit drop (%) ** 54% a 12% b 17% b 8% c (*) averages of shiss drop (%) that do not share the same letter are significantly different according to t-test analysis. (**) averages of fruit drop (%) that do not share the same letter are significantly different according to t-test analysis. table 2. percentage of drop generated in fruits and shiss of ‘khlass’, 12 days after the spray by ethephon at different concentrations. y = 0.0006x + 0.2829 r² = 0.9687 0% 20% 40% 60% 80% 100% 0 200 400 600 800 1000 sh iss dr op in kh la ss % ethephon concentration in ppm y = 0.0003x 0.0045 r² = 0.5556 0% 10% 20% 30% 40% 0 200 400 600 800 1000 fr ui td ro p in kh la ss ethephon concentration in ppm figure 4. correlation between the ethephon concentration and the percentage of shiss (a) and fruit drop (b) generated in ‘khlass’ date variety. ethephon concentration control (purewater)1000 ppm 800 ppm 600 ppm percentage of shiss drop (%) * 89% a 81% a 64% b 4% c percentage of fruit drop (%) ** 37% a 20% b 24% b 0% c (*) averages of shiss drop (%) that do not share the same letter are significantly different according to t-test analysis. (**) averages of fruit drop (%) that do not share the same letter are significantly different according to t-test analysis. 757757 a tla sj ou rn al of b io lo gy -i ss n 21 58 -9 15 1. pu bl ish ed by a tla sp ub lis hi ng ,l lc (w ww .a tla spu bl ish in g. or g) lower percentage of fruit drop. it seems that an ethephon concentration between 1000 ppm and 600 ppm (800 ppm for example) might be the optimal concentration to be tried in order to get more shiss drop and at the same time less fruit drop. for this reason, we replaced the concentration 400 ppm of ethephon by 800 ppm in the same season on the next experiment on ‘khlass’ that flowers after ‘sukkary’. on the other hand, when bunches of ‘khlass’ are sprayed with 1,000 ppm of ethephon, a total of 89% of shiss dropped together with 37% of fruits (table 2). at 800 ppm ethephon, 81% of shiss dropped, which is not significantly different from shiss percentage dropping in the case of 1000 ppm ethephon. the concentration 800 ppm ethephon also generated 20% of fruit drop, similar statistically to the fruit dropping in the case of 600 ppm ethephon (24%). at the concentration of 600 ppm ethephon, 64 % of the shiss dropped and 24 % of fruits. for the control where pure water is sprayed on the bunches, 4% of the shiss dropped, with no drop recorded in the fruits. in ‘khlass’, the ethephon concentration 800 ppm showed the optimum drop between shiss (81%) and fruits (20 %), as it generated similar shiss drop percentage generated by the highest ethephon concentration (1000 ppm) and kept an undesirable fruit drop percentage (20%) similar to the one generated by the lower 600 ppm ethephon concentration (24%). this has been translated by a strong correlation between ethephon concentration and shiss drop (r² = 0.9687) (figure 4a) and a low correlation between ethephon concentration and fruit drop (r² = 0.5556) (figure 4b). in a chemical thinning experiment, mohamed et al. (2015) sprayed ethephon ten days after pollination (10 dap) at 500 and 1,000 ppm on ‘khlass’ and ‘ruzeiz’ date varieties. when the evaluation is made after two months from pollination, they found out that the fruit drop in both ‘khlass’ and ‘ruzeiz’was not concentration-dependant. they reported a fruit drop in ‘khlass’ equal to 45.5 % and 38.6% respectively for 500 ppm and 1000 ppm of ethephon and in ‘ruzeiz’ equal to 16.3 % and 17.9% respectively for 500 ppm and 1,000 pm of ethephon. the effect of the increase in their ethephon concentration was only seen at the harvest when they evaluated the fruits retained on the bunch, which was in ‘khlass’ 63.9% and 44.9% respectively for the treatment 500 ppm and 1000 ppm of ethephon and was in “ruzeiz” 77.1% and 62.5% respectively for the treatment 500 ppm and 1,000 ppm of ethephon. ghazzawy et al., (2019), concluded on ‘khlass’ that the ethephon applied at different concentrations 5 days after pollination (dap) generated an average of 42.1 % of fruit drop against 43.4 % when applied 10 days after pollination. they also reported in both application times that the greater the concentration of ethephon is, the lower the fruit drop becomes. when the ethephon is applied 5 dap, the fruit drop is 50.1%, 40.2 %, 35.8 % and 36 % respectively for the control, 100 ppm, 200 ppm and 300 ppm of ethephon. conclusion to remove aborted fruits of date palm (known as tri-carpel or shiss) that remain on the brunch and exert a competition for water and nutrients, we sprayed ethephon in hababook stage at different concentrations on bunches of ‘sukkary’ and ‘khlass’ varieties. in ‘khlass’, the ethephon concentration 800 ppm showed the optimal drop (more shiss and less fruits) equal to 81% in shiss and 20% in fruits. while in ‘sukkary’, the concentration of 600 ppm was the optimal with a shiss drop equal to 44% and fruit drop equal to 12%. we consider that the concentration of 800 ppm at hababook stage might be the ideal concentration to generate optimal drop in shiss with reasonable percentage of fruit drop. we, therefore highly recommend a trial using this concentration on ‘sukkary’. acknowledgements the authors would like to deeply thank mr youssef jameel the chairman of yousef bin abdul latif and sons agriculture co. ltd for his financial support and for allowing us to use the farm and laboratory facilities of his company during this research. references abeles fb, pw morgan, and me saltveit jr. (1992) ethylene in plant biology,2nd ed.academic press, san diego, ca, pp. 264–296. arteca rn (1996) historical aspects and fundamental terms and concepts. in r. n. arteca (ed.), plant growth substances: principles and applications (pp. 1–27). new york, ny: chapman & hall. bakr ei, gm haseab, s el-kosary, and ty saber (2006) effect of chemical fruit thinning of samany 758758 a tla sj ou rn al of bi ol og yis sn 21 58 -9 15 1. pu bl ish ed by a tla sp ub lis hi ng ,l lc (w ww .a tla spu bl ish in g. or g) date palm cultivar. j. agric. sci. mansoura univ., 31 (10): 6385 – 6407. ebert a and f bangerth (1982) possible hormonal mode of action of three apple thinning agents. sci. hort. 16:343-356. el hamadi m, k ahmed, and aa el hamadi (1983) fruit thinning using ethephon. proceedings of the first symposium on the date palm.college of the agricultural sciences and food. king faisal university el hassa, saudi arabia. pp: 284-295. ghazzawy hs, mr alhajhoj, aa sallam, and m munir (2019) impact of chemical thinning to improve fruit characteristics of date palm cultivar khalas. iraqi j. of agri. sci. 50 (5): 1361-1368. al saikhan ms, and aa sallam (2015) impact of chemical and non-chemical thinning treatments on yield and fruit quality of date palm. j food res 4 (4): 18-29. https://doi.org/10.5539/jfr.v4n4p18. nicotra a (1982) growth regulators in pear production. acta hort. 124: 131-148. roberts ja, ka elliot, and zh gonzales-carranza (2002) abscission, dehiscence and other cell separation processes. ann. rev. plant biol. 53: 131-158. atlas journal of biology 2016, pp. 292–294 doi: 10.5147/ajb.2016.0142 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a rapid method for cannabis species determination by dna sequencing david a. lightfoot1,2,*, winston c. throgmorton3, and colton johnson3 1 genome and agricultural biotechnology llc, carbondale, il 62901, usa; 2 po box 0312, carbondale, il 62903, usa; 3 throgmorton attorney at law, 304 n. monroe street, marion, il, usa. tel: (618) 993-5379. received: september 19, 2016 / accepted: october 9, 2016 __________________________________________________ * corresponding author: ga4082@siu.edu 292 abstract determination of species within the genus cannabis has important legal medical and social implications. recent genome sequencing has shown that the genomes of c. sativa (recreational marijuana and hemp), c. indica (medical marijuana) and c. ruderalis (feral marijuana) can all be distinguished. however, hybridization among the species has occurred with widely varying outcomes in the percent of genome transmitted. the aim here was to determine if a simple assay based on the dna sequence of its2 could be used to distinguish among species. using sequences at genbank as a reference eighteen plant samples were sequenced and shown to be identical to c. indica sequence and different from c. sativa and c. ruderalis at 4 positions within the 25s rrna gene. this result and the geographic separation of the centers of genetic diversity argues strongly for polytypic origins of the 3 species. analysis of interspecific hybrids sequences at genbank suggested only the c. indica allele is transmitted preferentially. finally, a snp within the its could be used to distinguish two types within the eighteen plants. therefore this simple genetic test can be used for rapid plant identification and to assist in strain identification. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction internal transcribed spacer (its) refers to the spacer dna (non-coding dna) situated between the small-subunit ribosomal rna (rrna) and large-subunit rrna genes in the chromosome (coleman, 2007). there are two its’s in eukaryotes; its1 is located between 18s and 5.8s rrna genes, while its2 is between 5.8s and 25s (in plants) rrna genes. sequence comparison of the its region is widely used in taxonomy and molecular phylogeny because it a) is easy to amplify even from small quantities of dna (due to the high copy number of rrna genes), and b) has a high degree of variation even between closely related species (coleman, 2007; shultz et al., 2006). the success rates for using the its2 region to identify dicotyledons plants was 76.1%, at the species level (kress et al., 2006; yao et al., 2010). the its region is the most widely sequenced dna region in molecular ecology of plants and has been recommended among the universal barcode sequences (kress et al., 2006; yao et al., 2010) it has typically been most useful for molecular systematics at the species level, and even within species (e.g., to identify geographic races, varieties and ancestoral types). the standard its1 and its4 primers are used by most labs (white et al., 1990). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) determination of species within the genus cannabis has important legal medical and social implications. however, the literature to date has been confused on some key concepts because of the remarkable amount of genetic variation and phenotypic plasticity within the genus (schultes et al., 1974; sawler et al., 2015). selection, cultivation, hybridizations by both man and nature, crop abandonment and feral growth have all combined to confuse the identities and origins of the four crops, c. sativa (recreational marijuana and hemp), c. indica (medical marijuana) and c. ruderalis (feral marijuana). however, it seems possible they had polytypic origin since their centers of wild plant genetic diversity differ, east asia, india and russia for the 3 species respecctively. recent genome sequencing has shown that the genomes of c. sativa (recreational marijuana and hemp), c. indica (medical marijuana) and c. ruderalis (feral marijuana) can all be distinguished (sawler e al., 2015). however, hybridization among the species has occurred with widely varying outcomes in the percent of genome transmitted. observations of phenotypes suggest the c. indica type is dominant with preferential transmission of its alleles in known interspecific hybrids. preferential transmission of alleles is widely observed in interspecific crosses with one parent being dominant in all crosses made (guo et al., 1990). here is described a simple and rapid genetic test that distinguishes materials and methods sampling on february 10, 2015 eighteen samples from eighteen plants were collected by prof. david a lightfoot, principal scientist, gaab-llc, carbondale, il 62901, usa. an analysis of the samples species identity had been requested. they were provided as follows. dna extractions all eighteen samples were handled in the same way. gloves were worn by dr. lightfoot throughout. all vials and plastic ware were new and had been sterilized. the promega genewiztm kit was used for purifications. the kit was new. pcr conditions following white, et al (1990), primers were: 5’-agc cgc ctt cat ata tct gct t -3’ its1forward; 5’-tcc tcc gct tat tga tat gc -3’ its4 reverse. polymerase chain reaction (pcr) was performed using 25, 50 or 75 ng of genomic dna, 225 µl of supermix high fidelity enzyme (gibcobrl, grand island, ny), 5 µl h20, 5 µl its 1-forward, and 5 µl its4-reverse in a total volume of 20 µl. cycling conditions were: 950c for 1 min, then 450c for 1 min, then 680c for 1 min for 35 cycles finishing with a and 40c hold. the pcr products were prepared for sanger sequencing reactions by alkaline phosphatase and exonuclease 1 treatment. dna sequencing the dna in samples sent for forward dna sequencing using services provided by genewiz inc. (plainsfield, nj, usa) using the amplification primer its1. dna sequences were obtained that were found in the gene data repository at ncbi. the sequences were aligned with reference sequences for c. sativa (record identifier fj572045.1) and c. indica (record identifier kc292629.1) found at ncbi by blast searching. a new sequence for c. indica was deposited at genbank as accession number kx980526. 293 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) c. indica 652 gactagacccgtacgaccccaatgtgctgcgaacgcagtgccttcaacgcgaccccaggt 711 c. sativa 601 ........................................................c.ta 660 query_65399 560 ............................................................ 619 query_65400 563 ............................................................ 622 query_65401 564 ............................................................ 623 query_65402 560 ............................................................ 619 query_65403 562 ............................................................ 621 figure 1. a complex rearrangement in the 25s rrna gene distinguishes c. indica from c. sativa. c. indica 472 gggcgtcacacgccgttgcccccatgtgcactgccaaaagcgtgttcaggaggggcggag 531 c. sativa 421 ............................................................ 480 query_65399 380 ..................................................t......... 439 query_65400 383 ............................................................ 442 query_65401 384 ............................................................ 443 query_65402 380 ..................................................t......... 439 query_65403 382 ..................................................t......... 441 figure 2. a snp in the its2 region identified two strains of c. indica. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) results to date good sequences have been obtained from 14 of the 18 samples. in each case the sequences contained the tetra-nucleotide aggt between nucleotides 708 to 711 of the reference genome sequence of c. indica (record identifier kc292629.1). they all differed from the cgta at that position found in c. sativa (record identifier fj572045.1). an example alignment is shown in (figure 1). no further difference were seen between c. sativa and c. indica except a single a/t snp at position 522. the new sequence for c. indica was deposited at genbank as accession number kx980526. the t snp was unique to the samples tested and was not present in either reference sequence or on other genbank records. examination of the 12 homologous sequences found on genbank (figure 3) showed all but two to be c. indica though seven were miss-identified as c. sativa. one sample from house dust was also c. indica. not shown were several realted species including celtis sp. that aligned starting at the region of rrna transcription. in a remarkable example of convergent evolution sequence of a monocot dioscorea sansibarensis was well aligned with the canabis sp. too. alternately, dq267929 was a contaminated sequence submitted in error. conclusion therefore, it was concluded the tested plants were all c. indica. because the region sequenced is a primary diagnostic test of species and genus when used by systematists around the world the conclusion cannot be reasonably refuted (schultz et al., 2006; yao et al., 2010). the nature and position within the 25s rrna gene coding sequence suggests a separate origin for the two species which would agree with their separate centers of genetic diversity (shultes et al., 1974; sawler at al., 2015). references coleman aw (2007) pan-eukaryote its2 homologies revealed by rna secondary structure. nucleic acids res 35: 3322–3329 guo m, da lightfoot, mc mok, and dws mok (1991) analyses of phaseolus vulgaris l. and phaseolus coccineus lam. hybrids by rflp: preferential transmission of p. vulgaris alleles. theor. appl. genet. 81:703-710. kress wj, kj wurdack, ea zimmer, la weigt, and dh janzen (2005) use of dna barcodes to identify flowering plants. proc natl acad sci usa 102: 8369–8374. sawler j, jm stout, km gardner, d hudson, j vidmar, l butler, et al. (2015) the genetic structure of marijuana and hemp. plos one 10(8): e0133292. schultes re, wm klein, t plowman, and te lockwood (1974) cannabis: an example of taxonomic neglect. harvard university botanical museum leaflets 23: 337-367. schultz j, t muller, m achtziger, pn seibel, t dandekar, et al. (2006) the internal transcribed spacer 2 database a web server for (not only) low level phylogenetic analyses. nucleic acids res 34: w704– w707. white tj, t bruns, s lee, and jw taylor (1990) amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. pp. 315-322 in: pcr protocols: a guide to methods and applications, eds. innis ma, dh gelfand, jj sninsky, and tj white. academic press, inc., new york. yao h, j song, c liu, k luo, j han, et al. (2010) use of its2 region as the universal dna barcode for plants and animals. plos one 5: e13102. 294 figure 3. alignment of sequences at genbank. c.indica 661 cgtacgaccccaatgtgctgcgaacgcagtgccttcaacgcgaccccaggtcaggcggga 720 kc292629 661 ............................................................ 720 kf800374 811 ............................................................ 870 fj572045 610 ...............................................c.ta......... 669 jq230978 615 ............................................................ 674 dq267929 600 ............................c.............t................. 659 ab564722 623 ............................................................ 682 kf454086 266 ............................................................ 325 kf454085 266 ............................................................ 325 kf454084 266 ............................................................ 325 kf454083 266 ............................................................ 325 1. c. indica reference voucher sample 2. cannnabis sativa subsp. indica voucher 20121121-846 3. uncultured eukaryote clone cmh283 18s ribosomal rna gene found in house dust 4. cannabis sativa 18s ribosomal rna gene, industrial hemp 5. cannabis sativa voucher sbb-1163 18s ribosomal rna gene, india 6. dioscorea sansibarensis 18s ribosomal rna gene, probably a contaminant 7. cannabis sativa genes for 18s rrna, japan 8. cannabis sativa voucher 20120903569, china 9. cannabis sativa voucher 20120903568, china 10. cannabis sativa voucher 20120903567, china 11. cannabis sativa voucher 20120903566, china atlas journal of biology 2019, pp. 655–660 doi: 10.5147/ajb.v0i0.213 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) justification of urgent brain ct examinations at medium size hospital, jerusalem anan al-tell1, majdal hjouj2, muntaser s. ahmad3,*, and hjouj mohammad1,4 1 radiology and medical imaging department, maqassed hospital, jerusalem, palestine; 2 medical school, al-quds university, abu deis main campus, jerusalem, palestine; 3 department of medical physics and radiation science, school of physics, university sains malaysia, 11800 penang, malaysia; 4 medical imaging department, faculty of health professions, al-quds university, jerusalem, palestine. received: october 25, 2019 / accepted: december 8, 2019 __________________________________________________ * corresponding author: altell.anan@gmail.com. 655 abstract we studied requested urgent brain ct scan procedures justification. in addition, we addressed referrer’s (physician’s) awareness of radiation risks. in doing so, we considered two aspects to the issue. first, we reviewed the records of the requested urgent ct scan procedures for a sample of 339 patients at al-maqassed hospital, a major hospital in palestine. secondly, we surveyed a sample of forty-two referrer’s from the same hospital to test their awareness of radiation risks. our study shows that out of the 339 urgent brain ct requests, 69.6% were justified requests and 30.4% were unjustified. statistically, these observations implied the following general statement: at least, 25% of the ct requests at this hospital are unjustified with a p-value of 0.011. on the other hand, our survey on referrers shows that 42% of respondents knew the effective dose of a brain ct scan, 24% of respondents knew the radiation risks and 14% of respondents knew about radiation protection. although the study is limited, yet it shows the need to reduce the number of brain ct examinations and the need to improve their justification. consequently, the need for regular education and guideline implementation at least in this country is of paramount importance. keywords: radiation risk, ct scan, brain, ionizing radiation. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction diagnostic imaging modalities are increasingly applied in health care to diagnose the various injuries and diseases (merzenich et al., 2012). in spite of the improvements of the medical imaging modalities in the quality of diagnosis and treatment in various medical conditions in both paediatric and adult patients, by using different medical imaging modalities that utilizes ionizing radiation including conventional radiology, fluoroscopy, and computed tomography (ct). scientific studies proved that radiation has biological effects on the organism and it depends on the dose and exposure duration (squillaro et al., 2018). the ct has relatively high radiation doses compared with the other ionizing radiation modalities. thus, the protection against the hazard of radiation is an important issue (iaea,2009; remedios, 2011). the principle of radiation protection related to the international commission on radiological protection (icrp) for ionizing radiation is justification, optimization of protection and application of dose limits (agency, 2012). the justification process undergoes the base of risk versus benefit. this base is applied in various techniques, such as ionizing or non-ionizing radiation (malone et al., 2012). the justification involves using all of the medical team including referring physicians, radiographers, and radiologists (malone et al., 2012). all examinations in diagnostic radiology shall then be justified to avoid unnecessary irradiation of patients (remedios, 2011). the number of ct examinations is increasing every year (brenner et al., 2007, broder et al., 2006). referring physicians at the emergency room (er) who requested radiology examinations in a time-pressured environment need to know how dia tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) agnostic imaging is indicated as well as the necessity to know the accurate knowledge of the associated risks (keijzers and britton, 2010). by definition of justification, medical exposure is justified when the benefit of the examination is greater than the harm. previous investigations show that hundreds of examinations requested every year are not justified and most of them are not necessary at all; including the ct examinations((malone et al., 2012; teferi et al., 201 ; hobbs et al.,2018; vassileva et al., 2012). also, some of these studies show that there is a prevalent disregard of radiation doses among physicians (wong et al., 2012; thomas et al., 2006; rice et al.,2007; heyer et al., 2010). with the increase of radiation exposure towards patients, referring physicians play a greater role in the proper choice of investigation as well as deciding the right choice for the patient. there are some studies that have been conducted on the knowledge about radiation protection for health professionals who work with ionizing radiation (muhogora et al., 2010). however, they are a few studies that have been applied to the reference of a physician’s knowledge regarding the justification of the procedures (moifo et al., 2018). the goal of this study is to determine whether previous ct examinations done at makassed hospital in jerusalem on the patients who entered the emergency department were justified. to accomplish this, patient files of 339 urgent brain ct examinations were retrospectively reviewed. also, a questionnaire made up of 42 physicians was created to analyse the referring physician’s awareness of radiation risks and weather the requests of medical imaging procedures can be justified or not. materials and methods a sample of patients participants chosen for the study were patients who urgently referred from the er for 6 months from the first of january to the 30th of june 2018. approximately 339 patients with suspected or confirmed head injury with or without other major trauma were studied. the study referred to the archive of the hospital through the hospital information system (his) which records all observations before examinations are done. then, the results of those examinations are written and then compared. an institutional review approval was obtained for this prospective study. the patient consent was conceded, but the patient particularity was conserved by using the patient id without mentioning the name of the patient. emergency department patients were included, other departments were excluded. also, emergency department patients who during the morning working shift 7:00 am to 3:00pm as elective and scheduled were excluded while patients between 3:00 pm to 7:00 am were included. the data collection method included reviewing patient notes during emergency situations. we used the his archive which is used in the hospital. the data for each patient included age, gender, clinical data (obtained from the er physician), the exam indications and the findings written by radiologists. the 656 patients who have no written finding reports were also excluded. a sample of doctors fourty two (42) referring physicians from the hospital believe that they can justify at least ten brain ct requests of image procedures per week. thus, a plan has been developed to interview each potential respondent in the hospital. each meeting session concluded explanations that were provided from the respondent according to the objectives of the survey, the confidentiality of the responses and the anonymous nature of the survey model. once approved, the pre-screened questionnaire is administered to the respondent. sampling was continuous and easy going. radiologists and radiographers were excluded. the questionnaire consisted of a section on the person interviewed (gender, qualification, years of experience), a section on knowledge and the exercise of justification for the examination of the required medical radiation with emphasis on the effective dose of brain ct scans and radiation risks. other questions focused on the responsibility of justification and the daily practice of referring physicians on the justification of the required examinations and the principles of radiation protection. the main part of the questionnaire is shown in appendix 1. data analysis two experts in radiology with the new south wales ministry of health nsw health clinical practice guideline (haydon, 2013) wrote the following findings for each exam. it also assessed the justification of the performed examinations based on the referrals. the analyses of examinations were divided into two groups; justified and unjustified, and the percentage of the two groups were calculated. the guideline requirements for indicating the potentially significant mild head injury summarized in table 1. this scale can be applied with the time it took to deal with the patients. however, because our work is on the retrospective study; this scale cannot be applied. thus, researchers put a scale a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 1. initial management of adult closed head injury (nsw health algorithm). a-wptas, abbreviated westmead pta scale; gcs, glasgow coma scale. 1 gcs < 15 at 2 h after injury. 2 deterioration in gcs 3 focal neurological deficit 4 clinical suspicion of skull fracture 5 vomiting (especially if recurrent) 6 known coagulopathy or bleeding disorder 7 age >65 years 8 post-traumatic seizure 9 prolonged loss of consciousness (>5 min) 10 persistent post-traumatic amnesia (a-wptas <18/18 at 4 h after injury 11 persistent abnormal alertness/behavior/cognition 12 persistent severe headache 13 large scalp hematoma or laceration 14 multi-system trauma 15 dangerous mechanism 16 known neurosurgery/neurological impairment 17 delayed presentation or representation 657 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) that consists of four groups from 1 to 4 as follows: group 1 (g1): brain ct examination of cases related to head trauma and its involvement with road traffic accidents (rta), motor vehicle accidents (mva) and falling down cases. group 2 (g2): brain ct examinations of patients with vomiting, dizziness, seizure, and convulsion. group 3 (g3): brain ct examinations of patients suffering loss of consciousness, sleepiness, side body weakness, numbness, and severe headache. group 4 (g4): brain ct examinations after surgery (postoperative). the statistical analysis was obtained using the statistical package for social sciences. results as we mentioned, a total of 339 urgent brain ct request forms were received during the study period (6 months). of these; 209 (61.65%) were males and 130 (38.35%) were females. 111 (32.74%) were children less than 16 years, 228 (67.26%) were over 16. out of the total 339 ct request forms, 105 (30.98%) received were for patients in the er who stayed in different departments inside the hospital, while 234 (69.02%) brain ct requests were for patients who were at the er and who stayed under observation for 24 hours and were then discharged the next day. out of the 22 patients who were excluded from the study; 10 of them were not given a report due to their impatience. the remaining patients did not have an examination request. table 2 and figure 1 provide summary of the distribution of age, gender, and patients who were included as well as those were excluded in the study. as it is a retrospective study, it is difficult to justify every request based on clinical data only. in our study, we consider the exam retrospectively justified, if the note demonstrate that the patient belongs to one of the four group. table 3 and figure 2 show the distribution of the justified and non-justified requests. the study estimates that 236 (69.6%) requests of the total 339 was retrospectively considered a justified request, as the request fall in one of the four group, while 103 (30.4%) was not fulfilling the justification criteria applied to our study. statistically: the sample size is n=339, the number of unjustified requests is 103. let p denotes the proportion of the unjustified requests at this hospital in general, and pv denotes the proportion of the unjustified requests in our sample study. then, from the test ho: p ≤0.25 and ha: p>0.25, we have the following: a 95% confidence interval for p is 0.255 < p < 0.353 , and a p-value=0.011 as shown in figure 3. this strongly support the conclusion that p> 25% (figure 3). our two experts wrote the difference between the clinical data (indication) with the data findings (reports). out of the 339 patients, 23 (6.78%) had no radiology report, while 104 (30.67%) had normal results after the brain ct examination. this study estimates that 26 patients (7.66%) had mismatches between the indications and the correct findings after the examinations were done. keeping in mind, a brain ct indication is not a hard requirement to achieve from er physicians. 129 (38.05%) out of the 339 patients had matchings between the final reports with the clinical data, while 57 (16.81%) patients had mismatches between the final reports and exam indications. however, it’s still important to request brain exams from er physicians because the reports do show emergency findings inside these cases. in essence, to check the accuracy for brain ct examination requests from er physicians, while excluding radiologist redemographics january february march april may june total male split 26 28 26 42 40 47 209 female split 15 13 23 28 29 22 130 children patients <16 8 7 10 38 25 23 111 another patient >16 33 34 39 32 44 46 228 patients included 36 40 49 67 64 61 317 patients excluded 5 1 0 3 5 8 22 inpatient 15 12 23 22 15 18 105 outpatient 26 29 26 48 54 51 234 table 2. demographics of clinical study cohort. month j n.j %j %n.j t 𝑮𝟏 𝑮𝟐 𝑮𝟑 𝑮𝟒 january 41 5 11 2 6 17 58.53 41.46 february 41 9 3 14 3 12 70.74 29.26 march 49 10 3 15 10 11 77.56 22.44 april 70 22 6 15 7 20 71.43 28.57 may 69 23 4 6 10 26 62.32 37.68 june 69 29 9 8 6 17 75.37 24.63 total 339 98 36 60 42 103 69.6 30.4 table 3. documentation for justified and non-justified requests. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 658 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) ports, we distributed three categories of findings: a. true-negative: acts for the patients, who had a request to do an examination with a result. this category consists of both patients with matchings between the examination indication and the final report and those patients who have a mismatching between the clinical data with the radiology report. according to the reports, it was estimated that 54.86% show an importance for the exam to be made. b. true-positive: refers to the patients, an estimation of 30.67% who had a request to do the examination with normal results. c. the third category: consists of patients who had a request to do the examination, but the examination did not undergo the emergency exam, with an estimated 7.66%. from these results, we come to know that the accuracy of er doctors only reached 54.86%. table 4 summarized these results. there were 130 patients (38.34%) that received a brain ct without having the documented criteria required to justify the investigation based on the nsw health program. according to the questionnaire on doctors, the results for each question are shown in table 5. according to this survey, 18 referrers (42%) of the 42 questioned referrers know the effective dose of the brain ct scan. however, just 13 referrers (3%) has known that the effective dose of one brain ct scan is equal to the effective dose of 100 chest x-ray (a routine emergency radiological exam). in despite the that all referrers believe ionizing radiation is a risk factor for cancer development, only 24% of the them have known the exact excess risk of cancer from radiation. six referrers (14%) of all respondents knew the basic principles of radiation protection, and 10 referrers (23%) consider a local or an international protocol when request an urgent brain ct scans. discussion in this current study, the requests for brain ct examinations over a six month period are reviewed. approximately 30.4% was not justified, the degree of justification was only about 69.6%. our present study shows that the un-justification of brain ct examinations was statistically significant. for the children, the un-justification exams are at a significant high, with the total number of examinations for children being less than adult patients. this result is similar to a previous study done in germany (merzenich et al., 2012). the number of brain cts carried out on adult patients increased significantly, which were ordered after 15:00 o’clock comparing with the time before. no report normal report matching report with clinical data mismatching t f january 3 8 18 10 2 february 1 10 14 7 9 march 4 11 25 8 1 april 3 26 23 12 6 may 5 19 26 15 4 june 7 30 23 5 4 total 23 104 129 57 26 questions correct answer correct answer percent q1 c 42% q2 c 3% q3 d 24% q4 d 14% q5 a 23% table 4. the accuracy of ed physicians for order the brain ct exams. table 5. the questionnaire results. figure 2. distribution of the justified and non-justified requests during the period of study. figure 3. test on the proportion of the unjustified requests. figure 1. distribution of age, gender, and patients who were included as well as those were excluded in the study. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 659 despite the recommendations, radiologists were not regularly consulted before brain ct’s were requested for adult patients. the analysis of justification for a brain ct is also challenging because of the multiple and variable indications. according to the questionnaire on a sample of referring physicians, the level of knowledge of referring physicians regarding justification for the request of radiating examinations was not satisfactory. this is harmonious with many previous studies: o’sullivan et al which shows that 87% of the study population and 89% of controls considered that they had never been exposed to lectures or teaching focussed on radiation protection; soye and paterson which demonstrated that fiftyfour percent of students reported a moderate confidence in their knowledge about radiation and risks. borgen et.al study shows that the mean radiation knowledge score was 30.4/71. most respondents underestimated doses from high-dose imaging, e.g., barium enema (94.7%), chest ct (57.7%) and abdominal ct (52.7%). limited radiation knowledge was not compensated by using guidelines. only 20% of physicians and 72% of non-physicians used referral guidelines and the krille.et al systemic review which showed moderate to low knowledge among physicians concerning radiation doses and the involved health risks (o’sullivan, 2010; soye and paterson, 2000; zewdneh et al., 2012; borgen, 2010; krille,2010). this level of knowledge has not changed with professional experience or qualifications. the explanation might be the lack of initial training during medical studies and the absence of regular structured education in hospitals on radiation protection. there are several limitations to this study. the major limitation is that this study contains information from one institution only. the patient number selected for the justification evaluation is also low. the study dealt with brain ct examinations only. the data collection required to deal with the patients at the time of an examination to determine which patients fitted the criteria. also, the study concerned the er and not all hospital departments and it only focused on the patients after the 15:00 o’clock. in general, it is probably impossible to reach 100% justification. however, it is still essential to develop a justification process. regular updating of referral guidelines with the computer system used at the hospital to increase justification. continuous and easy communication system between referrers, radiographers and radiologists will improve the unjustifiable exam by feedback for each request. packs and his system make these communications easier and smooth. in conclusion, the study has shown that it is possible to reduce the number of brain ct examinations and to improve their justification by regular education, guideline implementation and to educate physicians about the ionizing radiations related to medical imaging, given their legal responsibility as dispensers under the ionizing radiation regulations (medical exposure) in the hospital. references agency hp (2012) annals of the icrp. 1 to 2005. borgen l, e stranden, and a espeland (2010) clinicians’ justification of imaging : do radiation issues play a role ? insights imagins 1 (3): 193-200. http://doi.org/10.1007/s13244-010-0029-4. brenner dj and ej hall (2007) computed tomography — an increasing source of radiation exposure. nejm 357 (22): 2277-2284. broder j and dm warshauer, (2006) increasing utilization of computed tomography in the adult emergency department, 2000 – 2005. emergency radiology 13 (1): 25–30. haydon nb (2013) head injury: audit of a clinical guideline to justify head ct. j. med. imaging radiat. oncol. 57 (2): 161-168. https:// doi.org/10.1111/1754-9485.12007. heyer cm, j hansmann, sa peters, and sp lemburg (2010) paediatrician awareness of radiation dose and inherent risks in chest imaging studies a questionnaire study. eur. j. radiol. 76 (2): 288-293 http://doi.org/10.1016/j.ejrad.2009.06.014 . hobbs jb, n goldstein, ke lind, d elder, gd dodd, and jp borgstede (2018) physician knowledge of radiation exposure and risk in medical imaging. j. am. coll. radiol 15 (1 pt a): 34-43, http://doi. org/10.1016/j.jacr.2017.08.034 . international atomic energy agency (iaea) (2009) report of a consultation on justification of patient exposures in medical imaging. radiat. prot. dosimetry 135 (2): 137–144. keijzers gb and cj britton (2010) doctors’ knowledge of patient radiation exposure from diagnostic imaging requested in the emergency department. mja 193 (8): 450-453. http://doi. org/10.5694/j.1326-5377.2010.tb03998.x. krille l, g hammer, h merzenich, and h zeeb (2010) systematic review on physician’s knowledge about radiation doses and radiation risks of computed tomography. eur. j. radiol 76 (1): 36-41. https:// doi.org/10.1016/j.ejrad.2010.08.025. malone j, r guleria, c craven, p horton, h järvinen, j mayo, g o’reilly, e picano, d remedios, j le heron, m rehani, o holmberg, and r czarwinski (2012) justification of diagnostic medical exposures: some practical issues. report of an international atomic energy agency consultation”, bjr 85 (1013): 523–538. https:// www.birpublications.org/doi/10.1259/bjr/42893576. merzenich h, l krille, g hammer, m kaiser, s yamashita, and h. zeeb (2012) paediatric ct scan usage and referrals of children to computed tomography in germany-a cross-sectional survey of medical practice and awareness of radiation-related health risks among physicians. bmc health serv. 12 (1): 1–7. moifo b, al edzimbi, h tebere, j tambe, rn samba, and jg fotsin (2014) referring physicians’ knowledge on justification of medical exposure in diagnostic imaging in a sub-saharan african country, cameroon. open j. radiol 4: 60-68. http://doi.org/10.4236/ ojrad.2014.41008. muhogora we, na ahmed, js alsuwaidi, a beganovic, o cirajbjelac, v gershan, e gershkevitsh, e grupetta , mh kharita, n manatrakul, b maroufi, m milakovic, k ohno, l ben omrane, j ptacek, c schandorf, shaaban ms, n toutaoui, d sakkas, js wambani, mm rehani (2010) paediatric ct examinations in 19 developing countries: frequency and radiation dose. radiat prot dosimetry 140 (1): 49-58. http://doi.org/10.1093/rpd/ncq015. o’sullivan j, oj o’connor, k o’regan, b clarke, ln burqouyne, mf ryan, mm maher (2010) an assessment of medical students’ awareness of radiation exposures associated with diagnostic imaging investigations,” insights imaging 1 (2): 86-92. remedios d (2011) justification: how to get referring physicians involved’,radiation protection dosimetry 147 (1-2): 47-51 . https:// doi.org/10.1093/rpd/ncr263. rice he, dp frush, mj harker, d farmer , and jh waldhausen,(2007) peer assessment of pediatric surgeons for potential risks of radiation exposure from computed tomography scans. j. pediatr. surg, 660 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 42 (7): 1157-64. soye ja and a paterson (2008) a survey of awareness of radiation dose among health professionals in northern ireland. bjr 81 (969): 725-729 http://doi.org/10.1259/bjr/94101717. squillaro t, g galano, r de rosa, g peluso, u galderisi (2018) concise review: the effect of low ‐ dose ionizing radiation on stem cell biology : a contribution to radiation risk. stem cells 36: 11491153. teferi s, d zewdeneh, and s bekele (2018) pediatric residents ’ and medical interns’ awareness about pediatric ionizing radiation dose from computed tomography and its associated risks in tertiary hospital in ethiopia. ethiopian journal of health science 28 (4): 383–392. thomas ke, je parnell-parmley, s haidar, r moineddin, e charkot, gb david, c krajewski (2006) assessment of radiation dose awareness among paediatricians. paediatric radiology 36 (8): 82332. vassileva j, mm rehani, h al-dhuhli, hm al-naemi, js alsuwaidi,k appelgate , d arandjic, eh bashier,a beganovic, t benavente, t bieganski, s dias, l el-nachef, d faj, me gamarrasánchez, j garcia-aguilar, l gbelcová, v gershan, e gershkevitsh, e gruppetta, a hustuc, s ivanovic, a jauhari, mh kharita, s kharuzhyk, n khelassi-toutaoui , hr khosravi, h khoury, d kostova-lefterova, i kralik,l liu, j mazuoliene , p mora,w muhogora, p muthuvelu,l novak, as pallewatte, m shaaban,e shelly, k stepanyan, el teo , n thelsy , p visrutaratna , a zaman , and d zontar (2012) iaea survey of pediatric ct practice in 40 countries in asia, europe, latin america, and africa: part 1, frequency and appropriateness. am. j. roentgenol. 198 (5): 1021-1031. http://doi. org/10.2214/ajr.11.7273 . wong cs, b haung, hk sin, wl wong, kl yiu, and t chu yiu ching (2012) a questionnaire study assessing local physicians, radiologists, and interns’ knowledge and practice pertaining to radiation exposure related to radiological imaging. eur. j. radiol., 81 (3): e264-8. http://doi.org/10.1016/j.ejrad.2011.02.022. yates d, r aktar, and j hill (2007) assessment, investigation, and early management of head injury: summary of nice guidance. bmj 335 (7622): 719–720. http://doi.org/10.1136/bmj.39331.702951.47. zewdneh d, st dellie, and t ayele, (2012) a study of knowledge & awareness of medical doctors towards radiation exposure risk at tikur anbessa specialized referral. iosr journal of pharmacy and biological sciences 2 (4): 1-5. http://doi.org/10.9790/30080240105. appendix 1. survey questionnaire with correct answers. q1: what is the effective dose of brain ct scan: 0.1msv 1 msv. 2 msv. 4 msv. 10 msv. q2: if we consider the chest x-ray effective dose as 1 unit, the effective dose of the brain ct scan will be 1. 10. 100. 1000 10000 q3: the excess risk of cancer from radiation is 1/10000 msv. 1/100000 msv. 1/100 msv. 1/4000 msv. no increase in cancer risk. q4: the radiation protection philosophy that promotes the use of the least amount of radiation possible for medical imaging is termed: ncrp nrc icrp alara q5: when you order a brain ct scan especially in trauma patients, do you consider any local or international guidelines. yes. no. atlas journal of biology 2 (3): 162–170, 2013 doi: 10.5147/ajb.2013.0130 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) month of seeding effect on low-input establishment of cool and warm-season turfgrasses in continental transition zone kenneth lynn diesburg and ronald f. krausz department of plant, soil sciences, and agriculture system, southern illinois university at carbondale, carbondale, il 62901 usa. received: june 30, 2013 / accepted: september 14, 2013 __________________________________________________ * corresponding author: diesburg@siu.edu 162 abstract this research was conducted to determine the degree of success, by month, in seeding establishment of tall fescue (festuca arundinacea schreb., kentucky bluegrass (poa pratensis l.), bermudagrass (cynodon dactylon [l.] pers. var. dactylon), and zoysiagrass (zoysia japonica steud.) at two locations in the moist, midwest, continental transition zone on a prepared seed bed without irrigation or cover. the four species were planted every month of the year starting in september 2005. starter fertilizer and siduron were applied the same day as seeding with no subsequent management except mowing. percent cover of living turfgrass was recorded in each of 24 months after seeding. tall fescue (80%) and bermudagrass (73%) provided the best percent cover over all planting dates. kentucky bluegrass provided 65% and zoysiagrass 24% cover. the cool-season grasses performed best in the julyto-march plantings; tall fescue 88% and kentucky bluegrass 72%. bermudagrass (94%) established best in the januaryto-april plantings, while zoysiagrass (32%) established best in the november-to-march plantings. germination and seedling survival after germination of all species were inhibited by limited moisture during summer. the warm-season grasses were further limited by winter kill in the august, september, and october seedings. these results emphasize the risk in spring-seeding as well as the value in dormant-seeding of both warmand cool-season turfgrasses for low-input, nonirrigated establishment. introduction research long ago established early autumn as the ideal time to seed cool-season species (defrance and simmons, 1951; harrison, 1944) and early summer for the warm-season species (johnson and thompson, 1961). hull (1948) reported that late fall seedings of cool-season grasses for southern idaho rangelands, while providing stands superior to spring seedings, resulted in stands the following growing season that were inferior to early fall seedings. the first mention of dormant seeding, regardless of coolor warm-season turfgrasses, was made by musser (1962). recent studies have addressed the best timing within seasons. diesburg (1986) reported that dormant seeding of kentucky bluegrass in central iowa in november produced better stands the following growing season than those of december, february, or march, while september seeding resulted in the best stands. reicher et al. (2000) found that dormant seeding of tall fescue and kentucky bluegrass in northern indiana during november, december, or march provided better stands the following august than those of april or may seedings. green et al. (1974) included tall fescue, kentucky bluegrass, and bermudagrass in a dormant seeding study of many species. done in both the mountains and coastal plain of virginia, they recorded favorable results from tall fescue and kentucky bluegrass but not from bermudagrass. the plots were covered with over 1,500 kg ha-1 wood fiber cellulose mulch. contrarily, shaver, et al. (2006) obtained favorable results from winter dormant seedings of bermudagrass. their plots were covered with six mm of sand. patton, et al. (2004) found in an irrigated study that bermudagrass and zoysiagrass seeded may 15 provided enough cover to survive the following winter. dudeck and peacock (1982) found in florida that winter dormant seeding of bahiagrass (paspalum notatum flugge) produced greater than a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 163 90% cover during the following growing season. in all seedling establishment, there is the necessity of soil surface moisture during emergence of the seed radical and the subsequent initial expansion of the seminal root system. without properly timed moisture, you risk losing the stand. there are many situations, however, where irrigation is not possible as in right-of-way, large lawn, range and pasture, and parks. these are common examples of an ever-growing topic in turf research, low input sustainable turf. even in conventional turf management, the professional would prefer to have a reliable amount of soil moisture to eliminate any need for irrigation. with these points in mind, it would be useful to know the degree of success possible with winter dormant seeding, not only which turfgrass species, but also which months within a region would be best suited to non-irrigated, low-input establishment. the objective of this study, therefore, was to compare establishment from monthly seedings, within an entire year, of the dominant turfgrass species in climates typical of the moist midwest transition zone of north america, which includes southeast missouri, southern illinois, and southern indiana. materials and methods two locations were chosen to represent climates and soils typical of the northern and central portions of the moist midwest transition zone. belleville, illinois (north location) is in the northern side of 6a hardiness zone (cathey, 1990) with a weir silt loam soil, and carbondale, illinois (south location) is in the southern side of 6a with a hosmer silty clay loam. both locations had been supporting tall fescue utility turf for several years prior to the experiment and had received no supplemental fertilizer for at least the previous four years. after a preliminary application of nonselective weed control with glyphosate, the sites were tilled and harrowed during august 2005 for the first monthly seeding in september. one cultivar of each of four species was chosen based on seed availability in that region and proven performance in the moist midwest transition zone (morris, 2005 to 2007). tall fescue (festuca arundinacea, schreb) ‘bingo’ at 391 kg ha-1 (8 lb 1000 sq ft-1), kentucky bluegrass (poa pratensis l) ‘north star’ at 146 kg ha-1 (3 lb 1000 sq ft-1), hulled bermudagrass (cynodon dactylon [l.] pers. var. dactylon) ‘mirage’ at 98 kg ha-1 (2 lb 1000 sq ft-1), and zoysiagrass (zoysia japonica steud.) ‘zenith’ at 146 kg ha-1 (3 lb 1000 sq ft-1) (mccarty, 2005), were seeded separately into 1.5 m2 plots each month from september 2005 through august 2006. at each monthly seeding the soil surface was loosened to a 3-cm depth with a garden rake. the tall fescue and kentucky bluegrass were incorporated lightly with the back of a spring-steel-tined leaf rake. the bermudagrass and zoysiagrass were left at the soil surface. incorporation of the bermudagrass seed (hulls removed) would have buried them too deeply due to their small size. zoysiagrass stands are severely compromised when the seed, having an irradiance requirement for germination, are incorporated into the soil. treatments were arranged in a split-plot design with three replications, species randomly assigned within a replication block as whole plots, and months of seeding randomly assigned within species as sub-plots. in order to inhibit initial competition from weed seedlings, the preemergent herbicide, siduron (tupersan), was applied to each plot the same day of seeding, except those of bermudagrass to which it is phytotoxic. field grade fertilizer (12-12-12, n-p-k) was chosen on the basis of availability as a starter fertilizer applied just before seeding at a rate of 24.4 kg n ha-1 (0.5 lb n 1000 sq ft-1). no additional inputs were applied at any point except to maintain turf at 5.1cm clip without removal of clippings. percent live cover was recorded monthly by visual estimation. monthly precipitation and evaporation rates at the two locations through the duration of seedings and recording of data are provided in figure 5 (illinois state water survey), showing the overall gain and loss of water through the seasons. average monthly maximum and minimum temperatures at two locations are provided in figure 6 (illinois state water survey). since there were only minute variations in average monthly temperatures between them, a single line represents both locations. means, analyses of variance, and tests of significance (α = 0.05) were calculated for each monthly set of data using statistical analysis systems (sas). the analysis of data combined from both locations revealed significant first and second-order interactions of species and month-of-seeding with location for every monthly set of data. therefore, data were analyzed separately by location. figures 1 to 4 provide graphic representation of establishment after monthly seeding. in several cases establishment from monthly seedings were so similar that they could not be distinguished from one another in the graph. correspondingly, there were no significant differences detected among those months. therefore, for the sake of clear presentation, the means of similar establishments after monthly seedings were averaged into one series of means and are represented by one line in a graph. results and discussion at both locations (tables 1 and 2) there were significant differences in turf percent cover among species and months-ofseeding. in addition, there was a significant species-by-month interaction during all months except october 2007 at the south location. tall fescue north location the september seeding (fig. 1a) provided excellent establishment (98% cover by june 2006) as did the june, july, and august seedings; june (94%), july (100%), and august (94%) by may 2007. germination of all those summer seedings occurred at the same time during september of that year. through the summer, rains were infrequent enough (fig. 5a), and subsequent soil moisture was fleeting enough to prevent their germination until fall. they were essentially summer-dormant seedings. germination from the october seeding although immediate, did not allow time for the tall fescue to mature enough to provide full cover (69%) through the winter months. this effect of reduced stand carried over through the duration of the study since tall fescue is not a spreading species. the winter dormant seedings a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 164 and march were similar to one another one year after seeding; november (81%), december (85%), january (78%), february (72%), and march (69%), and were thus, as effective as the october seeding. note that germination from all the winter dormant seedings occurred in march. summer covers after the april and may seedings were severely restricted by 9% to 21%. subsequent recovery from those months of seeding never surpassed 67% cover. south location the fall seedings (fig. 1b) performed similarly to those at the north location with the exception of their greater decline in percent cover during the summer months. the september seeding, having more maturity, declined an average of 24% from 95% in june to 71% in september 2006, then recovered to 90% by may 2007. the october seeding, with less maturity, declined 30% from 69% in june to 39% in july 2006, then recovered to 77% by may 2007. the winter dormant and march seedings behaved similarly to those of the northern location, again, with the exception of their more severe decline in response to summer stress (fig. 1b) showing 25 – 46% reductions in cover from may to august 2006. the april seeding differed from that of the north location, establishment behaving similarly to those of the dormant and march seedings. the may and june seedings failed, reaching only 8% and 32% cover, respectively due to temporarily inadequate soil surface moisture immediately following germination. the july and august seedings performed similarly to those at the north location site remaining dormant until september germination. kentucky bluegrass north location all of the monthy seedings (fig. 2a) displayed best percent cover during their subsequent april to july (51–91%). establishment from the september and october seedings was slow during fall 2005, but maximized to 83% and 71% cover by may 2006. the september cover subsequently sustained itself at 47% through a dry late summer and fall, while that of the october seeding thinned to 23%. the winter dormant and march seedings germinated in april expanding to covers of 51 – 72% by july. their cover also diminished through the dry late summer and fall to 28%. the april, may, june, and august seedings resulted in a mixture of dormant seeds and partially lost 2005 2006 source oct. nov. dec. jan. feb. mar. apr. may jun. jul. aug. sept. oct. species s) ** *** *** *** *** *** *** *** * ** ** ** ** mo. seeded (m) *** *** *** *** *** *** *** *** *** *** *** *** *** s x m *** *** *** *** *** *** *** *** *** *** *** *** *** lsd†: s x m 4.7 4.0 3.3 4.7 5.5 5.5 7.0 14.6 19.0 17.1 20.0 20.7 22.3 * significant f test at the 0.05 level of probability ** significant f test at the 0.01 level of probability *** significant f test at the 0.001 level of probability † least significant difference (α= 0.05) among species by month-of-seeding means table 1. tests of significance from analyses of variance of main effects and interactions of four turfgrass of seeding on percent turf cover at belleville, illinois (north location). 2005 2006 source oct. nov. dec. jan. feb. mar. apr. may jun. jul. aug. sept. oct. species s) *** *** *** *** *** *** *** *** *** *** ** ** ** mo. seeded (m) *** *** *** *** *** *** *** *** *** *** *** *** *** s x m *** *** *** *** *** *** *** *** *** *** *** *** *** lsd†: s x m 4.2 8.5 6.9 6.1 6.9 6.1 12.4 22.5 18.9 23.6 26.2 30.1 31.2 2006 2007 source nov. dec. jan. feb. mar. apr. may jun. jul. aug. sept. oct. species s) *** *** *** *** *** *** *** *** *** ** ** * mo. seeded (m) *** *** *** *** *** *** *** *** *** *** *** *** s x m *** *** *** *** *** *** *** *** *** *** * ns‡ lsd†: s x m 29.8 27.7 26.5 25.6 24.3 14.3 19.6 24.5 26.4 31.0 33.7 * significant f test at the 0.05 level of probability ** significant f test at the 0.01 level of probability *** significant f test at the 0.001 level of probability † least significant difference (α = 0.05) among species by month-of-seeding means ‡ ns, not significant at the 0.05 level of probability table 2. analyses of variance of main effects and interactions of four turfgrass species and twelve months of seeding on percent turf cover at carbondale, illinois (south location). stands through summer and fall. surviving plants established slowly through fall and winter and maximized by june 2007 at an average of 54% cover. the july seeding received enough timely rain to establish a 27% cover into winter which expanded rapidly during march to 90% cover by april 2007. south location expansion of cover was less focused in spring as it was at the north location due to more autumn rain events at the south location (figs. 5a, 5b). the september seeding (fig. 2b) reached 36% cover by november 2005, expanded gradually through the winter, and had 64% cover by may 2006, which it never surpassed throughout the rest of the study. the october through march seedings germinated during march established rapidly into may to an average maximum cover of 44%, then diminished severely through summer to an average of 12%. december seeding was unique in its maximum cover of 86% by may previous to its summer decline to 30%. fall 2006 growth was strong allowing expansion to 76% in the december seeding and 47% in the other months of seeding. the may and june seedings failed while the april seeding had enough surviving plants after summer stress to establish as much cover as those of the july and august seedings, averaging 32% by november 2006. those stands expanded further to 65% by june 2007. bermudagrass north location this species provided excellent cover from all the monthly seedings of october to june (fig. 3a). the september 2005 165 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 1. establishment of tall fescue at two transition zone locations, (a) north (belleville, il) and (b) south (carbondale, il), after monthly seedings through an entire year. lsd is the least significant difference between and among means for that month. 166 seeding established quickly reaching 77% cover by november. it survived the winter but never achieved the cover in the october to june seedings, reaching 90% by october 2006. the october to march seedings all germinated during april 2006, while the april, may, and june seedings germinated quickly within their respective month. almost full covers were achieved from the october to june seedings ranging from 81 to 100% through the subsequent fall to spring. the july seeding, while germinating quickly and covering to 94% by october, lost cover through winter. the august seeding failed with almost all juvenile plants dying through the subsequent winter. its few surviving plants spread enough to achieve 97% cover by august 2007. south location establishment was not as successful as at the north location. the a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) figure 2. establishment of kentucky bluegrass at two transition zone sites, (a) north (belleville, il) and (b) south (carbondale, il), after monthly seedings through an entire year. lsd is the least significant difference between and among means for that month. september and october 2005 seedings (fig. 3b) failed due to winter kill of juvenile plants. success of the dormant seedings was variable. all dormant seedings germinated in april with maximum cover achieved by october. average maximum cover of the november, december, and january seedings (66%) was not as great as the average of the february, march, and april seedings (96%). the may, june, july, and august seedings germinated in hot conditions with variable soil moisture, losing stand, achieving 56% by october. all seedings survived the 2006/2007 winter and recovered well in 2007. zoysiagrass north location all monthly seedings of zoysiagrass from september to april a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 167 (fig. 4a) germinated in april and achieved maximum covers by october 2006, as follows: september and october averaging 82%, november, january, february, march, and april averaging 61%, and december and may averaging 40%. all three summer seedings failed due to loss of seedling stand from soil surface drought. south location as with bermudagrass, seedings were not as successful as at the north location. september and october seedings (fig. 4b) germinated and died due to soil surface drought after seedling emergence. only a few seedlings established in the november, december, january, and february seedings, experiencing strong winter annual weed competition, reaching only 2% cover by may, then finally achieving 15% in the latter half of 2006 after the winter annuals had died. the march seeding was the most successful reaching 7% cover through the winter annual weed competition, then achieving 57% cover by november 2006. the april, may, june, july, and august seedings failed due to soil surface drought. conclusion figures 1a to 4b portray the quick and persistent dominance of tall fescue, the quick but winter-weak dominance of bermudagrass, the high degree of variability in kentucky bluegrass presence, and the slow but persistent increase of zoysiagrass toward dominance. these differences come as no surprise as they are characteristic of the species. the surprising thing is the months of seeding that brought about the best establishment. all of these four species have their place in low input sustainable turf in the transition zone. they have all persisted very well in the transition zone for decades at minimal to no figure 3. establishment of bermuda at two transition zone sites, (a) north (belleville, il) and (b) south (carbondale, il), after monthly seedings through an entire year. lsd is the least significant difference between and among means for that month. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 168 inputs except for mowing. within their respective niches, they are commonly found to provide turf of acceptable quality for a portion of the year. given that they are persistent species for low input sustainable turf, synchronization of management with growing seasons is paramount in order to optimize their capacities. for success in low-input establishment, timing is everything. the water and temperature conditions through the duration of this research, represented in figures 5 and 6, were typical for the region. precipitation through winter and spring is ample in recharging soil moisture. figures 5a and 5b indicate the north location receiving less winter precipitation than the south location in 2005/2006. even so, seed germination and seedling establishment in winter dormant seedings appears to have been unaffected between the two locations. the disparity between precipitation and evapotranspiration during summer months is common. the tall fescue and kentucky bluegrass results correspond with previous research, showing the advantage of seeding coolseason grasses in early fall, september, while the days are still warm, the nights are beginning to cool, soil moisture does not evaporate so rapidly, and rains will become more frequent during middle fall (fig. 5). the month previous to that time of year, august, provides excellent opportunity to prepare the seed bed since the soils are drier and rains more sparse. the data contradict, however, our recommendation to plant in spring as a second choice. from these transition zone data, the best second choice would be seeding any month other than april or may with march and june being marginal. during that early part of the growing season there is ample moisture to get the seeds germinated, but not enough to get the seedlings established and plants mature enough to withstand the rigors of summer. a safer approach is winter dormant seeding; getting the seed in place during any part of the moist, cold season, any month november to march, thus providing the critical two to three months needed after gerfigure 4. establishment of zoysia at two transition zone sites, (a) north (belleville, il) and (b) south (carbondale, il), after monthly seedings through an entire year. lsd is the least significant difference between and among means for that month. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 169 mination in early spring for at least some degree of plant maturation before summer. july and august are also recommended for summer dormant seeding. rains do occur during late summer in the transition zone, but with low frequency and short duration. that condition, combined with the rapid rate of water evaporation during the excessive summer heat (figs. 5 and 6), does not allow, in most cases, the seed enough time to germinate, and it remains dormant. the bermudagrass and zoysiagrass results contradict previous research which indicates the seeding of warm-season grasses when it is warm during late spring or early summer. that research contains the assumption that irrigation will be supplied if needed. low input establishment assumes no irrigation is applied. granted, the warmth is needed but not the pervasive and unpredictable droughts that accompany the warm season. additionally, by the time we seed in late spring or early summer, the warm-season annual weeds already have had a head-start in germination and can emerge over night, asserting competition against the vulnerable turfgrass seedlings, especially those of zoysiagrass. in this research, summer seedings provided, at best, marginal establishment, and at worst, complete failure. contrarily, winter dormant seeding provided, at best, excellent establishment, and at worst, marginal establishment. the ideal situation is to have the seed in place before the warm temperatures arrive in spring. evidence from this paper points to march as the best month for seeding bermudagrass and zoysiagrass in the continental transition zone. at that time, the soil temperatures needed for warm-season grass seed germination have not yet arrived and there is usually a break in the spring rains to allow seed bed preparation. the safest approach, however, would be to winter-dormant seed, taking advantage of the higher probability of utilizing favorable weather and soil conditions over a much longer period of time. among the species, tall fescue provided the most reliable low-input establishment throughout the year. its establishment was inadequate in only three of the nine months; april, may, figure 5. monthly precipitation and evaporation rates at two locations through the duration of seedings and recording of data, showing the overall gain and loss of water through the seasons. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) and june. bermudagrass performed as well as tall fescue at the north location with only three substandard months: july, august, and october. at the south site, however, there were no months of seeding with complete cover, with february, march, and april as its best months of seeding. greater moisture availability during the cool time of year interacted more reliably with the coolseason grasses than with the warm-season grasses. the slow germination and lack of vigor of kentucky bluegrass without ample soil fertility prevented it from providing complete cover, even though may and june were the only months of seeding with complete failure to establish. zoysiagrass was at an even greater disadvantage with its weak seedling and slow growth. at the north location, september and october were its best months of seeding while winter-dormant seeding and march seeding provided more relative success at both sites. june, july and august seedings failed. the long-term advantage of zoysiagrass for low-input turf could not be seen through the short duration of this study, since it takes longer for this species to become fully established. acknowledgments this research was partially funded by the illinois turfgrass foundation and the southern illinois golf courses superintendents association. references cathey hm (1990) plant hardiness zone map. revised, r. jordon, 2001. usda misc. pub. no. 1475. defrance ja and ja simmons (1951) relative period of emergence and initial growth of turf grasses and their adaptability under field conditions. proc. am. soc. for hort. sci. 57:439-442. diesburg kl (1986) dormant seeding study. 1985 iowa turfgrass res. rpt. publ. fg-451:65-66. dudeck ae and ch peacock (1982) dormant seeding of bahiagrass with cool season grasses in the winter. proc. fla. state hort. soc. 95:174-175. green jt, hd perry, jm woodruff, and re blaser (1974) suitability of cooland warm-season species for dormant winter seedings. p.557-568. in e.c. roberts (ed.) proc. int. turfgrass res. conf., 2nd, 19-21 june 1973. asa and cssa. madison, wi. harrison cm (1944) rough grasses for parks, highway, and recreational areas. greenkeeper’s reporter 12:30-31. hull ac jr (1948) depth, season, and row spacing for planting grasses on southern idaho range lands. j. amer. soc. agron. 40:960-969. illinois state water survey: http://www.isws.illinois.edu. johnson cm and wr thompson (1961) fall and winter seeding of lawns. mississippi farm research 24 (9): 4. mccarty lb (2005) best golf course management practices (2nd. ed). pearson education, inc., upper saddle river, nj. 868 pp. morris kn (2002) national turfgrass evaluation program. 1997 national bermudagrass test. ntep no. 02-7. usda, beltsville, md. morris kn (2005) national turfgrass evaluation program. 2000 national kentucky bluegrass test. ntep no. 05-6. usda, beltsville, md. morris kn (2006) national turfgrass evaluation program. 2001 national tall fescue test. ntep no. 06-5. usda, beltsville, md. morris kn (2007) national turfgrass evaluation program. 2002 national zoysiagrass test. ntep no. 07-2. usda, beltsville, md. musser hb (1962) turf management. mcgraw hill, new york. patton aj, ga hardebeck, dw williams, and zj reicher (2004) establishment of bermudagrass and zoysiagrass by seed. crop sci 44: 2160-2167. reicher zj, cs throssell, and dv weisenberger (2000) date of seeding affects establishment of cool-season turfgrasses. hortscience 35 (6): 1166-1169. shaver br, md richardson, jh mccalla, de karcher, and pj berger (2006) dormant seeding bermudagrass cultivars in a transition-zone environment. crop sci. 46:1787-1792. figure 6. average monthly maximum and minimum temperatures from two locations. there were only minute variations in average monthly temperatures between the two locations. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 170 atlas journal of biology 2018, pp. 583–591 doi: 10.5147/ajb.v0i0.177 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) confirmation of qtl that underlie resistance to soybean sudden death syndrome using nils and snps lee yc1,2, mj iqbal1,2,3, vn njiti1,4, sk kantartzi1,2*, pt gibson5, j anderson1,2, and da lightfoot1,2* 1 plant biotechnology and genomics core-facility, department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901, usa; 2 the illinois soybean center (center for excellence in soybean research, teaching and outreach), southern illinois university, carbondale, il 62901, usa; 3 present address: international programs of ca&es, environmental horticulture building room #1103, university of california, davis, ca 95616, usa; 4 dept. of biotechnology, alcorn state univ., lorman, ms 39096, usa; 5 present address: department of agriculture production, makerere university, p.o box 7062 kampala, uganda received: august 3, 2018 / accepted: october 20, 2018 __________________________________________________ * corresponding authors: kantart@siu.edu; ga4082@siu.edu 583 abstract soybean (glycine max (l.) merr.) cultivars differ in their resistance to sudden death syndrome (sds), caused by fusarium virguliforme (aoki). breeding for improving sds response has been challenging, due to the large number of known resistance loci (more than 43) and interactions among them. the aims here were to compare the inheritance of resistance to sds in a near isogenic line (nil) population that was fixed for 91.5% of the genome but appeared to segregated at loci underlying partial resistance to sds; to examine the interaction with the loci; and to identify regions containing candidate genes underlying qtl. used were; a nil population derived from residual heterozygosity in an f5:9 recombinant inbred line ef60 (lines 1-40). the sds disease index (dx) data were from two locations but two different years. there were 4 of 400 microsatellite and 456 of 5,361 snp markers tested that were polymorphic (8-10%). the snps clustered into 23 genomic regions. significantly associated with resistance to sds (0.005 < p > 0.0001) were regions from 2,788 kbp to 8,938 kbp on chromosome (chr.) 18 and 33,100 kbp to 34,943 kbp on chr. 20. the marker to trait association values suggested that the two closely linked loci on chr. 18 were really three loci (cqrfs1, cqrfs, and now rfs19). they were clustered within 20 cm of the rhg1 this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction sudden death syndrome (sds) of soybean (glycine max [l.] merr.) is one of the most devastating diseases in the midwest (wrather et al., 2009). sds is caused by a soil-borne fungus fusarium virguliforme (comma shaped spores; aoki et al., 2003; luckew et al., 2013; hartman, 2015). sds was first discovered in arkansas in 1971, it later spread to neighboring states and was found throughout most soybean producing states by the locus underlying resistance to soybean cyst nematode (scn; hgtype 7). an epistatic interaction between the chr18 loci and the chr 20 locus were inferred. therefore, qtl for resistance to sds were shown to be both internally complex and interacting. keywords: fusarium; resistance; soybean; glycine max; forrest; sds; near isogenic line; snp. abbreviations: receptor like kinase (rlk); soybean cyst nematode (scn); sudden death syndrome (sds); chromosome (chr.). a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) late 1990’s (hartman et al., 2015). there are several hypotheses about how sds spread to the us, it could be due to a species hop from common bean (phaseolus vulgaris l.) production; natural selection from common soil organisms; or in soil or dust introduced from south america (lightfoot, 2015). f. virguliforme infects the root causing a slightly tan to brown discoloration of the cortex, which leads to the loss of root mass and root nodules. the early above ground symptoms include; leaf mottling; yellowing between the major veins leading to necrosis; leaf abscission at the top of the petiole rather than the base; and finally early plant death. the diseases that comprise sds are favored by; cool and wet environments; early planting; soil compaction; meristem determinacy; and genotypes with high seed yield potentials. consequently, sds can lead to severe yield losses with average losses estimated to increase from $95 million a year in 1996 to $190 million a year in 2014 in the us midwest alone (luckew et al., 2013; lightfoot, 2015). the most efficient approach to try to control sds has been the use of resistant varieties (yuan et al., 2012; lightfoot, 2015; swaminathan et al., 2016). cultivars with higher genetic resistances may be the key for controlling soybean loss caused by sds. conventional breeding methods are time-consuming and labor-intensive; molecular methods can accelerate breeding programs and make selection for disease traits more effective. sds resistance is controlled by many (more than 43) quantitative trait loci (qtl; lightfoot 2015; swaminathan et al. 2016). restriction fragment length polymorphism (rflp) and random amplified polymorphic dna (rapd) markers were the first markers used to detect qtl underlying sds resistance in the 1990s (hnetkovsky et al., 1996; meksem et al., 1999; njiti et al., 1998, 2002). microsatellite markers (simple sequence repeat; ssrs) were later developed to identify qtl associated with sds (meksem et al., 1999; iqbal et al., 2001; kassem et al., 2006). with the availability of integrated ssr and snp maps of soybean, it has become possible to find many more qtl underlying sds resistance (wen et al., 2014; bao et al., 2015), although care must be taken with the phenotypic data collected (lightfoot, 2015). estimates of 18 30 loci underlying resistance across 12 populations have been made based on field data (lightfoot, 2015) or robust greenhouse data (bao et al., 2015; swaminathan et al., 2016). resistance to scn has been found to be linked or pleiotropic to resistance to sds in two regions (kazi et al., 2009; srour et al., 2012). up to 50% of resistance to sds appeared to be co-inherited with resistance to scn (gibson et al., 1994). three populations of recombinant inbred lines (rils) were used to map the loci affecting resistance to sds and soybean cyst nematode (scn) using phenotypic data in previous studies (iqbal et al., 2001; njiti et al., 2002; kazi et al., 2008). the soybean cultivars ‘forrest’, ‘hartwig’ and ‘pyramid’ showed a partial field resistance to sds whereas the cultivars ‘essex’, ‘flyer’ and ‘douglas’ were more susceptible to sds (lightfoot, 2015). the ten different ssrs that were polymorphic and linked to different sds qtl were selected to determine whether the qtl associate with four different disease assessments (disease incidence, foliar scorch, disease severity, and area under the disease progression curve; luckew et al., 2013). among the ten 584 tested, five qtl were strongly associated with at least one of the four disease metrics in multiple cross populations, providing more information on useful qtl for sds resistance breeding (luckew et al., 2013). the essex × forrest (e × f) population was developed to study the inheritance of sds resistance (gibson et al., 1994). a major limitation in using the essex × forrest (e × f) population in genomics research is the small population size (n = 100) that could preclude fine mapping (meksem et al., 1999). to overcome this problem, populations of near isogeneic lines (nils; n < 40) were developed from each ril (lightfoot et al., 2005). the residual heterozygosis present in the f5 seed was subsequently fixed as heterogeneity (but not completely) and captured in these nils. heterogeneity within the rils has been measured to be 8%, so each nil population was expected segregate for about 8% of the genome on a continuing basis (herein the f5:9:13 generation). residual heterozygosity was estimated at about 1% (triwitayakorn et al., 2005) based on the ability to find heterozygous plants at satt309. nil populations derived from e × f ril34 (ef34 lines 1-40) and e × f ril11 (ef11 lines 1-40). hundreds of their progeny, were used to fine map and isolate the receptor like kinase underlying cqrfs2 (triwitayakorn et al., 2005; srour et al., 2012). the forty nils were estimated to be equivalent to a 480 line ril population in the 8% of regions that were not fixed. however, to date many nil populations derived from lines within the e × f ril population have not been studied thoroughly. further field trials, greenhouse assays, and molecular marker techniques, including snps, could help to evaluate additional lines for resistance to sds and develop new cultivars highly resistant to sds. nine qtl were identified using the e × f population (lightfoot, 2015). among those, 4 qtl were mapped on linkage group g (lg g); chromosome 18 (chr,18), cqrfs, cqrfs1, cqrfs2, and cqrfs3, respectively. those 4 qtl were reported to be a cluster of loci for resistance to sds (triwitayakorn et al., 2005). meksem et al. (1999, 2001b) reported that each of the loci were located in 2to 5cm intervals and are mutually linked; the partial resistance beneficial alleles on lg g all derived from forrest (iqbal et al., 2001; triwitayakorn et al., 2005; lightfoot, 2008). loci cqrfs2 (cqsds002) and cqrfs3 were previously fine mapped in nil populations (meksem et al., 1999). loci cqrfs and cqrfs1 were not previously fine mapped in nil populations. another locus on chr.20 (lg i; cqrfs5) was reported (iqbal et al., 2001; kassem et al., 2006; de farias-neto et al., 2007; swaminathan et al., 2016). however, the qtl had low value for breeding selections for leaf symptoms but high value for root rot when multiple crosses involving large populations were analyzed (luckew et al., 2013). interestingly, the locus was effective against leaf scorch caused by fungal exudates (swaminathan et al., 2016). the locus cqrfs5 was not previously fine mapped in nil populations. here were reported; field trials on a new nil population ef60 lines 1-38; segregation data for ssr markers near cqrfs, cqrfs1 (closely linked on chr.18) and cqrfs5 (chr.20) that were associated with resistance to sds; and genome wide analysis of the nils using the 5,361 snp markers developed from song et a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 585 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) al. (2013). evidence of inter-locus interaction is presented. snp based maps of both genomic regions identified small regions encoding putative candidate genes. materials and methods plant material essex was crossed with forrest to generate 100 f5 derived rils (lightfoot et al., 2005). the rils were evaluated for sds resistance in many field trials from 1994 to 2015. the disease assessment methods used in the studies were described in njiti et al. (1996). briefly, disease incidence (di) was the percentage of plants showing sds leaf symptom (0-100%), disease severity (ds) was used to assess the severity of the disease (1-9 scale), the collected data were then converted into disease index (dx, ds × di/9), the data were collected during the r6-r7 stage (full green seed at upper 4 nodes, pods > 1.5 cm) of soybean growth and adjusted to the r6.5. several nil populations were selected and generated based on the performance of several of the rils (9, 11, 34, 60, 77) that appeared to segregate for ds based on a consistent di score in the range of 10-60% (matthews et al., 1991; njiti et al., 1998; meksem et al., 1999; triwitayakorn et al., 2005). some of those nils segregated for scn resistance (11 and 34) but some did not (9, 60 and 77). the ril60 was selected at the f5:9 generation to isolate 40 nil lines by collecting seed from 40 individual plants at random in disease free plots in 1994. the seed planted were extracted from ril60 at the f5:9 and advanced to the f5:9:13 generation following the method of njiti et al. (1998). after seed increase the lines were planted in several locations (carbondale, carmi, cora, harrisburg, ullin, and villa ridge) but significant disease was only found in the plots at the plantings at harrisburg, saline county, in 1998; and in carmi, white county, in 2000. the locations were selected based on historical appearance of sds symptom. the field trials used the random complete block design (rcbd) with 2 replications at each location. the nils were evaluated for disease performance using the disease assessment method previously described (njiti et al., 1996). dx data for two locations are presented herein. molecular marker analysis the nil population ef 60 (lines 1-40) were planted in the greenhouse in 2000 and the leaf tissues were collected for dna extraction at the f5:9:13 generation. the dna of the nils were extracted using the qiagen (hilden, germany) dnaeasytm kit from leaves of 10 plants per line. the dna was stored at -20 c until 2014. in early 2014 the dna samples were tested for polymorphisms with the 10 ssr markers described in luckew et al. (2013) to determine the polymorphisms among the nil ef 60 population. a step down polymerase chain reaction (pcr) was modified and performed based on the method described in luckew et al. 2013. the pcr products were electrophoresed on a 3% (w/v) agarose gel for 3 h and visualized using ethidium bromide. aliquots of the dna were shipped to the soybean breeding and genetics lab at michigan state university for the soysnp6k iselect beadchiptm analysis (illumina, san diego, calif. usa), which consists of 5,361 snps (song et al., 2013; akond et al., 2013). data analysis the snps significantly associated with the resistance to sds as judged by the phenotypic data were selected using the r software, basic packages for t-tests. data analysis was also performed by anova using jmp 11 (sas institute, cary nc, usa). the mean, standard error, and student t-test (p < 0.05) was recorded. two-way anova was used to look for interactions between the regions on chr. 18 and 20. all the phenotypic traits were analyzed for heritability following the methods described in kazi et al. (2008). table 1. snp markers and intervals significantly associated with the harrisburg sds data. snp intervals; allelic means and standard errors; probabilities; and variation explained are shown. markers and intervals lg disease index p > f r2 (% ) essex mean forrest mean ss715630114 ss715630131 g 17.2 ± 0.8 12.3 ± 1.1 0.0004 0.161 ss715630160 ss715630479 g 17.2 ± 0.7 11.8 ± 1.3 0.0007 0.175 ss715630520 ss715630660 g 17.3 ± 0.7 11.5 ± 1.2 0.0006 0.191 ss715630733 g 17.2 ± 0.7 11.9 ± 1.2 0.0006 0.164 ss715630903 ss715631000 g 17.3 ± 0.7 11.9 ± 1.2 0.0005 0.173 ss715631531 g 17.3 ± 0.7 11.5 ± 1.2 0.0006 0.191 ss715631642 g 16.3 ± 0.7 12.2 ± 1.5 0.0281 0.054 ss715632529 g 16.5 ± 0.8 13.0 ± 1.1 0.015 0.055 ss715632537 ss715632542 g 17.3 ± 0.7 12.5 ± 1.2 0.001 0.147 ss715632589 ss715632835 g 17.1 ± 0.7 13.12 ± 1.2 0.0067 0.097 ss715637419 ss715637459 i 0.16 ss715637485 ss715637550 i 17.0 ± 0.7 13.6± 1.2 0.02 0.07 ss715637647 ss715637657 i 16.7 ± 0.6 12.7 ± 1.7 0.0373 0.07 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 586 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg )table 2. snp markers and intervals significantly associated with the white county sds data. snp intervals; allelic means and standard errors; probabilities and variation explained are shown. figure 1. ranked nils showing a normal distribution of sds scores in harrisburg (a) and carmi, white county (b). markers and intervals lg disease index essex mean forrest mean p > f r2 ss715630114 ss715630131 g 0.09 ss715630160 ss715630479 g 28.9 ± 2.2 20.2 ± 3.4 0.0365 0.0563 ss715630520 ss715630660 g 30.5 ± 2.1 17.2 ± 2.9 0.0007 0.136 ss715630733 g 29.9 ± 2.0 17.2 ± 3.2 0.001 0.131 ss715630903 ss715631000 g 31.3 ± 2.3 16.4 ± 2.7 0.0001 0.187 ss715631531 g 29.9 ± 2.0 17.2 ± 3.2 0.001 0.131 ss715631642 g 0.0504 ss715632529 g 28.9 ± 2.1 20.1 ± 3.2 0.0292 0.047 ss715632537 ss715632542 g 31.1 ± 2.2 18.8 ± 2.6 0.0007 0.135 ss715632589 ss715632835 g 31.6 ± 2.2 18.9 ± 2.5 0.0009 0.149 ss715637419 ss715637459 i 30.9 ± 2.3 21.1 ± 2.6 0.0052 0.08 ss715637485 ss715637550 i 30.1 ± 2.4 21.1 ± 2.5 0.0091 0.074 ss715637647 ss715637657 i 29.6 ± 2.1 21.3 ± 3.4 0.0409 0.046 a b snp physical maps the snps that associated with the phenotypic data were selected to identify genomic regions and several candidate genes based on their positions in the soybean genome. those physical maps consisted of the snps that were segregating for the susceptible parent (essex) and the resistant parent (forrest), the monomorphic snps and the heterozygous or heterogenous snps. results sds dx trait data in nil60 lines 1-40 sds symptoms were less severe in the two locations in 1998, the highest dx value for harrisburg was 25 and arc was 15. the carmi field trial in 2000 had a more significant sds disease pressure, 58.1 being the highest dx value but ridgway had no detectable disease. therefore, this analysis only used the sds data from harrisburg and carmi, white county. trait data for sds showed non-normal distributions for dx (fig. 1). frequency distributions of traits showed there was evidence for bior tri-phasic distribution. traits representing field data, like dx, include some error variance that was reduced, but not removed, by replication. heritability values were moderate for dx (49 and 58%) respectively at each location. disease was more severe at carmi (where individual lines dxs ranged from 3-58) than harrisburg (where the individual lines dx ranged from 4-25). further, the genotype by environment interactions were significant, so means that pooled both locations data were not used. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 587 figure 2. ranked nils showing a normal distribution of sds scores in harrisburg (a) and carmi, white county (b). a b a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 588 dna marker analyses of polymorphic regions among 5,361 high quality snp scores made, just 49 of the chr.18 markers were polymorphic (supplemental table 1). most, 47-48, were significantly associated with the resistance to sds trait (fig. 2; table 1 and 2). they were physically mapped to one region of 73 markers. regions of monomorphism (15 encompassing the other 24 of the 73 markers within associated regions) were detected. these same regions were also monomorphic between the parents of the population, essex and forrest. therefore, the polymorphic region was divided into 14 regions, separated by the 15 monomorphic regions. eight of the polymorphic regions showed evidence of residual heterogeneity or heterozygosity suggesting heterozygosity was preserved allowing recombination to continue through many of the 13 generations. genotype 9 (ef 60-9) appeared to have preserved the most heterogeneity and/or heterozygosity. three of the regions were confirmed by microsatellite markers showing 2 bands on gels (not shown). recombination events had generated 10 rare genotype classes (g2-g11 in fig. 2 and 3) from the 2 common parental plant types (g1 and g12) developed from ril60, and still found in ril60. one of the common types (essex, susceptible type) was more abundant than the other (forrest, resistant type) suggesting non-intentional selection against resistance had occurred. three lines (ef 60-4, ef 60-11, and ef 60-17) were determined to be contaminants from another nil population based on the snp polymorphism analysis and so were excluded from the analyses. the marker data analyses indicated that there was a region of about 6.2 mbp where recombination events had occurred (g2-g11) on chr.18 (fig. 2; tables 1 and 2). there were 3 previously reported ssr markers within the region (reviewed by lightfoot 2015). they were satt570 (start position 3,162,724 end position 3,162,756 bp); satt130 (start position 4,639,943 end position 4,640,401 bp); and sat_403 (start position 6,169,553 end position 6,169,618 bp). satt570 was previously reported to be linked to cqrfs1. satt130 and a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 3. fine map of the region of chromosome 20 polymorphic in the ef60 nil populations in harrisburg (a) and carmi , white county (b). a b 589 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) sat_403 were previously reported to be linked to cqrfs. among the 5,361 high quality snp scores made just 12 of 21 were polymorphic on chr.20 in the region encompassing cqrfs5 (supplemental table 1). only 4-6 were associated with the sds trait (fig. 3; tables 1 and 2) at one or both locations. regions of monomorphism (4 encompassing 9 markers) were detected. essex and forrest parents were also monomorphic at these same snps. there were 5 polymorphic regions. two of those regions showed evidence of residual heterogeneity. recombination events had generated 3 rare genotype classes from the 2 common ef ril60 parental types. the marker data analysis indicated that there was a region of about 1.8 mbp where recombinant events had occurred on chr.20 (fig. 3). there was one ssr marker within the region, satt354 (start position 33,013,951 end position 33,430,010 bp). satt354 was previously reported to be linked to cqrfs5 (reviewed by lightfoot 2015). snp based sds trait association mapping on chr. 18 the snp to sds score association data from harrisburg appeared to show two regions associated with sds dx. one was large (2.7 mbp), from ss715630114 to ss715631531 (table 1; p < 0.007; n was 37). the region corresponded to the position of cqrfs1. the second was small, from ss715632537 to ss715632542, a 0.5 mbp region that corresponded to the position of cqrfs. however, at carmi there appeared to be three separated significant intervals, mapped between snp markers ss715630520 to ss715630540; ss715630903 to ss715631000; and ss715632537 and ss715632542 (table 2; p < 0.0001). the, second, middle putative qtl had not previously been detected. these finding suggested that there may be an additional qtl conferring resistance in this region. it might be named rfs19. it was mapped to a 0.06 mbp region. the first region was about 0.5 mbp and corresponded to the position of cqrfs1. the third region was about 0.05 mbp and corresponded to the position of cqrfs. on chr.20 the snp to sds score association data from harrisburg showed weak significance (table 1) across 2 of the 3 polymorphic regions. however, at carmi, white county, 2 of 3 polymorphic regions were associated with resistance to sds. one region was most strongly associated with sds dx in each location, but they differed. that suggested a 0.4 mbp region corresponded to the position of cqrfs5, but suggested that interactions were occurring. qtl interaction analyses the values of the 4 qtl segregating in the nil population were compared to determine the breeding value of each of the qtl. the loci cqrfs1 and the closely linked (0.2 mbp; 0.5 cm) rfs19 explained 14-19% (6-13 dx units) and (6-15 dx) 17-19 % of variation in trait respectively (table 1 and 2). cqrfs appeared to be a slightly weaker locus that explained just 14-15% of variation (5-12 dx) however it was only 2 mp (5 cm) away from the other 2 loci on chr.18. the cqrfs5 locus on chr.20 was weakest explaining just 4-9% of variation (7-8 dx units). beneficial alleles were all from forrest. interaction analyses were made using the common parental genotypes and treating the cluster on chr.18 as a single type (table 3). interactions appeared to be significant by two-way anova (p<0.05). the most resistant genotype had the forrest allele on both chromosomes (lgs g and i) when disease was severe. however, the chr.20 (i) locus appeared to have no value when disease was mild. notably, the genotype classes gf ie and ge if were rare (n =2 and 8 respectively at both locations). selection against those genotypes or drift in a small population may have occurred. caution should be exercised in interpreting the significance of the qtl interaction data. further experiments with larger populations are needed. discussion an archived nil population (ef60 lines 1-40) and its dna samples were used to confirm the mapping of three loci (cqrfs, cqrfs1 and cqrfs5) within two qtl previously found to underlie resistance to sds (iqbal et al., 2001; kassem et al., 2006; kazi et al., 2008; luckew et al., 2013) using newly available snp markers. however, an apparently additional interval (rfs19) with highly significant values was dissected within the chr.18 region based on data from carmi, white county in 2000. this may be due to the severe disease pressure in carmi, white county. nils showed good variation for sds dx because they were fixed to susceptible alleles at the 5 other known major loci for resistance to sds and scn in e × f (lightfoot et al., 2005; lightfoot 2008; 2015). snp mapping allowed a clearer picture of the recombination events in a nil population to emerge. the larger region of residual heterozygosity on chr.18 had the greater number of recombination events in it than the smaller region on chr.20. the recombination events allowed mapping of the qtl to small intervals (0.04-0.5 mbp) and suggested the existence of a new qtl in the cluster on chr.18. a map of cqrfs (between ss715632537 and ss715632542) was used to infer the location of 7 candidate genes (glyma.18. g070200 – glyma.18.g07800) that encoded; three as not yet named proteins with no pfams; a dna helicase tip49, which is also a tbp-interacting protein; a non-named protein containing an armadillo/beta-catenin-like repeat; a chaperonin-like wc epistasis gf ge if 16.9 (n=18) 34.2 (n=7) ie 19.9 (n=2) 28.6 (n=28) h epistasis gf ge if 11.6 (n=18) 17.1 (n=8) ie 10 (n=2) 17 (n=28) table 3. possible epistatic interactions among the loci on chromosomes 18 and 20 from the means of the two replicates. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 590 rbcx protein; and a heterogeneous nuclear ribonucleoprotein. a snp map of rfs19 (between ss715630903 and ss715631000) was used to infer the location of 5 candidate genes that encoded; a nodulin like major facilitator superfamily protein; a tetratricopeptide repeat (tpr)-like superfamily protein; an o-sialoglycoprotein endopeptidase in the serine threonine kinase family; an hxxxd-type acyl-transferase family protein; and a mitochondrial ribosomal death-associated protein 3 (glyma.18g055900 glyma.18g056300). none of these proteins were reported to be, or interact with, known partial resistance genes (triwitayakorn et al., 2005; srour et al., 2012). however, the genes that encode them may be good candidates for partial resistance genes and so will be tested in future experiments. the value of the loci for breeding on the two chromosomes was shown to be unequal, as reported by luckew et al. (2013). chr.18 loci were equal in the amount of dx they controlled, but were closely linked so only a few recombinant lines contributed to differences. chr.18 loci were not associated with resistance to fungal exudates (swaminathan et al., 2016) so the loci may not underlie toxin resistance. kazi et al. (2008) found loci in this region controlled root resistance (infection severity) but not leaf scorch in flyer by hartwig derived rils. however, in that population the locus rfs19 linked to satt130 was associated with dx but not root resistance. they noted the satt130 marker was not linked to any other chr.18 marker. genome sequence now shows the satt130 amplicon (gi:14969847) has strong paralogs (5.8e-2 >e<3.9e-4) on 7 other chromosomes (3, 6, 7, 8, 10, 11 and 9) all of which contain at least one qtl. therefore, it appears the loci detected by satt130 in e × f and f × h are not on the same chr. as was inferred previously by lightfoot (2015). the chr.20 locus cqrfs5 controlled much less of the variation in dx than cqrfs, cqrfs1 and rfs19. however, there were 18 loci associated with resistance to fungal exudates (swaminathan et al. 2016) in their cross made with a hartwig derived line (ls94-3207; schmidt et al., 2004). therefore, cqrfs5 may underlie toxin resistance. some evidence for epistasis was found. the chr.20 locus was only beneficial with the forrest allele present on chr.18. in fact the beneficial allele was change when the essex allele was present on chr.18. however, the low abundance of 2 of the 4 genotypes weakened those conclusions. the lower abundance of the gf ie and ge if might have been caused by drift or unintentional selection. in future we will isolate more recombination events in these nil populations from residual heterogeneity and heterozygosity found. the confirmed qtl have been sent to the soybean genetics committee requesting the assignment of names cqsds003-006. conflicts of interest none statement of contributions yi chen lee carried out the marker work, analyzed the data and made the first draft of the paper. victor njiti carried out the field work and made the sds scores. javed iqbal made the nil dnas. david. a. lightfoot and stella k. kantartzi edited the paper and added analyses and insights. stella k. kantartzi provided the snp data. paul gibson provided the e × f population to us in 1991 and developed the nil population strategy with his team. acknowledgements thanks are due to the field team at siuc from 1996 to date. references akond m, s liu, l schoener, ja anderson, sk kantartzi, k meksem, q song, d wang, z wen, da lightfoot da, and ma kassem (2013) snp-based genetic linkage map of soybean using the soysnp6k illumina infinium bead-chip genotyping array. j plant genome sciences 1 (3): 80–89. aoki t, k o’donnell, y homma, and ar lattanzi (2003) sudden death syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the fusarium solani species complex: f. virguliforme in north america and f. tucumaniae in south america. mycologia 95: 660–684. bao y, je kurle, g anderson, and nd young (2015) association mapping and genomic prediction for resistance to sudden death syndrome in early maturing soybean germplasm. molec. breed. 35: 128–130. de farias-neto af, r hashmi, me schmidt, 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effects of diseases on soybean yields in the united states 1996 to 2007. plant health progr doi:10.1094/php-2009-0401-01-rs. yuan j, r bashir, g salas, h sharma, ay srour, and da lightfoot (2012) new approaches to selecting resistance or tolerance to sds and fusarium root rot. j plant genom sci 1:10–17. atlas journal of biology 2 (2): 125–129, 2013 doi: 10.5147/ajb.2013.0083 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) pathogenic capacity of botrytis cinerea on leaves of pyrus mamorensis, an endemic tree of mamora forest in morocco zineb sellal, jamila dahmani, rachid benkirane, amina ouazzani touhami, and allal douira* laboratoire de botanique et protection des plantes, département des sciences de la vie, faculté des sciences, b. p. 133, université ibn tofail, kénitra, morocco. received: june 28, 2012 / accepted: september 15, 2012 __________________________________________________ * corresponding author: douiraallal@hotmail.com 125 abstract a survey in the mamora forest was done in the spring of 2010 and revealed that 67% of buds and 27% of leaves of pyrus mamorensis (trabut) samples collected had lesions with a gray felting. the pathogenic fungus was identified as botrytis cinerea by the filter – paper technic. koch´s postulate was verified by inoculating healthy leaves. the estimated disease severity on p. mamorensis leaves was respectively 75.56% and 68.81% for inoculation by conidial suspension and the mycelial disks. conidia production of botrytis cinerea on inoculated leaves by conidial suspension was 1.03.105 conidia.cm-2 and by mycelial disks was 0.60.105 conidia.cm2. this was the first report of gray mold disease of mamora pear caused by botrytis cinerea in morocco. keywords: morocco, pyrus mamorensis, botrytis cinerea, gray mold, inoculation. introduction mamora pear (pyrus mamorensis, trabut) is a perennial plant belonging to rosaceae. its abundant in mamora forest (morocco) is particular but does not form large stands. this species does not receive any conservation effort despite its endemism and rarity (fennane et al., 1998). in addition, the reasons of the declining health of this tree over the long term and especially in periods of drought where stress by human activity, livestock grazing and attack by pestalotia subcuticularis (yamni et al., 2006). other fungi were also isolated from leaves, flowers, fruits or trunk of this tree as aspergillus niger, a. fumigatus, a. versicolor, alternaria alternata, cladosporium herbarum, drechslera australiensis, mucor sp., rhizopus stolonifer, epiccocum nigrum, trichoderma harzianum, curvularia lunata, hysterium pulicare, h. asymetricum, and trematosphaeria pertusa (sellal et al., 2012). in the spring of 2010, the survey in the mamora forest revealed that 67% of 100 buds and 27% of 100 leaves collected from eight trees of pyrus mamorensis had lesions with a gray felting. leaf blights were gray with brown contour (fig. 1a). the other lesions were brown spots with a gray center (fig. 1b). the buds were brown with a gray felting (fig. 1c). this was the first time that these symptoms were observed on pyrus mamorensis, endemic species in morocco. the objective of this work was to achieve isolation of the pathogenic fungus from the diseased leaves and buds of pyrus mamorensis trabut and to verify the koch´s postulate. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 126 material and methods twenty leaves and ten buds showing lesions with a gray felting collected from eight trees of pyrus mamorensis were cut into fragments, washed in tap water, disinfected with alcohol and placed in petri dishes on filter paper moistened with sterile distilled water. after incubation at 22°c under continuous lighting for 2–3 days, leaves and buds fragments were examined using an optical microscope and the conidia emerging from the lesions were taken with a capillary tube and displayed on agar medium (15 g agar-agar, distilled water 1000 ml). a single spores were placed on psa medium (potato sucrose agar: 200 g potato, 20 g sucrose, 15 g agar-agar, distilled water 1000 ml) and incubated in the dark for five days at 28°c (benkirane, 1995). the developed colonies represented as a pure cultures, were retained and used for species determination. pathogenicity tests were realized by inoculating healthy leaves of pyrus mamorensis using two technics. the surface of sixty leaves was disinfected with 5% sodium hypochlorite, washed with sterile distilled water and dried on a filter paper. fifteen leaves were inoculated with mycelial disks of the fungus, the other fifteen by a conidial suspension adjusted to a final concentration of 105 conidia.ml-1 with sterile distilled water containing 0.05% tween 20 and 5% gelatin. thirty leaves were used as a control, the half of them were inoculated with distilled water containing tween 20 and gelatine and the other half with medium plugs. every bunch of five leaves was placed in 120-mm a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) petri dish containing small glass beads and sterile distilled water. inoculated leaves were incubated at 28 ± 1 °c under black plastic sheeting that was removed after 24 hrs. the disease severity was scored after 7 days of inoculation using the scale of stover modified by gauhl et al., (1995). the severity index (is) of disease was calculated using the formula: is= (σnb/(n – 1) x t) ×100 n = number of leaves for each degree of the scale b = degree of the scale n= number of the degrees used in the scale t= total number of the scored leaves the conidia production (conidia cm-2) of botrytis cinerea on the inoculated mamora pear leaves was estimated according to the technic of hill and nelson (1983). ten days after inoculation, the leaves those had shown lesions were cut into pieces of 1 cm2 and placed in 90 mm petri dishes on three filter paper discs moistened with sterile distilled water. the dishes were inclass 1 2 3 4 5 6 7 % diseased leaf area -0,5% of the limbus with symptoms 0,6 à 5% 6 à 15% 16 à 30% 31 à 50% 51 à 80% 81 à 100% a b c fig. 1. botrytis cinerea symptoms developed on leaves (a, b) and buds (c) of pyrus mamorensis. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 127 cubated for 48 hours under continuous fluorescent lighting. then each fragment was placed in a test tube containing 1 ml of sterile distilled water and agitated by a vortex mixer for 2 min. the conidia of the pathogen were counted using a malassez slide under an optical microscope at magnification × 100 with 10 counting of each sample. results and discussion on psa, the growing colonies of the isolated fungus were white at first, gray as it ages and whitish gray in reverse (fig. 2a). the mycelium was very large and dense, 12 µm of wide, cylindrical, often banded, especially when cultures are older. it appeared light brown to olive. the conidiophores were clear colored and shiny. their back always branched (fig. 2b) and supported small sterigmata. the conidia were ellipsoidal or obovoid, with slightly protuberant hilum colorless to pale brown 7-15 x 5-9 µm (fig. 2c) and abundant sclerotia developed as the fungus culture ages. based on the morphological description ellis (1971), the fungus was identified as botrytis cinerea pers. 1794. botrytis species were important pathogens of nursery plants, vegetables, ornamental, field and orchard crops, stored and transported agricultural products (elad et al., 2007). the botrytis cinerea pers. ex fr. caused gray mold disease in a wide variety of hosts (coley-smith et al., 1980; elad et al., 2004), and was a serious economic problem in crops such as table grapes, vines, strawberries, raspberries, lettuce, cucumbers, broad beans, tomatoes, beans, flowers, and forest plants produced in containers (elad et al., 2004). gray mold was an important sanitary problem in pinus radiata d. don and eucalyptus globulus labill. in chilean forest nurseries (butin and peredo, 1986). botrytis sp. and b. cinerea were also isolated from the young diseased plants of cedrus atlantica (endl.) manetti ex carriere respectively in morocco (bakry et abourouh, 1992) and in france (abourouh et morelet, 1999). b. cinerea seemed to prefer senescent leaves but were also found in other stages in quercus rotundifolia lam. in a holm oak forest (high atlas, morocco) (sadaka and ponge, 2003). two days after inoculation of leaves of pyrus mamorensis with conidial suspension, the first symptoms of botrytis cinerea appeared at the limbus and then covered the entire leaf surface. the lesions were brown to black (fig. 3b). the second type of lesions were black that appeared first at the tips then grew and covered the leaf surface (fig. 3c).the lesions appeared also on the petioles. the inoculated leaves with mycelial disks showed brown lesions, grayish to blackish in the disk and then cover leaf surface with blackening of the petiole (fig. 4b and c). seven days after inoculation, the estimated disease severity on leaves of p. mamorensis was 75.56% and 68.81% for inoculation by conidial suspension and the mycelial disks. no disease was observed on the control leaves (fig. 3a and fig. 4a). ten days after inoculation, botrytis cinerea produced conidia abundantly on leaves of p. mamorensis inoculated both by conidial suspension (1.03.105 conidia.cmˉ2) and the mycelial disks a b c fig. 2. botrytis cinerea on pda (a), conidiophores (b) and conidia (c) on cotton blue (×400). (0.60.105 conidia.cmˉ2). the pathogen was a common fungus that damaged flowers, leaves, stems, fruit and other parts of many plants (ellis, 1971; elad and shteinberg cited by o´neill et al., 1997). the fungus exists in different habitats as mycelia, microand macroconidia, chlamydospores, sclerotia, apothecia and ascospores and these are dispersed by diverses means (jarvis, 1980). if it is to infect, the fungus must conquer space (zadocs and scein, 1979) that is to move from the primary source and land on susceptible tissue. each part of the fungus thallus can serve as a dispersal unit. these propagules are dispersed by wind, rain and insect. botrytis cinerea was observed causing gray mold in pyrus communis (sommer et al., 1985; shiva, 1989; crous et al., 2000), p. serotina (cho and shin, 2004) , p. serotina var. culta and p. betulifolia (kobayashi, 2007). however, to our knowledge, this was the first report of gray mold disease of pyrus mamorensis caused by botrytis cinerea in morocco. 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k, n dohou, a outkoumit, a ouazzani touhami, and a douira (2006) first report of pestalotia subcuticularis on pyrus mamoremsis in morocco. phytopathologia mediterranea 45: 40-42. zadocs jc and rd schein (1979) epidemiolgy and plant disease managment. oxford university press inc., new york, usa, 427p. atlas journal of biology 2016, pp. 267–273 doi: 10.5147/ajb.2016.0140 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) influence of drought stress on several root traits and their correlation with seed protein and oil contents in soybean ambrocio zenis1, stella kantartzi2, khalid meksem2, and my abdelmajid kassem1* 1 plant genetics, genomics, and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc 28301, usa; 2 department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901, usa. received: may 2, 2016 / accepted: june 3, 2016 __________________________________________________ * corresponding author: mkvzant@siu.edu 267 abstract the important crop soybean [glycine max (l.) merr.] is cultivated worldwide and the us is its number one exporter. however, farmers face many challenges in cultivating soybeans, including drought and diseases that reduce yields drastically. the root system is very important for plants, including crops, because it receives water and minerals from the soil so that the plant/crop can photosynthesize, grow, and increase its yield. the objective of this study was to grow the ‘md 96-5722’ by ‘spencer’ recombinant inbred line (ril) population (n=86) in the greenhouse under normal (group i) and drought stress (group ii) conditions, and compare the root length (rl), root surface area (rsa), average root diameter (ard), and average root volume (arv) in the two groups of plants. whinrhizo software was used to measure the root traits and spsstm was used to evaluate population performance under normal and drought conditions. jmptm was used to compare the root traits under normal and drought conditions, and to analyze the correlation between root traits, protein and oil contents. the results showed that there is a huge variation in these traits among the parents ‘md 96-5722’ and ‘spencer’, and among their rils. for group i plants, the rl of parents and rils ranged from 20.67 cm to 2,327.88 cm; the rsa ranged from 4.57 cm2 to 1,176.79 cm2; the ard ranged from 0.38 mm to 4.04 mm; and the arv ranged from 0.08 cm3 to 47.34 cm3. for group ii plants, the rl of parents and rils ranged from 15.70 cm to 3,562.42 cm; the rsa ranged from 4.15 cm2 to 829.72 cm2; the ard ranged from 0.24 mm to 5.74 mm; and the arv ranged from 0.03 cm3 to 23.67 cm3. it is clear from the results that group introduction soybean [glycine max (l.) merr.] is a leguminosae crop grown for its oil, proteins, isoflavones, and other bioactive compounds. it also widely used in the food industry and as a biofuel (rosenthal et al., 2001; kinney and clemente, 2005). according to usda, north carolina planted soybeans in 1.82 million acres and produced 57.3 million bushels for a total of $487 million in 2015 (usda, 2015). in north carolina, soybean production amount was 31 million bushels from 1.4 million acres and a total value of $174 million (ncsuce, 2015). soybeans are usually planted in early soybean production system (esps) this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i plants have higher means of rl [572.58 cm vs. 537.33 cm], rsa [201.20 cm2 vs. 165.50 cm2], ard [3.96 mm vs. 1.45 mm], and arv [6.31 cm3 vs. 5.61 cm3] compared to group ii plants which demonstrates that drought-stressed plants have reduced overall plant growth and development. however, statistically, these differences were not significant; therefore, further studies with several replicates should be conducted both in the greenhouse and the field in order to determine the effects of drought stress on the ‘md 96-5722’ by ‘spencer’ rils. moreover, studies of quantitative trait loci (qtl) mapping of the root traits studied here are underway to genetically map qtl for these root traits in this soybean ril population. keywords: root length (rl), root surface area (rsa), average root diameter (ard), average root volume (arv), md 96-5722, spencer. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 268 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) (april) or conventional soybean production system (csps, mayjune) across the us (bowers, 1995; taylor et al., 2005; ouertani et al., 2011). soybean plants grown in esps have increased yield and are drought-tolerant compared to plants grown in csps (taylor et al., 2005; ouertani et al., 2011). plant breeders and researchers are trying to develop cultivars that are resistant to diseases and drought stress. drought stress reduces the ability of roots to absorb water and nutrients from the soil and it has been demonstrated that plants with vigorous and extensive root systems are able to cope with drought and become water deficit-tolerant (wdt) (pandey et al., 1984; benjamin and nielsen, 2006; reubens et al., 2006). during drought stress seasons, plants usually change the distribution of their roots and grow them deeper to absorb water and minerals as a mechanism of drought tolerance (pandey et al., 1984; benjamin and nielsen, 2006). in another study, drought-stressed soybean plants have a sharp decrease in their photosynthetic rates, leaf water potentials, starch concentration, and leaf sucrose contents, and pod growth (liu et al., 2004). defending themselves against drought stress, soybean plants close their stomata to escape dehydration but the overall photosynthetic rate is reduced by limiting co2 intake (via stomata), causing a reduced metabolism that might be reversible with enough supply or availability (kramer et al., 1995; specht et al., 2001; flexas and medrano, 2002; liu et al., 2004; gregory, 2006; mastrodomenico et al., 2013). the reduced photosynthetic rate has a drastic effect on reducing grain yield. the physiological effects of drought can be estimated by measuring the leaf relative water contents (rwc), root architecture, and reactive oxygen species (ros) (hunt et al., 1987; reddy et al., 2004; chiante et al., 2006; reubens et al., 2006; miller et al., 2010; comas et al., 2013). during seed fill and pod development stages, soybean plants, like other crops, need an adequate supply of water and any drought stress will decrease developmental and physiological functions that will lead to high decreased seed yields (kadhem et al., 1985; kramer et al., 1995; chiante et al., 2006; comas et al., 2013). to withstand drought stress, soybean plants and other legumes use several important mechanisms. among these are growing extensive root systems, water use efficiency, and high efficient nitrogen fixation (nunberg et al., 2006; mastrodomenico et al., 2013; hussain et al., 2014). it is known that drought-stressed plants tend to grow extensive root systems with continuous elongation at low root water potentials (westgate and boyer, 1985; kramer et al., 1995; bing et al., 2005; nunberg et al., 2006; mastrodomenico et al., 2013; hussain et al., 2014). the objectives of this study are to (1) grow the ‘md 96-5722’ by ‘spencer’ recombinant inbred line population (n=86) of soybean under drought stress in the greenhouse, study the influence of drought stress on several root traits such as root length (rl), root surface area (rsa), average root diameter (ard), and average root volume (arv), and (2) investigate the correlation between root trait measurements, protein and oil contents of ‘md 96-5722’ by ‘spencer’. materials and methods plant material in this study, the ‘md 96-5722’ by ‘spencer’ recombinant inbred line (ril) population (n=86) of soybean was used (akond et al., 2013). these seeds were obtained at the national soybean research laboratory (nsrl) in 2006 from dr. kantartzi. the parents of ‘md 96-5722’ were from a cross between ks4644 and ‘corsica’ ks4694, whose parents were ‘shermon and toano’. ‘spencer’ came from the crossings between a75305022 by ‘century’ (akond et al., 2013). experimental conditions the experiment was performed during 2013 (june 2013 to september 2013) in a greenhouse at fayetteville state university, fayetteville, north carolina. four seeds of parents, ‘md 96-5722’, ‘spencer’, and each ril (n=86; n is the population size) were sown at 1.5–2.0 inches deep in 2-gallon pots (20.32 x 17.78 cm) filled with potting soil of organic material (miracle grow potting mix) under daylight temperatures of 24–34o c. the pots were set on a brown sheet, avoiding water capillarity from the bottom. the pots were divided into two groups, a control group (n=86) for normal growth and a treatment group (n=86) for drought conditions. the treatment group would undergo drought stress during the entire growth development. first week, normal watering was given to each seedling, 3.76 liters of water. control group would receive normal watering throughout the whole study or when needed. treatment group was submitted through intense drought stress for two weeks after normal watering. afterwards, watering the plants at least once a week and at 1.5 liters of water. root extraction method after eight weeks of planting, roots were carefully extracted from the pots one by one, placed inside a tray filled with water and washed by hand. later, the roots were left to dry and have just enough moisture in order to separate the lateral roots from the primary root. roots were separated by using a plastic transparent wrap, the wraps texture allowed for lateral roots to separate easily without damaging the root and minimizing loss of fine lateral roots. afterwards, to preserve the roots, they were placed inside ziploc bags and placed in a refrigerator for later analysis. several root traits such as root length (rl), root surface area (rsa), average root diameter (ard), and average root volume (arv) were measured. protein and oil contents quantification seed protein and oil contents have been measured by nacer bellaloui at the usda-ars, stoneville, ms according to bellaloui et al. (2009). briefly, 25 g of seed from each plant were grinded using a lab mill 3600 (perten, springfield, il). near infrared reflectance was used for analyses using a diode array feed analyzer ad 7200 (perten, springfield, il) (bellaloui et al., 2009). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) root trait analysis the root traits root length (rl), root surface area (rsa), average root diameter (ard), and average root volume (arv) were measured in both groups (control and experimental groups) using an epson scanner and the whinrhizo software package 3.10 version (regent instruments inc. quebec, canada). the images were visualized and root data analyzed using the epson scanner and the whinrhizo software. root traits analyzed were root length (rl), root surface area (rsa), average root diameter (ard), and average root volume (arv). statistical analysis a distribution chart was constructed based on the differences in root trait measurements in (ril) population. spsstm statistical software package was used to evaluate population performance under normal and drought conditions. the evaluation was based on mean differences, estimation of variance and descriptive comparisons. jmptm was used to compare the root length (rl), root surface area (rsa), and average root diameter (ard) and average root volume (arv) under normal and drought conditions. also, jmptm statistical software package was used to analyze the correlation between root traits, protein and oil content. recombinant inbred lines of the parents were also compared along with the rils evaluation. t-test and pearson correlations were performed to compare the root traits in plants grown in the two conditions and between the root traits and protein and oil contents. results and discussion comparison of root traits data examples of scanned images of the root system of ril 2 under normal water regime and drought stress are shown in figure 1. measurements of root length (rl), root surface area (rsa), average root diameter (ard), and average root volume (arv) of ‘md 96-5722 by ‘spencer’ recombinant inbred lines root systems under normal (group i) and drought (group ii) growth conditions have been recorded. for control plants (group i), the root length (rl) of parents and rils ranged from 20.67 cm (ril 63) to 2,327.88 cm (ril 2); the root surface area (rsa) parents and rils ranged from 4.57 cm2 (ril 41) to 1,176.79 cm2 (ril 2); the average root diameter of parents and rils ranged from 0.38 mm (ril 80) to 4.04 mm (ril 43); and the average root volume (arv) of parents and rils ranged from 0.08 cm3 (ril 41) to 47.34 cm3 (ril 2). for drought-stressed plants (group ii), the root length (rl) of parents and rils ranged from 15.70 cm (ril 80) to 3,562.42 cm (md, 2,497.9 cm for ril 87); the root surface area (rsa) parents and rils ranged from 4.15 cm2 (ril 46) to 829.72 cm2 (md, 669.62 cm2 for ril 87); the average root diameter of parents and rils ranged from 0.24 mm (ril 7) to 5.74 mm (ril 80); and the average root volume (arv) of parents and rils ranged from 0.03 cm3 (ril 46) to 23.67 cm3 (ril 32). our results showed that both rl, rsa, ard, and ard have been significantly reduced by drought stress which is in agreement with several studies reporting the influence of drought on root traits (xiong et al., 2006, pandey and shukla 2015). for example, xiong et al. (2006) reported that drought stress reduced significantly lateral root growth in arabidopsis thaliana as a mechanism of drought adaptation (xiong et al., 2006). garay and wilhelm (1982) studies the effect of drought stress on root traits of two soybean isolines of ‘harosoy’ (harosoy normal (hn) and harosoy dense (hd)) and found that the root systems have been concentrated in the upper 0.15 m of the soil. however, after one month of drought stress, the root systems were denser at 0.90-1.2 m layer of the soil which means that the root system grew deeper into the soil to pump more water and mineral (garay and wilhelm, 1982). however, another study reported effect of drought stress on root distribution (benjamin and nielsen, 2006). statistical data analysis root traits data for the two groups (control plants – group i and drought stressed plants – group ii) were analyzed for the mean values, standard deviation, range, and coefficient of variation and the results are shown in table 1. in group i plants (normal water conditions), the mean of the average of root length (rl) was 572.58 cm with a coefficient of variance of (cv) of 0.95; the mean of the average of root surface area (rsa) was 201.20 cm2 with a coefficient of variance of (cv) of 1.13; the mean of the average root diameter (ard) was 3.96 cm with a coefficient of variance of (cv) of 6.01; and the mean of the average root volume (arv) was 6.3 cm3 with a coefficient of variance of (cv) of 8.02 (table 1). in group ii plants (water stress conditions), the mean of the average of root length (rl) was 537.33 cm with a coefficient of variance of (cv) of 1.25; the mean of the average of root surface area (rsa) was 165.5 cm2 with a coefficient of variance of (cv) of 1.01; the mean of the average root diameter (ard) was 1.45 cm with a coefficient of variance of (cv) of 7.89; and the mean of the average root volume (arv) was 5.6 cm3 with a coefficient of variance of (cv) of 1.03 (table 1). it is clear from the results that group i plants have higher means of root length (rl) [572.58 cm vs. 537.33 cm], root surface area (rsa) [201.20 cm2 vs. 165.50 cm2], average root di269 (a) (b) figure 1. scanned images of the root system of ril 2 under normal water regime (a) and drought stress (b). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ameter (ard) [3.96 mm vs. 1.45 mm], and average root volume (arv) [6.31 cm3 vs. 5.61 cm3] compared to group ii plants (table 1) which demonstrate that drought-stressed plants have reduced overall plant growth and development. a paired-samples t-test shown in table 2 was used to demonstrate whether there was a significant mean difference of root measurements (rl, rsa, ard, arv) between normal and drought growth in ‘md 96-5722’ by ‘spencer’ population planted under greenhouse conditions. levels of root trait measurements for root length elicited a difference of 58.60821 (95% confidence interval (ci), -167.15727 to 284.37370) cm when comparing normal and drought growth conditions, t (61) = .519, p > .005. the comparison for root surface area (cm2) displayed a difference of 42.32496 (95% ci, -34.88817 to 119.53809) and a t (61) = 1.096, p > .005. the average root diameter in comparison showed a difference of -.30523 (95% ci, -.63563 to .02518) mm and t (61) = -1.847, p < .005. last, when comparing average root volume, the measurements demonstrated a difference of 1.06339 (95% ci, -1.56043 to 3.68721) cm3 with a t (61) = .810, p > .005. there is no significant difference between root length, root surface area, and average root volume. however, there was statistical difference between normal and drought growth conditions for average root diameter. a paired statistic test was used to determine the different effects between two experimental conditions for each of the four root traits. in (table 2), results indicate a decrease for root measurements of length (cm) (529.14 -77.299), surface area (cm2) (167.5920.069), root volume (cm3) (5.660.719), and an increase in average diameter (1.42-0.140). in normal growth conditions there was a slight increase in measurements in length (587.75-72.141), 270 root traits n range minimum maximum mean sd cv% normal growth length (cm) 69 2307.201 20.679 2327.881 572.580 548.901 0.958 surface area (cm2) 69 1172.221 4.572 1176.793 201.208 227.754 1.131 average diameter (mm) 69 3.653 0.388 4.042 1.090 0.537 4.927 average root volume (cm3) 69 47.260 0.080 47.340 6.311 8.028 1.272 drought conditions length (cm) 75 2482.200 15.700 2497.901 497.001 582.135 1.171 surface area (cm2) 75 665.496 4.156 669.653 156.646 150.044 0.957 average diameter (mm) 75 5.499 0.245 5.745 1.449 1.154 7.961 average root volume (cm3) 75 23.638 0.039 23.677 5.610 5.796 1.033 table 1. comparison of root trait measurements of ‘md 96-5722 by ‘spencer’ rils under normal growth conditions and drought. n: population size (69 plants in group i and 75 plants in groups ii survived among 86 planted plants in total). root traits mean n sd pair 1 length-normal 587.753 62 568.043 length-drought 529.145 62 608.654 pair 2 surface area-normal 209.915 62 235.000 surface area-drought 167.590 62 158.028 pair 3 average diameter-normal 1.112 62 .556 average diameter-drought 1.417 62 1.105 pair 4 volume-normal 6.726 62 8.351 volume-drought 5.663 62 5.660 table 2. t-test showing summary of paired statistics for the two experimental conditions for each of the four root traits listed. for each condition we display the mean, number of participants (n), the standard deviation and standard error. n: population size. root traits n correlation significance level pair 1 length (normal)-length (drought) 62 -.141 .276 pair 2 surface area (normal) & surface area (drought) 62 -.165 .200 pair 3 average diameter (normal) & average diameter (drought) 62 -.131 .310 pair 4 volume (normal) & volume (drought) 62 -.053 .685 table 3. the pearson correlation between the two conditions, whether the difference between the means of the two conditions was large enough not to result, and last, 95% confidence interval was calculated. n: population size. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) surface area (209.91-29.845), volume (6.73-1.060) and a decrease in average diameter (1.11-.071). a paired sample correlation test was used to determine the significance between the two conditions and the root traits demonstrated no correlation between rl, rsa, ard, and arv under drought and normal conditions. there is no correlation when compared to parents of ril (tables 3 and 4). in a recent study, fenta et al. (2014) studied three soybean cultivars: a drought-sensitive (a5409rg), a drought-tolerant (‘jackson’), and an intermediate drought-tolerant cultivar (‘prima 2000’). they found that prima 2000 outperformed the other cultivars under drought stress with the highest grain yield, shoot biomass, and nodules. they also found a positive correlation between nodule size and seed yield and shoot biomass in both control and water stressed plants (fenta et al., 2014). in general, drought stress is a negative environmental condition affecting soybean production, growth and development (garay and wilhelm, 1982; benjamin and nielsen, 2006; du et al., 2009; fenta et al., 2014). this demonstrates that the carefully chosen root traits can be used to screen for drought tolerance in soybean. increasing water deficit tolerance (wdt) increases agricultural plant productivity. since the roots are the main organs for water and minerals uptake, they play a vital role in agricultural plant productivity. water stress or water deficit (wd) also decreases shoot biomass, leaf cuticular waxes, seed yield, nitrogen fixation, and seed development (purcell and king, 1996; serraj et al., 1999; kim et al., 2007). root systems are reported to be critical to cope with wd and increase yield. for example, deep and dense roots with high abilities of penetration and branching into the soil help rice plants to be drought tolerant (pandey and shukla, 2015). correlation between root traits and seed protein and oil contents a pearson correlations test was done between roots traits of group i (normal) and group ii (drought), protein and oil content as shown in table 5 and table 6. the frequency distribution charts for all root traits showed different measurement distributions. in group i plants, a positive correlation was observed between all root traits studied (rl, rsa, ard, and arv) and seed protein content while a negative correlation was observed between all these root traits and seed oil content (table 5). in group ii plants, a negative correlation was observed between rl, rsa, and arv and seed protein content while a positive correlation was observed between ard and protein content. a negative correlation was observed between rl, rsa, and arv and seed oil content while a positive correlation was observed between ard and seed oil content (table 6). several studies reported correlations between shoot traits and protein and oil contents in soybean (johnson et al., 1955; mansur et al., 1995; soybase, 2016); however, studies reporting correlations between root traits and protein or oil contents are non-existent our knowledge to date. in conclusion, we have demonstrated that water deficit decreased rl, rsa, and arv but not ard in soybean. it is very important to understand the root architecture and root traits under wd to be able to increase seed yield, seed quality, and crop productivity which will enhance the development of wdt variet271 root traits mean sd std. error mean 95% confidence interval of the difference t df sig. lower upper pair 1 length (normal) – length (drought) 58.608 889.006 112.903 -167.157 284.373 .519 61 .606 pair 2 surface area (normal) – surface area (drought) 42.324 304.045 38.613 -34.888 119.538 1.096 61 .277 pair 3 average diameter (normal) – average diameter (drought) -.305 1.301 .165 -.635 .025 -1.847 61 .070 pair 4 volume (normal) – volume (drought) 1.063 10.331 1.312 -1.560 3.687 .810 61 .421 table 4. paired samples test. the pearson correlation between the two conditions, whether the difference between the means of the two conditions was large enough not to result, and last, 95% confidence interval was calculated. sig.: significance level. n correlation sig. pair 1 length & protein 69 0.086 0.480 pair 2 length & oil 69 -0.240 0.047 pair 3 surface area & protein 69 0.113 0.354 pair 4 surface area & oil 69 -0.301 0.012 pair 5 average diameter & protein 69 0.084 0.492 pair 6 average diameter & oil 69 -0.293 0.015 pair 7 volume & protein 69 0.077 0.531 pair 8 volume & oil 69 -0.294 0.014 table 5. the pearson correlation between root traits from group i (normal growth), protein and oil content. sig.: significance level. n correlation sig. pair 1 length & protein 75 -0.225 0.052 pair 2 length & oil 75 0.221 0.057 pair 3 surface area & protein 75 -0.304 0.008 pair 4 surface area & oil 75 0.246 0.034 pair 5 average diameter & protein 75 0.029 0.804 pair 6 average diameter & oil 75 -0.026 0.827 pair 7 volume & protein 75 -0.233 0.044 pair 8 volume & oil 75 0.148 0.204 table 6. the pearson correlation between root traits from group ii (drought growth), protein and oil content. sig.: significance level. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ies and ensure food security. acknowledgments the authors would like to thank the department of defense (dod) for funding this work through the grant# w911nf-11-1-0178 to m.a.k and s.k. we thank ms. pam ratcliff and the rest of the undergrad students’ crew at fsu for taking care of the plants in the greenhouse and field, and dr. nacer bellaloui at usda-ars, stoneville, ms, for protein and oil analysis. references akond m, s liu, l schoener, ja anderson, sk kantartzi, k meksem, q song, d wang, z wen, da lightfoot, and ma kassem (2013) a snpbased genetic linkage map of soybean using the soys¬np6k illumina infinium beadchip genotyping array. journal of plant genome sciences 1 (3): 80–89, 2013. doi: 10.5147/jpgs.2013.0090. bellaloui n, jr smith, jd ray, and am gillen (2009). effect of maturity on seed composition in the early soybean production system as measured on near-isogenic soybean lines. crop sci. 49: 608–620. doi:10.2135/cropsci2008.04.0192. benjamin jg and dc nielsen (2006) water deficit effects on root distribution of 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westgate me and js boyer (1985) osmotic adjustment and the inhibition of leaf, root, stem and silk growth at low water potentials in maize. planta 164 (4): 540–549. xiong l, rg wang, g mao, and jm koczan (2006) identification of drought tolerance determinants by genetic analysis of root responses to drought stress and abscisic acid. plant physiology. 142:10651074. 273 atlas journal of biology 2016, pp. 308–312 doi: 10.5147/ajb.2016.0147 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) qtl underlying reniform nematode resistance in soybean cultivar hartwig yi-chen lee1,2, david a. lightfoot1,2, james anderson1,2, robert t. robbins3, and stella k. kantartzi1,2,* 1 plant biotechnology and genomics core-facility, department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901; 2 the illinois soybean center (center for excellence in soybean research, teaching and outreach), southern illinois university, carbondale, il 62901; 3 department of plant pathology, cralley-warren research center, university of arkansas, fayetteville, ar 72701. received: september 17, 2016 / accepted: december 1, 2016 __________________________________________________ * corresponding author: stella.kantartzi@siu.edu 308 abstract nematodes are one of the most destructive plant-parasitic pests in soybeans [glycine max (l.) merrill]. among the nematodes, soybean cyst nematode (scn, heterodera glycines ichinohe), southern root-knot nematode [rkn, meloidogyne incognita (kofoid and white) chitwood], and reniform nematode (rn, rotylenchlus reniformis linford and oliveria) are often the most problematic in soybean yield production. the plant introduction pi437654 has been used previously to map rn quantitative trait loci (qtl). however, ‘pi437654’ is non-domesticated. ‘hartwig’ was the first domesticated cultivar to introgress some of the resistances from pi437654. the aims here were to map qtl underlying rn resistance in hartwig. a cross between flyer and hartwig (n=92) was created to map qtl that underlie both scn and rn resistance. the f × h population was phenotyped at the nematology lab at the university of arkansas in 2014 and 2015 the f × h was genotyped with 140 polymorphic microsatellite markers (simple sequence repeats, ssr). in this study, 4 ssrs were highly significant (p< 0.001) associated by anova and composite interval mapping and each were determined to identify a qtl. there were qtl on chr. 12 (lg h, satt353), and 3 on chr. 18 (lg g, satt275, satt163, and satt309). the beneficial alleles all derived from hartwig. satt353 has previously been reported to link to sudden death syndrome (sds) qtl, and all three satt markers on lg g have been reported to link to rhg1.therefore, hartwig and cultivars derived from it (‘anand’, ‘ina’) may be used to address the growing rn problems. keywords: soybean, reniform nematode, resistance, hartwig. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction nematodes are one of the most destructive plant-parasitic pests in soybeans [glycine max (l.) merrill]. among nematodes, soybean cyst nematode (scn, heterodera glycines ichinohe), southern root-knot nematode [rkn, meloidogyne incognita (kofoid and white) chitwood], and reniform nematode (rn, rotylenchlus reniformis linford and oliveria) are often the most problematic in soybean yield production (robbin et al. 1994a; koenning and wrather 2010). nematode management options include nematicide application, rotation with non-host crop, and the use of resistant cultivars (jiao et al. 2015; lee et al. 2015). an estimation of $1 billion loss annually due to scn has been reported (wrather and koenning 2009), therefore breeding scn resistant cultivars has been the top priority. however, the continuous breeding for scn resistant cultivars may accelerate selection for other nematode pests, hence breeding cultivars with multiple nematode resistance is crucial (schmitt and barker 1988; lee et al. 2015). many cultivars that are resistant to scn but not rkn or rn have been reported. studies have shown that scnresistant soybean cultivars that derive resistance from ‘peking’ (like ‘forrest’) and ‘pi 437654’ (like ‘hartwig’) are potentially resistant to rn whereas resistance derived from ‘pi88788’ are not (caviness and riggs 1976; robbins el al. 1994a, 1994b; davis et al. 1996; robbins and rakes 1996; jiao et al. 2015). this indicates that there is a common or linked gene controlling the resistant for both scn and rn at or near the rhg1a allele (ha et al. 2007) but not the rhg1b allele. the rn was first observed in hawaii on cowpea roots in 1931 (robbins et al. 1999). rn was originally considered to be a tropical nematode pest but has since spread to the eastern half of the u.s. cotton belt, the rn has since become a major pest in a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) tropical, sub-tropical, and southern united states (robbins et al. 1999; jones et al. 2015). the rn host range includes up to 77 plant families, in the united states, common hosts include upland cotton, soybean, and pineapple (robinson et al. 2007; wubben et al. 2015). cotton yield lost due to rn infection could be greater than $100 million annually and soybean losses could exceed that in future (blasingame and patel 2012). numerous soybean breeding lines and cultivars have been tested for rn reproduction, resistance, and susceptibility, including lines from the arkansas and mississippi soybean variety testing program and lines submitted from extension nematologist (robbins et al. 1994; robbins et al. 2002). in addition, the genetic approach has been researched extensively to understand the quantitative trait loci (qtl) responsible for the resistance. two qtl have been reported in a cross between ‘bsr 101’ and pi 437654, on chromosome 11 (chr. 11, linkage group b1) and chr. 19 (lg l), using restriction fragment length polymorphisms, by pioneer hi-bred international, inc in 2000 (unpublished study). ha et al. (2007) reported two additional qtl in the bsr101 × pi 437654 population chr. 1and chr. 18 (lg b1, satt359 and lg g, sat_168) using simple sequence repeat markers (ssr). in addition, the positions of qtl previously identified by pioneer hi-bred international, inc were refined (chr. 19; lg l, sat_184 and satt513). jiao et al. (2015) reported two additional qtl on chr. 18 (lg g, barc-021459-04106) and chr. 11 (lg b1, barc-012237-01756). the pi437654, though non-domesticated and viney, has been used as a source to introgress neamatode resistances to domesticated cultivars including hartwig, ‘anand’ and ‘ina’ (kazi et al., 2008; 2010). a cross between ‘flyer’ and hartwig (n=92) was created to map qtl that underlie scn resistance (kazi et al. 2005; 2010). this population was used here to identify qtl that underlie resistance to rn. hartwig derives parts of its scn, rkn and rn resistances from the non-domesticated pi437654 but does so in a domesticated (forrest like) plant phenotype. the f × h population was phenotyped at the nematology lab at the university of arkansas in 2014 and 2015. the f × h population was genotyped with 140 polymorphic ssr markers (schultz 2007; kazi et al 2010). here the following is reported, 12 ssrs were identified to be significantly associated with the rn resistance trait, 5 out of the 12 ssrs were highly significant (p< 0.001) and were determined to identify qtl, this study further supports that there are common qtl or genomic regions controlling both scn and rn resistance. materials and methods plant material the genetic material used in this study consisted of the f × h recombinant inbred lines (rils; n = 92; yuan et al. 2002; kazi et al. 2007; 2008; 2009). populations were advanced to the f5:14 from 2003 to 2005 and seed were released in 2007 (kazi et al. 2007). the population was increased every 4 years since then and is now at the f5:16.the cross was selected because it showed segregation for seed yield and many pest resistances, including well characterized reactions to sds, rkn, rn and scn. hartwig was lower yielding but consistently resistant to nematode diseases and sds in most locations (wrather et al. 1995; njiti et al. 1997; 2001; mueller et al. 2002; kazi et al. 2010). it was strongly resistant to most hg types of scn, rkn and rn (anand 1992; niblack et al. 2003). flyer was higher yielding and resistant to phytophtora root rots but susceptible to most nematodes and to sds (mcblain et al. 1990; njiti et al. 1997; 2001; yuan et al. 2002; kazi 2005; et al. 2008). reniform assay the f × h population was sent to the nematology laboratory at the university of arkansas for reniform nematode assay, the assay is modified from robbins et al. 1994. the soil used in the study was fine sandy loam from the arkansas river, with a maximum of 4% clay, 85% sand and the rest was silt. the plants were germinated in vermiculite and transplanted to 8 ounce styrofoamtm coffee cups with drain holes at the cotyledon stage, and inoculated with 2,000 vermiform reniform nematodes on the same day. the assay included 5 repetitions per lines and the randomized complete block design (rcbd) was used in the study. to separate the resistant and susceptible lines more efficiently, the duration of the assay was set at 10 to 12 weeks. flyer, ‘braxton’, and ‘ellis’ were used as the susceptible checks and hartwig, and anand were used as the resistant checks. the plants were set out to dry for 3 to 4 days before harvest, the soybean roots were discarded and the number of reniform nematodes in the soil were counted. the average of the reniform nematode number on roots was calculated from the 5 repetition for each line. the data was converted into reproductive index, described as the average divided by the original inoculation (average/2000, ri). the assay was carried out in 2014 and 2015. the average of the ri from the two years was calculated. data analysis the f × h population was screened with 600 ssr markers, 140 ssr markers were selected based on the polymorphisms. the ril lines were scored by the genotypes. ri was used as the phenotypic trait for qtl mapping. a one-way anova was performed by using jmp 12 statistic software (jmp®, version 12, sas institute inc, nc, usa). the mean, standard error, and student-t test (p<0.05) was recorded. composite interval mapping was used to confirm the qtl. results polymorphism and linkage the linkage map used is described in kazi et al. (2008; 2010). briefly, one hundred and forty two markers were found to be polymorphic within a flyer × hartwig (f × h) ril population. there were 3-10 markers per linkage group and distance between markers was 10-25 cm except for lg g (meksem et al. 1999) and k that were tested with additional markers due to previous discoveries of scn resistance, sds resistance and seed yield qtl (yuan et al. 2002; kazi et al. 2008). sixty one mark309 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ers of the markers formed 17 linkage groups encompassing 534 cm. assuming 10 cm as a distance for qtl detection, the groups formed plus the 81 unlinked markers would allow the detection of qtl over 2,494 cm. the recombination distances and orders of markers in linkage groups (with 2-3 exceptions) and genome size (2,512 cm) agreed with those reported (song et al. 2004). variation of resistance within f × h rils to rn the means and standard errors of phenotypic variation of ri among the two parents and rils within ri data from both years were normal (fig. 1) indicating that the rn bioassays were useful for further analysis. transgressive segregation was observed in ri scores. the ril population mean was intermediate to the two parents for each hg type. narrow sense heritability estimates for ri had relatively high values (0.95, 0.96) in both years. correlations with scn trait data there was a significant positive correlation with resistance to scn (p<0.05; r2 was just 11%). interestingly the correlation with susceptibility to sds was significant (p<0.05; r2 was -27%). this is inverse to the correlation between sds and scn (p<0.05; r2 56%). dna marker analysis among the 140 markers only 12 ssrs with significant association were detected (fig. 2; table 1). the 11 ssr markers were detected on chr. 2 (lg d1b), chr. 5 (lg a1), chr. 8 (lg a2), chr. 12 (lg h), and chr. 18 (lg g). seven qtl had a p value greater than 0.001, whereas 4 qtl had p value less than 0.001 (highly significant). satt353 was detected on chr. 12 (p value=0.0076, r2=10%) (lg h, start position 1,682,557 end position 1,682,607), and the beneficial allele derived from 310 fig. 1. trait distribution for rn showing parental scores and limited transgressive segregation. table 1. potential qtl underlying resistance to rn. reproductive index ri marker chr lg flyer mean hartwig mean p>f r2 satt537 2 d1b 20.0 ±1.3 23.9 ±1.4 0.038 0.053 satt428 2 d1b 19.9 ±1.3 24.3 ±1.5 0.027 0.066 satt599 5 a1 19.7 ±1.7 25.5 ±1.5 0.014 0.11 b61p08b 8 a2 24.1 ±1.4 19.2 ±1.7 0.03 0.07 satt353 12 h 18.1 ±1.5 24.1 ±1.5 0.0076 0.1 satt181 12 h 19.3 ±1.4 24.4 ±1.8 0.02 0.078 satt275 18 g 24.7 ±1.2 19.2 ±1.5 0.0084 0.1 satt163 18 g 24.1 ±1.1 16.5 ±2.0 0.0028 0.135 satt309 18 g 24.1 ±1.0 16.4 ±2.0 0.0044 0.132 tmd1 18 g 24.1 ±1.1 17.6 ±2.0 0.0134 0.1 satt610 18 g 24.6 ±1.6 18.9 ±1.9 0.033 0.089 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 311 flyer. the 3 markers on chr. 18 (lg g) has previously been reported to link to scn 41-1, which is resistant to hg type 0 (kazi et al. 2010). the beneficial allele for all 3 markers derived from ‘hartwig’. satt163 was the most significantly associated with the trait (p value= 0.0028, r2=13.5%), followed by satt309 (p =0.0044 r2=13.2%), the least significant on lg g was satt275 (p = 0.0084, r2=10%). satt275 has been reported to be linked to satt309. discussion the three satt markers found on lg g had all been previously reported to be linked with scn qtl near rhg1 in the f × h population, satt275 linked to scn 41-1, satt163 and satt 309 linked to scn 41-2 (kazi et al. 2010). in addition, satt309 was very closely linked to rfs2/rhg1 allele a in the essex × forrest population (meksem et al. 1999). the, rhg1 resistance alleles a and b are required for all known hg type resistances to scn. however, only allele a appears to give resistance to rn (caviness and riggs 1976; robbins el al. 1994a, 1994b; davis et al. 1996; robbins and rakes 1996; jiao et al. 2015). these findings infer that reniform resistance qtl maybe closely linked to or pleiotropic with some of the genes underlying scn resistance qtl (cook et al 2012; srour et al 2012). the findings here on rhg1 agree with previous studies (ha et al. 2007) so are unlikely to be errors. which of the genes implicated in scn resistance (cook et al.2012; srour et al. 2012) also contributes to rn resistance, if any, will be the focus of future work with near isogeneic lines and transgenic lines (kazi et al. 2005; lightfoot 2015). based on linkage data, and correlations among rn, scn and sds traits it may be inferred the rn resistance gene(s) is or are linked to rhg1 and rfs2 on the telomeric side but at some distance. the one additional locus or qtl, on chr. 12 (lg h, satt353), had not been reported by 2016. many, but not all nematode resistance loci are found in cultivars derived from pi437654 (vierling et al. 1996; webb et al. 1996) like hartwig (kazi et al. 2010). the pi has superior resistance to cultivars derived from it. it may be hypothesized that the plant introduction contains many more resistance genes to nematodes than were yet discovered. since hartwig was parent to ina and anand, and many other cultivars, the qtl reported here may improve selections for resistance to rn for many breeding programs. acknowledgements this research was funded by grants from the united soybean board to sk and rr; and the family of ycl. the authors thank dr. p. gibson, o. myers jr. and m. schmidt for assistance with germplasm development and maintenance from 1991-2000. we thank j.h. klein iii for assistance with germplasm maintenance from 1991-2011. we thank ted ballard for assistance with the population from 2012 to present. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) d 2satt514 85.7 satt528 86.3 satt082 87.2 satt574 87.7 satt543 88.1 satt488 89.2 sat_001 92.1 satt301 93.7 sat_86 118.7 end 133.9 a 2 g h100b10b 47.8 a2d8 48.1 blt65 49.3 satt424 60.6 satt089 87.6 satt437 107.1 satt158 115.2 satt421 115.9 end 165.7 satt163 0 satt038_1 1.8 satt275 2.2 tmd1 4 satt309 4.5 satt038_2 9 satt610 10.9 satt570 12.7 satt130 23.1 sat_131 31.3 satt324 33.2 b09l01 35 satt115 43.8 satt566 49.9 satt352 50.5 satt427 51.7 satt594 52.9 satt564 57.3 b30o12 77 satt191 96.6 end 116.8 satt353 8.5 satt442 46.9 satt181 91.1 satt434 105.7 end 124 h fig. 2. linkage groups with significant qtl underlying resistance to scn hg types (black and stippled arrows) and rn (grey arrows) by anova and composite interval mapping. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, 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ibijbijen* environment and soil microbiology unit, faculty of sciences, moulay ismail university, b. p. 11201 zitoune, meknes, morocco. received: may 21, 2018 / accepted: august 4, 2018 __________________________________________________ * corresponding author: j.ibijbijen@fs.umi.ac.ma 546 abstract the presence of residual pesticides in the soil affect the microbial communities, as well the continuous use of pesticides exacerbates this problem. glyphosate is one of the most used herbicides in the world. up to date several studies have evaluated the tolerance and resistance of bacteria to glyphosate. nitrogen-fixing bacteria play an important role in soil fertility; thus, the alteration of these bacterial communities decrease soil fertility. the objective of this study was to evaluate the effect of glyphosate application on four bacterial strains pantoea agglomerans, rhizobium nepotum, rhizobium radiobacter, and rhizobium tibeticum. glyphosate was applied as the sole source of carbon at the rate (0 g/l, 0.5 g/l, 1g/l, 3g/l, 6g/l and 12 g/l) with two methods. microbial growth was measured by the colony forming units (cfus /ml) method. comparing with the control, our results showed that the growth of the four strains decreased by increasing the concentration of glyphosate. the four strains have shown resistance to glyphosate in the direct enrichment compared to the continued enrichment method. comparing strains with each other, rhizobium radiobacter is the most resistant strain to glyphosate. keywords: glyphosate; pesticide; nitrogen-fixing bacteria; tolerance. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction glyphosate [n-(phosphonomethyl) glycine], the active ingredient of the herbicide roundup, is a systemic, post-emergent, broad-spectrum herbicide, it is an inhibitor of aromatic amino acid synthesis (calvet et al., 2005). is introduced in agriculture in the 1970s (munira et al., 2016). the high efficiency in protecting crops against weeds has enabled the glyphosate to become the most widely used herbicide in the world (chlopecka et al., 2017), knowing that it occupied a quarter of the world’s herbicide sales (munira et al., 2016). glyphosate is foliarly applied. however, an important quantity of the herbicide may reach the soil. moreover, it will be leaching to groundwater. thereby, the study of interaction microorganisms/glyphosate in soil is very important. the soil is one of the largest reserves of the planet biodiversity. the nitrogen-fixing and phosphate-solubilizing bacteria are among the important bacteria in the soil, they play an important role in the nitrogen and phosphorus availability in the soil, and consequently on soil fertility. the bacterial nitrogen fixation phenomenon is an alternative to the high use of chemical fertilizers (bhattacharjee et al., 2008). previous studies have indicated that the glyphosate may modify natural ecosystem by affecting different components of the soil microbial community (carlise and trevors, 1988; ermakova et al., 2010). glyphosate can affect the fungi and bacteria in soil (imparato et al., 2015). whereas, numerous studies have shown microorganisms can degrade that glyphosate. several potential glyphosate degrading microorganisms have been isolated from organophosphates contaminated soils (shushkova et al., 2010) such as enterobacter cloacae (kryuchkova et al., 2014), geobacillus caldoxylosilyticus (obojska et al., 2002), pseudomonas spp. (dick and quinn, 1995), rhizobium sp. and a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) agrobacterium sp. (liu et al., 1991). due to its great impacts mainly on the ecology and the health, and based on previous studies in this field, we carried out this study whose objectives are to evaluate the glyphosate tolerance using two enrichment methods of four nitrogen-fixing bacteria, which were isolated from the nodules of a legume (bituminaria bituminosa). as well to assess the effects of glyphosate concentration on the growth of these four bacterial strains, using glyphosate as a sole source of carbon. materials and methods chemicals used the glyphosate used was a commercial roundup® (containing 360 g active ingredient/l of glyphosate, monsanto) purchased from a local dealer’s store in tanger, morocco. all the other chemicals were characterized by a high purity commercially available. to evaluate the tolerance or resistance of our bacterial strains to the glyphosate we have used it as a sole source of carbon and phosphorus, we used a mineral salt medium without carbon source (msmc). the composition of msmc in grams per liter of distilled water ph (7.0-7.2). was kh2po4 (1.5), na2hpo4 12h2o (1.5), nh4so4 (2), mgso4 7h2o (0.2), cacl2 (0.01), feso4 and 7h2o (0.001). the media was supplemented with glyphosate sterilized by filtration (0.2 µm filter). mineral salt medium (msm) with glucose as carbon source was used as control, the composition of msm control in grams per liter of distilled water ph (7.0-7.2) was c6h12o6 (10). kh2po4 (1.5), na2hpo4 12h2o (1.5), nh4so4 (2), mgso4 7h2o (0.2), cacl2 (0.01) and feso4 7h2o (0.001). bacterial strains four bacterial strains were selected to test their tolerance or resistance to glyphosate. these strains were originally isolated from nodules root of legume “bituminaria bituminosa” cultivated in the experimental station of the faculty of sciences, moulay ismail university, meknes. the selection of strains was based on their ability to fix nitrogen. the selected strains are pantoea agglomerans, rhizobium nepotum, rhizobium radiobacter and rhizobium tibeticum. preparation of inoculum inocula were prepared for the four strains by culturing the strains in 50 ml of nutrient medium for three days at 30°c under stirring conditions (150 rpm) until growth reached late exponential phase. cells were harvested by centrifugation at 4, 600 g for 5 min, washed with sterile saline solution 0.9% and were resuspended in 0.5 mcfarland standard (optical density of 0.108 at 625 nm), this suspension was used as inoculum. 547 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) treatments of used bacteria continuous enrichment glyphosate tolerance experiments were performed in flasks (250 ml) containing 100 ml of sterile msmc with 0.5 g /l of glyphosate. 2 ml of each inoculum was added to a sterilized flask and for each sstrain, three replicates were done, then all the flasks were incubated on a rotary shaker at 150 rpm for 7 days at 30 ° c. after 7 days 5 ml of each flask were transferred to fresh msmc containing 1g/l glyphosate and incubated for 7 days. three additional and successive transfers were made into media successively containing 3, 6 and 12 g/l of glyphosate. direct enrichment two (2) ml of each inoculum were added in flasks (250 ml) containing 100 ml of sterile msmc with 0.5g/l, 1g/l, 3g/l, 6g/l and 12g/l of glyphosate in each one. triplicate culture were incubated on a rotary shaker at 150rpm for 7 days at 30°c. enumeration of bacterial strains the bacteria were counted for each concentration (0.5, 1, 3, 6, 12 g/l) including the control, after 7 days of incubation; 1 ml of each sample was used to provide a series of dilutions (10-1, 10-2, 10-3, 10-4 and 10-5). moreover, 0.1 ml of each dilution was added to the plates containing the plate count agar (pca) medium, which its composition in gram per liter of distilled water, ph (7.0, 7.2) is: tryptone (5), yeast extract (2.5), glucose (1), agar (15). the plates were incubated at 28±2 °c, for 72h. statistical data analysis all variables were analyzed by anova (analysis of variance) and significant differences among treatments were determined using tukey post-hoc test. differences between treatments were considered statistically significant at p < 0.05. ibm spss statistics 20 was used for all above statistical analysis. results and discussion in order to assess the tolerance of bacterial strains (pantoea agglomerans, rhizobium nepotum, rhizobium radiobacter, and rhizobium tibeticum) to glyphosate, we evaluated the growth of each strain while increasing glyphosate concentration with two methods: continuous and direct enrichment. statistically, and comparing with the control, significant differences (p< 0.05) were observed for the four strains using both methods: direct enrichment (figure 1) and continuous enrichment (figure 2). bacterial load decreased highly while increasing glyphosate concentration in continuous enrichment, whereas in direct enrichment the bacteria showed a tolerance to glyphosate except rhizobium tibeticum strain. moreover, comparing the strains with each other in the continuous enrichment, no resistance was observed in the concentrations greater than or equal to 1 g/l of glyphosate, which is the recommended a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 548 figure 1. effect of increasing glyphosate concentration on the growth of bacterial strains in direct enrichment. figure 2. effect of increasing glyphosate concentration on the growth of bacterial strains in continuous enrichment. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) dose in morocco (figure 3). whereas in direct enrichment the behavior of the four strains was different (figure 3), rhizobium radiobacter and pantoea agglomerans strains tolerated concentrations up to 12 g/l (figure 1), while rhizobium nepotum tolerated concentrations up to 6 g/l (figure 1). whilst rhizobium tibeticum had no activity in all treatment (figure 1). the microorganism’s physiology and genetic’s play a key role on their tolerance or resistance to pesticides. generally, the microorganisms resistant to pesticide have a great potential to break the pesticides into simple products, which may be used by them as nutrient sources, such as carbon and phosphorus (cassigneul et al., 2016; myresiotis et al., 2012; bellinaso et al., 2003). according to the results of (wijekoon et al., 2018) which have shown that pseudomonas sp. and bacillus sp. are able to degrade glyphosate even at its high concentrations, these results are consistent with ours mainly for rhizobium radiobacter and pantoea agglomerans in direct enrichment and without carbon in the medium. as so, it can be suggested that these two strains used glyphosate as a source of carbon, while the behavior of these two strains was different in continuous enrichment. this result can be explained by the accumulation of the glyphosate, which make the medium toxic. thus, several studies have reported the eco-toxicity of glyphosate on specific soil microorganisms (allegrini et al., 2015; sihtmäe et al., 2013). on the other hand, according to the study made by (ahemad et al., 2012), the glyphosate affects negatively the growth of mesorhizobium strain mrc4, these results are consistent with those obtained for rhizobium tibeticum strain, as well as those of (ermakova et al., 2010) who claimed that none of the two achromobacter sp. and ochrobactrum anthropi bacteria could degraded glyphosate. overall, the effect of glyphosate on the bacterial community is different from one bacterial species to another. some strains are able to resist or tolerate glyphosate and use its metabolites as a source of nutrients and/or energy, whereas the glyphosate may be toxic for other strains; these findings was proved by several studies (sihtmäe et al., 2013; araujo et al., 2003; tsui et al., 2003). 549 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 3. comparison of glyphosate effect on four strains in two methods. our results showed that the continuous use of glyphosate affect nitrogen-fixing bacteria which play an important role in soil fertility. however, more research is needed in the field to better understand the effect of glyphosate on soil fertility. therefore, perspective research projects can focus on the genetics of the soil microbial community and its behavior towards glyphosate application. acknowledgments our special thanks to environment & soil microbiology unit, department of biology, faculty of sciences, moulay ismail university, meknes, morocco and all those who helped us to accomplish this work. references ahemad m, and ms khan (2012) ecological assessment of biotoxicity of pesticides 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author: mkvzant@siu.edu 249 abstract in the course of work on the taxonomy and agronomy of mirabilis expansa (ruiz and pav.) standl. (nyctaginaceae), it became clear that there is no thorough published review of the medicinal and other useful aspects of the family. this work illustrates the history and potential for use for nyctaginaceae at the generic level world-wide. several genera in this family have well documented potential as sources of medication, food, dye, lumber and adhesives. abronia, boerhavia, and mirabilis are reported most often for positive laboratory assay results. pisonia and neea have both been used as sources of lumber in the tropics. in addition, bougainvillea and mirabilis have been utilized for floriculture. included, is an updated analysis of the historical confusion between medicinal mirabilis jalapa l. and medicinal jalap in the convolvulaceae, and the disagreements over taxonomic names in both families which have contributed to this error and detracted from its resolution. known invasive issues for some nyctaginaceae species are discussed, having economic impact on agriculture. a recommendation is made that highly variant expression in nyctaginaceae taxa makes them strong candidates for investigation of polyploidy and epigenetic patterns, important for better understanding evolution and development in plants. introduction this is primarily a genus level overview of the economic and ethnobotany of the nyctaginaceae. details are only presented sufficiently to fill the void for a published world-wide look at the uses of the family. most of these uses were originally presented for particular species, growing in specific locations, in the literature. a separate review of the genus mirabilis, with species specific ethnobotanical and agricultural information primarily for m. expansa has been published as a separate paper (2016b) modified from kritzer van zant’s dissertation (2016a). asterisks next to source names below, indicate that they were mentioned in a secondary reference, and have not been directly evaluated by this author. there are four tables included of information summarized from the literature. they are: table 1. medicinal use of nyctaginaceae genera – anecdotal reports; table 2. food and forage uses of nyctaginaceae genera – anecdotal reports; table 3. lumber, floriculture and other uses of nyctaginaceae genera – anecdotal reports; and table 4. nyctaginaceae genera – results of laboratory analyses. methods book chapters, articles and journals obtained through library and on-line sources, and a napralert (natural products alert database) print out on the nyctaginaceae from the university of illinois-chicago, were analyzed and summarized. napralert uses its own standard coding for cross-comparing most of the data presented. however, ethnobotanical information is presented in each of the four tables by genus then source, as it was unclear to us if some seemingly technical terms were always used the same way among authors. there may be repetition among authors for some of the same reports of use. which were replicated and which were original could not be readily teased apart. therefore, they were left in the tables as is. also, this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 250 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. medicinal use of nyctaginaceae genera* -anecdotal reports. rearrangement even of napralert data saves little space, as instead of repeating technical terms for each location, it would have been necessary to repeat each location for each technical term. as previously mentioned, asterisks are used to mark dates given by other authors, for literature we have not examined first hand. as all of the information is in the tables, we grouped comments together as appropriate in the text. included below is an analysis of taxonomic problems involved in the confusion over m. jalapa and medicinal jalap in the convolvulaceae, for which many sources were used including the on-line ipni (international plant names index) database maintained by royal botanic gardens, kew. briefly mentioned below are both unpublished amino acid percentage profiles and negative cytotoxicity data for two m. expansa horticultural varieties originally from peru and ecuador, grown in sand plots in southern illinois (kritzer van zant, 2016a). the cancer cell cytotoxicity assay was executed by hee-byung chai (kritzer van zant, 2016a) in douglas kinghorn’s lab at ohio state university. methanol extracts of both horticultural varieties were utilized in the assay, made by this author in labouse genus plant part preparation tribe location source purgative boerhavia peru ruiz (1777-1788, 1940) against venereal infections boerhavia nyctaginaceae peru ruiz (1777-1788, 1940) purgative boerhavia roots malaysia bogle (1974) anthelmintic boerhavia roots malaysia bogle (1974) febrifuge boerhavia roots malaysia bogle (1974) asthma boerhavia leaves liquid extract s america bogle (1974) jaundice boerhavia leaves liquid extract s america bogle (1974) diuretic boerhavia leaves s america bogle (1974) emetic boerhavia leaves s america bogle (1974) expectorant boerhavia leaves s america bogle (1974) headaches boerhavia argentina napralert (1998) diuretic boerhavia argentina napralert (1998) antipyretic boerhavia argentina napralert (1998) urinary inflammations boerhavia bolivia napralert (1998) galactogogue boerhavia bolivia napralert (1998) childbirth, difficult boerhavia guinea-bissau napralert (1998) diuretic boerhavia india napralert (1998) jaundice boerhavia india napralert (1998) anti-inflammatory boerhavia india napralert (1998) cataracts boerhavia eye drops india napralert (1998) febrifuge boerhavia nigeria napralert (1998) laxative, mild for children boerhavia nigeria napralert (1998) anti-asthmatic boerhavia nigeria napralert (1998) anti-convulsant boerhavia nigeria napralert (1998) expectorant boerhavia nigeria napralert (1998) emetic boerhavia nigeria napralert (1998) menstruation, regulate boerhavia west africa napralert (1998) abortifacient boerhavia west africa napralert (1998) diuretic boerhavia india napralert (1998) dropsy, to treat boerhavia india napralert (1998) edema boerhavia india napralert (1998) anemia boerhavia india napralert (1998) jaundice boerhavia india napralert (1998) menstrual problems boerhavia india napralert (1998) heart disease boerhavia india napralert (1998) emetic boerhavia india napralert (1998) stomachic boerhavia india napralert (1998) laxative boerhavia india napralert (1998) diuretic boerhavia iran napralert (1998) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. continued. ratories at southern illinois university-carbondale. this negative cytotoxicity data, together with the specificity of published positive and negative anti-microbial data summarized below, indicate that there is unlikely to be a broad spectrum toxic micromolecule in the southern illinois grown m. expansa horticultural varieties. it should be noted that this kind of methanol extraction retains micro-molecules, though proteins are destroyed in the process. protein extracts from roots of m. expansa were found to have specific toxicity via a class i ribosome inhibitor, against certain species of soil fungi, in a series of publications by vivanco and colleagues, specified below. class i ribosome inhibitors are not known to affect humans (j.m. vivanco, personal communication, 2008). names, for the horticultural varieties of m. expansa from which data for the field and nutrition chapters in kritzer van zant (2016a) were taken, will be detailed in taxonomic papers also in preparation, including morphological keys and descriptions. these taxonomic papers await completion of analysis of the considerable and notoriously confusing contradictions for synonyms in the family, which has taken several years to clarify. these new assay results are mentioned below, in the overall look at laboratory assay results for the family, along with nutritional information concerning amino acids in m. expansa from assays done in collaboration with bill banz at siu-c (kritzer van zant, 2016a). details on how the extractions were made, and the results of the assays executed by dr. chai are also given in kritzer van zant (2016a). the unpublished taxonomic research on the mirabilis complex in the andes is the basis of acceptance of m. expansa (ruiz and pav.) standl., as the correct species name for both the wild type and crop. earlier peer reviewed publications 251 use genus plant part preparation tribe location source gonorrhea, for treating boerhavia iran napralert (1998) nephritis boerhavia iran napralert (1998) edema boerhavia iran napralert (1998) joint pain boerhavia iran napralert (1998) appetite stimulant boerhavia iran napralert (1998) aphrodisiac boerhavia new guinea napralert (1998) sterility, to induce boerhavia new guinea napralert (1998) hepatitis, to treat bougainvillea taiwan napralert (1998) cuts and sores, to heal commicarpus bahamas napralert (1998) laxative, mild mirabilis peru ruiz (1777-1788, 1940) purgative, mild mirabilis bogle (1974) dropsy, to treat mirabilis bogle (1974) poultices, to make mirabilis bogle (1974) boils, to treat mirabilis bogle (1974) abscesses, to treat mirabilis bogle (1974) scabies, to treat mirabilis bogle (1974) jalap source mirabilis roots resin pacific islands walker (1976) purgative mirabilis roots cabieses (1995) diuretic mirabilis roots peru napralert (1998) diuretic mirabilis roots west indies napralert (1998) herpes, to treat neea cuba napralert (1998) tooth decay prevention neea leaves chewed peru napralert (1998) tooth decay prevention neea leaves chewed columbia napralert (1998) rheumatic disease, to treat pisonia leaves decoction jamaica bogle (1974) venereal disease, to treat pisonia leaves decoction jamaica bogle (1974) rheumatic disease, to treat pisonia leaves decoction yucatan bogle (1974) venereal disease, to treat pisonia leaves decoction yucatan bogle (1974) fevers, to treat pisonia fruits mexico bogle (1974) dysentary, to treat pisonia rotuma napralert (1998) diuretic abronia diegueño california strike (1994) lactation, increase abronia mexico napralert (1998) fever, to treat allionia teton dakota missouri river region gilmore (1914*, 1919*, 1977) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. continued. on the crop also use the name m. expansa, and describe cultivars. however, these publications lack latin names for horticultural varieties. results from the literature are summarized from the tables, in the text. results anecdotal reports of use of the nyctaginaceae –general information. ruiz and pavon, who first named m. expansa, collected with french botanist dombey to carry through the first crown commissioned botanical expedition for spain in south america. both ruiz and pavon were primarily trained in spain as pharmacists, reflected in their collections and publications. ruiz’ anecdotal reports from the late 1700s, on the economic uses of nyctaginaceae taxa in south america, are included in this section along with reports from several later authors. gunther (1945) attributed information to stuhr (1933*) and haskins (1934*). bogle (1974) summarized information on use of mirabilis from bailey & bailey (1941*), chittenden (1965*), stemmerik (1964), and uphof (1968*). bogle (1974) states that in general, the nyctaginaceae have little economic value, except for widely cultivated species of abronia, mirabilis, and bougainvillea, and less commonly cultivated boerhavia and pisonia. bogle (1974) provides information on economic uses for mirabilis, boerhavia, and pisonia. bogle seems to have used a narrow interpretation of economic value, perhaps he was referring to reported value as cash crops. napralert (1998) has summarized numerous world-wide anecdotal reports for use of the nyctaginaceae. many of the more notable reports of use from napralert come from the equatorial region. north american anecdotal use from napralert is also covered in the anecdotal sections, followed by another section containing the results of laboratory assays reported from napralert and other sources. anecdotal information on use of nyctaginaceae genera by native americans is also included in tables 1 through 3 from moerman (1998) though only at the family level, as moerman is both extensive 252 use genus plant part preparation tribe location source vermifuge allionia combined w/ echinacea spp. oglala missouri river region gilmore (1914*, 1919*, 1977) swelling reduction, arms and legs allionia roots rub missouri river region gilmore (1914*, 1919*, 1977) wounds, to treat allionia externally ponka missouri river region gilmore (1914*, 1919*, 1977) sore mouths, to treat in babies allionia roots dry powdered pawnee missouri river region gilmore (1914*, 1919*, 1977) abdominal swelling, to reduce after childbirth allionia roots decoction pawnee missouri river region gilmore (1914*, 1919*, 1977) coughs, to treat bougainvillea mexico napralert (1998) sprains and swelling, to reduce mirabilis ojibwe smith (1932) muscles, strained mirabilis roots decoction ojibwa meeker et al. (1993) poultice for boils mirabilis roots beaten cherokee hamel and chiltoskey (1975) bladder problems mirabilis plant meskwaki king (1984) swelling reduction mirabilis ojibwa king (1984) fever, to treat mirabilis missouri river region tribes king (1984) worms, to treat mirabilis missouri river region tribes king (1984) swellings, to treat mirabilis missouri river region tribes king (1984) to keep new born babies healthy mirabilis decoction karok california strike (1994) purgative mirabilis decoction lusieño california strike (1994) stomach aches, to treat mirabilis decoction kumeyaay california strike (1994) lesions -dermato-mucosal , to treat neea guatemala napralert (1998) ringworm, to treat neea guatemala napralert (1998) fungal diseases of skin, to treat neea guatemala napralert (1998) gonorrhea, for treating neea guatemala napralert (1998) cough pisonia mexico napralert (1998) anti-diabetic salpianthus mexico napralert (1998) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. continued. and relatively easy to obtain. moerman (1998) summarized reported use by north american indigenous tribes, of many species in the genera abronia (which includes tripterocalyx), allionia, boerhavia, and mirabilis. inclusion of moerman (1998) demonstrates that widespread use of the family was not limited to the southern hemisphere and tropical countries. anecdotal reports of medicinal use of the nyctaginaceae medicinal uses of nyctaginaceae genera – anecdotal reports (table 1) includes information from ruiz (1777-1788 (1940), gilmore (1914*, 1919*, 1977), smith (1932), bogle (1974), hamel and chiltoskey (1975), walker (1976), king (1984), meeker et al. (1993), pelayo-benavides and anaya (1993), strike (1994), cabieses (1995), and napralert (1998), in addition to family level information summarized from moerman (1998). for abronia, anecdotal reports of medicinal use are attributed to the diegueño tribe in california, and to mexico. abronia has been reported anecdotally for medicinal use as a diuretic and to increase lactation (table 1). moerman (1998) summarized reported use by north american indigenous tribes, of many species in the genera abronia, which includes tripterocalyx. information on tripterocalyx is included at the family level below and in our tables, and is not distinguished here from abronia. for allionia, anecdotal reports of medicinal use are attributed to the missouri river region of the united states, and some uses are specified to the pawnee, teton dakota, oglala and ponka tribes (table 1). specifics, when given, are only for preparation from allionia roots. allionia has been reported anecdotally for medicinal use to reduce abdominal swelling after childbirth, to treat fevers, sore mouths in babies, for reduction of swelling of arms and legs, as a vermifuge, and to treat wounds (table 1). gilmore (1914*, 1919*, 1977) reports allionia was combined with echinacea spp. by the teton dakota. gilmore also seems to refer to the same combination of plants as used by the oglala as a vermifuge, stating that the oglala decoction will even expel large tapeworms. 253 use genus plant part preparation tribe location source diabetes, to treat salpianthus mexico napralert (1998) sedatives for children nyctaginaceae nat am n am moerman (1998) headaches, to treat nyctaginaceae nat am n am moerman (1998) neuralgia nyctaginaceae nat am n am moerman (1998) delirium nyctaginaceae nat am n am moerman (1998) dizziness nyctaginaceae nat am n am moerman (1998) fainting nyctaginaceae nat am n am moerman (1998) boils, to treat nyctaginaceae nat am n am moerman (1998) spider and insect bites nyctaginaceae nat am n am moerman (1998) astringent nyctaginaceae nat am n am moerman (1998) poultice to treat burns nyctaginaceae nat am n am moerman (1998) swellings, to treat nyctaginaceae nat am n am moerman (1998) sores nyctaginaceae nat am n am moerman (1998) sore mouth nyctaginaceae nat am n am moerman (1998) perspiring feet nyctaginaceae bath nat am n am moerman (1998) bruises, to apply to nyctaginaceae nat am n am moerman (1998) sprains, to apply to nyctaginaceae nat am n am moerman (1998) broken bones, to apply to nyctaginaceae nat am n am moerman (1998) dandruff nyctaginaceae as a shampoo nat am n am moerman (1998) snake bite treatment nyctaginaceae steam bath nat am n am moerman (1998) appetite, increase nyctaginaceae internal nat am n am moerman (1998) appetite, control nyctaginaceae internal nat am n am moerman (1998) effects of swallowing a spider, to neutralize nyctaginaceae nat am n am moerman (1998) cathartic nyctaginaceae nat am n am moerman (1998) sudorific nyctaginaceae nat am n am moerman (1998) emetic nyctaginaceae nat am n am moerman (1998) diuretic nyctaginaceae nat am n am moerman (1998) purgative nyctaginaceae nat am n am moerman (1998) febrifuge for eruptive fevers nyctaginaceae nat am n am moerman (1998) stomach cramps nyctaginaceae nat am n am moerman (1998) stomach disorders nyctaginaceae nat am n am moerman (1998) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) table 1. continued. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) for boerhavia, anecdotal reports of medicinal use are attributed to argentina, bolivia peru, south america, india, iran, malaysia, guinea-bissau, nigeria, and west africa (table 1). both roots and leaves of boerhavia have been specified as used in medicinal preparations. boerhavia has been reported anecdotally for medicinal use as an abortifacient, against venereal infections and anemia, as an anthelmintic, anti-asthmatic, anticonvulsant, anti-inflammatory, antipyretic, aphrodisiac, appetite stimulant, to treat asthma, cataracts, difficult childbirth, as a diuretic, to treat dropsy and edema, as an emetic, expectorant, febrifuge, galactogogue, to treat gonorrhea, headaches, heart disease, jaundice, joint pain, as a laxative mild enough for children, to address menstrual problems including regulation of menstruation, to treat nephritis, as a purgative, to induce sterility, as a stomachic, and to treat urinary inflammations (table 1). bougainvillea has been used medicinally in taiwan for treating hepatitis (table 1). commicarpus has been used in the bahamas to heal cuts and sores (table 1). for mirabilis, anecdotal reports of medicinal use are attributed to peru, the west indies, pacific islands, and california in the united states (table 1). mostly roots and the whole plant have been specified for mirabilis as used in medicinal preparation. mirabilis has been reported anecdotally for medicinal use to treat abscesses, bladder problems, boils, as a diuretic, to treat dropsy, fever, as a source of jalap (see discussion on jalap below), as a laxative also specified as mild, for treating strained muscles, boils, to make poultices, as a purgative sometimes specified as mild, to treat scabies, to reduce sprains and swellings, to treat stomach aches, to keep new born babies healthy and for worms (table 1). calyxhymenia and oxybaphus were included by us as mirabilis (table 1). oxybaphus was reported separately from mirabilis by king (1984) and smith (1932). king (1984) based her report on mirabilis nyctaginea (michx.) macmill. on information from densmore 1928*, gilmore 1919*, and smith 1928* and 1932. king gives oxybaphus nyctaginea (michx.) sweet as a synonym for m. nyctaginea, and says the plant is common in waste areas in illinois and introduced. however, gleason and cronquist (1991) separate m. nyctaginea’s native range, including illinois, from where it has since established in the eastern united states. for neea, anecdotal reports of medicinal use are attributed to columbia, peru, guatemala and cuba (table 1). only leaves of neea have been specified as used in medicinal preparation where reported. neea has been reported anecdotally for medicinal use to treat fungal diseases, gonorrhea, herpes, dermatomucosal lesions, ringworm, and leaves are chewed to prevent tooth decay (table 1). for pisonia, anecdotal reports of medicinal use are attributed to jamaica, yucatan, mexico and rotuma (table 1). leaves and fruits of pisonia have been specified as used in medicinal prepa254 use genus plant part preparation tribe location source fainting spells nyctaginaceae nat am n am moerman (1998) nausea nyctaginaceae nat am n am moerman (1998) coughs, to treat nyctaginaceae nat am n am moerman (1998) coughs, veterinary nyctaginaceae nat am n am moerman (1998) bladder problems nyctaginaceae nat am n am moerman (1998) gonorrhea nyctaginaceae nat am n am moerman (1998) kidneys, to support nyctaginaceae nat am n am moerman (1998) vermifuge nyctaginaceae nat am n am moerman (1998) snake bite nyctaginaceae nat am n am moerman (1998) swollen glands, particularly in the throat nyctaginaceae nat am n am moerman (1998) "life medicine" for health in newborns nyctaginaceae nat am n am moerman (1998) post-partum treatment nyctaginaceae nat am n am moerman (1998) laxative mirabilis pelayo-benavidez and anaya (1993) -nat am = unspecified native american tribes; n am = north america. -moerman (1998) is only cited here at the family level. as for all of the authors most information given at the genus level is available at the spp. level in the original source. -although some categories of use probably overlap, they were not compiled further as their exact meaning may have slightly different definitions in different sources. -an attempt was made to set up the coding of use so that similar descriptions of use would sort in succession, though this was not always possible. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ration, where reported. pisonia has been reported anecdotally for medicinal use to treat cough, dysentery, fevers, rheumatic disease and venereal disease (table 1). for salpianthus, anecdotal reports of medicinal use are attributed to mexico (table 1). salpianthus has been reported anecdotally for medicinal use as an anti-diabetic and to treat diabetes (table 1). moerman (1998) gave anecdotal reports of medicinal use of the nyctaginaceae attributed to native americans in north america (table 1). nyctaginaceae have been reported anecdotally for medicinal use by moerman as a “life medicine” for health in newborns, to both control and increase appetite, as an astringent, to treat bladder problem and boils, to apply to broken bones and bruises, as a cathartic, to treat coughs also for veterinary coughs, to treat dandruff and delirium, as a diuretic, to treat dizziness, to neutralize the effects of swallowing a spider, as an emetic, to treat fainting and fainting spells, as a febrifuge for eruptive fevers, to treat gonorrhea and headaches, to support the kidneys, to treat nausea, neuralgia and perspiring feet, as a post-partum treatment, for a poultice to treat burns, as a purgative, as sedatives for children, to treat snake bite, sore mouth, spider and insect bites, to apply to sprains, to treat stomach cramps and stomach disorders, as a sudorific, to treat swellings, swollen glands particularly in the throat, and as a vermifuge. anecdotal reports of food and fodder uses of the nyctaginaceae food and forage uses of nyctaginaceae genera – anecdotal reports (table 2) includes information from standley (1911), smith (1932), gunther (1945), cárdenas (1969), martínez (1969), bogle (1974), walker (1976), chang et al. (1983), popenoe et al. (1989), sperling and king (1990), cabieses (1995), basualdo et al. (1995), napralert (1998), ugent and ochoa (2006), and kritzer van zant (2016a), in addition to family level information from moerman (1998). food use for abronia has been reported for native american tribes including the makah and kallam from western washington state, and in the pacific coast region of the united states (table 2). only roots have been specified for food in reports for abronia (table 2). pigs dug allionia tubers for food (table 2). food use for boerhavia has been attributed to peru, south america and australia (table 2). boerhavia roots and leaves have been reported as food (table 2). boerhavia leaves have been used in soups (table 2). colignonia has been reported as food, attributed to peru (table 2). food use for mirabilis has been attributed to ecuador, peru, lake titicaca for both bolivia and peru, the andes, the southern ryuku islands, japan, okinawa, china and india (table 2). mirabilis roots, leaves, flowers and starch have been reported as food (table 2). mirabilis has been cultivated and cured to sweeten, and also cultivated as a sweet form. mirabilis roots have been dried and eaten like carrots, leaves have been used in soups and salads, and the starch in japan for specialty baking (table 2). mirabilis flowers have been used as a food dye in china (bogle, 1974). popenoe et al. (1989) and cabieses (1995) discuss use of cultivated m. expansa for food in the andes, and in particular for cabieses, in the region of lake titicaca for both peru and bolivia. both authors discuss m. expansa’s nutritional value, potential as a future crop, and note it has become rare in peru and the andes. our experience in ecuador is similar regarding m. expansa’s scarcity. cabieses says that fresh dug roots contain substances which irritate the mucosa of the mouth, but curing the plant in the sun transforms roots, giving them a sweet, attractive flavor. popenoe et al. (1989) says ecuadorian m. expansa is a sweet form, not astringent like m. expansa in peru, and discusses the plant’s use for forage. our nutrition research supports claims of high protein values for roots and leaves (kritzer van zant, 2016a). sperling and king (1990) describe m. expansa as one of the roots and tubers of the andean crop complex. the high density of raphides that we have seen in m. expansa, most likely made of calcium oxalate, may affect how mature leaves as well as roots of m. expansa and other nyctaginaceae taxa should be processed for use as food, particularly for humans. food use for pisonia has been reported, attributed to new guinea and the pacific and apparently malaysia (table 2). pisonia leaves were used as a pot-herb in the pacific and trees were cultivated, apparently for food, in malaysia (bogle 1974; table 2 below). pisonia has been used in new guinea as a source for salt (napralert, 1998; table 2 below). unspecified nyctaginaceae genera were mentioned as used for food in bolivia, paraguay and mexico, by cárdenas (1969), basualdo et al. (1995), and martínez (1969), respectively (table 2). this information was combined by us with information from moerman (1998) for an overview of food use for the nyctaginaceae at the family level (table 2). specific information was sometimes included for the nyctaginaceae, by moerman (1998) for roots, leaves, fruits and seeds, as well as the previous mention of starch for mirabilis by chang et al. (1983) (table 2). as for water in which m. expansa roots were boiled in the andes, leaves of nyctaginaceae were used by north american tribes for making refreshing cold drinks as well as for tea (moerman, 1998; table 2 below). anecdotal reports of other uses of the nyctaginaceae lumber, floriculture and other uses of nyctaginaceae genera – anecdotal reports (table 3) includes information from ruiz (1777-1788 (1940)), bogle (1974), hamel and chiltoskey (1975), walker (1976), olsson (1991), basualdo et al. (1995), armstrong (1998), napralert (1998), burger (2005), and agrebi et al. (2008), in addition to family level information from moerman (1998). boerhavia has been reported as used for flypaper (napralert (1998); table 3 below). pisonia has also been used for its sticky substances (see below). bougainvillea and commicarpus have been cultivated in okinawa and the southern ryuku islands, respectively (table 3). this is apparently for landscaping and/or floriculture. mirabilis has been reported for several uses in addition to food and medicine, attributed to japan, okinawa, the southern ryuku islands and north america (table 3). mirabilis starch has been used alone as a cell medium for extracellular protease production in bacillus spp. (table 3). m. 255 table 2. food and forage uses of nyctaginaceae genera* -anecdotal reports. jalapa tuber starch, by itself, has been shown to provide all carbon, nitrogen and salts needed by bacillus (agrebi et al., 2008). ground seeds of mirabilis have been used as a cosmetic powder and unspecified parts for face powder (table 3). varied parts of mirabilis have been used for dye (table 3). mirabilis nyctaginea (michx.) macmill. leaves were soaked in milk to make a fly poison (hamel and chiltoskey, 1975; table 3 below). as walker (1976) said for bougainvillea, m. jalapa was commonly cultivated on okinawa and the southern ryukyu islands, and though it is also not native there, it is frequently utilized in flower gardens, again particularly in the tropics (table 3). walker (1976) recounts that m. jalapa sometimes escapes, in one or both of these locations. neea fruits were used as a source of bright violet dye (table 3). neea leaves were chewed to blacken teeth to prevent tooth decay in columbia and peru (table 3). pisonia also has uses besides medicine and food. pisonia was used for lumber in argentina and jamaica (table 3). sticky substances are exuded from projections on the persistent calyx of pisonia in the pacific and western indian ocean (table 3). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 256 use genus plant part preparation tribe location source food boerhavia roots australia bogle (1974) food boerhavia roots peru bogle (1974) pot-herb boerhavia leaves soups south america bogle (1974) food colignonia peru ugent and ochoa (2006) food dye mirabilis flowers china bogle (1974) food mirabilis sometimes eaten okinawa walker (1976) food mirabilis sometimes eaten southern ryuku islands walker (1976) food mirabilis cultivated andes popenoe et al. (1989, 1990) food mirabilis cultivated, cured to sweeten peru, lake titicaca cabieses (1995) food mirabilis cultivated, cured to sweeten bolivian, lake titicaca cabieses (1995) food mirabilis cultivated sweet form ecuador popenoe et al. (1989) forage mirabilis andes popenoe et al. (1989) food mirabilis leaves soups, salads andes cabieses (1995) food mirabilis leaves soups, salads andes popenoe et al. (1989) food mirabilis roots dried, then used like carrots ecuador kritzer van zant (2016a) food mirabilis roots crop andes sperling and king (1990) food mirabilis peru ugent and ochoa (2006) food mirabilis roots india napralert (1998) food nyctaginaceae bolivia cárdenas (1969) food nyctaginaceae paraguay basualdo et al. (1995) food? pisonia leaves cultivated tree malaysia bogle (1974) food pisonia leaves pot-herb pacific bogle (1974) salt pisonia new guinea bogle (1974) food abronia roots sometimes eaten nat am standley (1911) food abronia roots large roots, sweet like sugar beets kallam western washington state gunther (1945) food abronia roots large roots consumed in fall makah western washington state gunther (1945) food abronia pacific coast region gunther (1945) food nyctaginaceae mexico martínez (1969) animal food allionia tubers dug up by pigs smith (1932) food nyctaginaceae roots nat am n am moerman (1998) food nyctaginaceae leaves for making tea and refreshing cold drinks nat am n am moerman (1998) food nyctaginaceae fruits nat am n am moerman (1998) food nyctaginaceae seeds nat am n am moerman (1998) food mirabilis starch specialty baking japan chang et al. (1983) -nat am = unspecified native american tribes; n am = north america. -moerman (1998) is only cited here at the family level. as for all of the authors most information given at the genus level is available at the spp. level in the original source. -although some categories of use probably overlap, they were not compiled further as their exact meaning may have slightly different definitions in different sources. -an attempt was made to set up the coding of use so that similar descriptions of use would sort in succession, though this was not always possible. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 257 ta bl e 3. l um be r, fl or ic ul tu re a nd o th er u se s of n yc ta gi na ce ae g en er a* an ec do ta l r ep or ts. u se g en us pl an t p ar t pr ep ar at io n tr ib e lo ca tio n so ur ce flo w er g ar de ns bo ug ai nv ill ea cu lti va te d o ki na w a w al ke r (1 97 6) flo w er g ar de ns c om m ic ar pu s cu lti va te d so ut he rn r yu ku isl an ds w al ke r (1 97 6) co sm et ic p ow de r m ira bi lis se ed s gr ou nd ja pa n bo gl e (1 97 4) fa ce p ow de r m ira bi lis o sh iro i-b an a / fa ce -p ow de r flo w er o ki na w a w al ke r (1 97 6) fa ce p ow de r m ira bi lis o sh iro i-b an a / fa ce -p ow de r flo w er so ut he rn r yu ku isl an ds w al ke r (1 97 6) dy e, b rig ht v io le t n ee a fr ui t pe ru ru iz (1 77 717 88 , 19 40 ) pr ev en t t oo th d ec ay n ee a le av es ch ew ed to b la ck en te et h pe ru n a pr a le rt (1 99 8) pr ev en t t oo th d ec ay n ee a le av es ch ew ed to b la ck en te et h c ol um bi a n a pr a le rt (1 99 8) ba rr el h oo ps pi so ni a br an ch es ja m ai ca bo gl e (1 97 4) bo xm ak in g pi so ni a w oo d a rg en tin a bo gl e (1 97 4) co ns tru ct io n pi so ni a w oo d a rg en tin a bo gl e (1 97 4) fly p oi so n m ira bi lis le av es le av es s oa ke d in m ilk c he ro ke e n a m h am el a nd c hi lto sk ey (1 97 5) dy e m ira bi lis va rie d pa rts n at a m n a m m oe rm an ( 19 98 ) da nd ru ff s ha m po o n yc ta gi na ce ae n at a m n a m m oe rm an ( 19 98 ) to ba cc o su bs tit ut e n yc ta gi na ce ae n at a m n a m m oe rm an ( 19 98 ) fly p oi so n n yc ta gi na ce ae n at a m n a m m oe rm an ( 19 98 ) m ed ic al d ia gn os is ai de n yc ta gi na ce ae ro ot s ha llu ci no ge ni c ai de n at a m n a m m oe rm an ( 19 98 ) pr ot ec tio n ag ai ns t w itc hc ra ft m ira bi lis le av es in fu sio n is m ad e to p ro te ct ag ai ns t w itc hc ra ft n at a m n a m m oe rm an ( 19 98 ) ce ll m ed ia ba ci llu s sp p. m ira bi lis tu be r sta rc h us ed a lo ne fo r ex tra ce llu la r pr ot ea se pr od uc tio n a gr eb i e t a l. (2 00 8) gl ue pi so ni a ca ly x pr oj ec tio ns pa ci fic a rm str on g (1 99 8) gl ue pi so ni a ca ly x pr oj ec tio ns w es te rn i nd ia n o ce an bu rg er ( 20 05 ) gl ue pi so ni a ca ly x pr oj ec tio ns to tr ap b ird s, us ed b y hu m an s v an ua tu a nd t he so lo m on is la nd s o lss on ( 19 91 ) fly p ap er bo er ha vi a n a pr a le rt (1 99 8) -n at a m = u ns pe ci fie d n at iv e a m er ic an tr ib es ; n a m = n or th a m er ic a. -m oe rm an (1 99 8) i s on ly c ite d he re a t th e fa m ily le ve l. a s fo r al l o f th e au th or s m os t i nf or m at io n gi ve n at th e ge nu s le ve l i s av ai la bl e at th e sp p. l ev el in th e or ig in al s ou rc e. -a lth ou gh s om e ca te go rie s of u se p ro ba bl y ov er la p, th ey w er e no t c om pi le d fu rth er a s th ei r ex ac t m ea ni ng m ay h av e sli gh tly d iff er en t de fin iti on s in d iff er en t so ur ce s. -a n at te m pt w as m ad e to s et u p th e co di ng o f us e so th at s im ila r de sc rip tio ns o f us e w ou ld so rt in s uc ce ss io n, th ou gh th is w as no t a lw ay s po ss ib le . table 4. nyctaginaceae genera* -results of laboratory analyses. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 258 genus plant part preparation compound assay target action source mirabilis seeds isolated peptides, two bacteria, 2 gram positive active against cammue et al. (1992) mirabilis seeds isolated peptides, two bacteria, gram negative inactive cammue et al. (1992) mirabilis seeds isolated peptides, two cells, cultured human inactive cammue et al. (1992) mirabilis seeds isolated peptides, two fungi, 13 pathogenic active against cammue et al. (1992) mirabilis seeds isolated peptides, two insects, pulse-transmission inactive, similar to spider neurotoxic peptides cammue et al. (1992) boerhavia extracted cells, hiv infected activity, weak napralert (1998) boerhavia extracted pseudomonas aeroguinosa (schroeter) migula active against napralert (1998) boerhavia extracted staphylococcus aureus (rosenbach) zopf active against napralert (1998) boldoa escherichia coli (migula) castellani and chalmers active against napralert (1998) boldoa propionibacterium acnes (gilchrist) douglas and gunter active against napralert (1998) boldoa extracted pseudomonas aeroguinosa (schroeter) migula active against napralert (1998) boldoa salmonella typhosa (zopf) white active against napralert (1998) boldoa sarcina lutea schroeter active against napralert (1998) boldoa shigella flexneri (flexner) castellani and chalmers active against napralert (1998) boldoa staphylococcus aureus active against napralert (1998) bougainvillea bacillus subtilis (ehrenberg) cohn active against napralert (1998) bougainvillea staphylococcus aureus active against napralert (1998) used for dispersal of fruits, this natural glue from pisonia was also used by humans to trap birds on vanuatu and the solomon islands (table 3). this glue has been reported to persist for many decades on pisonia herbarium specimens, and can be diluted with water (burger, 2005). boerhavia’s sticky surfaces have also been used for related purposes (table 3). branches of jamaican pisonia are utilized for barrel hoops, and wood in argentina is used for box-making and construction (table 3). moerman (1998) also reported uses other than medicine and food for the nyctaginaceae by native americans in north america (table 3). native americans used the nyctaginaceae for dandruff shampoo, fly poison and a tobacco substitute (table 3). there were mystical uses as well. nyctaginaceae roots were used as a hallucinogenic aide for medical diagnosis (table 3). leaves of nyctaginaceae were used for making an infusion to protect against witchcraft (table 3). laboratory analyses of nyctaginaceae nyctaginaceae genera – results of laboratory analyses (table 4) consists of results reported for chemical isolations and extracts from boerhavia, boldoa, bougainvillea, cryptocarpus, and mirabilis, utilized in assays. these assay results are summarized from published data in cammue et al. (1992), pelayo-benavides and anaya (1993), vivanco et al. (1997; 1999a, b), napralert (1998), vivanco and flores (2000), and vepachedu et al. (2003, 2005) (table 4). information on our update of the nomenclature for the target organisms is given after the summary of activity for these genera. chemical constituents of leaves of m. jalapa, apparently from india, were studied by behari, et al. (1975), though no details were given by them for individual assays, so there was insufficient information to include their results in table 4. boerhavia extracts are weakly active against hiv infected cells, and active against pseudomonas aeroguinosa (schroeter) migula and staphylococcus aureus (rosenbach) zopf (table 4). boldoa is active against escherichia coli (migula) castellani and chalmers, propionibacterium acnes (gilchrist) douglas and gunter, pseudomonas aeroguinosa (schroeter) migula, salmonella typhosa (zopf) white, sarcina lutea schroeter, shigella flexneri (flexner) castellani and chalmers, and staphylococcus aureus (table 4). bougainvillea is active against bacillus subtilis (ehrenberg) cohn, and staphylococcus aureus (table 4). cryptocarpus is active against neurospora crassa shear and b. o. dodge (table 4). mirabilis material has activity against enterobacter hormaeche and edwards spps., epidermophyton floccosum (harz) langeron and miloch., escherichia coli (migula) castellani and chalmers, salmonella typhosa, shigella flexneri, staphylococcus a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 259 ta bl e 4. c on tin ed . g en us pl an t p ar t pr ep ar at io n c om po un d a ss ay t ar ge t a ct io n so ur ce ry pt oc ar pu s n eu ro sp or a cr as sa sh ea r a nd b . o . do dg e ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis en te ro ba cte r ho rm ae ch e an d ed w ar ds s pp s. ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis ep id er m op hy to n flo cc os um (h ar z) la ng er on a nd m ilo ch . ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis es ch er ich ia c ol i ( m ig ul a) c as te lla ni an d ch al m er s ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis ex tra cte d sa lm on el la p ar at yp i (e x ka ys er ) ez ak i e t a l. 20 00 * a ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis sa lm on el la ty ph os a ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis sh ig el la fl ex ne ri ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis st ap hy lo co cc us a ur eu s ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis st re pt oc oc cu s py og en es r os en ba ch ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis tr ich op hy to n m en ta gr op hy te s (c .p .r ob in ) s ab ou r ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis vi br io c ho le ra [l ik el y v. ch ol er ae pa cin i] ac tiv e ag ai ns t n ap ra le rt (1 99 8) m ira bi lis tri go ne lli ne a lk al oi d ce ll cy cle , a lle lo pa th ic po te nt ia l ar re sts c yc le in g st ag e pe la yo -b en av id es a nd a na ya (1 99 3) m ira bi lis ch lo ro fo rm e xt ra cts lix iv ite s ro ot ti ps o f p ea s, al le lo pa th ic po te nt ia l su pp re ss m ito tic a cti vi ty pe la yo -b en av id es a nd a na ya (1 99 3) m ira bi lis ch lo ro fo rm e xt ra cts lix iv ite s ro ot ti ps o f p ea s, al le lo pa th ic po te nt ia l al te re d ce llu la r o rg an iz at io n pe la yo -b en av id es a nd a na ya (1 99 3) m ira bi lis re po rte d, s ou rc e un sp ec ifi ed co lin es ac tiv e -u ns pe cif ie d pe la yo -b en av id es a nd a na ya (1 99 3) m ira bi lis ro ot s ex tra cte d pr ot ei ns fu ng i, so m e ar e clo se re la tiv es to ac tiv e ag ai ns t sp p. in ac tiv e, in di ca te s sp ec ifi c ac tio n vi va nc o et a l. (1 99 7; 1 99 9a ,b ) vi va nc o an d fl or es (2 00 0) ve pa ch ed u et a l. (2 00 3, 2 00 5) m ira bi lis ro ot s ex tra cte d pr ot ei ns ba cte ria , so m e ar e clo se re la tiv es to ac tiv e ag ai ns t sp p. in ac tiv e, in di ca te s sp ec ifi c ac tio n vi va nc o et a l. (1 99 7; 1 99 9a ,b ) vi va nc o an d fl or es (2 00 0) ve pa ch ed u et a l. (2 00 3, 2 00 5) m ira bi lis ro ot s ex tra cte d pr ot ei ns ba cte ria , so m e sp ec ie s ac tiv e ag ai ns t vi va nc o et a l. (1 99 7; 1 99 9a ,b ) vi va nc o an d fl or es (2 00 0) ve pa ch ed u et a l. (2 00 3, 2 00 5) m ira bi lis ro ot s ex tra cte d pr ot ei ns fu ng i, se ve ra l s oi l p at ho ge ni c ac tiv e ag ai ns t vi va nc o et a l. (1 99 7; 1 99 9a ,b ) vi va nc o an d fl or es (2 00 0) ve pa ch ed u et a l. (2 00 3, 2 00 5) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 260 aureus, streptococcus pyogenes rosenbach, trichophyton mentagrophytes (c. p. robin) sabour and vibrio cholera [likely v. cholerae pacini] (table 4). an extract of mirabilis was active against salmonella paratypi (ex kayser) ezaki et al. 2000* a (table 4). two peptides of m. jalapa, isolated from seeds, are selectively active for two gram positive bacteria and inactive against cultured human cells of unnamed origin (table 4). extracted root proteins from mirabilis are selectively active against bacteria (table 4). a trigonelline alkaloid from m. jalapa arrests the cell cycle in the g2 stage (table 4). the same two peptides found active against two gram positive bacteria, are active against 13 pathogenic soil fungi (table 4). these same two peptides isolated from m. jalapa seeds were found to have chemistry similar to spider neurotoxins, yet were inactive for pulse transmission in unspecified insects (table 4). extracted mirabilis root proteins are selectively active against several soil pathogenic fungi, and this selectivity is sometimes for closely related fungal species (table 4). lixivites from chloroform extracts of mirabilis both suppressed mitotic activity and altered cellular organization, in the root tips of peas (table 4). colines from m. oxybaphus were reported as having unspecified activity (table 4). taxonomic updates of microorganism names author names were not given in napralert (1998) for microorganisms utilized in assays. these names were updated here as well as we could, based on ncbi (2011) and its considerable links, ebicbn (1958), and taxonomic literature for individual taxa. several points are added here about those taxonomic updates. once again, an asterisk indicates that we did not see the original source of information. salmonella paratypi is a rejected name for s. enterica kauffmann and edwards subsp. enterica (ex kauffmann and edwards) le minor and popoff 1987* serovar paratyphi a, though s. enterica (ex kauffmann and edwards) le minor and popoff 1987* is apparently an earlier rejection of s. enterica which has since been overturned, as this rejected version of the name is listed as a synonym of s. enterica (ncbi, 2011). it is also unclear if subsp. enterica is the currently accepted name for the subspecies or if subsp. is the new correct name, or who authored this last name or the names serovar or paratyphi in conjunction with the genus name salmonella (ncbi, 2011). these are questions for the original authors of the assay research and/or taxonomists specializing in those organisms. we also specified enterobacter hormaeche and edwards spps. enerobacter rahn which is also in the literature, though it does not appear to be a legitimate name. napralert (1998) had many more reports of inactivity than of activity for the nyctaginaceae, often indicating specific mechanisms when activity was found. reports of toxicity for mirabilis the single report in napralert, of human toxicity from consumption of m. jalapa, is from singapore, and was derived from an incomplete abstract that we have so far been unable to trace to the original source. it is also unclear if this toxicity is as benign as a mild stomach ache or refers to a poisoning resulting in death. it may refer to consumption of an improperly processed root, or toxicity due to some mitigating factor such as contaminated soils. the reports of using nyctaginaceae and m. nyctaginea in particular to make fly poison, may simply be a matter of concentrated oxalic acid crystals dissolving into liquid, or alternatively, the presence of a class i ribosome inhibitor protein which only affects insects. either way, these reports point to the need for proper preparation or utilization of mirabilis and other nyctaginaceae species for food, which may or may not only be safe to eat in certain stages of development or for certain parts of the plant, or both. there is a need for further investigation of toxicity before any mirabilis spp. should be adopted as a modern food on a wide scale. our own negative toxicity data for m. expansa extracts are highly preliminary, and insufficient to fully address this question. this data is from a small scale study on young plants grown in constructed plots in a single local, and only concerns micro-molecules and not peptides or proteins. however, our results do not indicate that there is a toxic micromolecule in m. expansa, which is encouraging for m. expansa’s future as a food source, even if some form of processing is required to remove or mitigate the oxalic acid. many plants utilized extensively for food have toxic parts, including carrots, potatoes and cashews. these crops have been proven to be a boon for agriculture, human and animal nutrition, and culinary purposes, once methods were established for their safe preparation. in addition, varieties of food plants with toxins or irritants have proven valuable for their insect and pest resistance as exemplified by the sweet versus bitter varieties of manihot esculenta crantz (euphorbiaceae) 1766 (ipni, 2011), also known as tapioca, manioc, bitter cassava, or yuca. again, it is a matter of knowledge of techniques for proper preparation. therefore, it may be that m. expansa and perhaps other nyctaginaceae species will prove useful as cash crops, with proper processing prior to consumption of more than small amounts for food. archeological significance of small starch particles in mirabilis m. jalapa starch has been used for specialty baking in japan, because of the extremely small size of the starch particles (chang et al., 1983). minute starch particles in m. jalapa were seen by ourselves under magnification of about 30,000 x, under both scanning and transmission electronic microscopes. both techniques require fixed material for imaging (bozzola and russell, 1999). we were unable to see m. jalapa starch grains under a standard microscope at 1000x. this lower magnification is used for archeological studies in which plants are identified using starch techniques. these techniques use starch which has not been fixed (ugent et al., 1986; ugent and peterson, 1988; ugent and cummings, 2004; torrence and barton, 2006). this may be why m. expansa is not reported from examination of excavated tools for preparing foods, found at archeological sites in the andes. it may also be why there have not been archaeological reports of m. jalapa starch for food in mexico. top soil over sand over gravel is characteristic of the plaza at the incan site of machu picchu in peru (nova, 2010). the garden terraces a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 261 leading up to machu picchu were of the same construction as the plaza (tpompo, 2012). sand over gravel is also characteristic of the plots used to successfully grow m. expansa outdoors in southern illinois (kritzer van zant, 2016a). it is plausible that m. expansa played a role in allowing the incas to occupy their highest altitude cities, even though this is not known from previous archeological research in the andes. update of taxonomic nomenclature relating to medicinal jalap mirabilis jalapa l. is the type for the family, and also the first nyctaginaceae species for which we have found published reported use by humans. to understand how medicinal jalap and m. expansa are intertwined it is helpful to understand the plant’s modern history, including its taxonomy. nyctaginaceae is a conserved name based on the genus name nyctago juss., which is a synonym of mirabilis l., according to article 19 of the 1966* icbn (international code of botanical names) (douglas and spellenberg, 2010). m. jalapa appears to have been distributed horticulturally in the british empire from the first quarter of the 16th century, which includes nearly the earliest years of the spanish conquest of mexico. clusius (1583) named a specimen from india admirabili peruana, which based on the description and included drawing, is clearly a synonym for m. jalapa l. peruana is either a species epithet, or clusius attributed mirabilis’ origin as a genus to peru, according to mirabilis literature and type descriptions of admirabilis, bryonia, jalap, jalapa, and jalappa. admirabili or admirabilis, is the earliest pre-linnaean latin synonym of mirabilis found in the course of taxonomic work on mirabilis by this author. bogle (1974) dates european horticultural use of m. jalapa to an even earlier date, 1552. bogle based this on a drawing in the badianus manuscript attributed to emmart [undated*]. bogle goes on to say that according to curtis (1797*) and showalter (1934), european introduction of m. jalapa by the spaniards occurred earlier still, in 1552. mirabilis jalapa l. was apparently given its specific name because of its presence in xalapa, mexico. the specific name jalapa was also used by linnaeus for a plant of similar appearance in the convolvulaceae (see below). this plant, like mirabilis, has a complex taxonomic history, and was the real source of medicinal jalap (felter and lloyd, 1898 (2010)). m. jalapa has been used as a substitute source of medicinal jalap. however, it was unclear to us if this substitution was real, or reported due to confusion over the names. walker (1976), while writing about the nyctaginaceae in okinawa and the southern ryuku islands, states that jalap resin has been extracted from m. jalapa roots, apparently in reference to historical extraction of jalap in mexico or spain. it was not clear from walker if m. expansa had ever been used for this purpose in the pacific as well. m. expansa’s identification as medicinal jalap was an “erroneous belief” according to bogle (1974). convolvulaceae is the family in which true medicinal jalap and several sometimes substituted relatives belong taxonomically. these relatives of true jalap are medicinally of inferior quality. several of these relatives have vernacular or common names including the term ‘jalap’. felter and lloyd (1898 (2010)) seem to be the source of much of the information and error found on a website for medicinal jalap (annonymous, 2010). annonymous (2010) also includes information on medicinal use of jalap from grieve (1931). this website steered us to felter and lloyd for detailed information on the use of m. jalapa as a substitute source for jalap syrup. prior to reading felter and lloyd, this author had only seen vague notes on m. jalapa as a substitute source of jalap, with little detail included. presented next is the story of jalap with updated notes on the taxonomy. bryonia mechoacana nigricans [no author given (felter and lloyd, 1898 (2010), annonymous 2010), was the first name applied to the true medicinal jalap plant. this latin name first appeared in 1609 (felter and lloyd, 1898 (2010)), annonymous 2010) and would actually be b. mechoacana nigrícans c. bauhin (1620). linnaeus (1753) used the spelling ‘nigracans’. the author name ‘bauh,’ which belongs to two different bauhins and was given with three citations by linnaeus (1753). linnaeus first citation is ‘bauh. pin. 293’, which was ‘pinax theatri botanici 1671’ by caspar (gaspard) bauhin (1560-1624). this author today is abbreviated as c. bauhin. the second citation is ‘prod. 135’, which was most likely ‘prodromos theatri botanici.’ this title was abbreviated in ipni (2010) as ‘prodr. (bauhin) edition 1 1620,’ also by c. bauhin. the earliest of linnaeus’ three 1753 citations; ‘bauh, hist. 2. p. 151’ is j.bauhin et al.’s ‘historia plantarum universalis nova, et absolutissima: cum consensu et dissensu circa eas. ...,’ published from 1650-1651* (ipni, 2010). j.bauhin is jean johannes bauhin (1541-1613) (ipni, 2010). next, ray named medicinal jalap convolvulus americanus [undated]* (felter and lloyd, 1898 (2010)), annonymous 2010). linnaeus’ author name for c. americanus appears as if it is spelled ‘raj.’ however, there is no author abbreviation spelled ‘raj.’ (ipni, 2010, brummitt and powell, 1992). ray is named author of c. americanus in both felter & lloyd (1898 (2010)) and annonymous (2010). this is john ray (1627-1705) (ipni, 2010). john ray (1682) includes latin descriptions of convolvulus and c. nigra, with the species name given on page 68, not page 67 as ray referenced in his index. we have not found a description nor mention of c. americanus by ray. ipni (2010) gives c. americanus (sims) j.w.louden 1844*, c. americanus greene 1898*, and c. americanus (sims) greene, for a variety of c. americanus named by sims in 1804*. however, c. americanus ray is not in ipni. as c. americanus ray is pre-linnaen, it cannot have been validly published by ray. linnaeus (1737) added after ‘convolvulus americanus’ the words “jalapium dictus” meaning ‘called jalapium’. no doubt linnaeus meant jalapium is the common name. but linnaeus (1737) also includes ‘jalapium & mechoacana nigra s.dale [1693*-1737*]’ as synonyms of mirabilis. felter and lloyd (1898 (2010)) and annonymous (2010) erred, in utilizing ‘convolvulus americanus jalapium dictus’ in full as ray’s name for medicinal jalap, slightly misinterpreting linnaeus (1737) and possibly ray as well. tournefort identified medicinal jalap as mirabilis, after being “deceived” by persons who claimed to know the plant (felter and lloyd, 1898 (2010); annonymous 2010). perhaps this was an honest mistake by tournefort’s sources as both plants have enlarged roots and large tubular flowers. also, frequently specimens of m. jalapa and many convolvulaceae taxa have cordate leaves. in addition, both m. jalapa and medicinal jalap a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 262 have been reported as purgatives. whether due to deliberate deception or innocent misidentification by others, tournefort’s misidentification led to further confusion about the identity of medicinal jalap and more names. balfour identified medicinal jalap as exogonium purga, though no author was given (felter and lloyd, 1898 (2010)) and annonymous (2010). e. purga lindl. (convolvulaceae) 1847 is given as a legitimate name in ipni (2010). linnaeus named true jalap convolvulus jalapa (felter and lloyd, 1898 (2010); annonymous 2010), which ipni (2010) dates as 1767. linnaeus had named the type specimen for m. jalapa in 1753 so he at least was not confused about the taxonomic identities of these two plants. differences of opinion continued until 1827, when j.r. coxe of philadelphia, obtained perfect flowers from roots of true jalap plants taken directly from their native mexican soils (felter and lloyd, 1898 (2010); annonymous 2010). felter and lloyd (1898 (2010)) and annonymous (2010), state that coxe’s new material was named ipomoea purga wender. & hayne. however, the earlier i. jalapa nutt. (convolvulaceae) at first appears more likely to be the correct legitimate name for medicinal jalap. ipni (2010) attributes i. purga 1833* separately to each author in the same publication in, “arzneyk. ann. 12: pls 33, 34”. however, ipni (2010) does not attribute this name to both authors together. perhaps one was the editor. in addition ipni (2010) has i. jalapa scheiede & deppe ex g.don, and i. jalapa pursh 1813*. however, the title page in both volumes of pursh is dated 1814. it may be that 1813 is the year of acceptance for the document and 1814 the year of publication. ipni (2010) also attributes to the same source i. jalapa (l.) pursh, with the date 1814*, and i. jalapa coxe 1829 (1830)* cited from “journ. am. med. sc. v....300.” however, i. jalapa nutt. is not currently found in ipni. common names for medicinal jalap (convolvulaceae), relevant for understanding m. jalapa references, include in english jalap, fusiform jalap, jalap bindweed, tampico jalap, true jalap, in germanjalap, swedishjalap, jalapa, jalaparot, portugesejalapa, spanishjalapa, and mechoacán (felter and lloyd, 1898 (2010)). four other plants in the convolvulaceae include jalap in their common names and are used as substitute sources of medicinal jalap, though they are of lower quality than true jalap. first is i. orizabensis (pellet.) ledeb. ex steud., with english common names fusiform jalap, jalap stalks, jalap tops, light jalap, male jalap, orizaba jalap, and woody jalap. second is i. pandurata (l.) g.mey, synonym c. panduratus l., with the english common name wild jalap. third is i. simulans hanb. with the english common name tampico jalap. fourth is operculina turpethum (l.) r.br., syn i. turpethum (l.) r.br., with the english common name indian jalap (felter and lloyd, 1898 (2010)). thus, the potential exists to find entanglements in the literature for m. jalapa with all of these species, as well as with true jalap. though we have laid out the story and partially clarified the references for medicinal jalap in the convolvulaceae, we did not confirm references marked with asterisks or evaluate many of the original sources beyond their presentation in ipni. the final decision as to the proper name of true medicinal jalap is left to those who study the convolvulaceae. what matters in this story for mirabilis taxonomy, is that the origin of several mysterious synonyms, including both genus names and species epithets, and of common names mixed into synonym lists or utilized as latin synonyms by some authors for m. jalapa in the literature, can now be understood in terms of their place in the convolvulaceae. examples are linnaeus’ (1737) inclusion of the genus names bryonia, jalapa, jalapium, mechoacana, and convolvulus as synonyms for mirabilis. as for economic application, reports of m. jalapa as a low quality substitute for medicinal jalap, could either be real, based on the uses reported above from many sources for this plant, or could be a simple case of mistaken identity based on inaccurate identification and confused taxonomy. m. jalapa contains calcium oxalate raphide crystals, true jalap does not (felter and lloyd 1898 (2010); annonymous 2010). it is these large calcium oxalate raphides, reported for mirabilis and at least some other nyctaginaceae, which may be responsible for some of the soothing and purgative effects of mirabilis reported in traditional medicine. still, the effects of proteins and peptides have been shown to go well beyond this, and there is potential for new anti-fungal and anti-bacterial compounds for both agriculture and medicine in the family. yet few people today are familiar with the uses of these plants. lawrence (1951, 1970) predates bogle (1974) in saying that the nyctaginaceae are economically of little domestic importance. this may be due to two linked issues, confusion over taxonomy, and insufficient research on useful taxa in the family. historical precedence for mirabilis spp. as experimental organisms in plant breeding and genetics there is a long history of plant breeding with mirabilis. at the time of conquest, europeans found indigenous peoples breeding m. jalapa for its flowers in mexico, and m. expansa for food in the andes. western science also has a long record of experimentation with breeding mirabilis. these experiments were among those that helped with the formulation of some basic theoretical concepts in plant genetics, and therefore have yielded information which became of economic value. in linnaeus’ dissertation, he recorded as his first experiment, emasculating flowers of m. longiflora l., then pollinating them with pollen from m. jalapa (roberts, 1929). linnaeus noted that ovules grew from this cross, though they did not mature (roberts, 1929). later, linnaeus pollinated m. longiflora flowers with pollen from other flowers of the same species, and observed that seeds matured. linnaeus then experimented further with pollen utilizing other taxa, leading him to conclude that such experiments demonstrate, “the generation of plants” (roberts, 1929). nineteenth century german doctor and plant breeder wilhelm olbers focke noted that hybrid formation does not always succeed equally well in both directions (roberts, 1929). focke included linnaeus’ crosses of m. jalapa and m. longiflora as an example of greater or lesser seed production depending on the direction of pollination (roberts, 1929). darwin echoed this concept in the origin of species, also referencing kolreuter’s crosses with m. jalapa in the late 1700’s (roberts, 1929). german botanist kolreuter counted pollen grains used for crosses with m. jalapa, relating their numbers to numbers of seeds produced (roberts, 1929). kolreuter also crossed red-flowered a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 263 with yellow-flowered m. jalapa and made the reciprocal cross, noting that an intermediate yellow-orange color resulted from both (roberts, 1929). kolreuter then back-crossed a yellowflowered individual to the hybrid, noting that the resulting flower color had a greater degree of yellow (roberts, 1929). these crossing experiments by kolreuter foreshadowed mendel’s work with garden peas. professor carl correns, who rediscovered mendel’s paper on the latter’s initial work with peas, and who also made his own crossing experiments with peas at the end of the 19th century, noted that mirabilis jalapa was, “one of my most fruitful objects of research” (roberts, 1929). mendel (1865) praised kolreuter and lecoq among others, in the introduction to experiments in plant-hybridisation. french botanist henri lecoq was said by his 19th century contemporary, dominique alexandre godron, to have observed that fertile hybrids of mirabilis grown with their parents were more likely to produce offspring which returned to the parent type (roberts, 1929). godron, also french and a physician botanist, made a general rule about hybrids returning to the parental forms, based in part on lecoq’s work with mirabilis (roberts, 1929). however, godron did not understand the true mechanisms involved (roberts, 1929). this author believes that mirabilis and other nyctaginaceae still have much to reveal about basic genetic principles. our own observations on the apparently independent development of structures in andean mirabilis indicate to us that one of the greatest social and economic values for these plants may be in their usefulness for identifying epigenetic and ploidy patterns which impact evolution, particularly timing of expression. poh gene’s varied effect on embryo development, via timing of activation of the gene, is now thought to be responsible for arthropod limb variation seen in the pre-cambrian fossil record (carroll, 2005). poh is one of many such genes and sometimes single alleles, known to have dramatically different effects based on the frequency and duration of their activity in early embryonic development in animals (carroll, 2005). research to identify similar genes in plants has been underway (grant-downton and dickinson, 2005; schaacke et al., 2010). phylogeny is being studied for the nyctaginaceae and several genera within the family (douglas and spellenberg, 2010; hernández-ledesma et al., 2010; hernández-ledesma et al., 2015). however, there does not seem to be any recent or current studies of epigenetic and/or ploidy mechanisms which are likely to underlie the visually obvious, real time variation of expression frequently found in individuals in the nyctaginaceae. discussion selective activity is important for drug discovery, which frequently begins with investigation of general cell toxicity. selective activity indicates that extracts and isolates are less likely to harm healthy cells, at least at the right dosages. selective activity is also important for agricultural application of molecules derived from biological materials, to crops intended for consumption by humans or livestock. as napralert (1998) reported more inactivity than activity in assays utilizing extracts and isolations from the nyctaginaceae, specificity may be very high for many nyctaginaceae active compounds. correct identification of any plant or other organism, is basic and necessary for meaningful extrapolation of research, from individuals or sample populations, to other members of the same taxon. therefore correct identification is a basic ingredient in getting biological research funded. confusing taxonomy has long been a problem for funding some types of research for many taxa in the nyctaginaceae. in addition, epigenetic and ecological effects may powerfully affect chemical production for these taxa. plants in many families show variation in production of bio-chemicals due to ecology, and probably also due to epigenetic factors. the wide range of ethnobotany reports, which include many unique descriptions of use, combined with high amounts of morphological plasticity, leads us to wonder if frequent profound biochemical plasticity occurs in the nyctaginaceae. morphological plasticity is frequently visible during development, for multiple structures on individual specimens in the family. as for any medicinal plants, it will be necessary to know if where and how plants are cultivated, impacts production of significant bio-chemicals, and if variation in production under different growth regiments, allows production to occur in a consistent manner. this understanding is necessary to set up successful commercial production for any organism, and has so far been little examined for the nyctaginaceae. food and forestry uses will also require ecological and agricultural studies. this includes a better understanding of each species’ potential to become invasive. this is an additional economic consideration for production of any species for use as crops, pesticides, dyes, adhesives, as well as for medical application. m. expansa is not listed by the usda as invasive. m. jalapa is a well-known invasive in the tropics. though it does not winter over in temperate climates, m. jalapa is capable of re-seeding as an annual under some conditions under cultivation. north american m. nyctaginea (michx.) macmill., is both native (mohlenbrock and voigt, 1959, 1974; spellenberg, 2006), and occasionally an invasive weed in the midwest (buckholtz et al., 1981, 1992). m. nyctaginea has also been reported as an “obnoxious weed” for some states (spellenberg, 2006). m. macfarlanei constance and rollins, is a rare endemic in idaho and oregon, and possibly limited by availability of pollinators in its range, especially the low incidence of hawkmoths (barnes, 1996, 1997). our observations of both m. jalapa and m. expansa, grown outside in southern illinois, showed that both species are visited by multiple potential pollinators. these include hawkmoths, spiders, and ants. we witnessed herbivory on m. expansa by wooly caterpillars and by caterpillars of hummingbird moths. thus, we believe it would be unwise to make assumptions about the behavior of nyctaginaceae species grown under new conditions from their behavior under different conditions, without testing. our work in southern illinois with m. expansa indicates it is highly limited there by drainage and has little resistance to freezing weather including early frosts. however, m. expansa is a perennial crop in the andean highlands (flores et al., 2003), where freezing temperatures are common at night. this could be due to the age of the plants, and whether they have both been propagated and allowed to mature in a particular climate a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 264 prior to strong seasonal changes. it may also be a function of better soil drainage and/or better snow cover in the andes. there are some questions to answer concerning food safety. there is a well-documented history of use as food for several members of this family. there is also indication of high nutritional value available for some nyctaginaceae species for direct human consumption. in addition, nyctaginaceae starch has been used as a medium for growing microorganisms for the production of valuable biochemicals. there is also potential to use some of these plants as sources of forage. toxicity to mammals is not seen in the nyctaginaceae literature, other than the single poorly substantiated report in humans, from m. jalapa, described above. toxicity from the nyctaginaceae has not been reported otherwise, even in taxa where high levels of biochemical activity have been reported. colleagues have told us that sustainable use of woody nyctaginaceae genera such as pisonia and neea would benefit from improved taxonomic work. lawrence (1951, 1970) includes abronia with mirabilis and bougainvillea as notable garden ornamentals, adding even more economic value to the family. highly plastic development, often seen in individual specimens of the nyctaginaceae, may offer a great deal of insight into flowering plant evolution including ploidy and epigenetics. conclusion clearly the nyctaginaceae has much to offer including sources of useful compounds and valuable agricultural material. these benefits should increase once their morphological taxonomy has been clarified. mirabilis’ and possibly other nyctaginaceae genera’s importance as paleo-foods requires further investigation, as the extremely minute size of starch grains in these plants would have prevented their recovery with current starch techniques, from tools for food preparation found at archaeological sites. nyctaginaceae genera, so far abronia, boerhavia and mirabilis in particular, have already been shown to have medicinal value through world-wide examples of traditional use from multiple cultures. the production of potentially useful biochemicals by particular nyctaginaceae species has been confirmed through published results of laboratory assays on specific compounds and their biochemical effects. certainly this family is a good candidate for fully funded studies of not only which genes are in which nyctaginaceae taxa, but also of morphological taxonomy and systematics, epigenetic and ploidy mechanisms, medicinal bio-chemistry, archeo-botany, and further agricultural research including forestry of woody species, floriculture of both woody and herbaceous taxa, plant breeding, use of substrate material for industrial biochemical production, as well as more information on food and forage nutrition, chemistry and safety. acknowledgements thank you to j.m. vivanco for his time. thank you also to bill banz, hee-byung chai, douglas kinghorn and david a. lightfoot for their advice and assistance, and for their help in producing some 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mohamed addi1, ahmed elamrani1, hana caid serghini1, aatika mihamou1, and malika abid1* ¹ laboratory of biology of plants and microorganisms, faculté des sciences oujda – université mohammed premier, bp-717 oujda, morocco; ² laboratory of wallonia center of agronomics researchs (cra-w), département sciences du vivant, bâtiment jean-baptiste de la quintinie, chaussée de charleroi, 234 b-5030 gembloux, belgium received: may 2, 2017 / accepted: june 1, 2017 __________________________________________________ * corresponding author: abidmalika@yahoo.fr 384 abstract for the first time eight local tomato cultivars collected from four different regions of morocco were assessed with rapd and ssr methods. most of rapd markers give monomorphic banding profiles. only opu03 marker showed a total of 4 polymorphic amplicons out of 8 recorded in figuig2 cultivar. the analysis with ssr markers gives more polymorphism. the number of alleles amplified assessed from 2 to 5 alleles among cultivars. the similarity matrix subjected by the unweighted pairgroup arithmetic method (upgma) clustering grouped the cultivars in four groups where figuig2 cultivar formed a separate and more distant cluster. in addition this cultivar holds the very high percentage of uniformity (99%) indicating that is an homogeneous traditional cultivar with high purity. this genotype can be conserved and used in breeding programs. more traditional moroccan cultivars must be collected in order to determine their genetic structure. keywords: moroccan tomato cultivars, rapd and ssr markers, genetic diversity. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction tomato belongs to solanaceae family and it is originated from the andean region, more exactly from ecuador, bolivia, colombia and chile. tomato seeds were introduced by spaniards from mexico to europe for domestication (peralta et al. 2006) and it was then referred as a cultivated plant in italy. at the end of the xixth century, tomato cultivars were self-pollinated and farmer saved seeds from a year to the other. new genotypes resulted from spontaneous mutations, natural outcrossing or recombination of pre-existing genetic variation (bauchet and causse, 2012). commercial itinerary has contributed to spread the species worldwide (diez and nuez, 2008). this has led to collect existing genetic diversity to preserve and to valorise it all over the world through public or private institutes of plant germplasm. the seed sector in morocco has known since the early 70s, development and sustained growth that enabled the creation of a national plant genetic quality. however, tomato has not yet benefited from this progress and we do not yet have a certified moroccan tomato cultivar. in morocco, the most tomato is grown in greenhouses under controlled climate. nevertheless, almost all the seeds were bought from foreigner countries. the use of certified seed by breeders is the means to exploit and disseminate the most effective advances in seed breeding. characterization of tomato germplasm is of great impora tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) tance for current and future agronomic and genetic improvement of the crop. furthermore, if an improvement programme is to be carried out evaluation is imperative, in order to understand the genetic background and the breeding value of the available tomatoes. morphological, biochemical and molecular characterization are used to specify genetic diversity between tomato varieties (garcia et al., 2004). morphological and biochemical characterization is used to evaluate many fruit quality traits such as diameter, height, shelf life, weight size, acidity, colour and firmness. those parameters do not always allow the quantification of genetic diversity in plants and are dependent of environmental factors (cooke, 1994). molecular markers are an efficient tool to investigate the genetic basis of agronomic traits and to make easier the transfer and accumulation of desirable traits between breeding lines. many molecular techniques including amplified fragment length polymorphism (aflp), restricted fragment length polymorphism (rflp), simple sequence repeats (ssr) and random amplified polymorphic dna (rapd) were used to set up genetic variation in tomato cultivar collections (bredemeijer et al., 1998; park et al., 2004; garcia-martinez et al., 2006). local tomato germplasm may provide some natural variation that is present in the species as a whole (brush, 2000; feuillet et al., 2008) since local varieties represent the main source of genetic variation in the cultivated species. they can offer big interest for their use in scientific studies and in breeding programs (chable et al., 2009). for this reason, eight local tomato genotypes and one french commercial tomato used as control were assessed using rapds and ssrs markers to examine the genetic variability, to establish their relationships and to compare the usefulness of these markers. materials and methods plant materials nine lots of tomato were studied in this investigation including one commercial variety of french origin “saint pierre” from vita company used as a control. the other lots were collected from four different regions of morocco and are listed as follow: from berkane region (northeast) two tomato lots noted berkane1 and berkane2, from figuig region (south east), two tomato lots noted figuig1 and figuig2, from rissani region (south west) three batches noted rissanib, rissanio and rissanin in which we found a difference in color between the seeds and finaly, one tomato lot from hoceima region (northern morocco). the seeds of each lot of tomato were sown in the greenhouse for germination and growth. after two weeks of sowing, the seedlings were transplanted and grown in green house at the nursery of the park lala aïcha with a regular watering. genomic dna isolation for total genomic dna extraction, fresh leave of each lot of tomato were grounded in liquid nitrogen and stored at -80°c. genomic dna isolation was performed following the procedure of the dneasy plant mini kit de qiagen. rapd and ssr primers ten rapd random primers and fourteen microsatellite markers were used for molecular characterization of moroccan tomato lots. all rapd and ssr primers were chosen among the highly polymorphic primers published on the literatures (suliman-pollatschek et al., 2002; areshchenkova and ganal, 2002) and have been applied successfully for assessing different plant genotypes. the used rapd primers are opc09, opu03, opa14, opu14, opa15, opb17, opb18, opc08, opg17 and opv19. the simple sequences repeat (ssr) primers are listed in table 2. rapd-pcr amplification pcr assays was performed in a 25µl final volume, containing 20ng of genomic dna, 0.2µm of operon random primer, 100µm dntps, 2.5mm mgcl2, 1mg /ml bsa, 5 x pcr reaction buffer, and 1 u taq dna polymerase (promega). the amplifcations were conducted with thermal cycler (applied system), with an initial 5 min at 94°c that was followed by 45 cycles of 1 min at 94°c, 1 min 30 s at 36°c, and 2 min 30 s at 72°c, ended by 7 min extension at 72°c. pcr product were electrophoresed on 1.4% agarose gel stained with ethidium bromide and observed under uv light and photographed. size of the amplicons was estimated with 1kb dna ladder which was resolved along with amplified product. reproducibility of the results was confirmed by repeating the amplification twice. ssr-pcr amplification for ssr analysis, among the relatively high number of ssr loci already reported in tomato, 14 ssr markers were selected from the published data (suliman-pollatschek et al., 2002; he et al., 2003) or on the website of solanaceae genomics network (http://solgenomics.net). pcr amplification was performed in a 20 µl total volume, containing 20 ng of genomic dna, 0.25 mm of each primer, 200 µm dntps, 1.5 mm mgcl2, 1 mg /ml bsa, 1 x pcr buffer, and 1 u taq dna polymerase (promega). the amplifications were conducted with thermal cycler (applied system), with an initial 5 min at 94°c that was followed by 35 cycles of 30 s at 94°c, 45 s at x°c, and 1min 30 s at 72°c, ended by 7 min final extension cycle at 72°c. the amplification products were separated and analyzed on a licor sequencer type (westburg) using a 6.5% acrylamide gel. the lengh of the alleles was determined by comparaison with marker loaded on adjacent gel traks. the raw data were collected and analyzed by the analysis software “gene imageir” (westburg). cluster analysis all 9 varieties were clustered based on the estimated genetic distance. the positions of a consistent rapd or ssr bands were scored and transformed into a binary character matrix “1” for the presence and “0” for the absence of a rapd and ssr band at a particular position. genetic similarities between genotypes 385 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 386 were calculated according to nei and li’s coefficient (1979). the similarity matrix was subjected to cluster analysis by the unweighted pairgroup arithmetic method (upgma; sneath and sokal 1973) and phylogenetic tree was created using the output data and the graphical module of the mvsp 3.1 software. results and discussion for the first time in morocco we studied the genetic variability of some local tomato cultivars collected from four different regions. nine lots of tomato were selected in this study including one commercial french variety “saint pierre” obtained by vita company used as a control. the other tomato lots were collected from farmers and were sourced from different geographic regions which differ greatly in their agro-ecological and ethnic compositions. the farmers were asked specific questions as the local names for each tomato lot and the location where they are cultivated. tomato genotypes collected from berkane (northeast) and hoceima (north) regions are adapted to semi-dry climate but humid. tomato genotypes from rissani and figuig regions are cultivated in a very dry environment. tomato seeds from these regions are collected from a year to the other to maintain germplasm. these lots are usually grown in small fields and reserved to self-consumption or to local markets. rapd markers rapd markers have great potential to evaluate genetic diversity within accessions and can provide much informations useful in breeding programs. rapd analysis is technically easy, simple and can generate polymorphic profile suitable for large scale germplasm characterization (rafalski and tingey, 1993). in our study, 10 rapd markers were chosen for the analysis of tomato cultivars variation from four different regions of morocco in a mixture of 5 plants per lot. the rapd fragments were scored for their presence (1) and absence (0) for each sample and genetic diversity among groups was calculated on the basis of nei & li’s coefficient (1979). out of these 10 primers, two primers (opg17 and opg19) did not achieve any molecular polymorphism. the same result was obtained with opg17 primer in 19 azerbaijan tomato genotypes (sharifova et al., 2013). the rest of the primers have amplified a total of 41 signals out of which 5 were polymorphic and 36 monomorphic. the number of signals amplified by these markers varied between 2 for opc08 marker and 8 for opu03. opc08 marker gives monomorphic banding patterns among all the cultivated cultivars (figure1). only random primer opu03 showed a total of 4 polymorphic amplicons out of 8 amplicons recorded in figuig2. in other study this percentage was much higher. for example, the same primer opu03 produced highest number of polymorphic bands (21 bands) in 19 tomato varieties (thamir et al. 2014). with this primer (opu03), only cultivated tomato figuig2 and berkane2 displayed a polymorphism showing 4 specific alleles for figuig2 and only one allele for berkane1. this marker discriminate cultivars figuig1, rissanib, berkane1 and hoceima who have the same profile from rissanin, and rissanio who share the same profile with the control cultivar saint pierre indicating that they could have common origin. these results indicated the existence of limited genetic variation within the studied tomato moroccan lots. many others markers showed highly monomorphic profile in the cultivated s. lycopersicum l. (labate and roberts, 2002). the values of pair-wise genetic distances ranged between 0,947 and 1 indicating low diversity in the studied tomato genotypes (table 2). the highest genetic distance (1) was observed between some cultivars like hoceima and figuig1 whereas the lowest genetic distance (0,947) was detected between berkane2 and figuig2 which is an evidence for a low genetic similarity value in the tomato germplasm studied. low degree of the genetic variability is often correlated to a weak discrimination of rapd markers. moreover, miller and tanksley (1990) estimated that only 5% of genetic variation exist within s. lycopersicum. nevertheless, these primers could dived moroccan tomato varieties into four groups: the first includes figuig1, rissanib, berkane1 and hoceima lots. the second includes rissanin, saint pierre and rissanio. the third includes only berkane2 figure 1. rapd electrophoretic pattern of tomato cultivars obtained by the primers opu 03 and opc 08 (from left to right) (a: figuig1 b: figuig2, c: rissanib, d: rissanin, e: saint pierre, f: rissanio, g: berkane1, h: berkane2, i: hoceima). right extreme lane represents 1kb dna. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 387 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ta bl e 2. s et o f sim pl e se qu en ce r ep ea ts (s sr ) p rim er s us ed in th is in ve st ig at io n. m ar ke r r éf ér en ce m ot if fo rw ar d r ev er se c hr om os om e t° h yb rid at io n ss r1 4 ht tp : / /s ol ge no m ic s.n et / (a ta )9 tc tg c a tc tg g tg a a g c a a g c tg g a tt g c c tg g tt g a tt t 3 55 ° ss r 2 2 ht tp :/ /s ol ge no m ic s.n et / (a t) 11 g a tc g g c a g ta g g tg c tc tc c a a g a a a c a c c c a ta tc c g c 3 50 ° ss r2 6 ht tp :/ /s ol ge no m ic s.n et / (c g g ) 7 c g c c ta tc g a ta c c a c c a c t a tt g a tc c g tt tg g tt c tg c 2 50 ° ss r6 3 ht tp :/ /s ol ge no m ic s.n et / (a t) 39 c c a c a a a c a a tt c c a tc tc a g c tt c c g c c a ta c tg a ta c g 8 55 °c ss r2 48 ht tp :/ /s ol ge no m ic s.n et / (t a )2 1 g c a tt c g c tg ta g c tc g tt t g g g a g c tt c a tc a ta g ta a c g 10 55 ° ss r5 78 ht tp :/ /s ol ge no m ic s.n et / (a a c )6 (a tc )5 a tt c c c a g c a c a a c c a g a c t g tt g g tg g a tg a a a tt tg tg 6 55 ° to m 23 623 7 su lim an -p ol la tsc he k et al .2 00 2 a t1 6 g tt tt tt c a a c a tc a a a g a g c t g g a ta g g tt tc g tt a g tg a a c t 9 47 °c to m 18 4 su lim an -p ol la tsc he k et al .2 00 2 (a tt t) 3 (a tt )7 c a a c c c c tc tc c ta tt c t c tg c tt tg tc g a g tt tg a a 4 45 to m 19 619 7 su lim an -p ol la tsc he k et al .2 00 2 (g a )1 4 c c tc c a a a tc c c a a a a c tc t tg tt tc a tc c a c ta tc a c g a 11 45 to m 21 021 1 su lim an -p ol la tsc he k et al .2 00 2 (a ta )1 5 c g tt g g a tt a c tg a g a g g tt ta a c a a a a a tt c a c c c a c a tc g 4 45 tm s 5 2 t. a re sh ch en ko va ·m .w . g an al .2 00 2 (a c )1 4 (a t) 18 tt c ta tc tc a tt tg g c tt c tt c tt a c c tt g a g a a tg g c c tt g 12 55 tm s5 6 t. a re sh ch en ko va ·m .w . g an al .2 00 3 (c t) 19 g a tc tc a a a g g a tg a a c a a ta c tc a tt a g g a g a tt c tt tg ta tc a 1 55 tm s6 3 a re sh ch en ko va an d g an al 20 02 (a t) 4( g t) 18 (a t) 9 g c a g g ta c g c a c g c a ta ta t g c tc c g tc a g g a a tt c tc tc 1 60 °c tm s6 5 t. a re sh ch en ko va ·m .w . g an al .2 00 2 (t a )2 5 (g a )2 0 a g c tt c a tc c a tt a c g c c a c g tg c a tc tg g c g ta c c ta c c 12 60 fi g u ig 1 fi g u ig 2 r is sa n ib r is sa n in r is sa n io be r k a n e1 be r k a n e2 h o c ei m a sa in t pi er re fi g u ig 1 1 fi g u ig 2 0, 96 1 r is sa n ib 1 0, 96 1 r is sa n in 0, 98 7 0, 97 3 0, 98 7 1 r is sa n io 0, 98 7 0, 97 3 0, 98 7 1 1 be r k a n e1 0, 98 7 0, 97 4 0, 98 7 0, 97 4 0, 97 4 1 be r k a n e2 0, 98 7 0, 94 7 0, 98 7 0, 97 4 0, 97 4 0, 97 4 1 h o c ei m a 1 0, 96 1 0, 98 7 0, 98 7 0, 98 7 0, 98 7 1 sa in t pi er re 0, 98 7 0, 97 3 0, 98 7 1 1 0, 97 4 0, 97 4 0, 98 7 1 ta bl e 1. p ai rw ise g en et ic d ist an ce s f ro m 1 0 ra pd m ar ke rs o f ei gh t t om at o cu lti va rs a nd o ne f re nc h co m m er ci al c ul tiv ar s ai nt p ie rr e. 388 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) primer name allelic size range (pb) no. of alleles polymorphic band ssr14 166-235 1 ssr22 208-214 2 2 ssr26 172-178 1 ssr63 206-248 4 4 ssr248 220-251 3 3 ssr578 290-299 1 tom236-237 154-210 5 5 tom184 163-206 2 2 tom196-197 206-214 3 3 tom210-211 216-222 3 3 tms52 148-178 5 5 tms56 102-126 3 3 tms63 154-181 3 3 tms65 288-298 4 4 table 3. total number of polymorphic bands, seize range, number of alleles for 14 ssr markers used on 8 selected moroccan tomato cultivars and one commercial variety. figure 2. (a) and (b) part of a gel obtained with the ssr primes tom 236-237 and visualized in a li-cor system. figuig1, figuig2, rissanib, rissanin, rissanio, berkane1, berkane2 and hoceima correspond to tomato local cutivars. sp correspond to saint pierre commercial cultivar. right and left extreme lanes represent 1kb dna. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 389 figure 3. dendrogram constructed from rapd and ssrs data showing relationship among 8 of local moroccan tomato cultivar and one french commercial tomato based on nei and li (1979) distance and the unweighted pairgroup arithmetic method (upgma). figure 4. rates of non-uniformity for moroccan tomato cultivars tested. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g)figuig1 figuig2 rissanib rissanin rissanio berkane1 berkane2 hoceima saint pierre figuig1 1 figuig2 0,512 1 rissanib 0,857 0,605 1 rissanin 0,723 0,588 0,766 1 rissanio 0,75 0,629 0,792 0,923 1 berkane1 0,638 0,588 0,681 0,579 0,615 1 berkane2 0,653 0,5 0,816 0,7 0,683 0,6 1 hoceima 0,741 0,634 0,852 0,622 0,652 0,8 0,723 1 saint pierre 0,651 0,6 0,698 0,882 0,857 0,588 0,667 0,585 1 table 4. pair-wise genetic distances from 14 ssr markers of eight tomato cultivars and one french commercial cultivar saint pierre. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 390 and the fourth group includes only figuig2. the latest genotype is clearly differentiated from the rest and could be used in tomato breeding program with specified objectives. simple sequence repeats markers simple sequence repeats (ssr) or microsatellite markers have been successfully used to discriminate varieties which are morphologically similar and genetically close (bredmeijer et al., 2002; he et al., 2003; frary et al., 2005; sarıkamıs et al., 2006, 2010). ssr markers may be adequate because of their high polymorphism, reproducibility, genetic co-dominance, easy detection, and multiallelic variation (ruiz et al., 2005). several studies have demonstrated the usefulness of ssr in cultivar identification (bredmeijer et al., 1998; he et al., 2003). in this investigation, fourteen microsatellite markers were selected from the published data or on the website of solanaceae genomics network (table 1) and used to screen genetic diversity and genetic relationships among nine tomato cultivars. three primers (21%) like ssr 14, ssr578 and ssr26 showed monomorphic profiles among all the screened tomato batches with only one amplified band (table 3). in other studies, this percentage is much higher 49% as reported by todorovska et al. (2014) and 25% by elawady et al. (2012). the other markers generated a polymorphic banding profile. the number of alleles amplified assessed from 2 alleles with primers tom 184 and ssr 22 to 5 alleles with markers tom 236-237 and tms 52 (figure 2) with a mean of 2.85 allele per locus. the scorable fragment sizes ranged from approximately 102 pb to 299 bp. limited allelic variation was also observed in a study of eight tomato varieties and lines with an average of 3 alleles per locus after testing 160 ssr loci (todorovska et al., 2014). el-awady et al. (2012) showed also a low level of genetic diversity with an average of 2.1 alleles per locus by using 20 ssr markers in ten tested tomato cultivars. to evaluate genetic relationship within the tomato lots, the data scored from the 14 ssr primers were analyzed on the basis of nei & li’s coefficient (1979). the relationship between tomato germplasm collected from different area in morocco is illustrated by the data in table 4. the genetic similarity estimated according to ssr data was scaled between 0.5 and 0.923 suggesting the potential of ssr markers in discriminating among plants compared to rapd markers. in other studies this coefficient is much higher. for example, archak et al. (2002) founded a similarity coefficients ranging between 0.610 0.976, fanjuan et al. (2010) showed a genetic similarity varying between 0.72 1, singh et al. (2014) reported a gene diversity from 0.65 to 0.97 underlying a weak genetic diversity in the tomato cultivars collection. the highest similarity value of 0.923 was shown between rissanin and rissanio while the lowest value 0.5 was observed between figuig2 and berkane2. figuig1 lot is distantly related to figuig2 (49%) and relatively closed to rissanib (85%). hoceima is close to berkane1 (80%) despite their diverse sources. rissanio and rissanib are distantly related to the certified control saint pierre 12% and 14% respectively suggesting an admixture of the control with those cultivated lots through hybridisation. the other lots are distantly related 30 to 42% to the control indicating a reduced germplasm diversity among the lot. the distance matrix based on rapds and ssrs data was combined and used to construct a dendrogram (figure 3). the dendrogram obtained can be divided into four main clusters, one contains only figuig2 which formed a separate and more distant cluster. the second main cluster contains two cultivars each one is presented in one branch hoceima and berkane1. the third one includes only berkane1. the fourth cluster is divided into two sub-clusters, in one is branched saint pierre, rissanio and rissanin cultivars and the second one contains rissanib and figuig1. traditional tomato cultivars rissanio, rissanin on the one hand, rissanib and figuig1 on the other hand are closely related to each other and seems to have common origin. this could be explained by the geographical situation of these region, rissani and figuig are both situated in the south of morocco and probably the farmers shared the same basis of the seeds. the dendrogram showed the average of at least 5 grouping dna samples per genotype. by calculating the rate of non-uniformity (figure 4) we found considerable heterogeneity within the traditional cultivars. the rate of non-uniformity varies between the largest percentage (35%) in figuig1 and the lowest (1%) in figuig2. the level of heterogeneity found in the most traditional tomato cultivars could be characterized by a higher level of heterozigosity in some loci. on the other hand, figuig2 possess a very high percentage of uniformity (99%) indicating that is a homogeneous traditional cultivar with high purity translating 100% homozygosity. this information could be efficiently used to establish a property rights and a germplasm conservation. conclusion ssr markers used in this investigation were more suitable in the eight moroccan local tomato cultivars as rapd system. ssrs marker are better identification of tomato genotyping because they are codominant (korir et al., 2014) while rapd highlight only the dominant alleles. except for figuig2 cultivar, the most genotype studied are closely related despite their geographic sources. only the local figuig2 cultivar shows the high degree of polymorphism since it was characterized with the highest number of unique bands (4) with rapd markers and (5) with ssr markers in comparison with the other genotype. furthermore, this lot hold the very high percentage of uniformity (99%) indicating that is a homogeneous traditional cultivar with high purity compared to the rest of tomato lots which are probably an admixture of commercial tomato. figuig2 cultivar is a farmerselected and adapted in area of local subsistence with semi-dry to dry climate and has low but stable annually yield. the polymorphism recorded in figuig2 cultivar can be exploited in the management of genetic resources collection in morocco and the establishment of property rights and protection. this genotype can be conserved and used in breeding programs and could offer gene combination to ensure adaptability and reproducibility in dried climate since genetic resources including landraces and wild relatives of crop species play an important role in breeding a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 391 programs (mccouch et al., 2013). in addition, a more accession survey will be necessary to evaluate a range of moroccan tomato germplasm with more informative marker system and to establish a core collection in the gene banks that enabled the creation of a national plant genetic useful in breeding programs acknowledgments this study was supported by the moroccan–belgian cooperation, wallonia brussels international project 2.9 and the ministry of education, higher education and scientific research in morocco. references archak s, il karihaloo, and a jain (2002) rapd markers reveal narrowing genetic base of indian tomato cultivars. cur sci 82: 1139-1143. areshchenkova t and m ganal (2002) comparative analysis of polymorphism and chromosomal location of tomato microsatellite markers isolated from different sources. theor and appl genet 104 (23): 229–235. bauche, g and m causse (2012) genetic diversity in tomato (solanum lycopersicum) and its wild relatives. pp. 133–162. in genetic 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molecular biology letters 7 (2a): 583–598. tabassum n, sk sony, sk bhajan, and mn islam (2013) analysis of genetic diversity in eleven tomato (lycopersicon esculentum mill.) varieties using rapd markers. plant tissue culture and biotechnology 23 (1): 49–57. thamir aj, ah al-saadi, and mc abbass (2014) genetic diversity of some tomato lycopersicon esculentum mill varieties in iraq using random amplified polymorphism dna (rapd) markers. journal of babylon university pure and applied sciences 9 (22): 2342–2351. atlas journal of biology 2018, pp. 592–602 doi: 10.5147/ajb.v0i0.191 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) the effect of environmental factors on the abundance of culturable nitrate reducing/denitrifying bacteria from contaminated and uncontaminated tallgrass prairie soil samer m. abubakr1*, kathleen e. duncan2, greg thoma3, and kerry sublette4 1 department of biological sciences, fayetteville state university, fayetteville, nc, 28301, usa; 2 department of microbiology and plant biology, university of oklahoma, norman, ok, 73019, usa; 3 department of chemical engineering, university of arkansas, fayetteville, ar, 72701, usa; 4 department of chemical engineering, university of tulsa, tulsa, ok 74104, usa received: january 16, 2019 / accepted: march 2, 2019 __________________________________________________ * corresponding author: sabubakr@uncfsu.edu 592 abstract various environmental factors have been proposed, such as soil moisture levels, carbon, and nitrate sources to affect the abundance of nitrate reducing (nr) and denitrifying (dn) bacteria. in this study, the strength of the association of the abundance of nr and dn bacteria with various environmental factors is estimated using multivariate statistical analysis. soil samples were collected from tallgrass prairie soils that had been contaminated with crude oil or brine (e.g. salt water) up to 10 years previously and from parallel uncontaminated sites. the sites had been subjected to remediation shortly after contamination and also more recently (e.g. current remediation treatments). the abundance of culturable nr and dn bacteria in the soil samples was estimated by 5-tube mpn method using nitrate broth, while total petroleum hydrocarbons (tph), sodium chloride, nitrate, and moisture were measured in the contaminated and the parallel uncontaminated sites. viable heterotrophic bacteria and nr and dn bacteria (>106/g soil) from all sites were obtained from samples with a broad range of soil moisture (from 10-30% water/g soil) regardless of the source (e.g. site) of the isolates. the abundance of nr and dn bacteria from the contaminated sites was not less than that from the uncontaminated sites. although mpn values for heterotrophs and nr and dn bacteria were similar over a broad range of moisture levels, the relative abundance of nr and dn bacteria had a wide range (e.g. 0% to 100%) in different samples with the same moisture level, which suggests that factors other than current levels of soil moisture controlled the % nr and dn bacteria. current remediation treatments of contaminated sites sometimes, but not consis this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction high levels of carbon, nitrate, and soil moisture (e.g. low oxygen concentration) are associated with higher denitrification rates when these environmental conditions occurred simultaneously (groffman et al., 1993). in fact, it is not known whether these conditions favorable for denitrification persisted in formerly oil-contaminated sites and produced a greater abundance of nr and dn bacteria. however, it has been shown that denitrifying bacteria are not uniformly distributed in soils where the spatial heterogeneity of denitrification is influenced by spatial heterogeneity of carbon and n substrates in soils, and that denitrifying populations from select sites can be much greater than that indicated by mpn procedures of bulk soil (murray et al., 1995; martin et al., 1988; harms et al., 2009). the soil microbial community structure is thought to be one of the most sensitive indicators of biological properties of soil and reflects changes in the soil ecosystem (kennedy and smith, 1995; yao et al., 2000). different environmental factors, such as soil moisture, affect microbial activities and composition in soil tently, were associated with greater abundance of nr and dn bacteria (2-way anova). therefore, no long-term effect of contamination on the abundance of nr and dn bacteria was shown nor were current remediation treatments effective in stimulating the numbers of nr and dn bacteria. keywords: nitrate reducing bacteria. denitrifying bacteria, soil moisture, crude oil and brine contamination, nitrogen. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) by affecting the physiological status of bacteria (harris, 1981). in fact, the osmotic status of bacterial cells is affected by water availability that can regulate diffusion of gases, substrate accessibility, soil temperature, and ph. moreover, moisture shortage may affect bacterial communities by stressing plants. drought causes changes in nutrient distribution below ground and the release of organic compounds from plant roots into soils (lynch and whipps, 1990). periods of moisture limitation may be a strong selective pressure on the structure and functioning of soil bacterial communities. also, it has been shown that soil moisture is involved in controlling fluxes of important greenhouse gases such as nitrous oxide, even if these fluxes are not totally the result of microbial activity (bollmann and conrad, 1998). sublette et al. (2007a) found that soil moisture had a strong influence on viable biomass of soil microbes when phospholipid fatty acid (plfa) concentrations from tallgrass prairie soil samples were measured. the plfa concentration reflects the viable microbial biomass (white et al., 1997). soil moisture also impacts nr and dn bacteria by controlling oxygen diffusion to sites of microbial activity. when the moisture levels increases up to 60% of moisture holding capacity, the number of most microorganisms increase. however, when soil moisture increases to more than 60% of moisture holding capacity, oxygen diffusion and availability to microbes will decrease. as a result, facultative anaerobic microorganisms will be stimulated to use alternative electron acceptors such as nitrate. these conditions are expected to increase the number of nr and dn bacteria with respect to that of strict aerobes, if these low oxygen conditions are maintained and nitrate is available (paul and clark, 1989). however, soil moisture is not the only environmental factor that could affect the abundance and activity of nr bacteria. the abundance of heterotrophic bacterial groups in any environment is related directly to the ability to utilize the available organic carbon sources as an energy source. in hydrocarbon-contaminated soils, hydrocarbons can be a source of carbon for those bacteria that possess pathways for hydrocarbon degradation. nitrate reducing bacteria are typically facultative anaerobes, and thus they may possess the aerobic pathways of hydrocarbon degradation such as that found in pseudomonas putida pg7, which contains the nah7 naphthalene-degradation plasmid (dunn and gunsalus, 1973). in addition, they may have an advantage over aerobes in being able also to degrade hydrocarbons under nr conditions if they possess a pathway active under nr conditions. pseudomonas fluorescens b-3468 may be such an organism, as it converted 2,4,6-trinitrotoluene under conditions of oxygen or nitrate respiration (naumova et al., 1988). various studies investigated the anaerobic degradation of polyaromatic hydrocarbons (pahs) under nr and dn conditions. one laboratory study (al-bashir et al., 1990) showed biodegradation of low molecular weight pahs when denitrifying organisms were grown. also, nitratedependent anaerobic degradation and mineralization of naphthalene by pure cultures of pseudomonas stutzeri and vibrio pelagius has been demonstrated (rockne et al., 2000). therefore, we hypothesized that the abundance of hydrocarbon-degrading nr and dn bacteria will increase in the crude oil-contaminated sites. however, the range of organisms that degrade most low593 molecular-weight components of mineral oil decreases with increasing salinity (kleinsteuber et al., 2006). therefore, soils contaminated with both brine and oil may be more difficult to be bioremediated, although it was shown that levels of tph were reduced in the presence of brine contamination (sublette et al., 2005). one more environmental factor that affects nr and dn bacteria in soils is the availability of nitrate. it has been shown that the addition of plant residues can lead to a long-term increase in potential nitrate reduction activity and size of a microbial community involved in nitrogen cycling. however, the effect of the type of plant residue itself was limited (chèneby et al., 2010). therefore, nitrogen-containing fertilizers are often used in bioremediation (pope and matthews, 1993).in fact, the fertilizers used in this study contained ammonium nitrate (nh4no3) (sublette et al., 2007a). considering the effect of these environmental factors one by one, and in combination, since the amount of water available in soil (soil moisture) plays such an important role in microbial activity and particularly for nr and dn bacteria by also affecting available oxygen levels, we hypothesize that bacteria numbers and the relative abundance of nr and dn bacteria increase with higher soil moisture. also, since crude oil contamination alters bacterial communities in soils by providing alternative carbon sources and killing sensitive members of the microbial community (sikkema et al., 1995) and many nr and dn are hydrocarbon degraders (al-bashir et al., 1990; sharak genthner et al., 1997; rockne and strand, 1998) we hypothesize that nr and dn bacteria will be more abundant in the crude oil-contaminated soils compared to that in the prairie uncontaminated soils, if the moisture levels are comparable. however, nr and dn bacteria will be less abundant in the sites that are contaminated with both oil and brine because salinity decreases the range of microorganisms that degrade most low-molecular-weight components of mineral oil (kleinsteuber et al., 2006). remediation treatments of nitrate fertilizer are expected to stimulate the activity and eventually increase the numbers of nr and dn bacteria. therefore, we hypothesized the best conditions for nr and dn bacteria are soils contaminated with hydrocarbons, and remediated by application of nitrate containing fertilizer and watered. however, it is unknown whether the potentially stimulating effect on nr and dn bacteria of nitrate remediation of oil contaminated soils persists over a period of several years. materials and methods the sampling sites used in this work are located in the tallgrass prairie preserve (tpp) in osage county, oklahoma. nitrate reducing and denitrifying bacteria were isolated from a total of 5 contaminated sites designated as g5, g7, lf, j6-f, and j6-nf and 4 adjacent uncontaminated sites (g5p, j6p, g7p, and lfp). leaks from oil pipelines had contaminated the sites with crude oil (j6-f, j6-nf, lf) or with a mixture of brine and oil (g5, g7). the sites had been partially remediated as described previously (sublette et al., 2005; sublette et al., 2007a; duncan et al., 1998; duncan et al., 1999). briefly, the contaminated sites were contaminated during 1996-2000 but still contained a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 594 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) low levels of tph and na+ and cl(tables 1.a and 1.b) when sampled for the current study. sites g7 and lf underwent a further round of treatment immediately prior to the course of this research as described in the materials and methods. the objective of this study was to determine the association of different environmental factors (e.g. tph, nacl, nitrate and soil moisture) with the abundance of nr and dn bacteria in long-term contaminated vs. uncontaminated prairie soils in order to determine which factors have an impact under field conditions. the following is a brief summary of the timeline of contamination and remediation of each site. the reader may consult the referred sources for more details. the g5 site was contaminated in the fall of 1999 by 3 separate breaks in the same line resulting in 3 lobes of contamination (e.g. g5n, g5m, and g5s). the amount spilled was unknown, but the water to oil ratio in this line was 10-15:1, and the brine had a total dissolved solids (tds) of 105,000 mg/l. the initial tph values were estimated as 1300-5200 mg/kg, na+ as 1100-1580 mg/kg, and clas 7802450 mg/kg (sublette et al., 2005). remediation, as described in sublette et al. (2005), was performed from june 2000 to august 2003 and consisted of hay and fertilizer containing nitrate, and installation of a drainage pipe downhill from the contaminated area. the j6 site was contaminated in january 1999 by a pipe break resulting in a spill of approximately 11 m3 of dewatered crude oil. the initial total petroleum hydrocarbon (tph) concentration (epa 418.1) was about 33,500 mg/kg (dry wt. basis) when remediation was initiated (following tilling) (sublette et al., 2007a). remediation, as described in sublette et al. (2007a), was performed from may 1999 to october 2001 and consisted of hay, fertilizer containing ammonium nitrate (nh4no3), diphosphorus pentoxide (p2o5), and potassium oxide (k2o), and tilling. j6 was divided into two sections, fertilizer was applied by tilling in four increments throughout the 2 year-period of remediation on j6-f, while j6-nf was tilled in parallel with j6-f but not fertilized. the g7 site was affected primarily by brine. the original spill was in 2000, remediation treatments of hay and nitrate-containing fertilizer were performed, ending in 2004, and the sodium and chloride levels were reduced, but large expanses still had no plants in 2005. sodium and chloride levels varied widely, with sodium ranging from 350-600 mg/ kg soil (sublette et al., 2004). on the other hand, lf, containing soil contaminated by a spill of crude oil, was remediated in 1996 and 1997 by mixing the contaminated soil with the uncontaminated soil plus nitrogen-containing fertilizer (duncan 1998, 1999). during that time, tph levels decreased to about a third of the original level and plants grew over most of the site, but they were primarily weedy, non-native, undesirable species. during the period of 2005-2006, treatments were performed on the g7 and lf sites in order to further decrease the contamitable 1. values for tph, na+, cl-, and no3near mpn sampling dates. site tpha sampling date # samples (mg/kg) (sdb) j6-nf april/june 2003* 2 1957.5 (958.13) j6-f april/june 2003* 2 959 (59.4) j6p april/june 2003* 2 0 (0) g5 april 2003** 3 315 (270) g5p april 2003 1 100 (n/a) g7 spring 2005 12 10941 (1068) lf spring 2005 34 14321 (3163) atotal petroleum hydrocarbons. bstandard deviation. *one sample was taken in april 2003, one in june 2003, and averaged. **one sample was taken from g5n, one from g5m, one from g5s, and averaged. n/a: not applicable. data from sublette et al. 2005, 2007, and unpublished data. site sampling date # samples na+ (mg/kg) (sd) cl(mg/kg) (sd) no3 (mg/kg) (sd) j6-nf june 2003 4 bdl bdl 4.18 (3.22) j6-f june 2003 5 bdl bdl 12.74 (6.73) j6p june 2003 4 bdl bdl 0.70 (0.12) g5 april/june 2003 3a or 26b 896 (402)a 1478 (859)a 28.12 (13.58)b g5p april/june 2003 1a or 9b 29a (n/a) 0a (n/a) 0.82 (0.17)b g7 july 2005 32 288 (79.88) 99.8 (42.98) 1.7 (0.74) lf august 2005 32 50 (9.09) bdl 12.4 (13.45) a: na+, clsamples (n= 3 for g5, n= 1 for g5p), samples were taken in april 2003. for g5, one sample was taken from g5n, one from g5m, one from g5s, and averaged. b: no3 samples (n= 26 for g5, n= 9 for g5p), samples were taken in june 2003. for g5, 9 samples were taken from g5n, 9 from g5m, 8 from g5s, and averaged. for g5p, 9 samples were averaged. bdl: below detected limits (e.g. the method detection limits were: 10 mg/kg for tph, 1 mg/kg for brine components. n/a: not applicable. data from sublette et al. 2005, 2007, and unpublished data. table 1a. total petroleum hydrocarbons (tph). table 1b. brine components and no3-. nation levels. the areas treated were approximately 45.72 m x 15.24 m (g7) and 27.43 m x 25.91 m (lf). restoration treatments were started in the spring of 2005 by ripping the soil to a depth of 30 cm and tilling. each site was divided into 4 blocks, and soil within each block was homogenized by mixing before being distributed into 36 enclosures per block. the homogenized soil was placed inside a bag of bridal veil material, inside a 30 cm diameter pvc pipe enclosure with slots drilled into it for drainage. each 30 cm enclosure was centered in 2 m x 2 m area within the block (qapp: quality assurance project plan, 2004). the soil inside each enclosure received one of the 4 following treatments: hay alone, fertilizer alone, hay plus fertilizer, and the fourth treatment being no fertilizer and no hay. the fertilizer was mixed into the top 15 cm of soil, and the hay was placed on the soil surfaces. each block contained 4 repetitions of each treatment, and one of each treatment per block was destructively sampled on each sampling. an additional enclosure from lf, g7, or both, was collected on most sampling dates for quality control purposes giving a total of 33-34 samples. soil for an mpn series was obtained from each destructed sample. after adding the amendments, the entire site was covered with hay. the enclosures were installed and the amendments added in may 2005 and watering began (sublette et al., 2004). all samples were obtained from the tallgrass prairie preserve in osage county, oklahoma. soil samples were collected from the following sites: g5 (brine/oil contaminated) (sublette et al., 2005), j6-f (crude oil contaminated, treated with fertilizer) (sublette et al., 2007a), and j6-nf (crude oil contaminated, not treated with fertilizers) (sublette et al., 2007a) in march, 2005. in addition, soil samples were obtained from g7 (brine/ oil contaminated) in july 2005, october 2005, and june 2006, and from lf (crude oil contaminated) in august 2005, october 2005, and june 2006. samples from adjacent uncontaminated sites (g5p, j6p, g7p, and lfp) were collected at the same time as for contaminated sites. briefly, for g5 and j6 sites, a trowel was used to remove 5 scoops of soil from 5 widely spaced points within a 25 m radius from approximately the top 10 cm of soil, beginning just below any loose litter layer, and placed in whirl-pac® bags. then, these soil samples from g5, j6-f, j6nf and their parallel uncontaminated sites were homogenized in autoclaved beakers using autoclaved spoons. the enclosures in lf and g7 were pulled out and the soil poured into stainless steel bowls, mixed and subsamples were taken out for a variety of assays. approximately 300 g of homogenized soil from each of 33 or 34 enclosures was subsampled from lf and g7 sites for mpn assays (see materials and methods for more detailed information). nitrate broth medium for detection of nr and dn bacteria consisted of 2.5 g of nano3 added to 500 ml of nutrient broth (difco inc., detroit, mi) before autoclaving. the antifungal agent, cycloheximide (sigma chemical company, st. louis, mo) (100 µg/ml final plate concentration) was added after autoclaving. nitrate reduction broths containing beef extract, pancreatic digest of casein, or peptone, are commonly used to distinguish facultative heterotrophic bacteria based on whether they are able to reduce nitrate to nitrite or produce n2 gas (atlas, 1993). control strains were used to confirm that the test conditions (e.g. detection of nr and dn bacteria in the presence of soil) correctly identified nitrate reducers and denitrifyers. these controls were: pseudomonas aeruginosa atcc 27853 (denitifyer), p. putida f1 atcc 17485 (negativeneither a denitrifyer nor a nitrate reducer). p. aeruginosa s1-1 (a denitrifying bacterium isolated from tallgrass prairie), and p. putida pg7 (neither a denitrifyer nor a nitrate reducer). p. aeruginosa strains were grown at 37°c and p. putida strains were grown at room temperature (23-25°c). soils were initially mixed in whirl-pac® bags using autoclaved spoons in the field and later in the lab were further homogenized in autoclaved beakers by mixing with autoclaved spoons. after homogenization, 2.00 g soil (wet weight) from each sample was added to a sterile 50 ml centrifuge tube containing 18 ml of sterile isotonic saline (0.85% nacl), vortexed for 1 minute and serially diluted (1:10) to 10-8 for all samples. twenty microliters of each dilution was used to inoculate 180 µl of the nitrate broth (see a. medium) in 5-fold replicate, making a five tube mpn series (rodina, 1972). the inoculated microtiter plates were incubated at room temperature (23-25°c) in the dark for 14 days (jones et al., 1991). all wells were examined for evidence of growth (turbidity) after 14 days, and those that were turbid were scored positive for aerobic/facultative heterotrophic bacteria. subsequently, half of the volume of the wells was transferred to a fresh microtiter plate and tested for the presence of nr and dn bacteria using griess reagents (baron and finegold, 1990; smibert and krieg, 1994). briefly, 2 drops of reagent 1 (sulfanilic acid) and 2 drops of reagent 2 (n, n-dimethyl-1naphthylamine) (biomérieux vitek, inc., hazelwood, mo) were added to every well. the presence of nitrite is detected by a red color after the addition of reagents 1 and 2, e.g, indicating nitrate reduction. if the medium remained colorless, e.g. no nitrite formed, it is due either to lack of reduction of nitrate, e.g. no nitrate reduction or the reduction of nitrite to other products, such as no, n2o, or n2, hence denitrification. if the medium remained colorless, zinc metal dust (mallinckrodt chemical works, st. louis, mo) was used to detect if unreduced nitrate was present by reducing nitrate to nitrite and turning the medium pink or red. therefore, pink or red after zinc dust indicates neither nr nor dn bacteria. if colorless after zinc dust, the well was assumed to contain dn bacteria. however, since only the loss of nitrate and nitrite was demonstrated, bacteria in the colorless wells could be nitrite reducers rather than performing the entire denitrification pathway. calculation of # of viable cells from mpn reading used the 5-tube mpn tables (rodina, 1972), and were corrected for the % soil moisture to give estimated # cells/g soil (dry weight). to test whether the presence of soil interferes with the ability to detect the reduction of nitrate/nitrite in the microtiter plate test format, the four pseudomonas control strains of known phenotype previously described (see materials and methods) were tested. tests with p. aeruginosa strains were performed at 37°c, those with p. putida at room temperature (23-25°c). a small colony from each pseudomonas strain was resuspended in 1.0 ml of sterile isotonic saline (0.85% nacl) and vortexed. the inoculated saline was added to 1 g of autoclaved soil that came from g5p site. the inoculated saline and soil were added a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 595 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 596 to 8 ml of sterile saline to produce a 1:10 dilution (e.g. 10-1). an additional 1:10 dilution was performed and 20 µl diluted cell suspension added to wells of a microtiter plate containing 180 µl of nitrate broth. the inoculated microtiter plates were incubated at room temperature (23-25°c) in the dark for 14 days (jones et al., 1991) and scored for growth and for the reduction of nitrate or nitrite as described previously. soil moisture for all samples was determined from the original soil mass by gravimetric measurements of two 10 g samples (wet weight) after oven drying (wollum, 1982). the % soil moisture was calculated as % soil moisture = (g water/g wet soil) x 100. samples were taken from both the contaminated and the prairie sites. g7 and lf were regularly irrigated throughout the study. the other sites received only natural rainfall. soil moisture for g5, j6-f, j6-nf, g5p, and j6p was measured for march 2005. soil moisture for g7 and g7p was measured for july 2005, october 2005, and june 2006 at the time of sampling. soil moisture for lf and lfp was measured for august 2005, october 2005, and june 2006 at the time of sampling. brine components (na+, cl-) were extracted from oven-dried soil with deionized water, and concentrations were determined by ion chromatography (harris, 1998). continental laboratories (salina, ks, usa) performed tph analysis of soil samples by using environmental protection agency (epa) method 418.1. nitrate was measured in oklahoma state university laboratory (stillwater, ok, usa). metadata was collected from different studies that include tph, na+, cl-, and no3 in contaminated-treated (g5, g7, lf, j6-f, and j6-nf) and prairie (g5p, g7p, lfp, and j6p) sites (duncan et al., 1999; sublette et al., 2005; sublette et al., 2007a). mean, standard deviation, coefficient of variation, and linear regression were calculated using microsoft excel version 2007. also, one-way anova, 2-way anova, and duncan’s multiple range tests were used to analyze the data using statistical package for the social sciences (spss for windows version 19.0, ibm, chicago, ill, usa). in addition, student’s t test and welch’s test were used to compare data using graphpad prism ver. 3 (graphpad software, san diego, ca, usa). the data were considered significantly different if the two-tailed p-value was <0.05. mpn values were converted to log10 values for statistical analysis in order to normalize the data for parametric statistical tests. results contaminant levels at the beginning of the study metadata was collected from different published studies and summarized in table 1 to show the levels of tph, na+, cl-, and no3 for sampling dates closest to those representing the beginning of the current study. samples for the metadata were taken prior to site manipulation for g7 and lf. the metadata we collected show that there was a significant difference (p < 0.05) between tph in g7 (primarily brine-contaminated) and that in lf (crude oil contaminated) when sampled in spring 2005 with g7 being lower (table 1.a). (welch’s approximate t = 5.417, 43 d.f., mean difference = 3380.0, 95% ci = 2121.7 4638.3). on the other hand, there was no significant difference (p > 0.05) between tph in j6-nf and that in j6-f during the period of april/june 2003. (welch’s approximate t = 1.471, 1 d.f., mean difference = -998.50, 95% ci = -9623.3 7626.3). nitrate-containing fertilizer was applied once in may 2005 to selected enclosures in the g7 and lf sites. nitrate-containing fertilizer was last applied in june 2000 to j6-f and samples taken in june 2003. fertilizer was applied in april 2001 to g5 and samples taken in april/june 2003. nitrate levels varied in different sites (table 1.b). when we compared nitrate levels in j6-nf, j6-f, and j6p, there was a significant difference (p < 0.05) in nitrate concentration among those 3 sites. nitrate level in j6-f was higher than that in j6-nf or j6p (p < 0.05), with no significant difference between j6-nf and j6p (tukey-kramer multiple comparisons test. mean difference (j6-nf vs j6-f), = -8.560, 95% ci = -17.034 to -0.08612; mean difference (j6-nf vs j6p) = 3.480, 95% ci = -5.452 12.412; and mean difference (j6-f vs j6p) = 12.040, 95% ci = 3.566 to 20.514). finally, nitrate levels were significantly higher (p < 0.05) in g5 (e.g. g5n, g5m, and g5s) than that in g5p (welch’s approximate t = 10.248, 25 d.f., mean difference = -27.300, 95% ci = -32.786 -21.814). mpn assays table 2 summarizes mpns for each site and each sample date, % nr and dn bacteria (e.g. “#nr and dn/ #heterotrophs” x 100), and soil moisture percentages. although the mpn assays correctly indicated pseudomonas control strains as denitrifyers in the absence of soil, when autoclaved soil was added to the dilution series, control dn strains were scored as nitrate reducers (nr), e.g. nitrate was consumed but not nitrite. therefore, nr bacteria were not reliably distinguished from dn bacteria when scoring mpns from the soil, and wells showing loss of nitrate and/or nitrite were reported as nr and dn bacteria. the association of soil moisture levels with nr and dn bacteria: j6 and g5 sites the j6-nf, j6-f, j6p, g5 (includes the n, m, and s lobes), and g5p sites were analyzed together as they represent contaminated and parallel uncontaminated sites (eg. j6p, g5p) that were not treated during the period of study and were sampled on the same dates. table 2 shows the % soil moisture [= (g water/g wet soil) x 100] in the j6 and g5 soils sampled in march 2005 (figure 1, one-way anova: f (4, 9) = 89.988, p = 0.000). the uncontaminated prairie samples bounded the range of % soil moisture, with g5p being the lowest (13.6%) and j6p the highest (21%). post hoc tests (e.g. duncan’s multiple range test) group together the homogenous subsets showing that soil moisture in g5p was significantly lower than % soil moisture in the others, j6-nf next lowest, g5 and j6-f grouped together, and j6p with the highest % soil moisture (figure 1). estimated viable counts of either heterotroph or nr/dn bacteria varied little with the % soil moisture (table 2, figure 1), with the exception of j6-f. it was estimated there were 1.07 x 107 culturable heterotrophic bacteria/g soil in g5p (13.6% soil 597 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) moisture) and 1.78 x 107 culturable heterotrophic bacteria/g soil in j6p (21% soil moisture) but 4.72 x 107/g soil in j6-f (17. 5% soil moisture). estimates of dn/nr bacteria were about one quarter to one third that of the heterotrophic bacteria (table 2), again with the exception of j6-f, in which estimates of dn/nr bacteria were about 70% that of the heterotrophic bacteria. . soil moisture association with nr and dn bacteria: g7 and lf sites figure 2 plots average and standard deviation of % soil moisture values in g7 and lf sampled at different seasons during the experiment. bars with the same numbers form homogeneous sets (duncan’s multiple range test). our moisture results showed that highest % soil moisture values were obtained in july 2005 (g7, fig. 2a) and in august 2005 (lf, fig. 2b). note that the uncontaminated samples (g7p, lfp) were drier than the samples from the contaminated sites collected at the same time since the contaminated sites were watered throughout the study. figures 2.a and 2.b also show that in spite of attempts to control water content, g7 generally was wetter than lf. multiple samples were obtained from each site for every sampling date, which allowed us to more closely investigate the association between mpn values and soil moisture. repeatedly, for each site and sampling date, our moisture versus mpn results in g7 and lf sites showed that the slope of the regression line was very nearly zero indicating similar mpn values over a fairly broad range of moisture levels (figures 3a and 3b, table 2). in addition, the abundance of heterotrophic and dn/nr bacteria did not show a consistent pattern of association—the bivariate correlation between the number of heterotrophic and dn/nr in a particular sample ranged from -0.49 to +0.79 (table 3). comparing mpn for g7 and lf across all sample dates g7 mpn values (table 2) for heterotrophic bacteria, nr and dn bacteria, and %nr and dn bacteria for all sample dates of g7 and g7p were analyzed together. the abundance of heterotrophic bacteria with respect to g7 and g7p soils sampled on different dates varied significantly (one-way anova: f (4,103) = 11.292, p = 0.000). duncan’s multiple range test, a post-hoc test which groups together homogeneous subsets of a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) site date n % soil moisture (sd) nr and dn* bacteria (sd) heterotrophs mean (sd) % nr and dn bacteria (sd) g5p mar-05 1 13.6 (0.14)** 2.80 x 106 (n/a) 1.07 x 107 (n/a) 26.17 (n/a) j6-nf mar-05 1 16.55 (0.07)** 5.60 x 106 (n/a) 1.44 x 107 (n/a) 38.89 (n/a) j6-f mar-05 1 17.45 (0.35)** 3.40 x 107 (n/a) 4.72 x 107 (n/a) 72.03 (n/a) g5 mar-05 3 18.02 (0.51)** 5.10 x 106 (5.2 x 105) 1.50 x 107 (3.49 x 106) 35.08 (7.90) j6p mar-05 1 21 (0.14)** 5.00 x 106 (n/a) 1.78 x 107 (n/a) 28.09 (n/a) g7p oct-05 2 12.85 (0.21) 2.02 x 106 (7.78 x 104) 1.96 x 107 (0) 10.28(0.40) g7p jun-06 4 13.7 (1.49) 1.54 x 106 (9.79 x 105) 6.94 x 106 (2.61 x 106) 26.90 (19.89) g7 jul-05 34 25.8 (2.78) 5.76 x 106 (6.04 x 106) 1.96 x 107 (2.68 x 107) 51.92 (39.92) g7 oct-05 34 23.3 (2.78) 7.29 x 106 (9.10 x 106) 3.66 x 107 (2.87 x 107) 22.40 (24.67) g7 jun-06 34 22.8 (2.17) 2.14 x 107 (2.39 x 107) 4.02 x 107 (2.42 x 107) 55.2 (27.40) lfp oct-05 2 12.95 (0.07) 1.66 x 106 (1.06 x 106) 1.40 x 107 (1.44 x 107) 16.96 (9.84) lfp jun-06 4 13.50 (1.74) 2.60 x 106 (2.09 x 106) 3.52 x 106 (1.56 x 106) 69.95 (35.11) lf aug-05 34 20.7 (4.51) 4.92 x 106 (7.07 x 106) 7.06 x 107 (2.51 x 108) 31.52 (33.65) lf oct-05 34 18.9 (4.17) 1.06 x 107 (1.26 x 107) 6.12 x 109 (3.23 x 1010) 24.44 (29.52) lf jun-06 34 18.9 (5.31) 1.46 x 107 (1.70 x 107) 2.77 x 107 (2.71 x 107) 55.50 (30.29) date: soil sampled collected for mpn estimation. n: number of samples. n/a: not applicable. *average per gram dry weight soil estimated from 5-tube mpn (rodina, 1972). **moisture %: the average of 6 samples for g5, and an average of 2 for each j6-nf, j6-f, g5p, and j6p from soil wet weight for g7 and lf, n= the value listed. % soil moisture = (g water/g wet soil) x 100. soil moisture was calculated from 2 soil samples per sample. sd: standard deviation. nr and dn: nitrate reducing and denitrifying bacteria. table 2. most probable numbers (mpns) of nr and dn bacteria, heterotrophic bacteria, %nr and dn bacteria, and % soil moisture. figure 1. abundance of heterotrophic and nr/dn bacteria with respect to soil moisture (sites g5, j6). the log10 # viable counts (mpn) for heterotrophic (open squares) and nr/dn (black circles) are plotted on the y axis versus the % soil moisture (% water/g soil wet wt) for samples taken from the g5 and j6 sites march 2005. see tables 1 and 2 for additional information about the sites. n=1 for mpn values and n=2 (averaged) for each soil moisture value. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 598 the data, grouped together g7p (june 2006) and g7 (july 2005) as having the lowest abundance of heterotrophic bacteria, g7 (july 2005), g7p (october 2005), and g7 (october 2005) as the group with the next to the lowest abundance, and g7p (october 2005), g7 (october 2005), and g7 (june 2006) as the subset with the highest abundance of heterotrophic bacteria. the log10 #nr and dn bacteria varied significantly among the sites, with g7 (july 2005), g7p (october 2005, june 2006) lower than that in the remaining samples. one-way anova (nr and dn: g7, g7p): f (4,103) = 9.142, p = 0.000. the % nr and dn bacteria varied greatly among samples taken from the same site, with the least variation and lowest value in g7p (october 2005, oneway anova (%nr and dn: g7, g7p): f (4,103) = 6.588, p = 0.000. for lf samples, the log10 #heterotrophic bacteria value in lf october 2005 was significantly higher than that in the remaining samples (one-way anova (heterotrophs: lf, lfp): f (4,102) = 10.923, p = 0.000). however, the log10 #nr and dn bacteria value in lfp october 2005 is significantly lower than the remaining samples. one-way anova (nr and dn: lf, lfp): f (4,102) = 5.639, p = 0.000. finally, the % nr and dn bacteria varied greatly among samples taken from the same site, with the least variation and lowest value in lfp october 2005. one-way anova (%nr and dn: lf, lfp): f (4,102) = 5.915, p = 0.000. coefficient of variation (cv) the % coefficient of variation (cv) was calculated (table 4) for the g7 and lf sites in order to estimate whether subsamples within a sample were more variable for numbers of dn/nr bacteria than for heterotrophs. values of the cv for log10 # nr table 3. bivariate correlation between log10# heterotrophic and dn and nr bacteria. site / date correlation g7/july2005 0.17 g7/october 2005 0.05 g7/june 2006 0.63 g7p/june 2006 -0.46 lf/august 2005 0.22 lf/october 2005 -0.49 lf/june 2006 0.79 lfp/june 2006 0.74 table 4. % coefficient of variation (cv) of nr and dn and heterotrophic bacteria in g7, g7p, lf, and lfp sites. site sample date mean (sd) log10 nr and dn % cv log10 nr and dn mean (sd) log10 heterotrophs % cv log10 heterotrophs g7 jul-05 6.43 (0.82) 13 7.06 (0.43) 6 lf aug-05 6.40 (0.57) 9 7.23 (0.56) 8 g7 oct-05 6.51 (0.66) 10 7.47 (0.29) 4 g7p oct-05 6.30 (0.02) 0 7.29 (0.00) 0 lf oct-05 6.72 (0.59) 9 8.03 (0.91) 11 lfp oct-05 6.17 (0.30) 5 6.98 (0.57) 8 g7 jun-06 7.2 (0.31) 4 7.5 (0.32) 4 g7p jun-06 6.1 (0.40) 7 6.8 (0.18) 3 lf jun-06 7.0 (0.43) 6 7.3 (0.37) 5 lfp jun-06 6.3 (0.35) 6 6.5 (0.19) 3 sd: standard deviation. % cv: % coefficient of variation = (sd/mean) x 100%. (2a.) 0 5 10 15 20 25 30 35 g7 july 2005 g7 october 2005 g7p october 2005 g7 june 2006 g7p june 2006 so il m oi st ur e (% ) 3 2,3 1 2 1 0 5 10 15 20 25 30 lf august 2005 lf october 2005 lfp october 2005 lf june 2006 lfp june 2006 so il m oi st ur e (% ) 3 2,3 1 2,3 1,2 figure 2. moisture % in g7 and lf sites. g7 july 2005, g7 october 2005, g7 june 2006, lf august 2005, lf october 2005, and lf june 2006: n=34. g7p june 2006 and lfp june 2006:n=4. g7p october 2005 and lfp october 2005:n=2. bars indicate average values. error bars indicate ±1 standard deviation. (2.a.) 1, 2, 3: homogenous subsets (duncan’s multiple range test). one-way anova: f (4,103) = 31.341, p = 0.000. (2.b.) 1, 2, 3: homogenous subsets (duncan’s multiple range test). one-way anova: f (4,103) = 3.420, p = 0.011. (2b.) 599 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 5. two-way anova: summary of significant treatment effects. site sample date treatment dependent variable f value sig. g7 oct-05 hay log10 nr and dn 3.592 0.068 g7 oct-05 hay %nr and dn 3.916 0.057 g7 jun-06 hay x fertilizer interaction %nr and dn 4.725 0.038 lf oct-05 hay log10 nr and dn 3.721 0.063 lf oct-05 hay %nr and dn 2.924 0.098 lf oct-05 fertilizer %nr and dn 4.436 0.044 lf jun-06 fertilizer %nr and dn 4.453 0.043 significant treatment effects are defined as p<0.050. figure 3. the relation between soil moisture and the log10 number of heterotrophic bacteria or log10 number of nr and dn bacteria in g7 or lf on different dates. circles: heterotrophic bacteria. diamonds: nr and dn bacteria. open shapes: soils from contaminated sites (n= 33 or 34). filled shapes: soils from uncontaminated prairie sites (n= 2 or 4). r2 values for linear trend lines fitted to the data are shown on the figures. (3.a.) g7 samples; (3.b.) lf samples. (3.a.) (3.b.) 600 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) and dn bacteria and for log10 # heterotrophic bacteria ranged from 0-13%. the cv was higher for log10 #nr and dn bacteria than that for the log10 # heterotrophic bacteria for 2 sample dates for g7, the same for june 2006, higher for 2 sample dates for lf and lower for lf october 2005. this indicates that there was proportionally more variation in abundance of nr and dn in lf and g7 than for heterotrophic bacteria sampled at the same time. not as many samples were obtained from the uncontaminated sites, but in 2 out of 4 samples, the cv was higher for log10 #nr and dn than that for log10 # heterotrophic bacteria, the same for one sample and lower for one sample (lfp october 2005). two-way anova: effect of hay and/or fertilizer on bacterial numbers in g7 and lf hay and/or fertilizer were applied may 2005 to the soil in selected enclosures in the g7 and lf sites (materials and methods, “restoration phase”). two-way anova calculations were made to determine if there was a significant effect of applying hay and nitrate-containing fertilizer amendments on the abundance or proportion of nr and dn bacteria. our multivariate analyses (e.g. 2-way anova) showed the effect (p= 0.038) of hay and fertilizer together on the relative abundance of nr and dn (%nr and dn) bacteria sampled from g7 in june 2006 (table 5). enclosures where no hay and no fertilizer were applied had the highest %nr and dn (69.5%), followed by plus hay plus fertilizer (56.7%), then plus hay no fertilizer (49.1%), and the lowest was the no hay plus fertilizer treatment (38.3%) (data not shown). enclosures with fertilizer addition had higher %nr and dn bacteria sampled from lf site in october 2005 (p= 0.044) (table 5) where %nr and dn was 13.9% with no fertilizer added and was 34.0% in enclosures when the fertilizer was added (data not shown). the opposite was true for june 2006, the greatest relative abundance of nr and dn bacteria sampled from lf site was 66.3% from enclosures with no fertilizer added and was 45.1% in enclosures when fertilizer was added in may 2005 (p= 0.043) (table 5) (data not shown). there was no significant effect on the hay and fertilizer treatments on the number or relative abundance of nr and dn bacteria for the remaining sample dates nor on the abundance of heterotrophic bacteria for any sample date. discussion this study investigated the effect of the long-term (5-10 years) residual tph and brine contamination on the abundance of nr and dn bacteria. our results suggested that the residual tph and brine levels were not associated with differences in abundance of nr and dn bacteria beyond that predicted by soil moisture levels in most cases. our j6 study sites (tph contaminated) sampled at an earlier stage after contamination showed that nitrogen mineralization rates in a contaminated non-fertilized soil (j6-nf) were significantly lower than that in a contaminated fertilized soil (j6-f, sublette et al., 2007b). these low rates of nitrogen mineralization (e.g. tendency toward net immobilization) reflect reduced availability of inorganic nitrogen to nr and dn bacteria but mpns were not performed. nitrogen mineralization was not measured in the current study, but our mpn results show that there were no fewer nr and dn bacteria in the contaminated sites compared to those in the uncontaminated sites 5-10 years post contamination. indeed, there was a higher % and greater number of nr and dn bacteria in j6-f than in j6nf or g5 sites (table 2, figure 1). although mpn values for heterotrophs and nr and dn bacteria from the lf and g7 sites were similar over a broad range of moisture levels, the relative abundance of nr and dn bacteria had a wide range (e.g. 0% to 100%) in different samples with the same moisture level, which suggests that factors other than current levels of soil moisture controlled the % nr and dn bacteria. however, previous studies (martin et al., 1988; murray et al., 1995, harms et al., 2009) showed that denitrifying bacteria are not uniformly distributed in soils and that samples from bulked soils are not representative of the actual distribution. in fact, denitrifying populations from selected sites can be much greater than those from other sites. note that, as explained in the materials and methods, our soil samples from g7 and lf sites were homogenized within each block and placed in the enclosures, and further mixing was performed before sampling from enclosures. this multi-step homogenization might be expected to lower the heterogeneity in the distribution of denitrifying bacteria. however, the heterogeneity in log10 abundance of nr and dn bacteria and heterotrophic bacteria were shown by estimates of the coefficient of variation to be greater for g7 and lf samples (% cv average for log10 of nr and dn = 8.5%, for log10 of heterotrophic bacteria = 6.3%, n = 6) than for the 4 samples taken from the uncontaminated sites (% cv average for log10 of nr and dn = 4.25%, for log10 of heterotrophic bacteria = 3.5%, n = 4). even after soil homogenization, cv was still greater for nr and dn bacteria than that for heterotrophic bacteria, and greater for contaminated sites than the cv for the uncontaminated sites. the different treatments (e.g. nitrate, hay) added to the enclosures may have contributed to spatial variation among enclosures. since no subsamples were taken from within individual enclosures, we do not know the degree of variation at a smaller scale within enclosures. the g7 and lf sites were irrigated resulting in increased soil moisture and the concentration of oxygen in those sites was expected to be low or even anaerobic conditions created. in fact, higher moisture was associated with greater heterotrophic and nr and dn abundance but the effect was slight. the amount of oxygen available in hydrocarbon contaminated soils determines the pathway by which those contaminates are degraded. for example, the biodegradation of pahs has been studied under both aerobic and anaerobic conditions. the biotransformation of pollutants by microbial communities has been observed under anaerobic conditions (coates et al., 1996a, 1996b). our results showed that although moisture levels varied (range 11.6-37.2%) among sites and among sampling dates, mpn values for nr and dn bacteria did not vary greatly. however, a different study showed that high soil moisture (29.76% ± 3.75), organic matter, and available nitrogen increased denitrifyer populations (peralta et al., 2010). in addition, it was shown that phylogenetic composition of the archaeal and bacterial community and nitrogen cy601 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) cling functional genes (e.g. nirs and nosz) were tightly coupled with soil moisture and with seasonal alterations in labile carbon and nitrogen pools where nirs was more abundant at higher moisture levels (rasche et al., 2010). support for the effect of environmental parameters such as nitrate was shown by the higher nitrate level in j6-f that was accompanied with higher net nr and dn bacteria and % nr and dn bacteria in j6-f. other comparisons are suggestive (e.g. higher net nr and dn bacteria and %nr and dn bacteria in g5 than in g5p march 2005) but measurements showing a higher nitrate level in g5 than in g5p were obtained in 2003, none were made in march 2005. the amount of nitrate in soil (tiedje, 1988) and soil moisture (xia et al., 2004) are well-known to be important regulators of denitrification. it was shown that a higher nitrate concentration in soil indicates a higher denitrification activity (yu et al., 2012). in addition, soil moisture was identified as an important factor that increased denitrification activity (orr et al., 2007). the study of denitrification in a tallgrass prairie in central kansas (groffman et al., 1993) emphasized that when multiple factors occur simultaneously, denitrification is promoted. these factors are high levels of water, nitrate, and available carbon source (groffman et al., 1993). however, in this study, nitrate was applied only one time, the easily degraded hydrocarbon may have been already depleted, and moisture levels were generally low. therefore, the stimulation efforts may not have been successful since not all factors required for denitrification were provided simultaneously. in addition, we are measuring the abundance of nr and dn bacteria. it may take a longer period of favorable conditions to increase the number of dn cells than to stimulate expression of denitrification. a different study on the effect of environmental factors on denitrification of saline wetlands in semi-arid regions showed that a high exchangeable sodium percentage decreased the denitrification rate, apart from organic carbon, nitrate, and denitrifying bacteria (huibin et al., 2012). another study investigated the processes that inhibited the decomposition of organic material and affected the dynamics of mineral nitrogen (dendooven et al., 2010). dendooven et al. (2010) found that the high electric conductivity and ph of alkaline soil inhibited the decomposition of organic materials such as glucose. also, the authors found that the reduction of nitrate and the formation of nitrite and ammonia in the glucose-amended alkaline soil was a result of aerobic assimilatory nitrate reduction (dendooven et al., 2010), not dissimilatory nitrate reduction. however, our mpn results show that brine-contaminated sites did not have a lower abundance of nr and dn bacteria. a previous study showed a decrease in the range of organisms that degrade most lowmolecular-weight components of mineral oil with increasing salinity (kleinsteuber et al., 2006). this study provides an overview of the effect of several environmental factors such as hay and nitrate-containing fertilizer on the abundance of nr and dn bacteria in these soils. in fact, hay and fertilizer were shown to improve soil fertility while increasing the rate of salt leaching from the soil (harris et al., 2005). however, the excessive use of fertilizer may increase salinity in arable soils (quantin et al., 2008). we showed that in some cases, hay only, fertilizer only, or the interaction between hay and fertilizer were associated with lower abundance of nr and dn bacteria. this suggests that the carbon source provided (e.g. hay) may have increased the abundance of other groups of bacteria and/or fungi which, as a result of competition, decreased the relative abundance of nr and dn bacteria. since the amendments (e.g. hay and/or fertilizer) were associated with a greater abundance of nr and dn bacteria in some cases and lower in other cases, there was no conclusion about the effect of these amendments on the abundance of nr and dn bacteria. however, our results showed that sites that were contaminated 5-10 years previously by crude oil and/or brine did not have a lower abundance of nr and dn bacteria, therefore, the biological potential for denitrification was present despite residual 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s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) evaluation of exposure index values for conventional radiology examinations: retrospective study in governmental hospitals at west bank, palestine muntaser s. ahmad1*, mohammad shareef1, mohammad wattad1, nora alabdullah1, mouath d. abushkadim1, and ammar a. oglat2 1 department of medical imaging, faculty of applied medical health, palestine ahliya university, dheisha, bethlehem, palestine; 2 department of medical imaging, faculty of applied medical sciences, hashemite university, zarqa, jordan. atlas journal of biology, 2020, pp. 724-729 doi: 10.5147/ajb.vi.219 abstract in the digital radiology system, radiologic technologists (rts) can choose imaging parameters include kvp and mas. the rts received a feedback after acquisition an image in the form of exposure index (ei). the aim of the current study was to check if the ei values are within the range values recommended by the manufacturer (mrei) for radiological examinations that include the chest, abdomen, pelvis, spine, and extremities. data was collected from 3,000 adult x-ray examinations taken from several government hospitals in palestine. the information included patient gender, kvp, mas, ei values, and the examination time. all examinations included in the study used grid. while the study excluded all images that contained implant or prosthesis. descriptive statistical analysis was used to analyse the data, while the mann–whitney u test was used to detect statistically significant differences, p < 0.05. some examinations showed the ei values outside the mrei ranges. the eis in the chest ap examination was higher in the female group than males while other examinations have no difference between males and females. the eis out of working hours were higher than in working hours, especially in chest (p<0.0001), abdominal (p<0.0001), pelvic (p =0.02) and spine (p =0.0005) exams. in the summary it has been proven that some of the examinations are outside the mreis, with differences between the patient gender and the time of the examination. the retrospective study for the exposure index is very important in reducing the risk of radiation to patients. keywords: ei, mrei, kvp, mas. received: june 23, 2020 / accepted: december 12, 2020 _____________________________________ *corresponding author: wmuntaser@gmail.com this is an open access article distributed under the terms of the creative commons attribution license (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 724 a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) introduction the development of radiation technologies, digital photography (dr) has become the most used technique compared with conventional radiology in the past two decades (paulo, 2015)(muntaser s. ahmad, rumman, malash, et al., 2018). dr system allows to use varying values in the technical parameters kilo-voltage peak (kvp), milliampere (ma), and time(s). the dr system has a wide dynamic range of exposure latitude compared to conventional x-rays without affecting on the produced image quality (vañó et al., 2017)(moey & shazli, 2018). to remove the noise level on image in dr system, the radiologic technologists (rts) is forced to raise the image parameters. however, the increase in the image parameters produce a readable image (takaki et al., 2016)(muntaser s ahmad et al., 2019). the extent of the increase in parameters increase radiation dose to patients. this phenomenon is known as exposure creep (mc fadden et al., 2018)(american college of radiology, 2017). therefore, the rts should work to balance the imaging factors without overexposing to the patient at the same time the produced image be readable. thus, there is a need to monitor the technical parameters to reduce the patient radiation dose which is exposed. over time, the exposure creep increases by the rts. therefore, the x-ray manufacturers have created an exposure indicator (ei), and that means the rts indicates an overexposure or underexposure in the produced image (american association of physicists in medicine, 2009). the ei is a numerical value that describes the estimated absorbing dose of the detector, which depends on the sensitivity and efficiency of the image receptor (ir) (peck et al., 2015)(delis et al., 2017). specifically, the ei does not display the actual absorbed dose that patient was exposed (shantel lewis et al., 2019a)(seeram & brennan, 2016). however, it gives an impression of that amount (s. lewis et al., 2019). the ei is used as a quality control tool to check the image quality, because the ei is proportional to the square signal to noise ratio (snr) (takaki et al., 2016)(martin et al., 2017). the ei values for radiological examinations vary according to the manufacturer, each of manufacturer has specific values for the ei. therefore, an international standardized ei was developed by international electrotechnical commission (iec) and the american association of physicists in medicine (aapm) in association with dr system manufacturers (scott et al., 2016). by standardizing the ei values, a linear relationship was designed between the detector exposure and index value (takaki et al., 2019). given these scenarios, the aim of the current study was to monitor the ei of government hospitals in west bank to evaluate the rts who working in these hospitals for choosing the suitable radiologic technique for helping to reduce exposure to the patient. by doing a retrospective evaluates of the actual ei obtained from the abdominal, chest, pelvic, and spine, and extremities examinations for real patients and comparing them with the manufacturer recommended standards. 725 materials and methods equipment the current study was performed on taking x-ray images from three government hospitals in the west bank, palestine. the hospitals were distributed in different areas of the west bank. all government hospitals use the same dr system and the same full picture archiving and communication system (pacs) environment. the authors preferred to keep the names of the hospitals anonymous for not revealing the names of rts in those hospitals. examination details for abdominal, chest, pelvic, spine, and extremities images were extracted using the philips dr digitaldiagnost system, which contains flat panel detector. data collection a total of 3,000 adult patient samples were obtained from januarys to march 2019. the data (age, gender, examination type, exam projection, kvp, mas, actual ei, and manufacturer-recommended ei (mrei) were recorded for each case over 90-day. recorded examinations consist of chest (n = 1305), abdomen (n = 261) and pelvis (n=66), spine (n=63), and extremities (n = 1305). all exposures are adjusted from the rts point of view and do not follow the rules automatic exposure control (aec). all examination details were retrieved from the hospital pacs. all patients under the age of 18 were excluded, and all cases that contained implants or prosthesis were also excluded. the actual ei were compared to the mrei. all selected examinations were used grid with the grid ratio [r] 8:1, and line frequency [n] is 36 lines/cm. ethics an ethical permit was obtained from the palestinian ministry of health to allow patients information to be taken in order to apply the study while keeping the patient's names anonymous. statistical analysis the data obtained was statistically analysed using ibm ssps v.25. descriptive statistics were used to summarize the characteristics of each data. a mann–whitney u test was used to find the statistically significant differences between male versus female patients and in hours versus out of hours working. the value percentage of not adhering to mrei was calculated. the significance level was 5%, median; tables were used to present and the sociodemographic data using pie chart. a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) 726 results descriptive statistics the sample were contained on adult male (1687) and female (1313) patients. all the data was used in the ei analysis of the examinations were shown in the table 1. most of the examinations showed a deviation from mreis where the table appears all the examinations contain overexposure and underexposure ratios. figure 1 shows that 52.7% of the actual eis fall within the mr standard. the other half, 32.8% of the examinations were overexposure and 14.5 % underexposures than mreis. eis which has values less than 250 were included in the overexposure values, while values greater than 630 were included in the underexposure (shantel lewis et al., 2019a). the highest results for the overexposure were represented in upper extremities (lat) examination while the lowest examinations were in shown in chest (pa). on the other hand, the chest lat showed the highest results in underexposure and the upper extremities (lat) examination the lowest percentage in the underexposure. statistically significant results patient gender there is no clear difference in ei between the patient genders, this is what table 2 shows. most of the examinations showed that female median is higher than males. chest anterior–posterior (ap) exam showed a significant difference between both gender where p-value less than 0.05 (p= 0.04). moreover, female patients showed has overexposures than males in extremities, pelvis, abdomen and chest lat examinations. in hours and out of hours despite, the number of cases withi-n working hours is greater than out of hours, the median ei values for out of hours is higher than in working hours in most examinations; were chest ap (p < 0.0001); chest pa (p=0.01); abd ap (p<0.0001); and spine ap (p=0.0005). however, the median ei values for pelvic examination within working hours is higher than out of hours (p=0.02) (see table 3). discussion the aim of the current study was to determine the extent of rts commitment within the mrei ranges for various radiological examinations, including chest, abdomen, spine, pelvis, upper and lower extremities. also, for determining whether the patient's gender and the time of its imaging have an effect on the ei values. the results showed that most of the examinations fall outside the mrei ranges. upper extremities (ap) and (lat) were the highest rates of overexposure ratio by 51.1% and 51.9%, respectively. followed by chest ap examination with an 40.3% overexposure rate. these results are consistent with previous studies, and this indicates that the transition from conventional imaging to the use of techniques cr and dr can result to increases patient exposure (vaño et al., 2007)(nassef & kinsara, 2017). pelvic ap examinations show the lowest underexposure rates where the ratio indicates to 10%. however, this value should be shown with caution due to the small sample size. the high percentage of overexposure, which is the upper extremities, causes great anxiety, because these examination are among the second most common radiographic examinations after chest exam (paper, 2015)(muntaser s. ahmad, rumman, hjouj mohammad, et al., 2018). the increased rate of the overexposure examinations in the upper extremities is related to the use of grid in imaging rather than not using it compared to examination number (n) median exposure % over % under chest anterior–posterior (ap) 385 275.5 40.3 17.7 chest lateral (lat) 280 259 23.5 25 chest posterior–anterior (pa) 640 392 15.8 19.7 abdomen erect (ap) 261 305 28.6 19 pelvis (ap) 66 284.5 30 10 spine (ap) 20 304.5 37.5 12.5 spine (lat) 43 319 22.2 11.1! upper extremities (ap) 442 191! 1.15 ! 6.4! upper extremities (lat) 443 195! 51.9! 3.7 lower extremities (ap) 210 285 33.8 15.4 lower extremities (lat) 210 293 26.9 19.2 total 3000 32.8 14.5 table 1. descriptive statistics of exposure index value distributions for the examinations included in this study. 32% 54% 14% actual ei (n=3000) over exposure optimal exposure under exposure figure 1. actual exposure indicator compared to manufacturer recommended standards. a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) 727 table 2. exposure index values for each patient gender and examination type included in this study. examination in hours (i)/out of hours (o) number median exposure % over % under p-value chest anterior–posterior (ap) i 257 226 0.6 5.3 <0.0001! o 128! 251 1.9! 0.8! ! chest lateral (lat) i 191 322 16.5 12.7 0.44! o 89 344 20.2! 14.9! ! chest posterior–anterior (pa) i 323 374 16.9 17.9 0.01! o 317 418 6.6! 18.7! ! abdomen erect (ap) i 177 220 11.5 22.5 <0.0001! o 84 239 15.5! 26.5! ! pelvis (ap) i 55 307 10.8 1.5 0.02! o 11 297 32.2! 27.1! ! spine (ap) i 11 268 36.5 18.5 0.0005! o 9 353 25.2! 5.8! ! spine (lat) i 27 244 26.5 8.5 0. 55! o 16 334 9! 11! ! upper extremities (ap) i 266 257 42.9 5.6 0.71! o 176 142 54.4! 3.8! ! upper extremities (lat) i 266 245 46.5 4.1 0.36! o 177 154 52.1! 1.5! ! lower extremities (ap) i 138 294 27.6 10.4 0.12 o 72 281 35.6 20.2 lower extremities (lat) i 138 273 24.3 15.2 0.35 o 72 371 21.5 23.5 total 3000 table 3. in and out hours exposure index values for all examinations type included in this study. examination gender number median exposure % over % under p-value chest anterior–posterior (ap) m 254 320 34.1 26.8 0.04! f 131! 184 2.45 ! 0! ! chest lateral (lat) m 188 320 20 14.2 0.723! f 92 340 23.7! 16.4! ! chest posterior–anterior (pa) m 326 372 20.4 19.4 0.532! f 314 416 10.1! 20.2! ! abdomen erect (ap) m 87 238 15 24 0.582! f 174 218 19! 28! ! pelvis (ap) m 8 305 14.3 0 0.202! f 58 295 35.7! 28.6! ! spine (ap) m 13 266 40 20 0.820! f 7 348 28.7! 7.3! ! spine (lat) m 24 243 30 10 0.355! f 19 330 12.5! 12.5! ! upper extremities (ap) m 263 255 46.4 7.1 0.91! f 179 140 57.9! 5.3! ! upper extremities (lat) m 254 241 50 5.6 0.26! f 189 151 55.6! 0! ! lower extremities (ap) m 135 292 31.1 11.9 0.28 f 75 275 39.1 21.7 lower extremities (lat) m 135 271 27.8 16.7 0.535 f 75 374 25 25 total 3000 a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) 728 film screen combination (fsc) and computed radiography (cr). in addition to the short distance between source image receptor distances (sid) (sheridan & mcnulty, 2016)(pomerantz, 2013). moreover, the reason may be due to the increased pressure on the rts in imaging for a large number of patients, thus the rts are forced to increase x-ray parameters on the patient to avoid returning the image again (al-tell, 2019)(imaging & journal, 2007). the relationship between focus to skin distances (fsd) and higher entrance skin doses (esd) was explained. that’s when it's changing the exposure factors and checking its effect on examination dose reductions (gibson et al., 2011). it should be reduced the eis by improving the exposure parameters on the dr device. it is important to take into account that the current study did not focus on the use of aec and its effect on the ei. it is important to observe ei to ensure quality assurance within the radiology units. in this study, the focus was on the effect of the patient's gender and time of imaging on performance on ei values. as for the gender of the patient, all the examinations except for the chest examination (ap), there is no difference between males and females on the ei. however, a chest ap exam showed an increase in females than in males. there is an unclear justification for this high exposure pattern in female patients. an earlier study suggested that inaccurate patient exposure patterns could lead to higher ei values and it was recommended improving exposure charts to reduce the dose of detector (lanc & silva, 2008). improving the exposure chart can neutralize this bias of gender. it is noted in the previous literature that differences in male and female doses are closely related to monitoring aec systems: the smaller differences in detected mean doses means the more sensitive aec and it will be changed in the attenuation characteristics of the different patient groups (web-based tools for quality assurance and radiation protection in diagnostic radiology, n.d.). the relationship between eis and the increase in female patients should be checked in future studies under using dr devices. all other examinations were used aec technique, while the chest ap examination did not used it, and because the location of the image plate under patients back directly especially patient on stretcher" non-ambulatory patient". regarding the timing of patient image, the study showed that there are statistically significant differences between working within hours and out of hours. as the ei values were increasing out of hours working. the reason for this is due to a shortage number of rts in out of working hours, which start from 3 pm to 8 am next day. rts may have to increase exposure values to avoid repeating images again under intense pressure. this is what was determined in the previous study (shantel lewis et al., 2019b). also, the reason may be related to the number of experience years for rts, as most of the rts on the out of working hours have a little experience. however, the increase in the ei must be monitored to achieve the best results. the aim of this study was to retrospectively analyse the ei for all examinations used in normal x-rays. it is not intended to assess the ei of the manufacturer or assessment the rts in their performance, or assessment the patient doses. it was not possible to determine the size of the patient and the body part thickness because the study was a retrospectively. this would clarify some of the gender-related findings and their eis. conclusion in the summary, the results of the study show that there are many examinations that fall outside the framework recommended by the manufacturer. however, to determine the correct eis from the manufacturer, an improvement should be made to the current system. it can be said with confidence that it is difficult to determine the inevitable reasons for the high exposure index commensurate with the patient gender and the time of the imaging, and the correct strategies must be taken in addressing this imbalance. the current study highlights the exposure that can be used to reduce a patient's dose. conflict of interest all authors declared no conflict of interests related to this article. disclosure the authors did not receive any type of commercial support in forms of either compensation or financial support for this study. references paulo gdnn (2015) optimisation and establishment of diagnostic 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spu bl is hi ng .o rg ) estimating the sequestration potential of organic carbon in forest soils in the central middle atlas: a tool to fight climate change mohamed el mderssa1*, hassan benjelloun2, hafida zaher2, omar zennouhi1, laila nassiri1, and jamal ibijbijen1 1 faculty of sciences, moulay ismail university, b.p. 11201, zitoune, meknes, morocco; 2 national school of forest engineering, sale-tabriquet, morocco received: january 16, 2019 / accepted: march 2, 2019 __________________________________________________ * corresponding author: elmderssa.enfi@gmail.com 603 abstract soil organic carbon sequestration is an important matter in international negotiations to fight climate change through a reduction in greenhouse gas (ghg) emissions. this study was carried out in the forests of the moroccan central middle atlas, with the objective of determining the storage of carbon in the high organo-mineral layer of soils (<30 cm deep) and under different spontaneous forest species in this area. it shows that the soil organic carbon stock (socs) in these forest ecosystems is higher in green oak and deciduous zeen oak forest stands, recording 264.73 and 251.54 t/ ha respectively on a basaltic substrate followed by cedar (resinous species) (178.07 t/ha). mixed forest stands come at last with low socs (109.02 t/ha). this variation may be attributed to several factors, mainly the type of vegetation, the physical and chemical soil properties and soil type of the studied area. keywords: sequestration, central middle atlas, organic carbon, stock. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the quantification of the organic carbon stock in forest ecosystems is to well understand the general carbon cycling in these ecosystems. indeed, this quantity, in forest soil, is the result of the balance between the net primary production of vegetation and the decomposition of organic matter (liski & westman, 1997). these two processes depend on climatic conditions, mainly temperature and humidity. however, forest soils may be a significant source of co2 as a result of global warming, which lead to a mineralization of organic matter higher than the net primary production of vegetation (liski, 1999, bernoux et al., 2005). every change in the organic carbon reservoir in the soil can significantly affect the concentration of co2 in the atmosphere, since the soil contains twice as much carbon as the atmosphere (schlesinger 1977; post et al., 1982; watson et al., 1990). in addition, each tree species has different functional characteristics that allow it to have a unique influence on its environment, including the soil on which it grows. for example, depending on differences in shade tolerance, foliage persistence (evergreen vs. deciduous leaves), evapotranspiration capacity, and composition of the herbaceous stratum, soil moisture and temperature conditions may vary depending on stand type (laga tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) anière et al., 2011). apart from the influence that a single tree species can have on its environment, mixing two or more tree species in mixed stands could have an impact on carbon sequestration in soils. the present study aims to partially fill this knowledge gap, especially with regard to the quantification of carbon stock in forest soils as a result of the forest composition comprising four natural species, namely cedar, maritime pine, green oak and zeen oak in three types of forest stands in the moroccan central middle atlas, namely the south of jbel aoua , azrou and jaaba forests. materials and methods description of the study areas this study was carried out in three natural forests of the central middle atlas, namely the forests of south of jbel aoua, azrou and jaaba (figure 1). the south of jbel aoua forest the south of jbel aoua forest covers an area of 7865.42 ha. it is composed of a green oak coppice, a maritime pine grove and a cedar grove. the geological formations of the area are dominated by lower jurassic (or lias) formations, which include sandy dolomites and dolomitic limestones. two types of soils are described in the region. soils developed on hard limestone namely fersialitic red soils and soils developed on sandy dolomite which are para rendzine that are relatively rare. the climate in the area is of a mediterranean type, the drought period is 5.5 months (mid-may late october) and that the bioclimate is subhumid of cold variant (hceflcd, 2007). 604 azrou forest located on the northern edge of the middle atlas plateau, the azrou forest covers an area of 17806.79 ha. this forest is characterized by a contrasted relief with very variable altitudes where one may observe the inversion of the altitudinal layers of vegetation (case of michlifene and jbel habri) due to the oppositions of the slopes and the accumulation of cold air in the closed depressions. from a bioclimatic point of view, the azrou forest is of the humid bioclimatic type with a cold variant or sub-humid type with a temperate variant. the climate is also characterized by major storms, making it more favourable for the establishment and development of the cedar grove. the composition of the plant formations of the azrou forest includes pure cedar stands, mixed cedar, green oak stands, pure green oak stands and reforestation stands of cedar and cypresses (laaribya, 2016). jaaba forest the jaaba forest has a total area of 10449.16 ha. it receives an annual average rainfall varying between 810 and 976 mm over a 30-year climatic period with a bioclimatic type ranging from cool sub-humid to wet very cold. in this forest, forest stand typology is defined by the combined effects of the nature of the substrate and the topographic position. indeed, zeen oak stands develop on the basalt substrate and in depressions, while on carbonate rock (limestone and dolomite), the type of forest stand is based on green oak. the jaaba forest includes three types of forest formations: pure green oak, pure zeen oak and a mixture of green oak zeen oak (hceflcd, 2007). a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 1. map of the studied area. 605 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) methodological approach at the level of each forest, a representative plot was chosen taking into account the topo-climatic position and the type of forest stand. using a geographic information system (gis) and field validation visits, nine (09) different forest stands were identified (table 1). at the level of each stand, soil samples from the higher organo-mineral soil (<30 cm deep) were collected from six (06) soil site replicates. according to benjelloun (1997), this layer is the one where root concentration is higher and exchanges between the soil and plant roots are important (figure 2, 3 and 4). after sieving the soil samples through 2 mm meshes, we carried out the following physical and chemical analyses: grain size, bulk density, organic matter, carbon, total nitrogen and acidity (ph (h2o), ph (kcl)). these analyses were carried out in the soil and environmental microbiology laboratory of the faculty of science of meknes. determination of soil carbon stock the percentage of carbon and the bulk density allow the determination of carbon stocks and organic matter per unit area using the following formula: q(i) = 0.1 x ei x da(i) x ci where: q (i): soil corganic content (t/ha), ei: depth (i) (cm), da(i): apparent density of the fine fraction (< 2 mm) in depth (i) (g.cm-3), ci: concentration of organic carbon in fine soil for depth (i) (g.kg-1). mo = c org x 1.724 (t/ha) statistical data analysis this step of the work consists of analyzing the data obtained and processing them statistically. indeed, an analysis of descriptive statistics, mainly means, standard deviations and coefficients of variation, was carried out to explain the variability of sites within the same forest formation (forest stand). comparisons of formation means for the different studied variables were obtained through a single-criteria analysis of variance (anova 1). the anovas were conducted to verify whether the average carbon stocks of the different strata were significantly different from each other. the assumptions of this test, which are the normality of the distribution and the homogeneity of the variance, were verified beforehand. results a. physical properties the results of the physical properties are presented in table 2. texture because texture, directly or indirectly, conditions other physical properties of the soil, it plays an important role in porosity, drainage and especially carbon stocks in the soil (jessica, 2009). the textural classes of the studied soils are represented in the textural diagram (figure 5). the most represented textures of these soils are fine silt (s1, s2, s8 and s9), clayeysandy silt (s4 and s6) and clayey-sandy silt (s3, s5 and s7). thus, most of the forest soils have a rather coarse texture linked to the lithological nature of the parent material. the results of the single-criterion anova show that the proportion of clays is significantly different from one site to another (p=0.04) for a significance level of 5%. table 1. description of the identified sites. site no. localisation coordinates exposition slope (%) altitude (m) parent rock forest vegetation 01 jaaba forest long=-5°10’28’’ lati=33°33’7’’ nw 14 1593 basalt mixture of an adult forest of zeen oak and green oak 02 jaaba forest long=-5°10'38’’ lati=33°33’16’’ north 10 1562 basalt mature zeen oak forest 03 south jbel aoua forest long=-5°01'33’’ lati=33°34’26’’ ne 13 1745 sandy dolomite mature green oak forest 04 south jbel aoua forest long=-5°01'26’’ lati=33°34’21’’ ne 15 1750 sandy dolomite young cedar forest 05 south jbel aoua forest long=-5°00'54’’ lati=33°34’10’’ ne 18 1740 sandy dolomite mature maritime pine forest 06 south jbel aoua forest long=-5°00'51’’ lati=33°34’03’’ ne 20 1768 sandy dolomite mixture of a young green oak forest and a mature cedar forest 07 azrou forest long=-5°08'31’’ lati=33°29’57’’ ne 20 1690 basalt pure cedar forest 08 azrou forest long=-5°08'39’’ lati=33°29’34’’ sw 10 1720 basalt mature zeen oak forest 09 azrou forest long=-5°08'43’’ lati=33°29’30’’ sw 25 1710 basalt mature green oak forest apparent density (da) the apparent density of the soil is a necessary parameter for calculating carbon stocks. values range from 0.74 to 0.85 g/ cm3. thus, we should say that the studied soils have a good structure allowing them to have a good aeration. the results of the single-criterion anova show that there is no significant difference in the average site density for a significance level of 5%. there is also a high degree of homogeneity within the sites between replicates, expressed by the values of the coefficients of variation, which vary from 3 to 19%. b. chemical properties the results of the chemical properties of the soils studied are presented in table 3. carbon and organic matter (om) concentrations the average organic carbon content varies from 4.69% (s6) to 11.10% (s9), and the distribution of organic carbon is relatively heterogeneous across all sites based on the values of the coefficient of variation ranging from 29 to 74%. according to a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 606 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 2. distribution of soil sampling sites (replicates) in the forest stands of the jaaba forest. figure 3. distribution of soil sampling sites (replicates) in the forest stands of the south of jbel aoua forest. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 607 the anova results, the averages are significantly different (p = 0.008), hence the influence of the site or forest stand on soil carbon sequestration and thus on soil organic matter reserve. the variation in the percentage of organic matter in the different sites follows the same trend as the carbon concentration since mo = c orgx 1.724 (soltner, 1988). total nitrogen the total soil nitrogen in the studied soils (higher soil layer) varies from 0.20% (s6) to 1.76% (s9). these values are recorded under mixed forest stands of cedar and green oak growing on a sandy dolomite substrate and under pure green oak stand figure 4. distribution of soil sampling sites (replicates) in the forest stands of the azrou forest. figure 5. textural diagram of the studied soils. 608 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 6. carbon and organic matter stocks in the studied forest stands in decreasing order. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 2. physical characteristics of the soils studied. table 3. chemical properties of the soils studied. s1: green oak stand mixed with zeen oak (jaaba forest), s2: zeen oak stand (jaaba forest), s3: green oak stand (jbel aoua forest south), s4 : cedar stand (south of jbel aoua forest), s5: maritime pine stand (south of jbel aoua forest), s6: cedar stand mixed with green oak, s7 : cedar stand (azou forest), s8 : zeen oak stand (azrou forest),s9 : green oak stand (azrou forest), values in brackets indicate coefficients of variation in %, similar letters indicate that the values are not significantly different. s1 s2 s3 s4 s5 s6 s7 s8 s9 pa rt ic le si ze clay (%) 34,65 (32) a 36,12 (32) a 20,15 (53) b 29,17 (46) ab 22,28 (60) c 29,92 (46) ab 17,12 (17) d 26,72 (25) ab 27,45 (24) ab thin silt (%) 14,28 (30) 13,30 (24) 9,02 (84) 11,27 (64) 9,05 (74) 12,05 (46) 13,18 (39) 13,83 (39) 12,62 (38) coarse silt (%) 26,05 (12) 31,33 (21) 24,32 (60) 21,68 (54) 18,15 (76) 25,65 (38) 29,37 (40) 33,97 (20) 34,82 (38) thin sand (%) 11,30 (44) 9,15 (26) 23,83 (53) 20,20 (79) 22,78 (59) 15,30 (73) 20,60 (44) 13,78 (37) 11,02 (41) coarse sand (%) 12,72 (25) 10,10 (33) 22,68 (51) 17,68 (88) 27,75 (67) 17,03 (90) 19,77 (48) 11,72 (35) 14,10 (36) texture silty thin silty thin sandy-clay sandy clay silt sandy-clay sandy clay silt sandy-clay silty thin silty thin apparent density (g/cm3) 0,74 (10) 0,83 (15) 0,80 (19) 0,85 (18) 0,87 (15) 0,85 (9) 0,83 (18) 0,78 (14) 0,79 (3) s1 s2 s3 s4 s5 s6 s7 s8 s9 organic matter (%) 8,58 (38) ab 10,80 (74) ab 12,04 (39) ab 10,39 (44) ab 8,83 (64) ab 8,08 (57) a 12,87 (29) ab 18,89 (41) b 19,14 (31) b carbon (%) 4,98 (38) ab 6,27 (74) ab 6,99 (39) ab 6,03 (44) ab 5,12 (64) ab 4,69 (57) a 7,46 (29) ab 10,96 (41) b 11,10 (31) b total nitrogen (%) 0,3 (25) a 0,60 (107) ab 0,70 (37) ab 0,63 (48) ab 0,52 (70) ab 0,20 (121) ab 0,89 (137) ab 0,28 (131) ab 1,76 (38) b c/n 16,6 (43) 10,45 (121) 9,98 (89) 9,57 (81) 9,84 (175) 23,45 (101) 8,38 (103) 39,14 (120) 6,31 (97) ph (h2o) 6,52 (3) 6,46 (2) 7,23 (2) 7,21 (4) 7,37 (4) 7,09 (6) 6,51 (3) 6,10 (3) 6,33 (6) ph (kcl) 5,84 (2) 6,11 (8) 6,81 (3) 6,61 (4) 6,98 (3) 6,52 (5) 5,98 (1) 5,50 (5) 5,37 (3) s1: green oak stand mixed with zeen oak (jaaba forest), s2: zeen oak stand (jaaba forest), s3: green oak stand (jbel aoua forest south), s4 : cedar stand (south of jbel aoua forest), s5: maritime pine stand (south of jbel aoua forest), s6: cedar stand mixed with green oak, s7: cedar stand (azou forest), s8: zeen oak stand (azrou forest), s9: green oak stand (azrou forest), values in brackets indicate coefficients of variation in %, similar letters indicate that the values arenot significantly different. 264.73 251.54 178.07 174.5 160.86 144.22 134.06 119.13 109.02 456.39 433.65 306.99 300.84 277.32 248.64 231.12 205.38 187.95 0 50 100 150 200 250 300 350 400 450 500 1 2 3 4 5 6 7 8 9 s to ck o n t/h a s ite o rganic matter o rganic carbon 609 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) on basalt parent rock, respectively the results, obtained by the anova using only one criterion, generate a statically significant difference between the different sites (p =0.04). in terms of fertility, the recorded values showed that all sites are considered to be very rich in total nitrogen according to the standard found by dabin (1963) ranging from 0.15 to 0.25%. c/n ratio the carbon-nitrogen ratio of a soil is determined as the weight ratio between the amount of organic carbon and nitrogen (gobat et al. 2003). it allows the evaluation of the quality of organic matter as well as the estimation of the importance of humification and mineralization processes acting in soils (jessica 2009). lafond et al. (1992) and akselsson et al. (2005) mentioned the c/n ratio as a good indicator for assessing carbon sequestration in soils. a high c/n ratio represents a low rate of carbon decomposition since decomposing organisms use nitrogen which quickly becomes limiting. a low c/n ratio indicates a high nitrogen concentration and a high degree of decomposition. the results obtained from the c/n ratio range from 6.31 (s9) to 39.14 (s8). taking these values into account, the majority of which are less than 10, it can be said that the biological activity is good and that the nitrogen mineralization process outweighs the immobilization process by soil microorganisms. acidity the ph (h2o) analysis results (table 3) show that the studied soils have ph values ranging from 6.10 to 7.37 indicating a weakly acidic to neutral chemical environment. the ph (kcl) is, always lower than the ph (h2o), and varies between 5.50 and 6.98. according to the single-criterion anova, there is a very significant difference between the means (p= 0.000) showing the effect of forest composition on soil acidity, with a high homogeneity within sites between repetitions which values of coefficients of variation vary between 1 and 6%. soil organic carbon stock (socs) the ecological variables as well as the physical and chemical properties of the soil may influence the accumulation of organic carbon in the soil. the results of socs in the studied soils are presented in table 4 and highlighted in figure 6. the anova results generate a significant difference between sites (p = 0.012) in socs, hence the effect of the site and the forest type on the variation in socs. the quantities of carbon sequestered in the studied soils ranged from 109.02 to 264.73 t/ha. the highest stock is found under green oak and zeen oak deciduous forest trees (s8 and s9) on a basaltic parent rock, followed by cedar resinous trees on a basaltic substrate (s7). this mixing of stands may have led to the variability in soil bed quality, rooting depth of tree species and soil chemistry, which may also influence carbon storage. a student t test comparing the means for independent samples was performed, showing a significant difference (p = 0.012) between the means of socs in a basaltic and sandy dolomite substrate, hence the effect of the nature of the lithological material on the variation in socs under the same forest species. this is the case for s3 (green oak on sandy dolomite with a scos=174.50 t/ha) and s9 (green oak on basalt with a socs=264.73 t/ha). discussion the forest soils contribute to about 70% of the carbon dioxide exchanges between the biosphere and the atmosphere. these quantities are mainly produced by the decomposition of organic matter and root respiration (jessica 2009). these processes vary according to several parameters including climate, vegetation type, soil physico-chemical characteristics (conant and paustian 2001), frequency of natural disturbances and development. it is therefore difficult to accurately estimate organic carbon stocks at the regional level and to obtain quality empirical data that accurately represent this heterogeneity (kulmatiski et al. 2003 in jessica 2009). the obtained results show that the forest soils of the central middle atlas have a high carbon sequestration capacity. indeed, the average organic carbon stock at the level of the studied forests is about 231.45 t/ha in the azrou forest, 147.14 t/ha in the south of jbel aoua forest and 126.62 t/ha in the jaaba forest in the first thirty centimetres of soil. this variation depends on the nature of the soil in the study area. the carbon stocks found in this layer under the green oak stands are higher than those observed by boulmane (2010), which are of the order of 63 t/ha and 47 t/ha respectively in the forests of tafechna and reggada considered relatively degraded. this scos value is still higher than that recorded under cork oak stands in the maamora forest (78 t/ha) (oubrahim 2015) and in the moroccan rif (100 t/ha) (sabir et al., 2002). these strong potentialities of the studied table 4. soil organic carbon stock (scos) at the studied soils. s1: green oak stand mixed with zeen oak (jaaba forest), s2: zeen oak stand (jaaba forest), s3: green oak stand (jbel aoua forest south), s4: cedar stand (south of jbel aoua forest), s5: maritime pine stand (south of jbel aoua forest), s6: cedar stand mixed with green oak, s7: cedar stand (azou forest), s8: zeen oak stand (azrou forest), s9: green oak stand (azrou forest), values in brackets indicate coefficients of variation in %, similar letters indicate that the values are not significantly different. s1 s2 s3 s4 s5 s6 s7 s8 s9 oct stock (t/ha) 109,02 ad 144,22 ad 174,50 abc 160,86 abc 134,06 abc 119,13 abc 178,07 abc 251,54 be 264,73 ce organic matter stock (t/ha) 187,95 248,64 300,84 277,32 231,12 205,38 306,99 433,65 456,39 cv (%) (32) (60) (60) (57) (75) (56) (18) (40) (34) 610 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) forest soils in terms of carbon sequestration can be explained by the large quantities of litter returned to the ground by the existing forest formations in the studied sites, and by the presence of allophanes resulting from the alteration of basaltic rocks (jaaba and azrou sites) and calcium (ca++) resulting from the alteration of limestone and dolomite (jbel aoua site), which have the ability to fix and retain organic matter in a stable state in the soil (benjelloun 2017). a similar study by eglin (2005) and lecointe et al (2005) in british forests estimated an socs in the organic layer (depth< 30 cm) of 136 and 153 t/ha respectively is still below the socs found in the study area. a socs was evaluated in a canadian forest by jessica (2009) below 5 types of plant formations with values ranging from 61 to 133 t/ha, the highest of which is still observed under hardwood in the first organic layer of the soil (<30cm), and highlighted the effect of plant material on soil organic carbon accumulation. conclusion the purpose of this study was to determine carbon stocks under different forest formations in the central middle atlas. with regard to the results obtained, it can be said that there is spatial variability in carbon stocks in the study area and that this may be explained by the different nature of the forest formations concerned. however, several factors contribute to the understanding of these stocks and it will be essential to consider them in subsequent analyses. indeed, the best socs are observed below the hardwoods (green oak and zeen oak) followed by cedar-based softwoods on a basaltic substrate. mixed strata showed low carbon accumulations. the presence of allophanes and exchange earth bases (ca++ and mg++) in the soil contributes to the fixation and maintenance of soil organic matter in a stable state. references akselsson c, b berg, v meentemeyer, and o westling (2005) scaling up carbon sequestration 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[in french] watson rt, h rhodhe, f oeschger, and u siegenthaler (1990) greenhouse gases and aerosols. in: climate change, the ipcc scientif assessment. j.t. houghton, g.j. jenkins & j.j. ephraums (eds), cambridge university press, p. 40. atlas journal of biology 2019, pp. 619–627 doi: 10.5147/ajb.v0i0.206 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a breeding study of some grain quality characters in rice (oryza sativa l.) nessreen n. bassuony1 and david a. lightfoot2* 1 rice research section, field crops research institute, a.r.c.,giza, egypt; 2 department of plant, soil, and agricultural systems, mc 4415, southern illinois university, carbondale, il 62901, usa. received: july 21, 2019 / accepted: august 24, 2019 __________________________________________________ * corresponding author: ga4082@siu.edu 619 abstract the present study was conducted at the farm of the rice research and training center, (rrtc), sakha, kafr el-sheikh, egypt, during the 2016, 2017 and 2018 seasons. six rice cultivars (oryza sativa l.) that differed in their grain quality characters were used as parents to produce six generations of three crosses of rice namely ‘giza178’בegyptian yasmine’, ‘giza177’×’upr82-1-7’ and ‘iet1444’בpusa basmati1’. generation mean analysis was used to estimate types of gene actions, heterosis, inbreeding depression, heritability and genetic advances for milling%, grain dimensions and cooking and eating characters. the results indicated that the scaling test was significant for one scale in all the crosses for all the traits, indicating an inadequacy of the simple additive dominance model. segregation analysis indicated partial dominance for grain quality characters for almost crosses except gel consistency and amylose content percentage. this suggested over dominance for most traits in most crosses. moreover, evaluation of genetic parameters indicated the importance of additive gene effects. that were significant and positive in the inheritance of grain quality characters for the three crosses. exceptions were grain length and grain shape which were significant and negative. different types of gene action were detected, varying by the characters and the crosses under study. epistasis was observed among the crosses. heritability estimates for milling and eating and cooking quality were low to moderate indicating that selection exercised in late generations may be effective. heritability estimates for grain dimensions were high, suggesting that selection in early generation will be effective in future. keywords: oryza sativa, back cross, grain quality. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction rice (oryza sativa l.) is an important staple food crop in many parts of the world. (manful 2010). it feeds more than 50% of earth’s inhabitants especially in developing countries yield is the most noticeable characteristic to farmers while the crop is in the ground, but when the product of the crop, the milled rice, reaches the market, quality becomes the key determinant of its sale-ability .thus rice breeders presently pay great attention to rice quality improvement (gnanamalar and vivekanandan, 2013). grain quality is one of the most important quantitative traits in rice. it is a multi-faceted trait involving physical and biochemical aspects relating to milling, appearance, cooking and eating quality and nutrition (fitzgerald et al., 2009; bao, 2014). the grain and cooking characters which are influenced by physico-chemical properties of starch play an important role in determining the texture and consumer acceptance of cooked rice. many of the physico-chemical quality characters follow a complex polygenic mode of inheritance. the characters gel consistency and head rice recovery percentage contributed maximum towards genetic divergence (chamundeswar, 2010). the generation mean analysis has been considered to be one of the best methods for estimating the different components of genetic variance and presence or absence of epistasis. the concept of generation mean analysis was developed for the estimation of genetic components of variation. additive and non-additive gene action were found for amylose content swain and (nagaraju, 2004). additive and non-additive gene action for grain shape (kumar et al., 2006; nayak et al., 2007) while studying genetics of quality characters in three crosses found that kernel length was controlled by both additive and dominance effects, kernel length/breadth ratio was controlled by additive, dominance and epistatic effects in all crosses. for alkali spreading value, additive, dominance and all three interactions were important. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) cooked kernel length was controlled by additive (d), and additive x additive (i), additive, dominance (h) and additive and additive (i) were significant. additive, dominance and epistatic gene actions of additive x additive and dominance x dominance and duplicate type of interaction controlled the elongation ratio. amylose content was under the control of duplicate epistasis. lakshmi (2009) studied generation mean analysis in two crosses and found that both additive ([d]and [i]) and dominant type ([h] and [l] components were significant for hulling percent, gelatinization temperature for both the crosses. additive variance was available for selection for head rice recovery and kernel elongation . before launching any breeding program, surveys of genetic variability are absolutely necessary to start an efficient breeding program. heritability and genetic variability are prerequisites for carrying out selection based improvements. the information about these help breeding programmes by broadening the gene pool of rice and gives an indication about the efficiency of transformation of characters into future generations (selvaraj et al., 2011). the detection and estimation of epistasis would also enable the breeders to understand the genetic cause of heterosis with greater reliability. lacking information about these breeding parameters, the present study was planned to investigate the genetics of some grain quality characters by using six-generations of the three crosses under normal conditions. materials and methods the present study was conducted at the farm of the rice research and training center, (rrtc), sakha, kafr el-sheikh, egypt, during 2016, 2017 and 2018 seasons to study the genetic behavior of some grain quality characters of rice cultivars . six rice cultivars that differed in their grain quality characters ‘giza 178’ and ‘giza 177’ (both short grain) and ‘egyptian yasmine, ‘iet1444’, ‘upr82-1-7’ and ‘pusa basmati’ (all long grain) were used as parents to produce six generations of three crosses of rice. namely giza 178×egyptian yasmine, giza 177×upr82-1-7 and iet1444 × pusa basmati 1 were used to apply generation mean analysis. reciprocal crosses were carried out following the technique proposed by jodon (1938) and modified by botany (1961) in the 2016 season. hybrid seeds along with the parents were grown in the next season (2017). some of the f1s were left for self-fertilization to produce seeds of f2 plants and some were back crossed with the parents to produce of bc1 (f1xp1) and bc2 (f1xp2). in 2018, parents, f1, f2 and backcross (bc1 & bc2) generations were raised in the field. experiments were laid out in a randomized complete block design with three replications. each replicate contained a single row for parental lines, and f1’s, with eight rows for each of the backcrosses and 15 for the f2 generation. the length of each row was one meter. row to row and plant to plant distance was 20 cm. normal agronomic practices were followed for growing the crop. at maturity five plants per replication for f1 and each parent, 50 for each of the backcrosses and 100 for the f2 generation were selected to harvest grain rice on individual plant basis. about 150 grams (three replication) of rough rice for all samples were taken and well mixed and cleaned. 620 all samples were analyzed for the following grain quality characters: 1. milling characters: hulling%, milling% and head rice% were determined according to adair (1952) by using satake testing machines. 2. physical characters: grain length, width and shape were measured for milled rice grain according to khush et al. (1979). 3. cooking and eating quality characters: gelatinization temperature (g.t.), amylose content and gel consistency test (g.c.) were estimated for milled rice samples following the methods of little et al., (1958). juliano (1971) and cagampang et al. (1973) respectively. elongation ratios were calculated according to azeez and shafi (1966). genetic parameters as broad and narrow sense heritability’s were estimated according to powers et al. (1950) and warner, (1952), respectively. genetic advance in percent of mean and phenotypic and genotypic coefficients of variations were estimated using the procedure suggested by burton, (1952). the following genetic parameters were estimated for the studied characters, heterosis and degree of dominance followed mather and jinks, (1971), scaling test for adequacy of additive and dominance model and genetic components followed mather, (1949) and expected genetic advance (gs %) after johnson et al. (1955). results and discussion 1. scaling tests table 1 showed scaling test for adequacy of additive and dominance model of milling characters, grain physical characters, cooking and eating characters in the three studied crosses. the scaling test was significant for one scale in all the crosses for all the quality traits studied, indicating inadequacy of simple additive dominance model. 2. genetic parameters 2.1. degree of dominance milling characters and grain dimensions (table 2) showed partial dominance in all crosses except cross 1 for hulling % and cross2 for head rice % were over dominance. according to sivasubramanian and mahadevamenon (1973), hulling percentage was independent of grain size and shape and all these traits showed partial dominance. kernel elongation and gelatinization temperature indicated partial dominance in all crosses except cross 3 for kernel elongation and cross 2 for gelatinization temperature were over dominance. while gel consistency and amylose content % indicated over dominance except cross 3 for amylose content% was partial dominance. it was remarkable that partial to complete and/or over dominance were both detected for all crosses for the studied characters depending on the crosses as well as the character. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 621 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 2.2. additive genetic variance and dominance genetic variance table 2 indicated that additive genetic variance (½d) were higher than that of the dominance genetic variance (¼h) in three cross under study for milling characters, grain dimensions and all cooking and eating quality, but the dominance variance was the highest value in cross 1 and cross 3 for the milling %. the results suggested that early generation selection may be effective in improving of milling % in these crosses cross 1 and cross 3. these results were observed indicated that additive that variance played an important role in the inheritance of milling characters in these crosses. the same results for grain length, grain width and grain shape characters in the three crosses. similar results were obtained by kishore et al. (2008). in cross 2 both additive and dominance variance in milling % hulling % and cross 1 for head rice were the biggest value. both additive and non-additive gene effects were important for amylose content with predominance of additive effects (chamundeswari, 2010). 2.3. broad and narrow sense heritability it is clear from table 2 that hulling percentage in cross 2 recorded the highest broad sense heritability (78.92%) and narrow sense (63.85%) (medium) compared to the other crosses studied (table 2). broad sense heritability and narrow sense heritability estimates were lowest for milling % in cross1.the highest broad sense heritability was observed for cross 3 and the same cross gave 38.07 for narrow sense. heritability, the broad sense value, was high. while , heritability estimates in the narrow sense was moderate values in all study crosses for head rice %. moreover, the results in table 2 indicated that heritability broad sense values were high for grain dimensions. on the other hand, heritability estimate in the narrow sense was moderate. these results were in agreement with those obtained by partitioning of the genetic variance. these results also were in agreement with those reported by bharadwaj et al. (2007). high heritability values were expected, because most likely such traits are controlled by additive gene effects (bagati et al. 2016). table 1. scaling test for adequacy of additive and dominance model of all tested characters in the three studied crosses. characters crosses a b c hulling% milling % head rice % cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 66.312±3.361** 63.932±4.71** 59.702±4.66** 73.182±1.336** 71.251±0.983** 68.311±o.888** 12.831±0.361** 14.546±0.451** 13.477±0.989** 60.162±0.511** 63.177±461** 60.272±0.611** 70.364±0.812** 71.453±o.793** 68.433±0.731** 11.543±1.361** 13.612±2.666** 14.876±1.782** 49.932±0.723** 51.872±0.872** 32.733±0.549** 65.983±0.783** 71.974±0.830** 71.991±0.772** 9.875±0.655** 10.812±0.763** 9.755±0.876** grain length grain width grain shape cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 0.923±0.012** 0.836±0.23 ** 1.341±0.046** 0.069±0.078** 0.076±0.034** 0.076±0.091** 0.735±0.041** 0.899±0.038** 0.754±0.056** 0.936±0.213** 0.886±0.179** 0.995±0.119** 0.106±0.057** 0.093±0.049** 0.087±0.032** 0.981±0.041** 0.889±0.061** 0.0902±0.522** 0.765±0.0231** 0.807±0.119** 0.931±0.291** 0.244±0.065** 0.497±0.031** 0.466±0.072** 0.874±0.034** 0.763±0.044** 0.7755±0.066** grain elongation gelatinization temp gel consistency amylose content% cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 28.119±0.836** 21.816±0.903** 15.940±0.776** 2.664±0.236** 2.912±0.117** 3.092±0.269** 15.723±0.859** 7.441±0.913** 18.762±1.25** 4.933±0.298** 5.664±0.342** 0.339±0.369** 22.736±1.036** 18.546±0.921** 7.833±0.711** 1.352±0.254** 2.667±0.254** 1.445±0.336** 16.345±1.514** 8.711±1.362** 20.933±0.561** 3.091±0.664** 1.283±0.451** 4.237±0.955** 17.851±0.833** 22.789±0.729** 8.134±0.727** 2.493±0.336** 2.576±0.256** 3.122±0.364** 21.333±1.23** 17.879±2.06** 8.65±0,994** 1.696±0.664** 0.997±0.367** 2.367±0.489** *significant at 5% level **significant at 1% level, respectively. where( cr1 ), giza 178xegyptian yasmine; cr2 ,giza 177xupr 82-1-7 and cr3.iet1444 x pusa basmati 1. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 622 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) heritability estimates in broad sense for all cooking and eating characters was higher than narrow sense heritability indicating that the selection in early generation may be not effective in improving such characters (salem et al., 2015). the estimates of heritability help the plant breeder in selection of elite genotypes from diverse genetic population, hence prior knowledge about the heritability of the traits is a prerequisite for any selection program (singh et al., 2011). broad sense heritability explains both fixable (additive) and non-fixable (dominant and epistatic) variances which helps in estimating the inheritance of a character (nirmaladevi et al., 2015). on the basis of heritability the traits are classified into 3 categories: highly heritable (>70%), medium heritable (50 70%) and low (< 50%), (robinson, 1966). 2.4. genetic advances genetic advance as percent mean was categorized as low (0-10%) in all crosses for milling characters, grain length, kernel elongation, gel consistency and amylose content%. low genetic advance values were gotten in studied crosses with low narrow sense heritability for all milling character could be expected because this trait is under polygenic control. genetic advance for grain width, grain shape and gelatinization temp was medium for all crosses under study except cross 2. grain width and grain shape ga was low except in cross 2 where high genetic advances were seen. 3. genetic component of generation means 3.1. milling characters: 3.1.1. hulling percent the estimated values of the parameters m, d, h, i, j and l of six parameter model are shown in (table 3). the mean values for this trait ranged from 77.985% to 79.690, cross 2 recorded highest hulling recovery of 79.690% while cross 1 recorded table 2. estimation of genetic parameters for all tested characters in the three crosses. characters crosses degree of domains genetics variance heritability g.s% ½d ¼h broad sense narrow sense hulling% milling % head rice % cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 -5.5131 -0.6329 0.7617 0.4020 0.5271 0.6089 0.5665 1.7864 0.9952 0.0642 0.1982 0.0827 0.2077 0.3794 0.1861 0.5912 0.4930 0.3486 0.0175 0.0368 0.0217 0.2246 0.1923 0.3132 0.2117 0.1317 0.1259 67.49 78.92 78.37 65.92 71.16 82.87 81.01 68.70 76.79 53.01 63.85 62.09 31.66 47.22 38.07 59.52 54.22 56.79 0.487 0.920 0.591 0.741 1.195 0.922 1.8284 1.6268 1,6882 grain length grain width grain shape cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 -0.2602 0.2916 0.0480 -0.3529 -0.1340 0.1612 -0.1895 0.1736 -0.1865 0.0534 0.0336 0.0657 0.0471 0.0282 0.0609 0.0429 0.0106 0.0427 0.0183 0.0094 0.0266 0.0303 0.0127 0.0510 0.0213 0.0054 0.0334 71.04 70.84 81.42 92,46 83.81 93.50 88.05 76.36 85.31 52.87 55.35 57.97 56.27 57.79 50.88 58.89 50,66 47.45 4.745 6.235 6.235 14.27 9.358 15.629 13.767 7.177 10.449 kernel elongation gelatinization t temp gel consistency amylose content% cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 0.2014 -0.9706 -1.2577 0.1594 1.5900 -8.660 1.3406 2.2727 5.2350 1.5593 -1.1343 -.05726 4.7975 4.7411 4.2547 0.3546 0.4286 0.3524 1.5908 5.6670 6.3623 0.2938 0.3040 2.3020 1.4820 4.7240 2.1841 0.2244 0.1794 0.6874 5.7855 17.441 5.2121 0.1331 0.1433 1.0400 51.85 52.56 60.52 74.81 70.34 98.45 79.28 82.54 75.39 45.07 50.46 51.22 39.61 26.33 39.99 45.81 49.58 33.37 17.10 20.24 41.44 31.01 34.30 35.29 4.299 3.905 5.04 18.128 22.503 14.939 1.310 2.658 4.252 3.267 3.368 7.014 (cr1), giza 178xegyptian yasmine; cr2, giza 177xupr 82-1-7 and cr3.iet1444 x pusa basmati 1. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 623 the lowest one (77.985) for hulling percentage, additive[d] gene effects were significant and positive in all crosses (table 3). highly significant and positive dominance [h] gene effects and were observed in cross3 and highly significant and negative dominance [h] gene effects were observed in cross 1 and cross 2. additive x additive [i] interaction effects were significant and negative in crosses 1 and 2. the magnitude of additive x additive [i] effects were highest in the cross 1. dominance x dominance [l] effects were positive and significant in crosses 1, 2 and 3 of was found to be significant and negative. two crosses (1and 2) recorded significant and positive additive x dominance [j]epistatic effects whereas anther cross expressed negative significant effects. in three crosses dominant effect and dominant x dominant effects were displayed with opposite signs and then indicated the presence of duplicate epistasis (non-allelic gene interactions). 3.1.2. milling percent data in table 3 showed that the mean of cross 2 was superior in milling % comparing with the other two crosses. two crosses (cross 1 and cross 2) recorded positive and significant, while cross 3 recorded negative and significant additive, [d] gene effects. the role of dominance [h] effects were observed for cross 3, it exhibited highest positive and significant effects (table 3). additive x additive [i]interaction effects were significant and positive in cross 3 whereas cross 1 and cross 2 recorded negative and significant effect. additive x dominance [j] was positive and non-significant in case of cross 2, while it was negative and significant in cross 1 and cross 3. negative and significant dominance x dominance [l] effects were observed in cross 3, whereas cross 1 and cross 2 exhibited positive and significant effects for milling percentage. in two crosses 1and 2 dominance effects and dominant x dominant effects were displayed with opposite signs that indicated the presence of duplicate epistasis. the presence of epistatic gene effects causes an upward bias in the estimates of both additive and dominance genetic variance (hayman, 1957). when epistasis is of major importance, it is impossible to obtain unbiased estimates of additive or dominance genetic effects. therefore epistatic components cannot be ignored in formulating breeding programs to develop varieties. (paul et al., 2003). 3.1.3. head rice recovery data indicated also that mean of cross1 was superior in head rice % comparing with the other cross. additive [d] gene effects, dominance [h] gene effects and effects additive x additive [i] gene effects were significant and positive for all crosses under study (table 3). additive x dominance [j] effects were positive and significant in cross 1 and cross 2, while negative and significant in cross 3. all the three crosses exhibited negative and significant dominance x dominance [l] effects (venkanna, 2014). 4. physical characters data in table 3 indicated that mid parent value for grain length, (m) was highly significant in all crosses. the highest estimated value was recorded in cross 3 (6.45) followed by cross 2 (5.920) then cross 1 (5.590). moreover, additive effect was highly significant in negative direction in all crosses but cross 1 had the highest value. in addition, the genetic parameters, dominance effect, additive x additive type of gene interaction were highly significant in negative direction in cross 2, but the same parameters were highly significant in positive direction in crosses 1 and cross 3. in contrast, the additive x dominance type of gene interaction was found to be highly significant in cross 3. whereas, dominance x dominance type of gene interaction was found to be highly significant in positive direction in crosses 2. these results indicated that all types of gene interaction were played an important role in the inheritance of grain length according to the cross itself. these results were in agreement with abd el-lattef et al. (2012) and hassan et al. (2013). the classification of epistatis largely depends on the parameters dominant effect (h) and dominant x dominant (i). according to mather and jinks (1971), if dominance effect and dominant x dominant are significantly different from zero and have opposite signs, then duplicate epistasis is indicated. the same two parameters here, were significant, had same sign and, thus, indicated the presence of complementary epistatis. the estimates of mean (m) were highly significant for all the crosses for grain width (table3). additive effects were found significant in cross 2 only, but additive effects were observed as non-significant in cross 1 and cross 3. non-significant in those cases may be ascribed to large error variances (edwards et al.,1975).whereas dominance effect was significant for all cross. additive x additive (i) interaction was significant for all crosses, additive x dominance (j) interaction was significant for cross1 and cross 2, whereas dominance x dominance (1) interaction was also significant for cross 1 and cross 2. the data in table 3 indicated that the estimates of mean (m) were highly significant for all the crosses for grain shape. the additive effect was negative significant in cross 2 and cross 3, while the dominance effect was positive significant for crosses 2 and 3. additive x additive (i) interaction was significant for crosses1 and 2, additive x dominance (j) interaction was significant for cross 2 and cross 3, whereas dominance x dominance (1) interaction was also negative significant for cross 3. the components were opposite in direction indicating duplicate epistasis for this trait for which bi-parental mating was suggested. duplicate types of epistasis was also reported by nayak et. al. (2007). kernel length, kernel width, length/width ratio, elongation ratio, were controlled by additive gene action. vivekanandan and giridharan (1995). 5. cooking and eating characters 5.1. elongation table 3 indicated the highest mean value of elongation was found with cross 1. results obtained from regression analysis 624 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 3. genetic component of generations mean all tested characters in the three rice crosses. showed that additive gene effect (d). dominance gene effect (h) and epistasis (i), (j) as well (i) were significant for grain elongation for all crosses under study except additive x dominance gene effect (j) for cross 2 (table 3). the duplicate type of epistasis can be effectively utilized in pedigree breeding by delaying the selection and it is easier to exploit duplicate type than the complementary type of epistasis. nayak et.al. (2007) reported predominant role of additive gene effect. 5.2. gelatinization temperature the analysis of gene effects in six-parameter model showed that dominance gene effect (h) was significant and positive in crosses 2 and 3and additive x dominance gene effect (j) for cross 2 while additive x additive was significant and positive in cross 3 ,whereas dominance x dominance were significant for three crosses for this character. as shown in table 3, some of the additive effects were negative. the negative or positive signs for additive effects depend on which parent is chosen as p1 (cukadar-olmedo and miller, 1997). tomar and nanda (1985) reported duplicate epistasis for gelatinization temperature. 5.3. gel consistency the mean value for all characters were soft . the results obtained from regression analysis showed that additive gene effect as well as dominance gene effects and epistasis were significant for three crosses of gel consistency (table 3). in the role of the inheritance of gel consistency, direct selection may not be useful. the corresponding fixable gene effects (additive gene effects) were greater than the magnitude of non-fixable gene effects (additive x dominance gene effects, kamara et al., 2017). 5.4. amylose content the mean values for cross 1 and cross 2 were low for amylose content while cross 3 had high amylose content % (table 3). from results it was clear that the additive effects were greater than the dominance effects for this characters. the contribution of the parents to dominance effects varied according to trait. the sign for dominance effect is a function of the f1 mean value in relation to the mid-parental value and indicates which parent is contributing to the dominance effect cukadar-olmedo characters crosses m d h i j l hulling% milling % head rice % cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 77.985** 79.690** 78.961** 71.270** 72.944** 71.089** 66.833** 65.473** 54.114** 0.996** 2.507** 1.112* 1.224** 2.686** -2.415** 4.317** 4.659** 02.312** -3.692** -2.547** 4.174** -3.619** 0.779 11.748** 4.167** 18.303** 27.077** -2.848** -1.202** 0.044 -4.356** -0.576 7.814** 3.034** 15.418** 20.408** 0.843** 0.382** -4.311** -0.611** 0.121 -8.875** 2.317** 3.044** -9.013** 7.401** 4.494** -9.241** 4.797** 3.220** -23.516** -1.134 -9.297** -4.458** grain length grain width grain shape cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 5.590** 5.920** 6.450** 2.350** 2.810** 2.32** 2.379** 2.107** 2.805** -0.960** -0.578** -0.507** 0.053 0.280** 0.097 -0.446 -0.434** -0.365** 0.835** -1.504** 0.984** 0.316* -0.582** 0.348* 0.027 -0.080** 0.695** 1.092** -1.664** 0.934** 0.360** -0.560** -.0394** 0.120 -0.136* 0.866** 0.030 -.028 0.533** -.032 0.120** -0.192** 0.048 -0.109** 0.552** -1.045** 2.031** -0.936** -0.420* 0.667** 0.255 -.005 0.183 -0.450* grain elongation gelatinization temp gel consistency amylose content% cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 66.047** 58.922** 53.263** 4.580** 4.220** 4.729** 82.00** 83.00** 78.667** 19.030** 19.750** 26.443** 7.592** 9.281** 9.834** -0.050 0.006 0.057 -5.837** -7.666** 12.667** -0.849** 0.865** 2.992** 21.967** 50.748** -38.365** 0.115 1.153** 2.737** 41.260** -14.418** 6.832** 0.243 -6.530** -0.483 19.615** 58.340** -27.900** 0.060 0.420 3.170** 36.993** -20.668** -8.002** -0.198 5.558** 0.144 -4.089** 1.460 1.514* 0.295 0.467** 0.107 -2.654**10.416** 9.836** -0.567** 1.722** 1.897** -89.278** 120.944** 36.376** 2.250** 3.110** 1.610* -13.521** 8.836* 19.669** 1.647* 11.879** 3.366 * significant at 5% level **significant at 1% level, respectively. where( cr1 ),giza 178xegyptian yasmine; cr2 ,giza 177xupr 82-1-7 and cr3.iet1444 x pusa basmati 1. and m, mean of f2 ;d ,additive effect ; h, dominance effect ; i , additive x additive ; j , additive x dominance ; l, dominance x dominance. 625 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 4. mean estimates of parents , f1 generation and heterosis as deviation from m¯p and b¯p for rice of all tested characters in three studied crosses. characters crosses mean performance heterosis % p¯1 p¯2 f¯1 m¯p b¯p hulling% l.s.d 5% 1% milling % l.s.d 5% 1% head rice % l.s.d 5% 1% cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 78.986 83.010 80.030 71.757 75.350 73.610 69.500 71.030 66.570 77.680 78.760 69.185 68.088 70.220 60.690 65.500 67.800 53.168 77.990 79.540 78.738 70.660 74.137 72.084 68.633 72.300 66.538 -1.070* -1.663** 5.536** -0.946 1.272 1.055* 1.858** 5.859** 0.895 1.204 1.679** 4.156** 11.139** 0.580 0.780 -1.262* -4.180** -1.614** 1.092 1.469 -1.528** -1.610** -3.432** 1.o33 1.390 -1.247** 1.788** -0.048 0.670 0.901 grain length l.s.d 5% 1% grain width l.s.d 5% 1% grain shape l.s.d 5% 1% cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 5.014 4.965 5,618 2.518 2.868 2.452 1.991 1.731 2.294 6.993 5,676 7.698 2.348 2.547 1.875 2.978 2.382 4.128 5.746 5.676 6.708 2.403 2.686 2.210 1.991 1.731 2.294 -4.289** 2.910** 0.751** 0.346 0.586 -1.233** -0.794** 2.149** 0.046 0.062 -3.763** 2.747** -5.325** 0.057 0.076 14.599** 14.320** 19.402** 0.503 0.676 -4.567** -6.346** -0.869** 0.053 0.072 -19.711** -11.293** -26.357** 0.065 0.088 grain elongation l.s.d 5% 1% gelatinization temp l.s.d 5% 1% gel consistency l.s.d 5% 1% amylose content% l.s.d 5% 1% cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 cr1 cr2 cr3 64.039 69.473 61.959 4.800 4.360 6.883 91.800 71.500 75.000 18.840 19.570 28.795 40.679 53.830 45.319 5.490 5.302 6.302 98.166 69.000 69.333 19.405 21.283 26.605 54.711 54.060 43.175 5.200 5.571 6.500 99.250 78.000 87.000 19.563 19.455 27.073 4.492** 12.314** 19.509** 3.340 4.492 1.069** 15.142** -6.245** 0.464 0.624 4.192** 8.711** 20.555** 2.619 3.523 2.304* 4,756** 2.264* 2.241 3.015 -14.566** -22.186** 30.317** 3.857 5.187 -5.282** 5.130** -6.917** 0.536 0.721 1.104 4.698** 16.000** 3.025 4.068 3.838 -.588 1.759 2.588 3.481 * significant at 5% level **significant at 1% level, respectively. where( cr1 ),giza 178xegyptian yasmine; cr2 ,giza 177xupr 82-1-7 and cr3.iet1444 x pusa basmati 1. and m, mean of f2 ;d ,additive effect ; h, dominance effect ; i , additive x additive ; j , additive x dominance ; l, dominance x dominance. 626 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) and miller, (1997) preponderance of additive gene action in the inheritance of the trait amylose content. estimates of genetic components showed both additive and non-additive gene effects to be important. swain and nagaraju (2004). 6. heterosis 6.1. milling characters data presented in table 4 indicated that highly significant positive values of heterosis were recorded for milling and head rice % characters as a deviation from mid-parent in three cross paramasivam et. al. (1996) and cross 3 in hulling %. also data in (table4) showed that the heterosis values for head rice % of cross 2 only was positive highly significant as deviation from the better parent (1.788) , while the remaining crosses recorded negative significant mean heterotic for this traits. these results agreed with surender (2002). 6.2. physical characters in table 4 for grain length, the heterosis as a deviation from mid-parent and better parent were significant and positive in three cross but only in cross 1recorded significant and negative heterosis as a deviation from mid-parent. both positive and negative significant heterosis for grain length were reported (singh and singh, 1985). for grain width and grain shape, three crosses showed significant and negative relative heterosis and heterobeltiosis. the exceptions were heterosis as division in mid parent that was significant and positive in cross 3 for grain width and cross 2 for grain shape. the same trends for grain shape were previously observed (gnanamalar and vivekanandan 2013). 6.3. cooking and eating characters 6.3.1. elongation percent data presented in table 4 indicated that highly significant positive values of heterosis were recorded as a deviation from mid-parent in cross 1. also data in table 4 showed that the heterosis values for elongation % of cross 3 was positive and highly significant as deviation from the better parent (30.317), while the remaining crosses recorded negative significant mean heterosis for this trait. linear elongation without breadthwise expansion is considered a highly desirable trait in rice quality (gnanamalar and vivekanandan, 2013). 6.3.2. gelatinization temperature cross 2 showed positive and significant heterosis as a deviation from mid-parent and better parent ,whereas cross 1 gave the same as a deviation from mid-parent . 6.3.3. gel consistency three crosses recorded significant and positive heterosis and heterobeltiosis for gel consistency except cross 1 heterosis as deviation better parent was not significant . the maximum heterosis is was recorded by cross 3 (20.555 and 16.000 per cent respectively) and the minimum of 4.192 per cent was found in cross 1 as deviation mid-parent standard heterosis this trait (gnanamalar and vivekanandan, 2013). 6.3.4. amylose content data presented in table 4 indicated that highly significant positive values of heterosis were recorded for this character a deviation from mid-parent in the three cross. these values of heterosis were ranged between 2.264% for the cross 2 and 4,756% for cross 3. also data in table 4 showed that the heterosis for values amylose content % of three crosses were no significant as deviation from the better parent. conclusion finally, it can be concluded that broad sense heritability estimates were high for milling, grain dimensions, gel consistency and gelatinization temperature in three crosses under study. low to moderate narrow sense heritability estimates were evident for grain 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laboratory of microbial biotechnology and plant protection, faculty of sciences, ibn zohr university, agadir 80000, morocco received: august 1, 2019 / accepted: september 6, 2019 __________________________________________________ * corresponding author: saidbaroud@gmail.com 611 abstract in this study, we have evaluated the effects of three brown algae (cystoseira gibraltarica, bifurcaria bifurcata, and fucus spiralis) as biostimulants on the germination and in vitro growth of pepper (capsicum annuum) as well as in greenhouse. two different treatments (amendment/spray) have been tested accordingly, both with varied concentrations (0.5%, 1% and 2%) for extract and (c1=0.5 g/pot, c2=1 g/pot and c3= 2.5 g/pot) for amendment). results showed that the seeds which were treated by c. gibraltarica, and b. bifurcata with a low concentration (0.5%) have slightly germinated with a high length of hypocotyls. similar results have been obtained for the radicle length. on the other hand, f. spiralis extract improved the percentage of germination, the length of the radicle, hypocotyls, and seedling length and dry weights for the three tested concentrations. also, all concentrations have a favorable effect on seedling biomass. in the greenhouse, the growth rate (roots’ length, aerial part length, total length, and dry weights) was higher for the plants which received higher concentrations of aqueous extracts of the three species b. bifurcata, f. spiralis, and c. gibraltarica. additionally, the maximum quantity of chlorophyll ‘a’ has been recorded in the plants treated with b. bifurcata and c. gibraltarica at 2% and f. spiralis extracts at 0.5%. in regard to the protein content, the maximum values were recorded at the level of the plants that were treated this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the fertilizers amelioration have become nowadays, one of the most serious issues for the whole countries of the world due to the fast growth of the population as well as the negative impact of using chemical fertilizers on the soil’s fertility, and its risks on the environment, too. a significant portion of the algae which is produced annually is used as a nutrient supplement or plants growth regulator, fertilizer or pesticide (khan et al., 2009). several studies have shown that the use of products based on seaweed improves significantly the productivity and quality of crops, even when they are used at low doses. according to (marfaing and lerat, 2007), algae is a very rich source of multiple nutrients such as (fiber, minerals and proteins), vitamins and fatty acids which are occupying a unique place in nutrition compared to other plants and more generally to other with f. spiralis extract at 0.5%. this study has provided important information about b. bifurcata and f. spiralis species could be considered as a good biostimulant to improve pepper’s growth. keywords: algae extracts, amendment, biostimulants, germination, growth. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) sources of nutrients. a chemical analysis of algae and their extracts revealed the presence of a wide variety of plant growth promoting substances such as auxins, cytokinins, and betaines (khan et al., 2009). these substances can influence the development of the stem and the root’s system of a plant (stirk et al., 2004). in addition, macronutrients and micronutrients can help improve the growth of various vegetables, fruits and other crops, (blunden, 1991; crouch and van staden 1993; möller and smith, 1998). similarly, various researches maintain that organic fertilization with seaweed has tremendous effects on the qualitative and quantitative productivity of various plant species (augier and santimone, 1978). indeed, market gardening is a staple food for the populations by contributing to the satisfaction of consumption needs. according to fao (2017) statistics, world production of pepper in 2014 is estimated at about 32.3 million tonnes for an area of 1.9 million ha yielding an average yield of 17 t/ha. accordingly, we have tested the effect of two treatments (algal extract and amendment) with brown algae on a socio-economically important plant which is pepper. materials and methods three species of brown algae, cystoseira gibraltarica, bifurcaria bifurcata and fucus spiralis had been collected during tide time at the coastal zone cap ghir, (30°38’37”n, 09°53’20”o), located about 43 km west of agadir, morocco. in the laboratory, the algae species were being carefully cleaned of parasites they contained; they were also dried and then transformed into a fine powder. treatments preparation aqueous extracts five grams of each algal species were added to 100 ml of sterile distilled water. the obtained mixture was left for 24 hours to macerate and then centrifuged at 3000 tour/minute for 15 minutes. the recovered supernatant was filtered on whatman nbr 1 paper. also, the obtained extracts were stored at 4°c for different analysis and preparation of various watering concentrations. amendments the obtained powder was used for the preparation of the three concentrations, c1 (0.5 g/pot), c2 (1g/pot) and c3 (2.5 g/pot) based on the concentrations used in organic farming 25kg/100 m2. germination tests the experiments were carried out using pepper seeds (capsicum. annuum) of the roldan variety. ten treatments were tested with 100 seeds divided into four petri dishes (25 seeds per a dish). before treating the seeds with the aqueous extracts, they were treated with a solution of sodium hypochlorite 6% for 612 3 minutes. the tested seeds were placed on a paper filter in petri dishes and then treated with 5 ml of distilled water (control) or different concentrations of the aqueous extracts (0.5%, 1% and 2%). the dishes are incubated at 24 ± 1°c. the germination is under a daily observation over a period of eight days. the variables which were measured, were the germination percentage (gp), the germination index (gi), the mean germination time (mgt) and the seedling vigor index (svi), as well as the length of the hypocotyl, the length of the radicle, the total height of the plant and dry weight of the seedlings. the effect of aqueous extracts on seed germination and plant growth was measured after eight days of germination. greenhouse growth the two hundred pepper plants that were cultivated in the greenhouse had been selected and planted in pots that contain a mixture of soil and peat (3:1). the plants were watered with aqueous algae extracts (50 ml per week), meanwhile, the fertilization with the amendment was done at the time of plant transplantation with the three different concentrations, determined previously. all pots were irrigated separately with water every two days. the potted plants were growing for seven weeks in the greenhouse, each treatment was represented by ten repetitions. the plants of each treatment were chosen randomly for diverse analyses. after 45 days of transplantation, the plants were removed and analyzed at the level of their growth parameters (length of root, length of the aerial part, total length, dry weight) and their biochemical parameters (pigments, proteins, total sugars). algae’s physicochemical contents the determination of the content of the three algae in mineral elements is carried out according to page et al. (1982). dosage of total sugars the dosage of total sugars was carried out according to dubois et al. (1956). briefly, twenty mg of dry plant material was homogenized with 2 ml of 70% ethanol (v/v), the mixture was centrifuged at 2000 rpm for 10 min. the supernatant was recovered and the pellet was rinsed twice with 70% ethanol (v/v). for the supernatants, they were combined; 16 ml of distilled water was added to them. 200 µl of a solution to be dosed and added to 200 µl of a 5% aqueous phenol solution, then 1 ml of concentrated sulfuric acid was rapidly introduced into the reaction medium. the homogenized vortex mixture was allowed to stand for 10 minutes and then placed in a water bath, for 10 to 20 minutes at a temperature of 30 ° c. the optical density is taken at 490 nm using the visible ic 6400 spectrophotometer. dosage of chlorophylls and carotenoids a quantity of 0.5 g of fresh frozen leaves are crushed, then homogenized with 50 ml of acetone 90% (v/v), the extract is then centrifuged at 3000 rpm. the od (optical density) was a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 613 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) read at different wavelengths: 470 nm for carotenoids, 645 nm for chlorophyll “b” and 663 nm for chlorophyll “a”. the concentrations were calculated on the basis of the following formulas (lichtenthaler, 1987): chlorophyll “a” (chl “a”) (mg/g fm) =(11.75×do6632.35×do645)×50/500 chlorophll “b” (chl “b”) (mg/g fm) = (18.61×do6453.96×do663)×50/500 carotenoids (mg/g fm) = ((1000×do470)-(2.27×chl “a”)((81.4×chl “b”))/227)×50/500. dosage of proteins the dosage of protein content was carried out according to lowry et al. (1951). briefly, the lowry method consists in forming a complex between the peptide bonds and the copper sulphate, in an alkaline medium. this complex after reduces the phosphomolybdic and phosphotungstic acids of the folinciocalteu reagent, to give a second blue complex, as measured by spectrophotometer (frolund and griebe, 1995). the assay reagent (solution r) is prepared from three solutions in the order of addition of the reagents and stirring after each addition: -solution a: sodium carbonate (na2co3) at 20 g/ and sodium hydroxide (naoh) 0.1 mol / l. -solution b: sodiumtartrate/potassium (na/k) at 20 g/l. -solution c: copper sulphate at 10 g/l. an amount of 0.1 g of leaf powder is milled in 1 ml of lysis buffer to extract the proteins. the extract is centrifuged at 13000 g for 10 min. the lysis buffer is prepared by mixing 8 ml of 1m tris-hcl ph = 6.8, 2 ml of β-mercaptoethanol, 10 ml of sds and 80 ml of water. 10 μl of the supernatant are added, 990 μl of water and 5 ml of solution r (3 ml of solution c, 3 ml of solution b and 300 ml of solution a). the tubes are incubated for 10 min in the dark, then 0.5ml of a solution of folin-ciocalteu 50% (v/v) reagent is added, the mixture is vortexed. stabilization of the color takes a few minutes. the intensity of the coloration obtained is evaluated by measuring the absorbance at 750 nm using visible spectrophotometer ic 6400. statistical analysis the data has been processed by the statistica software, version 6.0. the analysis of variance (anova) was used to determine the degree of significance. the averages were compared using duncan’s tests at the probability level (p <5%). results physicochemical analysis the results obtained from the physicochemical analysis of the aqueous extracts of the three brown algae (c. gibraltarica, b. bifurcata and f. spiralis) showed the presence of various micro and macroelements (table 1). the concentration of n and ca is higher in b. bifurcata (1.75 and 3.24 g/100g dm, respectively), whereas p and na are rather high in f. spiralis extract (0.2 and 1.85 g/100g dm respectively). the potassium is well-represented in the extracts of the three algae with a higher content in b. bifurcata (5.02 g/100g dm) followed by f. spiralis (4.96 g/100g dm) and c. gibraltarica with a value of (4, 12 g/100g dm). in contrast to k, p is poorly concentrated in all three algae (table 1). the concentration of fe, cu and organic matter is higher in both f. spiralis and c. gibraltarica algae (55.94, 25.76, 73.74 mg/kg dm respectively). while the concentration of zn is higher in b. bifurcata extract (13.6 mg/ kg dm). the mn is higher in c. gibraltarica and b. bifurcata extract (18.44 mg/kg dm), the same result is revealed for mg which represents a high concentration in f. spiralis (1.03 g/100g dm) (table 1). on the other hand, the ph values for the three aqueous algae extracts are greater than 6 except for the aqueous extracts of b. bifurcata at 1 and 2% (table 2). the value of the electrical conductivity (ec) increases proportionally with concentration of the aqueous extracts of the three algae (table 2). the same finding is recorded for salinity, which increases with increasing concentration of the extract and is also lower for f. spiralis extract (table 2). effect of aqueous extracts of algae on seed germination and growth of pepper seedling the pepper plants treated with the aqueous extracts of these three brown algae showed an improvement in the germination percentage (gp), germination index (gi), mean germination time (mgt) and seedling vigor index (svi) (table 3). three brown seaweeds b. bifurcata, c. gibraltarica, and f. spiralis with the exception of the 2% concentration of b. bifurcata and c. gibraltarica showing a minimum value of the germination percentage and the vigor index of the seedlings respectively (table 3). according to statistical calculations the aqueous extract of b. bifurcata at 0.5% obtained significantly high results in the other extracts. the maximum value of the germination percentage is presented by the concentrations 0.5 and 1% of f. spiralis and b. bifurcata (87.3), whereas the minimum value g/100g mg/kg species mo n ca k mg p na fe mn cu zn b.b 69,68b 1,75 ±0,1a 3,24±0,3a 5,02±0,02a 0,66±0,06c 0,16±0,01b 1,65±0,4b 37,28±0,4b 18,44±0,1a 12,87±0 08b 13,6±0,1a c.g 73,44a 1,38±0,1b 2,46±0,1b 4,12±0,03c 0,74±0,1b 0,12±0,01c 0,98±0,2c 55,92±0,5a 18,45±0,1a 25,76±0,09a 5,45±0,1b f.s 73,74a 1,33±0,8c 2,34±0,2c 4,96±0,01b 1,03±0,08a 0,20±0,02a 1,85±0,3a 55,94±0,9a 9,22±0,1b 25,76±0,08a 5,46±0,2b the values represent the mean ± sd (n = 3). bb: b. bifurcata, cg: c. gibraltarica, and fs: f. spiralis. table 1. minerals elements content of the three algae studied. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 614 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) is recorded by the aqueous extract of b. bifurcata at 2% (table 3). similarly, all the aqueous extracts of the algae show a significant difference to the control with the exception of the 2% aqueous extract of f. spiralis which has a low germination index (8.9), whereas the maximum value is shown by the aqueous extract of b. bifurcata at 0.5 % (16.1) (table 3). while all concentrations of these three brown algae recorded a mean germination time, not significantly different from the control. the vigor index of the seedlings is significantly improved by all the aqueous extracts of the algae with the exception of the concentration 2% of b. bifurcata and c. gibraltarica (261 and 265 respectively), the maximum value of the seedling vigor index is recorded by the aqueous extract of b. bifurcata at 0.5% (490.1) (table 3). in addition, whatever the alga or its concentration, there is a positive effect on the parameters of the growth of pepper seedlings (table 4). it should also be noted that low concentrations are more effective than high concentrations for all three algae. thus, the highest mean radicle length is recorded by the aqueous extract of all b. bifurcaria, c. gibraltarica and f. spiralis algae at 0.5% (3.06 and 2.83 and 2.96 cm, respectively) (table 4). in addition, these results indicate that the aqueous extracts of these three algae promote the growth of hypocotyl length with the exception of the aqueous extract of b. bifurcata and c. gibraltarica at 2% which show an inhibitory effect on the length of the hypocotyl (1.41 and 1.31 cm, respectively). the length of the highest mean hypocotyl is presented by the aqueous extracts of c. gibraltarica and b. bifurcata at 0.5% (2.59 and 2.57 cm, respectively) (table 4). the maximum length of the seedlings is recorded by the aqueous extract of b. bifurcata at 0.5% (5.66 cm), while the minimum length is presented by the concentration 2% of c. gibraltarica and b. bifurcata (3.07 and 3.22 cm, respectively) (table 4). aqueous extracts of all algae showed a significant effect on the dry weight of pepper seedlings. the maximum dry weight is shown by the aqueous extract of f. spiralis at 0.5 (3.72mg) (table 4). effect of algae on pepper seedling growth in greenhouse after germination the seedlings are transported under greenhouse and we noticed that all the aqueous extracts of the three algae showed an improvement in the length of the aerial part, the root and the total length of the plant (table 5). plants treated with aqueous extracts of b. bifurcata at 1% and c. gibraltarica at 2% showed an increase in the length of the aerial part (24.55 and 24.35 cm, respectively). aqueous extract of b. bifurcata at 2% recorded high values of root length and total plant length (10.32 and 34.35 cm, respectively) (table 5). fertilization by amendment shows an improvement in the length of the aerial part with the exception of c3 amendment of c. gibraltarica and two concentrations c2 and c3 of f. spiralis which show low values (table 5). the c1 amendment of b. bifurcata has the maximum length of the aerial part (22.39 cm), while the minimum length of the aerial part is recorded by c3 amendment of c. gibraltarica (15.5 cm). all amendments show an improvement in root length with the exception of c. gibraltarica at c3 amendment and f. spiralis at c1 amendment which shows a minimum length (6.4 cm) (table 5). no noticeable effect was detected by the amendments, except for the c1 and c2 concentrations of b. bifurcaria and c. gibraltarica which show significantly different values to the control (table 5). the maximum total length is recorded by the c1 amendment of b. bifurcaria (32.69 cm) (tab 5). regarding the dry weight, we noted that the aqueous extract of c. gibraltarica at 2% has a table 2. physicochemical characterization of aqueous algae extracts from treatments of b. bifurcata (bb), c. gibraltarica (cg), and f. spiralis (fs). treatments (%) ph ec ( ds/m) salinity (g/l) control 7,0±0,01 a 0 e 0,05±0,01 e bb 0,5 6,02±0,01 f 2,16±0,02 g 1,12±0,01 g bb 1 5,9±0,01 h 4,3±0,02 d 2,24±0,01 d bb 2 5,82±0,02 g 7,98±0,01 a 4,32±0,02 a cg 0,5 6,22±0,11 c 2,01±0,01 h 1,03±0,01 h cg 1 6,13±0,01 d 3,7±0,1 e 2,01±0,01 e cg 2 6,09±0,01 e 6,59±0,01 b 3,53±0,01 b fs 0,5 6,29±0,01 b 1,46±0,02 k 0,76±0,01 k fs 1 6,29±0,02 b 2,80±0,02 f 1,44±0,03 f fs 2 6,29±0,03 b 4,6±0,04 c 2,44±0,01 c the values represent the average ± sd (n = 3). ec: electrical conductivity. aqueous extract (%) gp (%) gi mgt (days) svi control 82,3±3,11 ab 9,2±1,00 a 8,5±0,30 a 281,4±28,6 ab bb 0,5 86,3±1,11 a 16,1±2,97 c 8,6±0,65 a 490,1±9,9 c bb 1 87,3±3,77 a 14,6±3,6 b 8,7±0,78 a 398,4±29,3 bc bb 2 78,3±1,11 b 9,3±2,65 a 9,1±0,73 a 261,9±20,2 a cg 0,5 86,3±3,77 ab 13,6±2,60 ab 8,7±0,65 a 430,1±10,0 c cg 1 82,3±1,11 ab 14,4±1,27 ab 8,5±0,32 a 387,6±12,4 abc cg 2 84,3±3,55 ab 13,6±1,69 ab 8,8±0,49 a 265,6±14,8 a fs 0,5 87,3±1,11 a 12,5±1,46 ab 8,8±0,39 a 453,6±30,7 bc fs 1 82,6±3,11 ab 10,7±3,14 a 8,7±0,10 a 370,2±26,4 abc fs 2 82,3±1,11 ab 8,9±3,49 a 8,9±0,03 a 383,5±15,2 abc the values represent the average ± sd (n = 300 seeds). the values indicated by a different letter are significantly different p≤0.05. bb: b. bifurcaria, cg: c. gibraltarica,fs:f. spiralis. table 3. effects of aqueous algae extracts (aaes) treatments on the germination parameters of pepper seeds: germination percentage (gp), germination index (gi), mean germination time (mgt), and seedlings vigor index (svi). aqueous extracts (%) radicle length (cm) hypocotyl length (cm) seedling length (cm) dry weight (mg) control 1,78±0,07 a 1,69±0,02 ab 3,47±0,13 abc 2,61±0,03 a bb 0,5 3,06±0,51 b 2,59±0,36 a 5,66±0,84 d 3,28±0,18 ab bb 1 2,77±0,44 ab 2,37±0,51 ab 5,16±0,05 abd 3,19±0,32 ab bb 2 1,73±0,31 a 1,48±0,22 ab 3,22±0,73 ac 3,67±0,54 b cg 0,5 2,83±0,25 ab 2,57±0,54 a 5,49±0,07 bd 3,50±0,33 ab cg 1 2,47±0,41 ab 2,18±0,69 ab 4,62±0,12 abcd 2,96±0,64 ab cg 2 1,74±0,35 a 1,31±0,58 b 3,07±0,15 c 2,95±0,59 ab fs 0,5 2,96±0,69 b 2,16±0,43 ab 5,14±0,04 abd 3,72±0,27 b fs 1 2,7±0,47 ab 1,87±0,38 ab 4,62±0,10 abcd 3,15±0,14 ab fs 2 2,68±0,73 ab 1,99±0,54 ab 4,68±0,12 abcd 3,33±0,42 ab the values represent the average ± sd (n = 300 seeds). the values indicated by a different letter aresignificantly different p≤0.05. table 4. effects of aqueous algae extracts c. gibraltarica (cg), b. bifurcata (bb) and f. spiralis (fs) on the length of the radicle, length of the hypocotyls and dry weight of pepper seedlings at different concentrations (0.5%, 1% and 2%). a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 615 spraying plant length (cm) dry weight in (g) ae (%) aerial part root total length aerial part root total weight control 17,32±0,49 b 6,52±0,88 c 23,84±0,41 d 0,21±0,03 d 0,08±0,02 c 0,29±0,06 d bb 0,5 23,35±1,27 a 7,30±0,38 b 30,65±0,31 bc 0,49±0,01 c 0,11±0,01 bc 0,60±0,01 c bb 1 24,55±2,6 a 7,39±0,65 b 31,91±1,05 bc 0,64±0,03 ab 0,2±0,01 ab 0,85±0,1 ab bb 2 24,03±2,47 a 10,32±,32 a 34,35±0,72 a 0,63±0,01 ab 0,23±0,02 a 0,86±0,05 a cg 0,5 23,91±2,73 a 9,56±0,65 b 33,47±0,97 a 0,59±0,02 b 0,20±0,07 ab 0,8±0,02 ab cg 1 23,93±1,91 a 8,79±0,70 b 32,72±2,12 bc 0,64±0,01 ab 0,21±0,05 a 0,86±0,01 a cg 2 24,35±2 a 8,58±0,77 b 32,93±2,01 ab 0,65±0,02 a 0,24±0,01 a 0,90±0,05 a fs 0,5 23,54±1,82 a 8,09±0,73 b 31,63±1,24 ab 0,6±0,04 ab 0,22±0,05 a 0,82±0,01ab fs 1 22,95±1,27 a 7,79±0,84 bc 30,74±1,46 c 0,54±0,07 c 0,20±0,06 ab 0,74±0,16 b fs 2 23,62±2,64 a 7,50±0,48 bc 31,12±0,83 c 0,59±0,07 b 0,23±0,02 a 0,83±0,01ab amendment control 17,32±0,29 ab 6,52±0,36 bcd 23,84±0,77 de 0,21±0,01 cd 0,08±0,01 ed 0,29±0,02cd bb c1 22,39±0,85 c 9,95±0,68 a 32,69±1,26 a 0,39±0,05 a 0,19±0,02 a 0,58±0,01 a bb c2 20,03±0,91 bc 8,73±0,88 ab 28,50±0,78 b 0,31±0,03 b 0,12±0,01 bc 0,44±0,01 b bb c3 17,78±0,89 ab 6,97±0,75 cd 24,62±0,78 ef 0,31±0,01 b 0,11±0,01 bcd 0,43±0,01 b cg c1 18,76±0,75 b 8,35±0,22 bc 27,17±0,67 c 0,34±0,02 b 0,14±0,01 c 0,48±0,01 b cg c2 18,73±0,50 b 7,71±0,35 d 26,14±0,32 e 0,33±0,01 b 0,14±0,01 c 0,47±0,02 b cg c3 15,50±0,24 a 6,40±0,98 d 21,83±0,79 g 0,20±0,01 d 0,07±0,01 de 0,28±0,01 d fs c1 18,09±0,50 ab 6,4±0,54 cd 24,02±0,32 f 0,26±0,01 c 0,09±0,01 de 0,35±0,01 c fs c2 15,79±0,61 a 7,13±0,31 cd 22,13±0,64 g 0,19±0,01 d 0,06±0,01 e 0,24±0,01 d fs c3 15,88±0,64 a 6,60±0,30 d 22,33±0,62 g 0,20±0,01 cd 0,09±0,02 bde 0,30±0,01cd the values are the average ± sd (n = 10). the values indicated by a different letter are significantly different p≤0.05. c1 (0.5 g/pot), c2 (1g/pot) and c3 (2.5 g/pot). table 5. effect of aqueous algae extracts bifurcaria bifurcata (bb), cystoseira gibraltarica (cg), and fucus spiralis (fs) treatments applied as soil spraying and soil amendment on the length of the aerial part, the length of the roots, total length and biomass of pepper plants at different concentrations. table 6. effect of aqueous algae extracts b. bifurcata (bb), c. gibraltarica (cg), and f. spiralis (fs) treatments applied as soil spraying and soil amendment on protein content, the quantity of total sugars and chlorophyll pigments content of pepper leaves at different concentrations. spraying proteins total of sugars pigments (mg/g fm) ae (%) mg/g dm mg/g dm chl a chl b carotenoids chl total control 27,1±1,51 ef 18,47±1,16 c 0,50±0,13 cd 0,30±0,07 bc 0,10±0,02 e 0,81±0,20 bc bb 0,5 24,65±1,04 f 22,33±1,05 b 0,45±0,02 d 0,26±0,01 c 0,16±0,02 cd 0,71±0,03 b bb 1 35,84±0,92 c 30,08±0,55 a 0,50±0,04 cd 0,32±0,08 abc 0,15±0,02 cd 0,82±0,13 bc bb 2 32,78±1,31 d 18,16±0,38 c 0,72±0,10 abcd 0,42±0,07 abc 0,22±0,03 ab 1,15±0,17 abc cg 0,5 43,54±1,57 a 14±0,88 d 0,62±0,17 abcd 0,39±0,12 abc 0,19±0,05 abc 1,02±0,30 abc cg 1 35,32±1,18 c 18±0,27 c 0,62±0,10 abcd 0,29±0,02 bc 0,16±0,02 bcd 0,92±0,11 abc cg 2 27,54±1,31 e 9,36±0,7 e 0,80±0,18 abc 0,46±0,11 ab 0,20±0,20 abc 1,26±0,29 ac fs 0,5 44,67±2,62 a 23,44±0,96 b 0,88±0,09 a 0,50±0,07 a 0,25±0,03 a 1,38±0,16 a fs 1 27,45±0,92 e 23,36±0,46 b 0,82±0,21 ab 0,44±0,16 abc 0,20±0,04 abcd 1,26±0,37 ac fs 2 40,39±1,04 b 22,75±0,27 b 0,55±0,19 bcd 0,29±0,14 bc 0,13±0,04 d 0,85±0,13 bc amendment control 27,1±1,51 c 18,47±0,46 e 0,50±0,01 b 0,30±0,03 c 0,10±0,01 ab 0,81±0,02 ab bb c1 31,03±1,06 b 21,13±0,46 d 0,63±0,09 a 0,35±0,03 abc 0,09±0,01 b 0,98±0,1 a bb c2 31,73±1,31 b 35,19±0,42 b 0,67±0,09 a 0,38±0,07 a 0,13±0,02 a 1,05±0,16 a bb c3 26,75±1,04 c 44,47±1,25 a 0,46±0,03 bc 0,27±0,04 bde 0,09±0,01 b 0,74±0,07 abcd cg c1 22,55±1,04 d 35,5±0,05 b 0,65±0,15 a 0,27±0,09 bde 0,09±0,01 ab 0,93±0,1 abc cg c2 23,08±2,62 d 13,88±0,31 f 0,40±0,02 c 0,24±0,01 e 0,07±0,01 b 0,64±0,04 d cg c3 13,98±1,51 e 11,88±0,92 g 0,4±0,11 c 0,23±0,06 e 0,07±0,01 b 0,62±0,17 d fs c1 28,15±1,32 c 21,94±0,7 d 0,41±0,08 c 0,25±0,05 de 0,09±0,01 b 0,66±0,14 d fs c2 22,47±1,06 d 25,61±0,14 c 0,45±0,17 bc 0,25±0,09 de 0,07±0,01 b 0,7±0,27 bd fs c3 38,47±1,09 a 26,72±0,18 c 0,43±0,03 bc 0,26±0,01 de 0,10±0,03 ab 0,69±0,04 d values average is ±sd (n = 3). the values indicated by a different letter are significantly different p≤0.05. c1 (0.5 g/pot), c2 (1g/pot) and c3 (2.5 g/pot). 616 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) significant effect on the dry weight of the aerial part, root and on the total weight (0.65, 0.24 and 0.9 g, respectively) (table 5). the fertilization by amendment showed a marked improvement of the aerial biomass, the root part and the total weight of the pepper plant with the exception of c2 amendment of f. spiralis which has low values of dry weight (table 5). the c1 amendment of b. bifurcaria has a maximum dry weight of the aerial part, the root part and the total weight (0.39, 0.19 and 0.58 g, respectively) (table 5). effect of algae on protein content, total sugars, and chlorophyll pigments all aqueous extracts of these three brown algae improved the protein content of pepper plants with the exception of b. bifurcata at 0.5%, which is the lowest level (24.65 mg/g dm). the plants watered with the aqueous extracts of the three brown algae had significantly higher levels than the control up to 44.67 mg/g dm (table 6). f. spiralis with its three concentrations has the highest values, followed by the three concentrations of c. gibraltarica. the fertilization by amendment has affected the protein content less, indeed only the concentrations c1 and c2 of b. bifurcata and c3 of f. spiralis are significantly higher than the content of the control. the highest value is obtained by f. spiralis at c3 (38.47 mg/g dm), while the minimum content is presented by c3 amendment of c. gibraltarica (table 6). the aqueous extracts of the three brown algae improved the total sugar content of the pepper plants with the exception of c. gibraltarica at 0.5 and 2% (14 and 9.36 mg/g ms, respectively) which had the lowest levels, significantly different from the witness. the plants watered with the aqueous extracts of the three brown algae had significantly higher levels than the control up to 30.08 mg/g dm (table 6). but unlike proteins, the aqueous extract of c. gibraltarica has no effect on the total sugar content. fertilization by amendment has also improved the total sugar content. indeed, all the treatments by amendment show significantly different values of the control except for once again c2 and c3 of c. gibraltarica which have the lowest levels (13.88 and 11.88 mg/g ms respectively). the highest values are obtained as for the algal extracts, by the three concentrations of b. bifurcata (table 6). the maximum value of the total sugar content is recorded by c3 amendment of b. bifurcata (44.47 mg/g dm). all aqueous extracts of these three brown algae showed an improvement in the amount of chlorophyll ‘a’, chlorophyll ‘b’, carotenoids and total chlorophyll (table 6). plants irrigated with 0.5% aqueous extract of f. spiralis had significantly higher levels of up to 0.88 mg/g mf for chlorophyll ‘a’ 0.50 mg/g mf, chlorophyll ‘b’ 0.25 mg/g mf carotenoids and 1.38 mg/g mf total chlorophyll (table 6). the amendment treatments have no generally noticeable effect on chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids content with the exception of b. bifurcata at c1 and c2 which have significantly different values from control. the maximum quantity of chlorophyll ‘a’, chlorophyll ‘b’, carotenoids and total chlorophyll are obtained by amendment c2 of b. bifurcata (0.67, 0.38, 0.13 and 1.05 mg/g mf respectively) (table 6). discussion the physicochemical analysis of bifurcaria bifurcata, cystoseira gibraltarica, and fucus spiralis extracts revealed the richness of these brown algae in macroelements (ca, k, p, na, n), which was similar to the previous work of (hong et al., 2007) (kalaivanan and venkatesalu, 2012), (hernandez-herrera et al., 2013). likewise, these extracts have a ph which is slightly acidic and electrical conductivity and salinity which increased with the increase of the concentration of the algal extract. these last two parameters can influence tomato’s germination and growth (booth, 1969; henry, 2005). thus, the highest germination rate of pepper seeds was obtained with the low concentrations of the extracts of the three algae which have a conductivity that does not exceed 2.16 ds m/1. according to reinhardt and rost, (1995), most plants are sensitive to salinity during germination and seedling growth. however, a low concentration of salts in the medium enables the seeds to imbibe water and thus favor the rate of germination. numerous studies have shown that the low concentrations of algal extracts have no harmful effect on plant germination and growth (sridhar and rengasamy, 2010; kumari et al., 2011; kalaivanan and venkatesalu, 2012; kumar et al., 2012; ganapathy selvam et al., 2013). however, aqueous extracts of b. bifurcata and c. gibraltarica algae at a higher concentration of 2% (7.98 and 6.59 ds/m, respectively) showed a negative effect on seeds germination, inhibiting them to absorb water. similar results have been reported by (hernandezherrera et al., 2013). whatever the type of treatment (extract or amendment), the two algae of b. bifurcata and c. gibraltarica show maximum values of the length of the aerial part, the root, the total length and the biomass the pepper plant. according to crouch et al., 1990, aqueous extracts of algae improved nutrient uptake in the roots. this stimulates root activity, increasing the absorption of water and nutrients, which improves plant growth and vigor. whatever the type of treatment (extract or amendment) b. bifurcata had a significant effect on the length of pepper’s root, this probably happens due to the richness of this alga in macroelements (n, ca, k, p, mg, na). algae contains macronutrients and microelements, amino acids, vitamins, cytokinins, auxins and abscisic acid that affect the cellular metabolism of treated plants, resulting in increased growth with crop yield (crouch and van staden, 1993; stirk et al., 2004). in addition, the presence of polysaccharides in aqueous extracts of algae as known sugars can enhance plant growth in a similar manner to hormones (rolland et al., 2002). the aqueous extracts of brown algae contain various betaine type compounds (blunden et al., 1986; ghoul et al., 1995). this molecule acts as a compatible solute that alleviates salinity induced osmotic stress, and functions as a nitrogen source when provided in a low concentration and as an osmolyte at higher concentrations (naidu et al., 1987). our study showed that treatment with aqueous extracts is more effective than amendment treatment for improving photosynthetic pigments in pepper leaves. similar results have been shown by whapham et al., 1993, where aqueous extracts of ascophyllum nodosum improved chlorophyll content in toma617 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) to leaves. similarly, the aqueous extract of sargassum was effective for the improvement of chlorophyll synthesis on two zea mays and phaseolus mungo plants (lingakumar et al., 2004). a large content of chlorophyll contained in leaves treated with a low concentration of aqueous extracts of algae might be the consequence of the absorption of magnesium, which is a major constituent of chlorophyll (whapham et al., 1993). the protein content of the plants that were treated with the aqueous extracts is greater, compared to the treatment with an amendment. all aqueous extracts of these three brown algae improved the protein content of pepper plants. the maximum value of the protein content is obtained by f. spiralis at c3. such an increase in protein content could be attributed to the increased availability and uptake of elements (n, k, ca, na, mg, cu, and zn) which algal fertilizers contained. the same results were obtained in zea mays and phaseolus mungo which had high protein content when treated with concentrations 0.5% and 1% of the aqueous extract of sargassum. sp (lingakumar et al., 2004). other results showed that the aqueous extracts of sargassum johnstonii resulted in an increase in the protein content (kumari et al., 2011), the aqueous extracts of hydroclathrus clathratus at 1.5% also lead to a significant increase at the level of sorghum vulgare proteins (ashok and douglas, 2004). the increase in protein content in low concentrations of aqueous extract may be due to the absorption of most necessary elements (n, k, ca, na, mg, cu, and zn) for seedling growth (anantharaj and venkatesalu, 2001). the plants watered with the aqueous extracts of the three brown algae showed significantly higher total sugar contents than the control. fertilization by amendment has also improved the total sugar content. the highest values for total sugars are obtained by c3 amendment of b. bifurcata. aqueous algae extracts stimulate various biological processes that increase carbohydrate levels in tomato plants (kumari et al., 2011). similar observations were recorded at vigna catajung treated with aqueous extracts of caulerpa racemosa (anantharaj and venkatesalu, 2001). a significant amount of total sugars was also observed in the plants treated with the aqueous extract of sargassum johnston (kumari et al., 2011). conclusion the study highlighted the positive effect of algae fertilizers on germination, growth parameters and biochemical composition of pepper. plants which were treated with low concentrations (0.5%) of three brown algae b. bifurcata, f. spiralis and c. gibraltarica showed greater growth. similarly, the chlorophyll content was higher when the plants were treated with these 0.5% algal extracts. meanwhile, the quantity of total sugars reached the maximum when fertilized with amendment or extract of b. bifurcata. the maximum growth parameters (root length, length of the aerial part, total length, and dry weight) were recorded in the plants treated with the aqueous extracts of the three brown algae b. bifurcata, f. spiralis and c. gibraltarica at high concentrations. in addition, the plants treated with aqueous extract of f. spiralis showed maximum values in the content of proteins. based on these results, both f. spiralis and b. bifurcata algae can be used as fertilizer for pepper or other vegetables, but at low concentrations to increase agricultural production. these results also had shown that a small content of aqueous algae extract can be used or even mixed with available fertilizers exist in the marketplace to enhance plant growth. moreover, we found that treatments with algal extract were more effective than the one conducted by amendment. in conclusion, b. bifurcata and f. spiralis had also shown outstanding results in relation to c. gibraltarica. therefore, more studies are needed to determine the potential of b. bifurcata and f. spiralis as growth biostimulants that can be used as organic biofertilizers in morocco. acknowledgements authors would like to thank plant biotechnology lab members for supporting this work. references anantharaj m and v venkatesalu (2001) effect of seaweed liquid fertilizer on vigna catajung seaweed res utiln 23:33-39. ashok sk and mr douglas (2004) counting flux vacua journal of high energy physics 2004:060. augier h and m santimone (1978) composition en azote total, en protéines et en acides aminés protéiques de fertilisant foliaire «goémar», à base d’algues marines botanica marina 21:337-342. blunden g (1991) agricultural uses of seaweeds and seaweed extracts seaweed resources in europe:65-81. blunden g, a cripps, s gordon, t mason, and c turner (1986) the characterisation and quantitative estimation of betaines in commercial seaweed extracts botanica marina 29:155-160. 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the multigeneic rhg1/ rfs2 locus: polymorphisms underling alterations of root development and pest resistance melody n. hemmati1, matthew j. b. geisler2, naresh pola3, khalid meksem1, and david a. lightfoot1,2* 1 department of plant, soil science and agricultural systems, southern illinois university, carbondale, il 62901-4415, usa. present address; fermentation unit, monsanto, des moines, iowa, usa; 2 department of plant biology, southern illinois university, carbondale, il 62901, usa; 3 department of electrical and computer engineering, southern illinois university, carbondale, il 62901, usa. received: july 22, 2017 / accepted: october 28, 2017 __________________________________________________ * corresponding author: ga4082@siu.edu. 407 abstract soybean (glycine max (l.) merr.) suffers yield loss due to root infection from soil infestation by heterodera glycine i. (soybean cyst nematode scn) and fusarium virguliforme (aoki; sudden death syndrome (sds)). the major locus for scn and sds resistance has previously been identified as rhg1/rfs2 (chr18; lg g) (site reference). the objective of this experiment was to compare the sanger dna sequence of a resistant cultivar (‘forrest’) and two susceptible cultivars (‘williams 82’ and ‘asgrow a3244’). sequences were downloaded from genbank for williams 82, phytzome for a3244 and a newly sequenced bac-b73p06 (82,157 bp) encompassing the rfs2/rhg1 locus. using the resistant cultivars, 800 single nucleotide polymorphisms (snps) and 57 indels were identified. in contrast, the susceptible cultivars had just 12 snps and no indels between them. polymorphisms were clustered within 59 kbp, divided into three sections. there were 5 predicted recombination breakpoints. the third and fourth breakpoints were located before gene 3 and after gene 5 (glyma18g02680; the rlk at rhg1/rfs2) which were therefore inferred to be derived from peking, within the rhg1/rfs2 region. comparisons of snps identified in illumina sequences from 31 semi-domesticated genomes showed 80% of the total snps in forrest were found among the genomes. annotation and gene prediction showed the bac gene prediction encoded 9-10 genes. there were 31 snps within exons and 137 among introns. just 11 this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. snps caused amino acid changes. there were 5 snps in cis regulatory elements (cres) and 14 in promoters. polymorphisms indicated the regions that were introgressed from peking had defined limits. proteins across the region were highly conserved compared to non-coding regions, suggesting purifying selection occurred. keywords: snp; indel; introgression; recombination; gene annotation; bacs. abbreviations: receptor like kinase (rlk); soybean cyst nematode (scn); sudden death syndrome (sds); bacterial artificial chromosome(bac); maff genebank system, national institute of agrobiological sciences, tsukuba, japan; nrrl, the agriculture research service culture collection, national center for agricultural utilization research, usda/ars, peoria, il usa. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) introduction soybean (glycine max (l.) merr.) cyst nematode (scn; heterodera glycine i.) and sudden death syndrome (sds; caused by fusarium virguliforme aoki) are two of the major pathogens causing yield loss in soybean. the genetic basis of resistance has been studied using genome sequences (hague et al., 2000; schmutz et al., 2010) and genetic transformations (cook et al., 2012; srour et al., 2013; liu et al., 2017). root infections caused by soil infestations by soybean cyst nematode (scn) have been severe since the crop was first domesticated, consequently scn has become the world’s most widespread and damaging soybean pathogen (wrather, 2001). losses to scn have been reduced by almost half since 1990, but it still remains a major problem in soybean (lightfoot, 2015). scn causes plant and root stunting and leaf chlorosis. sudden death syndrome (sds) has been shown to be a facultative hemibiotrophic fungus (li, 2009; roy, 1997) that causes yield loss in soybean. the amount of loss has doubled every decade in the us, since 1990 (wrather, 2001; lightfoot, 2015). sds, as a syndrome, is disease complex of both a root rot and a leaf scorch. studies have shown that the response of the root system to the pathogen has been associated with light, temperature, soil moisture and genetics of both the host and the pathogen, (arelli, 1994; lighfoot, 2005) suggesting a complex genetic control. partial resistance to scn and sds was significantly associated with the rhg1/rfs2 region at a sub-telomeric region of the soybean chromosome 18, molecular linkage group g (ruben et al. 2006; srour et al. 2013). the locus has been shown to be responsible for resistance to all hg types (previously races niblack et al. 2003) of scn and about half of the total variation in resistance to sds in resistant by susceptible crosses. it has previously been reported the resistance forms of the rhg1/rfs2 region were associated with delayed seedling development, lower root mass and reduced seed yield (afzal et al. 2012) . ‘forrest’ has one of the 3 types of resistance encoded by the rhg1/rfs2 locus (hague et al. 2000; ruben et al. 2005). it was one of several high yielding cultivars developed during the second cycle of intercross breeding and selection from ‘peking’ by edgar e. hartwig at mississippi agricultural and forestry experiment station in 1972, a product of a usda breeding program (hartwig and epps, 1973; lightfoot, 2008). it originated as an f5 line selected from the cross ‘dyer’ × ‘bragg’ and was highly resistant to scn hgtype 2.5.7, hgtype 0 and hgtype 7 (niblack et al. 2004; previously one type of race 1 and the two kinds of race 3). the forrest cultivar alone prevented crop losses of about $450 million from 1975-1980 as it was one of the first cultivars released with resistance to scn (lightfoot, 2008). bac b73p06 has been shown to encompass most of the rhg1/rfs2 locus(ruben et al. 2005). this locus works along with the rhg4 locus (liu et al., 2012; lakhssassi et al., 2017) encompassed by bac b100b10 (zatskayera et al., 2017). ‘williams 82’ has one of the 5 types of susceptibility encoded by the rhg1/rfs2 locus (hague et al. 2000; ruben et al. 2005). it originated as a composite of four lines resistant to phytophthora root rot (bernard and cremeens, 1988). it was selected from a ‘williams’ 3 × ‘kingwa’ bc6f3 (six backcross generations). kingwa was used as the donor parent to introgress phytophthora root rot resistance into the recurrent parent williams. williams 82 experienced one generation of single-seed descent following the six back-cross generations. haun et al. (2012) showed heterogeneity had persisted, but not on chromosome 18, so not within the rhg1/rfs2 region. asgrow soybean variety ‘a3244’ has a different one of the 5 types of susceptibility encoded by the rhg1/rfs2 locus (hague et al., 2000; ruben et al., 2005). it is a non-transgenic conventional variety. it is known for its superior agronomic characteristics and high-yield. the rhg1/rfs2 region was sequenced from a bac contig (hague et al., 2000). the objectives of this study were: to compare the sanger sequences from the region encompassed by bac b73p06 from a domesticated cultivar resistant to scn and sds with two susceptible cultivars; and to compare those sequences to the single nucleotide polymorphisms (snps) found by illuminatm sequencing among the 31 semi-domesticated and domesticated genomes reported by lam et al. (2010); then to use those sequence comparisons to infer which regions were introgressed into resistant domesticated cultivars from semi-domesticated cultivars; where the recombination breakpoints were; and which region, genes and polymorphisms were likely to be part of a multigenic rhg1/rfs2 locus. materials and methods cultivars compared to perform a comparative genomic study, sanger based dna sequence of three cultivars at the rhg1/rfs2 region were selected. forrest was selected to represent the cultivar resistant to scn and sds. twenty plants were used to make the bac library (meksem et al., 2000). one plant provided a molecule that was cloned in the bac b73p6. bac library development, physical mapping and screening candidate genes were described previously (triwitayakorn et al., 2005; ruben et al., 2006; srour et al., 2012). a3244 and williams 82 were chosen to represent the susceptible cultivars based on sequences available at genbank and phytozome prior to 2009. the sequences were downloaded. the williams 82 sequence was derived from dna isolated from multiple plants in puc18 (schmutz et al., 2010). predicted gene models had been made where there was transcript data to support the models. the a3244 sequence was derived from several overlapped bacs each derived from a single region but likely from separate plants (hauge et al., 2006). gene models had been predicted previously (triwitayakorn et al., 2005). bac sequencing methods the resistance alleles that might be derived from the rhg1/ rfs2 region from forrest were analyzed by sequencing the entire bac b73p06, which was derived from one region (parental alleles) of a single plant. the sequencing accuracy was high due to an 8 fold redundancy. the sequence of the insert (82,157 bp) was submitted to genbank as hq008938 (srour et 408 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 409 al. 2012). the sequencing method was the dideoxy chain-termination method using an abi big dye cycle sequencing kittm, on abi3730 automated dna sequencers at the j. craig venter institute (jcvi; ruben et al. 2006; afzal et al. 2012; srour et al. 2012). annotations the annotation methods are summarized in figure 1. there were four areas: structural annotation, functional annotation, searches for sequence variations and comparison of snps (supplemental table 1). structural annotations for structural annotation of genes, eukaryotic genemark (borodovsky et al. 2005) and a semi computational annotation, was applied to the forrest sequence to predict gene models de novo. the accuracy of eukaryotic genemark was checked versus other gene predictor software: augustus, genscan, geneid, fgenesh-m, fgenesh and snap at dna subway (http:// dnasubway.iplantcollaborative.org). pairwise alignment based methods were applied to the forrest bac b73p06 sequence versus all available ests (express sequence tags) to support the predicted genes. master, an exceltm-based genomic snp and indel comparative tool (hemmati et al., 2014) was utilized for analysis of polymorphisms in and around the predicted genes. signals and cis regulatory elements the 112 most highly conserved cis-regulatory elements in plants (dr. matt geisler, unpublished) were sought within the bac sequence. these cis-regulatory elements were found in common between arabidopsis and rice near (<500bp) promoter and enhancer regions. they were compared with the patterns of transcript abundance across many microarray experiments and were found in the 500 bp upstream region (not the 5’utr) of arabidopsis thaliana. each of these 112 conserved cis-regulatory elements (enhancer like motifs) were 8 bp, they were all searched and mapped within the forrest sequence by short sequence search software blat and patmat. the best result was prepared by using the alignment analysis tool “matcher” (matcher, 2011). repetitive elements and genetic markers microsatellites (simple sequence repeats; ssrs) were marked and mapped in the master file for further evaluations. dozens have previously been identified in earlier studies (srour et al. 2012); new markers were also identified and listed. promoter prediction tssp / prediction of plant promoters, regsite plant db, softberry inc, (plant-promoters, 2011) and putative eukaryotic pol ii promoter sequences (pol-ii-promoter, 2011) were used to predict the promoter regions which best matched with the predicted gene models. the best matches were mapped and recorded in master and in the data table submitted to genbank. functional annotations the putative genes were translated to amino acids. the translated regions were analyzed for functional domains and prediction of function by homology searches to closely related species. gene ontology, protein searches, domain searches each predicted protein was analyzed by blast in nbciblastp and wu-blast. pfam was also used for confirmation of possible domains and their functions. information about figure 1. flow chart of the experimental plan. 410 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) each possible protein, domain and its functions was recorded in the data table submitted to genbank. homology based predictions of function ncbi-blastp search between forrest versus the viridiplantae resulted in top hits with genes from ricinus communis l., populus trichocarpa l., medicago truncatula l., and fragaria vesca l. sequence similarities were recorded in the data table submitted to genbank. searches for sequence variations all variations including single nucleotide polymorphisms (snps) or insertions and deletions, either geneic (within gene coding region) or non-geneic (within non-coding regions) were recorded in the data table submitted to genbank for forrest, a3244 and williams 82 sequences. the snp frequency was calculated per 500 bp region (figure 2). snp density was used to infer the distribution of recombination breakpoints within the forrest sequence by using rdp4 (recombination detection program version 4), a windows 95/xp program (martin et al. 2005). also, protein variation effect analyzer (provean) software (choi et al. 2012) was applied to predict if a protein sequence variation had an impact on protein function. comparison of snp results with 32 genomes of re-sequenced-snps using the snpi-tool master the snp motifs of 31 genomes (lam et al., 2010) could be added by reference to the bp position of williams 82. where misalignments had occurred they were clear from the snp motifs disagreements and could be logically nudged to the nearest, most likely position. once aligned the snps were de-convoluted to single columns, each corresponding to a single genotype (supplemental table 2). data management feature table the annotation was deposited in a feature-table-format according to the instructions provided by genbank and published in genbank database (benson et al. 2013). visualization the master file contained all the predicted genomic features. it was a useful tool to actually observe and simultaneously perform data mining on any part of sequences of forrest, a3244 and williams 82. results bac b73p6 composition the bac b73p6 insert was shown to encompass 82,157 bp and predicted to encode 9 genes. these genes comprised of 57 exons (2–8 per gene). sequence composition (figure 3) showed that 52%, was non-regulatory-non-coding regions. intron regions comprised 24.5 %. exon regions occupied 21.5% of the sequence. enhancer, promoter and satellite regions together accounted for 2.3% of the sequence. the sequence composition a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 2. snp density per 500 bp among the predicted genes and their intergenic regions. the x axis indicates b73p06 sequence (1-82,157 bp). snp number was binned for every consecutive 500 bp intervals. the y axis shows density of snps per 500 bp region. black arrows are indications of the approximated positions of the predicted gene coding regions (total of 10 genes). a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 411 was similar to earlier reports for a3244 (hague et al. 2000) and williams 82 (schmutz et al. 2010). polymorphism frequencies however, sequence comparisons showed that across the entire bac there were exactly 800 snps between the resistance region in forrest and the sequences of both regions associated with sds and scn susceptibility. the number of snps found was as large as the number found among the 31 re-sequenced genomes in the same region (lam et al. 2010). a few snps were found in the promoter and enhancer regions of all 9 genes (5 in potential cis-regulatory elements and 14 in core promoter regions). however there were only 31 snps within genes and only 11 of the 31 that caused amino acid changes. there were amino acid changes in just 6 of the 9 proteins caused by those 11 snps. therefore, all of the protein coding sequences were inferred to be subject to purifying selection. there was evidence for a large and highly polymorphic region within the bac, 743 snps and 54 indels in 59 kbp (from 1,500-60,500 bp). a highly polymorphic region was expected to be a characteristic of the region introgressed into forrest from peking. equally, relatively monomorphic regions were common when comparing sequences of us cultivars. in fact, the same two regions of williams 82 and a3244 were nearly identical with just 12 snps. on the basis of high frequencies of polymorphism and transgenic plants, gene 5, glyma18g02680, the receptor like kinase (rlk) (srour et al. 2012; afzal et al. 2013) was shown to be part of the rfs2 allele and influence the rhg1-a region derived from peking. here, on the basis of high rates of polymorphisms, 4 genes were inferred to be derived from peking: gene 1 glyma18g 2650, gene 2 glyma18g 2660, gene 3 glyma18g 2670, and gene 10 glyma18g 2720. the coding regions were supported by 101 ests outside the 59 kbp central region. for example the region that encompassed gene 10 had 5 snps, across 18.25 kbp, among the 3 alleles. however, snps did not alter protein sequence, so there were no alloproteins. functional snps among the 800 snps between forrest and the sequences of susceptible cultivars, there were only 31 snps within geneic regions. exactly 20 of those 31 snps (64.5%) did not change amino acid sequence (synonymous snps). only 11 snps ( 35.5%) caused amino acid changes (non-synonymous snps). only one of these non-synonymous snps (c6615t / a152v), located at gene 1, arogenate dehydrogenase, was predicted to have a deleterious effect on protein function in susceptible cultivars. interestingly, there were also 2 snps in the promoter region of gene 1 (figure 4). recombination break point prediction there was a large and highly polymorphic region within the bac, 743 snps in 59 kbp, from 1.5–60.5 k bp. figure 5, depicts all 9-10 genes that may be inferred from sequence and transcripts, including the genes located in rhg1/rfs2 region. the 5 recombinant breakpoints predicted within rhg1/rfs2 are shown. the only deleterious snp located at gene 1 is marked as well. remarkably, comparing the result of 1,068 snps found among 17 semi-domesticated and 14 domesticated genomes (lam et. al. 2010), showed that 79% of those identified by the forrest to williams 82, comparison were found. some snps were unique to forrest and others to williams 82, depending on the sequencing method used. overall 63% of the 1,063 snp were identified. the similarity between the snps in the current research and snps from lam et al. (2010; figure ; supplemental table 2) and polymorphism frequency in this region (figure ; supplemental table 1) supports the breakpoints for the regions identified in peking. discussion this research study was able to shed more light on the complex rhg1/rfs2 region by analysis of structural and functional annotation. the existence of predicted recombinant breakpoints in this region and the similarity between the snps in the current research and snps from 31 genomes indicated the regions are likely derived from peking. it appears most of the snps in this region were introgressed into forrest from the semi-domesticated peking. in addition to the rlk proven involved by srour et al. (2012) and the copy number variation in snap ( cook et al. 2013; 2014; liu et al.2017) there are several other changes in the defense related gene alleles (matsye et al. 2012; matthews et al. 2013) and many may have been introgressed from peking. possible functions and roles in defense mechanisms were found for all 9 predicted genes. gene 1 (glyma18g02650) encoded an arogenate dehydrogenase-like protein (agdh; ec 1.3.1.43, ncbi reference sequence: xp_003552195.1) which is one of the enzymes in the shikimate pathway (wink, 1999; rippert and matringe, 2002). the rna atlas at soybase figure 3. compositions of the bac 73p06 dna encoded regions. each portion of the pie chart indicates the percentages of each type of region encoded (exon, intron, enhancer, promoter, satellite dna (sat), non-regulatory-non-coding regions (non)). all the genes are potentially involved in resistance. p7306 layout exon intron enhancer promoter sat non 52% 24.5% 21.5% 2.3% a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 412 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 4. snp detection flow chart. figure 5. ideogram of the structure of the regions predicted to be introgressed to forrest from peking. the black arrows indicate the positions of the 9-10 genes located in the bac b73p6 rhg1/rfs2 region. putative gene functions are indicated. recombinant breakpoints are shown as green arrows. a predicted deleterious snp and 2 snps in the promoter of gene 1 are shown as blue arrows. showed high transcript abundances in roots, flowers, pods and leaves (supplemental table 3). the products of shikimate pathway are precursors for many of aromatic and phenolic secondary metabolites. these metabolites are involved in numerous vital processes such as plant defense, formation of structural biopolymers and cell wall components including lignin, lignols, defense related phenolics and the synthesis of hormones and vitamins. phenolic compounds are involved in pcd (program cell death) in response to scn and f. virguliforme infections (mahalingam and skorupska, 1996; yuan et al. 2002; iqbal et al. 2005; iqbal et al. 2008; kandoth et al. 2011). gene 2 (glyma18g02660; ncbi cg_3016.1) encoded two putative conserved domains; the duf 3411 superfamily (pfam 11891) and the duf 399 (pfam 04187) superfamily both of which were domains of unknown functions. however, the transcript is abundant in flowers, nodules, roots, pods and leaves (supplemental table 3). gene 3 was a biotin-carboxyl-carrier-protein (glyma18g02670; ncbi np_567035.1). biotin was particularly important in the scn pathogenesis process (liu et al. 2012). biotin is a critical coenzyme which is needed for cell growth, the production of folate, the production of fatty acids, and the metabolism of fats and amino acids. pathogens have a high demand for the macroand micro-nutrients for their survival and proliferation during infection. higher expression of proteins required for de novo biotin synthesis, uptake and metabolic adaption may be processes that allow pathogens to survive during infection. biotin is required for both folate uptake and metabolism (pendini et al. 2013) and folate starvation appears to be a key defense strategy of scn resistant soybeans (liu et al. 2012). gene 4 was unusual. it has no transcript support and did not present any similariies to known functional domains. however, a psi-blast search through 8 iterations found 72 genes in the ortholog family, which inferred that the gene might be involved in cell and protein interactions. in addition, 20 of the 25 snps in that gene were in the c-terminal region, an ankyrin pfam. therefore, gene 4 might also be part of the rhg1/rfs2 locus and the rhg1-a allele derived from peking. gene 5 (glyma18g02680, ncbi: aci05083.1, gmrlk18-1, gene id, symbol loc547641) was a receptor-likekinase shown to confer partial resistance to scn and sds in stable transgenic plants (srour et al. 2012). the protein was shown to bind proteins and peptides in the nematode and plant secretomes (afzal et al. 2013). plants lack extra cellular antibodies for defense against pathogen attacks, but, they are able to establish other strategies for detection and adaptation to environmental changes. these strategies include a wide range of receptors both at cell surfaces and within the cells (gomezgomez and boller, 2000; thordal-christensen, 2003; afzal, 2007; haffani et al. 2004). the leucine-rich repeat extracellular domains of these receptors function as detectors and provide an early warning signal for the presence of potential pathogens. they commonly activate protective signaling cascades in plants and/or alter plant development (matsushima and miyashita, 2012). they often lead to activation of various host defense responses, including a specialized type of (pcd) programmed cell death known as the hypersensitive response (hr; tao et al. 2000). predicted genes 6 and 7 (glyma18g02690) were both parts of the diphenol oxidase laccase enzyme transcript (iqbal et al. 2008). this enzyme (ec.1.10.3.2) is a blue copper-containing oxidase found in plants, fungi, bacteria and arthropods. diphenol oxidase laccase enzymes catalyze the oxidation of wide variety of organic and inorganic substrates such as polyphenolic compounds. phenolic compounds are synthesized from remote precursors as a response to pathogen attacks. some antibiotic phenolics are stored in plant cells as inactive bound forms (preformed antibiotics) and are convertible into biologically active antibiotics by plant hydrolyzing enzymes in response to pathogen attacks. the normal anticipated amount of preformed antifungal phenolics in healthy plants may increase as a response to pathogens attack (lattanzio et al. 2006). moreover, diphenol oxidase laccase may participate in cell wall lignifications, by which lignifications of plant cell wall may contribute to reduction the frequency of scn feeding site development (lattanzio et al. 2006; iqbal et al. 2008). genes 8 and 9 were ion antiporters (glyma18g02700, glyma18g02710). na/h antiporters are key transporters in maintaining the homeostasis of actively metabolizing cells. the activity of na+/h+ antiporters, na+/h+ concentration and cell volume is critical for the viability of all cells. typically, na+/ h+ antiporters are located at the plasma membrane (uniport, 2012). action of na+/h+ antiporters is necessary for the uptake of most metabolites, plant growth and development and also for plant response to environmental stresses (hunte et al. 2005). gene 10 was predicted to be a helicase (glyma18g02620; dexdc ,cd00046; helicc, cd00079). helicases are categorized as enzymes that use energy derived from the hydrolysis of a nucleotide triphosphate to unwind double-stranded structures. increasing evidences suggest that the dead-box helicases play an important role in plant growth and development processes, possibly by regulation of rna metabolism and gene expression (wang et al. 2000; li et al. 2008). on the basis of copy number variation and transgenic hairy roots, 3 genes just distal to b73p06 were predicted to be part of the rhg1-b region (cook et al. 2012; meksem et al. 2014; liu et al. 2017). the genes glyma18g 2570 and glyma18g 2590 were shown to be present at 1 copy in susceptible cultivars but at 10 copies in cultivars with the rhg1-b allele. at the rhg1-a allele there appeared to be 3-5 copies of the 3 genes. there were again many snps, insertions and deletions that changed amino acid sequences of the alloproteins from forrest compared to williams 82 and essex (meksem et al. 2014). using both virus induced gene silencing and hairy root assays these changes were shown to underlie part of the resistance to scn. the alpha-snap gene had been identified to alter scn resistance in hairy roots among dozens of other genes from whole genome screens (matsye et al. 2012; matthews et al. 2013; meksem et al. 2014). finally, some studies found an rhg1/rfs2-like region at other locations in a few scn resistant pis (concibido et al. 2004). locations included linkage group (lg) b1, mid lg g and lg b2 (chromosomes 11, 18 and 14; vierling et al. 1996; wang et a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 413 al. 2001; yue et al. 2001). the findings suggest the existence of functional paralogs of rhg1/rfs2 among the duplicated regions of the soybean genome. therefore, the rhg1/rfs2 region is predicted to be multigeneic, each gene having paralogs, but to encompass several genes and polymorphisms in the region from glyma18g 2570glyma18g 2720 in forrest. data depositions tmd1 marker, fj520231; corrected rlk at rhg1/rfs2 gene af506516 and mrna af506517; siuc-satt122, bankit1155667; bac pb73p06 complete sequence hq008938 (corrected jn597009). three supplementary tables will be published online and links to 5 sequences at genbank are provided. conflicts of interest none statement of contributions christopher d. town of jcvi provided the bac sequences from a bac library made by khalid meksem. naghmeh hemmati, and david. a. lightfoot analyzed bac sequences and generated figures and supplemental tables, wrote the manuscript and edited the final version. naresh pola extracted the snps for the 32 genomes. matthew geisler provided the list of cres ahead of publication. acknowledgements the physical map location of b73p06 was supported by the nsf under grants no. 9872635. usb supported the sequencing of the bac. thanks are due to christopher d. town of jcvi for sequencing and eliza ruben for bac isolation. any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the nsf. references afzal aj and da lightfoot (2007) inclusion bodies contain rhg1/ rfs2 folding intermediates: a novel refolding protocol for protein purification. protein expr purific 53: 346–355. afzal aj, srour a, saini n, hemmati n, el shemy ha, lightfoot da (2011) recombination suppression at the dominant rhg1/rfs2 locus underlying soybean resistance to the cyst nematode. theor appl genet 124: 1027-1039. afzal aj, a srour, a natarajan, n saini, mj iqbal, m geisler, ha el shemy, and da lightfoot (2012) resistance to soybean cyst nematode: rhg1. j plant genom sci 2012 39: 39-45. afzal aj, aj wood, and da lightfoot (2008) plant receptor-like serine threonine kinases: roles in signaling and plant defense. molec plant microb int 21: 507-517. arabidopsis.org. 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is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) coiling during male-male combat in snakes: differences be-coiling during male-male combat in snakes: differences between vipers and other groups, and between constrictors and tween vipers and other groups, and between constrictors and non-constrictorsnon-constrictors ishmel j. lock, kaitlin e. zalewski, and philip j. senter* department of biological and forensic sciences, fayetteville state university, fayetteville, nc 28301, usa received: november 17, 2022 / accepted: february 20, 2023 __________________________________________________ * corresponding author: psenter@uncfsu.edu 759 abstractabstract during male-male combat (mmc) in snakes, combatants of-during male-male combat (mmc) in snakes, combatants often coil around each other. to determine whether there are ten coil around each other. to determine whether there are differences in this behavior between different snake groups, differences in this behavior between different snake groups, we examined video footage of 100 instances of mmc in 49 we examined video footage of 100 instances of mmc in 49 snake species from six families. results show that in viperi-snake species from six families. results show that in viperidae, mmc involves smaller numbers of loops than in the oth-dae, mmc involves smaller numbers of loops than in the other three clades considered here (pythonidae+loxocemidae, er three clades considered here (pythonidae+loxocemidae, elapidae+pseudoxyrhophiidae, and colubridae). in viperi-elapidae+pseudoxyrhophiidae, and colubridae). in viperidae, coiling also seems to be an accidental result of other dae, coiling also seems to be an accidental result of other movements and does not involve coil tightening, whereas movements and does not involve coil tightening, whereas in the other three clades it appears to be deliberate and is in the other three clades it appears to be deliberate and is often enhanced by coil tightening. the duration of coiling is often enhanced by coil tightening. the duration of coiling is shorter in the clade elapidae+pseudoxyrhophiidae (most of shorter in the clade elapidae+pseudoxyrhophiidae (most of which are non-constrictors) and viperidae (non-constrictors) which are non-constrictors) and viperidae (non-constrictors) than it is in colubridae (many of which are constrictors) and than it is in colubridae (many of which are constrictors) and the clade pythonidae+loxocemidae (constrictors). it is also the clade pythonidae+loxocemidae (constrictors). it is also shorter in non-constricting colubrids than in constricting shorter in non-constricting colubrids than in constricting colubrids, although the number of loops does not differ be-colubrids, although the number of loops does not differ between the two groups. we conclude that coiling is of lesser tween the two groups. we conclude that coiling is of lesser importance for mmc in viperidae than in the other three importance for mmc in viperidae than in the other three clades, and that maintaining loops during mmc is of lesser clades, and that maintaining loops during mmc is of lesser importance in non-constricting groups than in constrictors. importance in non-constricting groups than in constrictors. these results show that differences in the amount of coiling these results show that differences in the amount of coiling during mmc in snakes follow phylogenetic lines. the videos during mmc in snakes follow phylogenetic lines. the videos used in this study were collected from social media and dem-used in this study were collected from social media and demonstrate that social media can be useful in collecting data for onstrate that social media can be useful in collecting data for scientific studies.scientific studies. keywords:keywords: behavior; combat; constriction; social media; viperidae. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introductionintroduction in snakes of the clade afrophidia (the clade that is phylogenetically bracketed by the boa and python clade and the clade caenophidia—see vidal et al., 2007), males engage in a combat ritual that often involves combatants coiling around each other and raising their foreparts, with each combatant attempting to push the other’s foreparts down (carpenter, 1977; senter et al., 2014; abu baker et al., 2021; senter, 2022). detailed descriptions of male-male combat (mmc) have been published for numerous snake species (reviewed in carpenter, 1977; shine, 1978, 1994; senter et al., 2014; abu baker et al., 2021; senter, 2022), but the number of loops in the coil is usually not reported. in published illustrations of mmc in snakes, the number of loops is usually as few as one to three in members of viperidae (shaw, 1948; carpenter et al., 1976; carpenter, 1977; nishimura et al., 1983; andrén, 1986; schuett and gillingham, 1989), whereas it is often greater than three in members of other families (fleay, 1951; bogert and roth, 1966; turner, 1992; almeida-santos et al., 1998; muniz-da-silva et al., 2013; guedes et al., 2019; valencia et al., 2020; abu baker et al., 2021). if this difference is due to a tendency among vipers to use fewer loops during mmc, and not just an artifact of having been photographed at moments when fewer loops were used than is usual for vipers, then coiling may be a component of mmc that is of lesser importance in viperidae than in other snake clades. if so, this would be a major behavioral difference in mmc between vipers and other snakes. such a difference would be an aspect of the evolution of snake behavior that has not previously been reported. we therefore sought to determine whether vipers use fewer loops during mmc than is the case in other snake clades, by examining footage of mmc in vipers and other snakes. coiling is an integral part of constriction, and previous studies have recorded that at least some snakes that use constriction to dispatch prey also employ active constriction during mmc (martin, 1976; barker et al., 1979; guedes et al., 2019). we therefore also sought to determine whether differences exist in the degree of coiling during mmc in constrictors versus non-constrictors. materials and methodsmaterials and methods videosvideos previous studies have demonstrated that social media can be useful in the collection of data for scientific study (miranda et al., 2016; liberatore et al., 2018; paterson, 2018; maritz and maritz, 2020; abu baker et al., 2021). applying this principle, we searched youtube (www.youtube.com) for footage of mmc in snakes. many youtube videos of snakes have misleading titles in which the species is misidentified, courtship is mistaken for combat (or vice versa), or an interaction is misidentified as combat when one snake is merely treating the other as an inanimate obstacle during locomotion in a confined space. we were therefore careful to include a video in the study only if we could confirm (or correct) the species identification, and only if it recorded combat. combat can be distinguished from courtship in afrophidian snakes in that it usually includes attempts by the combatants to push down each other’s raised heads (in non-lampropeltine snakes) or to pin each other’s heads to the ground (in lampropeltines) (senter, 2022). in contrast, courtship usually lacks such elements and includes a different suite of behavioral elements that usually includes chin-rubs and jerking of the head or body, often with the male performing such behaviors while his head moves along the dorsum of the female toward her anterior (senter, 2022). as shown in table 1, the footage included in the study comprises 100 instances of mmc in snakes of 49 species in six families: 29 instances in the family viperidae, one instance in the family loxocemidae, 12 instances in the family pythonidae, 18 instances in the family elapidae, one instance in the family pseudoxyrhophiidae, and 39 instances in the family colubridae. analysisanalysis we compared both the number of loops and the duration of coiling during mmc between viperidae and three other clades: pythonidae+loxocemidae (henceforth, p+l for concision), elapidae+pseudoxyrhophiidae (henceforth, e+p for concision), and colubridae. we considered pythonidae and loxocemidae together as a single clade, because the two are closely related (reynolds et al., 2014). likewise, we considered elapidae and pseudoxyrhophiidae together as a single clade, because the two are closely related (zaher et al., 2019). to count the number of loops in each instance of mmc, we considered a single loop to be a complete turn (360º, as viewed down the long axis) of one snake’s body around the other snake’s body (fig. 1). during mmc, the number of loops continuously changes as the snakes roll about their long axes, generating new loops near the head while uncoiling near the tail, and it occasionally happens that coiled snakes suddenly decouple with a violent whipping motion, reducing the number of loops to zero. in our comparison of the number of loops, we therefore considered only the maximum number of loops in each video (table 1). for each of the four clades, we calculated the mean and standard deviation of the maximum number of loops among the included instances of mmc (table 2). to determine whether a significant difference exists in the maximum number of loops between clades, we ran a one-way anova. to compare the duration of coiling between clades, we first calculated values that we called a, b, and c. for each clade, a is the total duration of all mmc footage in all the included videos of that clade, added together. for each clade, b is the total duration of mmc footage with coiling, in all the included videos of that clade, added together. for each clade, c is the total duration of mmc footage without coiling, in all the included videos of that clade, added together. for each clade, b / a × 100% = the percentage of mmc footage in which the combatants are coiled. we recorded that percentage for each clade in table 2. to determine whether a significant difference exists in that percentage between clades, we used the pearson chi-squared test to compare a and c between each pair of clades (table 3). to determine whether our data reveal a difference in the degree of coiling between colubrids that use constriction to dispatch prey (in our sample: dolichophis, hierophis, lampropeltis, pantherophis, pituophis, spilotes, zamenis) and non-constricting colubrids (in our sample: dispholidus, drymarchon, masticophis, philothamnus, ptyas), we repeated the analyses delineated above, comparing those two groups of colubrids. we did not repeat the analyses to compare constricting vs. 760 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) figure 1. figure 1. method of counting loops. the white snake is coiled around the gray snake with four loops. the smaller bracket shows one loop of the white snake around the gray snake. the larger bracket shows 1.5 loops of the white snake around the gray snake. 761 table 1. table 1. data on coiling during male-male combat in snakes, in the videos used in this study. dcoi = duration of coiling in the footage. dcom = duration of combat in the footage. mnc = maximum number of loops in the footage. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) family species mnc d com d coi reference (author and year) xenopeltidae xenopeltis unicolor 2 18 s 18 s xtclueck, 2009 pythonidae aspidites melanocephalus 5 123 s 123 s nq dry tropics nrm, 2016 morelia spilota > 2 51 s 3 s afg boii, 2018 morelia spilota > 3 141 s 94 s tomonews us, 2016 morelia spilota 4 20 s 20 s browne snake removals, 2014 morelia spilota > 3 105 s 30 s coldblooded revolutions, 2015 morelia spilota 5 37 s 37 s andre, 2016 morelia spilota 4 146 s 146 s forgreenies, 2021 morelia spilota 6 118 s 118 s noosafifi, 2016 morelia spilota 7 639 s 639 s tisdall, 2017 morelia spilota 5 50 s 50 s new york post, 2018 morelia spilota 6 46 s 46 s linnett, 2014 morelia spilota 6 555 s 555 s majikfaerie, 2016 viperidae agkistrodon contrortrix and a. piscivorus 1 200 s 30 s living alongside wildlife, 2016 agkistrodon contortrix 0 96 s 0 s louisiana amphibian and reptile enthusiasts, 2018 agkistrodon piscivorus 1 136 s 4 s water possum, 2015 agkistrodon piscivorus 0 104 s 0 s bruggemann, 2017 bitis arietans 0 93 s 0 s kruger sightings, 2016 bitis arietans > 2 226 s 10 s cape snake conservation, 2017 causus defilippi 0 12 s 0 s williams, 2016 daboia palaestinae 0 165 s 0 s reptiles, 2020 daboia russelii > 2 83 s 4 s (very loose loops) searider1949, 2020 macrovipera lebetina 3 123 s 60 s cityfreepress, 2019 montivipera xanthina 1 30 s 3 s gkousios, 2020 crotalus adamanteus 0 104 s 0 s browning, 2019 crotalus atrox > 3 624 s 120 s ringo999999, 2009 crotalus atrox 2 54 s 3 s dana, 2010 crotalus atrox 0 83 s 0 s fox 5 atlanta, 2016 crotalus atrox 3 254 s 10 s moser, 2018 crotalus atrox 2 65 s 2 s seanblue622, 2011 crotalus durissus 2 88 s 76 s franco, 2015 crotalus horridus 3 75 s 68 s bauer, 2020 crotalus horridus 2 228 s 10 s thatanimalguy, 2018 crotalus mitchellii 0 24 s 0 s desert museum, 2015 crotalus oreganus 3 57s 50s nature picture library, 2019 crotalus oreganus 0 28 s 0 s san jacinto trail report, 2017a crotalus oreganus 2 33 s 6 s san jacinto trail report, 2017b crotalus oreganus 2 69 s 62 s crabbe, 2018 crotalus ruber 1 35 s 1 s greg l, 2014 crotalus viridis 3 73 s 73 s paul goins, 2014 crotalus viridis 2 51 s 30 s joshua anderson, 2017 sistrurus miliarius 2 417 s 165 s crotalusco, 2008 762 table 1. table 1. continued. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg )family species mnc dcom dcoi reference (author and year) elapidae demansia psammophis 10 129 s 129 s still waters, 2012 demansia vestigiata 3 68 s 68 parksaustralia, 2013 dendroaspis angusticeps 8 30 s 30 s best of africa, 2017 dendroaspis angusticeps 9 880 s 540 s beach bumz, 2016 dendroaspis polylepis 5 65 s 50 s kruger sightings, 2015 dendroaspis polylepis 5 71 s 25 s kruger sightings, 2017 micrurus frontalis 10 137 s 137 s peret, 2017 micrurus ibiboboca 6 47 s 47 s kemp, 2015 naja mossambica 3 185 s 50 s daily mail, 2018 naja naja > 7 530 s 180 s kumar, 2018 notechis scutatus 5 98 s 27 s snake master, 2019 ophiophagus hannah 4 73 s 30 s madras cr. bank trust, 2019 ophiophagus hannah 1 40 s 1 s felis creations tv, 2014 ophiophagus hannah > 7 757 s 60 s dinkelman, 2018 pseudechis guttatus 6 51 s 51 s mh outdoors, 2019 pseudechis porphyriacus 5 98 s 48 s maximus marcus, 2017 pseudechis porphyriacus > 15 183 s 80 s caters clips, 2019 pseudonaja textilis > 3 1384 s 300 s meek-and-wild, 2016 pseudoxyrhophiidae leioheterodon madagascariensis 10 219 s 166 s trebbor frog, 2015 colubridae dispholidus typus > 8 64 s 64 s nathan, 2020 dolichophis jugularis > 5 158 s 158 s reptiles, 2021a dolichophis jugularis ? 17 s 17 s reptiles, 2021b dolichophis jugularis 4 20s 19 s reptiles, 2021c dolichophis jugularis 5 ? ? reptiles, 2021d dolichophis jugularis 5 103 s 103 s reptiles, 2021e dolichophis jugularis 5 14 s 14 s reptiles, 2021f dolichophis jugularis 5 12 s 12 s reptiles, 2021g dolichophis jugularis 4 23 s 23 s reptiles, 2021h dolichophis jugularis > 3 97 s 97 s reptiles, 2021i dolichophis jugularis 4 36 s 36 s reptiles, 2021j drymarchon corais 0 22 s 0 s georgiawildlife, 2014 drymarchon corais 0 48 s 0 s smith, 2011 hierophis viridiflavus 9 314 s 296 s bramham, 2008 hierophis viridiflavus > 6 92 s 60 s gary and rachel, 2015 hierophis viridiflavus 4 164 s 164 s canary honey, 2014 lampropeltis californiae 0 118 s 0 s wetherbee, 2017 lampropeltis californiae 6 181 s 181 s douglas collins, 2016 lampropeltis getula getula 11 48 s 48 s cimarronsc, 2008 lampropeltis holbrooki 5 184 s 152 s martin, 2016 lampropeltis triangulum > 3 120 s 20 s lukeovcrashcourse, 2017 lampropeltis triangulum 2 153 s 153 s vegged out, 2020 masticophis flagellum 0 164 s 0 s favor, 2018 masticophis flagellum 7 196 s 182 s jacquiisaacson, 2008 pantherophis alleghanensis > 5 247 s 40 s lewis, 2014 philothamnus semivariegatus 4 17 s 16 s marais, 2013 pituophis catenifer > 5 60 s 25 s easter, 2016 pituophis catenifer 0 61 s 0 s webetubing, 2007 pituophis catenifer 3 91 s 91 s brennadl, 2019 pituophis catenifer 6 776 s 776 s robinson, 2014 ptyas mucosus > 5 351 s 351 s nilaview, 2015 ptyas mucosus > 4 143 s 20 s nat geo wild, 2018 ptyas mucosus 7 22 s 22 s randadath, 2012 ptyas mucosus 0 44 s 0 s krishnan, 2016 ptyas mucosus 7 124 s 124 s chavhan, 2015 ptyas mucosus 5 129 s 119 s dhinith s, 2018 ptyas mucosus 5 121 s 113 s reddy, 2020 spilotes pullatus 8 154 s 154 s mebert, 2017 zamenis longissima 5 618 s 420 s living zoology, 2020 763 non-constricting members of e+p, due to a low sample size of constricting species in our sample of the clade. the e+p footage used here includes one instance apiece of mmc in two elapid species that are known to use constriction to dispatch prey: pseudonaja textilis and demansia psammophis (shine and schwaner, 1985). however, in the latter species, only juveniles are known to constrict prey (shine and schwaner, 1985), which reduces our sample size of videos of e+p species that constrict prey as adults, to only one video. resultsresults during the collection of data from videos, it became evident that coiling in vipers is often an accidental result of the movements involved in the combat dance, that coil tightening is rare, and that active attempts to maintain a coil are also rare. in contrast, coiling in the other clades often appears to be deliberate, coil tightening is common, and the combatants often actively attempt to maintain large numbers of loops around each other, especially posteriorly. as shown in table 2 and fig. 2, the maximum number of loops per instance of mmc tends to be lower in the viperidae than in the other three clades (x̅ = 1.4 in viperidae; x̅ = 4.5 in p+l; x̅ = 6.4 in e+p; x̅ = 4.5 in colubridae) (table 2). one-way anova revealed that a significant difference exists in the mean number of loops between at least two clades (f(3, 95) = 18.9407, p = 1.0389e-09). tukey’s hsd test for multiple comparisons found that the mean number of loops is significantly different between viperidae and p+l (p = 0.0011826 < 0.01), between viperidae and e+p (p = 0.0010053 < 0.01), between viperidae and colubridae (p 0.0010053 < 0.01), and between e+p and colubridae (p = 0.0199122 < 0.05). it found no significant difference in the mean number of loops between p+l and e+p (p = 0.0994088) or between p+l and colubridae (p = 0.8999947). pearson’s chi-squared test found that between every pair of clades considered here, there is a significant difference in the duration of coiling during mmc (table 3). our analysis uncovered no significant difference in the a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) table 2. table 2. data on coiling during mmc for each group. table 3. table 3. results of pearson’s chi-squared tests comparing a and c (see materials and methods) between pairs of clades. figure 2. figure 2. box-and-whisker graph of maximum number of loops in each instance of mmc, for each clade. outliers are represented by small circles beyond the box and whisker. 764 number of loops between constricting and non-constricting colubrids. for the maximum number of loops per instance of mmc, the mean is 4.0 in non-constricting colubrids and 4.72 in constricting colubrids (table 2). one-way anova found no significant difference between the maximum number of loops between the two groups (f = 0.6461, p = 0.4268). however, pearson’s chi-squared test found a significant difference in the duration of coiling between constricting and non-constricting colubrids (χ2 = 50.4899, p < 0.0001). discussiondiscussion previous studies have shown that there are differences in mmc among different clades of snakes. for example, in the colubrid clade lampropeltini, high head-raising (present in other afrophidian clades) is absent from the mmc repertoire, and dorsal bowing of the body (absent in other afrophidian clades) is present (abu baker et al., 2021; senter, 2022). biting during mmc is also more prevalent in lampropeltini that it is in other snake clades (abu baker et al., 2021; senter, 2022). also, mmc is often absent in snake clades that subdue prey by means other than venom or constriction (schuett et al., 2001). however, previous studies have not explored differences in the degree of coiling between snake clades. the results of this study therefore elucidate an aspect of snake mmc that has not previously been elucidated. as this study shows, the lesser importance of coiling in vipers is a real difference in mmc between viperidae and other snake clades. furthermore, this study shows that the duration of coiling during mmc differs between snake groups, with the highest durations occurring in constrictors (pythonidae+loxocemidae and colubrid constrictors) and lower durations in e+p and non-constricting colubrids. this study is the first to document and quantify these differences. as such, it adds to current knowledge of variation in mmc across the clade serpentes. it is interesting that in applying constriction to their opponents, constrictors incorporate their main prey-killing technique into mmc. in contrast, vipers usually do not incorporate their main prey-killing technique (a venomous bite) into mmc (senter, 2022). it is also interesting that in colubrid mmc, constrictors remain coiled longer than non-constrictors but do not use a greater number of loops than non-constrictors. this suggests that if coiling during mmc has become reduced through evolution in non-constricting colubrids, the duration of coiling is more vulnerable to evolutionary reduction than is the number of loops in a typical coil. it further suggests that those two parameters may undergo separate evolutionary trajectories and are therefore not constrained to evolve in tandem. conclusionsconclusions our results show that vipers tend to use fewer loops than do members of the other three clades considered here. vipers also tend to spend less time coiled around opponents during mmc than do members of the other three clades considered here. in short, coiling is of less importance for mmc in vipers than in other snake clades. the predominantly accidental nature of the coiling in vipers and their apparent unconcern for maintaining or tightening the loops underscores the lack of importance of coiling as a component of combat in viperidae. our results also indicate that the maximum number of loops per instance of mmc is not significantly different between p+l and colubridae, nor between p+l and e+p. however, they also indicate that the duration of coiling during mmc differs between all three of these clades, with longer durations in clades in which constriction is common (p+l and colubridae). likewise, our results indicate a significant difference in the duration of coiling between constricting and nonconstricting colubrids (the duration is longer in the constrictors), but not in the maximum number of loops per instance of mmc. further, this study demonstrates that social media can play an important role in the collection of data for scientific study. previous studies that have incorporated data collection via social media have also confirmed that social media can be useful for that purpose (e.g. miranda et al., 2016; liberatore et al., 2018; paterson, 2018; maritz and maritz, 2020; abu baker et al., 2021). this study provides another instance of it. acknowledgments:acknowledgments: we thank the photographers of the videos of the snakes in combat. we also thank the reviewers for helpful comments that improved this paper. we also thank the fsu-rise (research initiative for scientific enhancement) program, which provided funding for ishmel j. lock. references:references: abu baker ma, m al-saraireh, zs amr, and pj senter (2021) male-male combat in the large whip snake, dolichophis jugularis (serpentes: colubridae). herpetol notes 14: 735–744. afg boii (2018) pair of fighting snakes fall through family’s bedroom ceiling. 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is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) 767 watch?v=phxufwbh9di (accessed 25 february 2021) xtclueck (2009) two snakes mating? https://www.youtube.com/ watch?v=jsst32hkwpm (accessed 25 february 2021) zaher h, rw murphy, j camilo arredondo, r graboski, p roberto machadofilho, k mahlow, gg montingelli, a botallo quadros, nl orlov, m wilkinson, y zhang, and fg grazziotin (2019) large-scale molecular phylogeny, morphology, divergence-time estimation, and the fossil record of advanced caenophidian snakes (squamata: serpentes). plos one 14(5:e0216148). doi: https://journals.plos.org/plosone/article?id=10.1371/journal. pone.0216148 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l lc (w w w .a tla spu bl is hi ng .o rg ) layout 1 a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) effect of germination on improving grain quality, chemical composition, antioxidants, and phytic acid in rice (oryza sativa) nessreen n. bassuony 1*, eman n. m. mohamed2, and ekram h. barakat3 1 rice research department sakha-kafr el-sheikh, field crops research institute, arc, egypt; 2 seed technology research department, field crops research institute, arc, egypt, 3 depatement of home economics, faculty of specific education, kafr el-sheikh university, egypt. atlas journal of biology, 2021, pp. 741-751 https://doi.org/10.5147/ajb.vi.227 abstract this study aimed to study the effect of the germination process on grain quality, chemical composition for brown rice and comparing them with white rice for use it is on a commercial scale . three rice varieties namely sakha 104( japonica), giza 178( japonicaindica), and giza 182(indica) were used in this study. and the three statuses (milled rice, brown rice, and germinated brown rice). a completely randomized design in the factorial arrangement was used in this experiment to determine some cooking and eating quality characters i.e. gelatinization temperature, amylose content and elongation %, water uptake, hardness, chemical composition: phytic acid, total antioxidant capacity, and panel test evaluation for rice samples. the results indicated that there were significant differences in amylose content and gelatinization temperature among the three rice statuses and no significant difference in these characters with the three rice varieties under study. germinated brown rice showed the lowest amylose content (15.58%), followed by brown rice (17.26%) and white rice (18.39%). brown rice gave the highest temperature followed by germinated brown rice then milled rice. a maximum elongation ratio was observed in giza178 ( japonica indica). white rice gave the maximum elongation (47.18%) followed by germinated brown (24.56 %) then brown rice (16.08 %). japonica rice exhibited lower hardness than indica rice. the strongest value (4.82) was recorded at brown rice, while the weakest value (3.64) was in white rice. the indica rice variety giza181 had the highest protein and fat%. the germinated brown rice had the highest value of protein, crude fiber, and fat (7.27, 1.98, and 2.87%, respectively), compared with compared to brown rice and white rice. white rice had lower phytic acid (%) followed by germinated brown rice, then brown rice. japonica rice cultivar (sakha 104) has a higher antioxidant level than indica rice cultivar (giza 182). brown rice contains the highest value of antioxidant followed by germinated brown rice, while white rice gave the lowest level. germinated brown rice recorded the second two good tastes after white rice. so, this study recommended that we can increase the nutritional value of rice by germinated brown rice with little change in taste. keywords: germinated brown rice; protein; amylose; antioxidant. received: may 14, 2021 / accepted: june 15, 2021 ________________________________________ *corresponding author: nnazmy4@yahoo.com this is an open access article distributed under the terms of the creative commons attribution license (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 741 introduction rice (oryza sativa l.) is one of the important food crops that feed more than 50% of the world's population, (thuengtung et al., 2018). white rice is the most common type of rice that humans consume. however, ground rice serves as a major source of carbohydrates for your daily energy needs. brown rice offered additional health benefits (bassuony and el abed 2016). a comparison to white and brown rice, brown rice is a rich source of many bioactive compounds, such as γ-oryzanol, tocopherol, tocotrienol, amino acids, dietary fibers, and minerals. the advantages for health with the consumption of brown rice mainly come from the phytochemicals found in its bran layers (ravichanthiran et.al, 2018). consumption of brown rice is less than milled rice because it is more difficult to cook than milled rice due to slow absorption in water, and the palatability of brown rice is the lowest of milled rice. (ohtsubo et al., 2005), while the soaking process improves the nutrients in brown rice easily to be digested and brown rice texture is better. (wu et al., 2013). starch digestibility, the extent rate of starch hydrolysis by amylolytic enzymes. flours prepared from germinated grains were documented to obtain better nutritional values than those of ungerminated flours. grains germination has changes in enzyme activity and subsequent changes in composition. the production of germinated brown rice grain has gained big attention as a way to improve the eating quality and potential health-promoting functions of cooked brown rice (cornejo et al., 2015). accordingly, grain brown rice has become popular among health-conscious consumers due to its bioactive compounds (cho and lim, 2016). phytic acid is regarded as a potent inhibitor of minerals in plants. (raboy, 2003). phytic acid has also been reported to form stable complexes with proteins, which may result in decreased protein solubility, enzymatic activity, and proteolytic digestibility (ravindran et al., 1995). great efforts have been made to reduce the amount of phytate in foods through various processes, including the addition of exogenous enzymes. germi742 nation has been reported to reduce phytic acid and increase inorganic. it could thus improve the bioavailability of minerals in cereals (ghavidel and prakash, 2007). the consumption of germinated brown rice is increasing because of its improved quality of palatability and potential health functions (phattayakorn et al., 2016). several nutrients and total protein were increased while sugar was reduced in germinated brown rice compared with ungerminated brown rice (trachoo et al., 2006). germinated brown consumption rice is associated with human health-improving due to a big range of biological properties (sutharut and sudarat, 2012). the aim of this study is 1-study changes in total starch, sugars, and physicochemical properties as well as being affected by the germination process and comparing them with milled rice and brown rice. 2study the possibility of enhancing the antioxidant of germinated brown rice. 3-evaluating the possibility of increasing the antioxidant properties of germinated rice. materials and methods three rice varieties namely sakha 104, giza 178, and giza 182 were used in this study (table 1). the samples were used from freshly harvested grains of paddy rice at 14% moisture content from 2020 season planting. brown rice: was prepared by removing a husk of the ungerminated paddy rice using an experimental huller machine (satake). milled rice: brown rice was consequently milled using mcgill miller no. 2. the sample was milled for 60 sec. germinated brown rice (gbr): paddy rice (5kg) for all cultivars was soaked in tap water at room temperature for 24 h and water was changed every a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) no. cultivar parentage types origin 1 sakha 104 gz 4096/gz 4100 japonica egypt 2 giza 178 giza 175/millyang 49 japonica – indica egypt 3 giza 182 giza181/ir39422//giza181 indica egypt table 1. parentage, types, and origin of the three cultivars used under study. 743 7-8 h. all rice cultivars were distributed that soaked in baskets of plastic by cheesecloth covered and germinated in locker germination of for 48 h at 2830°c and 90-95% relative humidity. after germination, the germinated grains were dried at 50°c to approximately 10% of moisture content (fig. 1). the hulls, shoots, and roots were separated using an experimental huller machine (satake). the milled were prepared and brown rice and germinated brown were dehulled at the grain quality lab., rrtc. sakha, egypt (fig. 2). samples were taken in random sampling (three replications for three cultivars) completely randomized design in the factorial arrangement was used in this experiment. about 150 grams (three replication) of rough rice for all samples(brown rice and germinated brown) were taken and well mixed and cleaned for the gelatinization temperature, water uptake, hardness, and elongation % analyses. grind 10 whole milled rice grains of all samples to a fine powder to obtain rice flour and use in the analyzes of amylose content, chemical component, phytic acid, and total antioxidant capacity. cooking and eating quality characters i.e. gelatinization temperature, amylose content, and elongation % were estimated for rice samples following the methods of little et al., (1958). juliano (1971), azeez and shafi (1966) respectively. water uptake was calculated according to the procedure of singh et al.,(2005). hardness: the universal testing machine (utm) (model: lr five series, m / s llyod instrument, england) was employed to measure the hardness of raw rice chemical composition: flours were analyzed for the following chemical compositions protein content (nx 5.95), lipids content, ash content (%), crude fibbers content, and total carbohydrate content (%) following the method described by a.o.a.c. (1990). phytic acid: a method by wheeler and ferrel(1970). total antioxidant capacity: the total antioxidant capacity of the extracts was evaluated by the method of banerjee et al., (2005) using phosphor molybdenum reagent (3.3 ml sulphuric acid, 335 mg sodium phosphate, and 78.4 mg ammonium molybdate in 100 ml of distilled water) with 0.1ml of the extract. the absorbance of the samples was measured at 695 nm after boiling in a water bath for 95 ° c for 90 min against an empty reagent, which included the appropriate volume of the same solvent instead of samples. panel test evaluation: brown rice, milled rice, and germinated brown rice samples (1kg) were cooked and were served to a panel of 10 judges for evaluation. the cooking recommendation was to use the same water volume temperature and time of cooking. the samples were evaluated translucency and oder then after cooking for, kernel expansion, cooked kernel hardness, stickiness, odor, whiteness, expansion, hardness. stickiness and taste according to julino (1965). all collected data were presented for analysis of variance according to gomez and gomez (1984). treatments means by which duncan's multiple range test were compared (duncan, 1955). all statistical analysis was performed using variance technique using "mstat" software package. a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) fig. 1. rice cultivars (sakha 104, giza 178, and giza 182, respectively after germination. fig. 2. milled rice, brown rice, and germinated brown for three cultivars under study. 744 results and discussion analysis of variance (anova) data shown in table 2 represent the mean square (ms) of the sources of variance for characters under study. the analysis of the mean square showed there was a very highly significant difference among the cultivars for all characters evaluated except amylose content%, gelatinization temperature, ash (%), fiber, and fat (%), while the mean square estimates showed highly significant differences among the three statues for all characters under study. cultivars and statues interaction for the mean square was significant for all the characters except for fat (%), carbohydrate (%), and fiber. these findings indicated the presence of large variation among cultivars and statues under study. cooking and eating quality characteristics amylose content (%): the data shown in table 3 a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) source of variance d.f ms amylose content% gelatinization temperature (gt) elongation % hardness water uptake ash (%) genotype 2 0.046 0.083 38.27** 0.365** 1.63** 0.004 statues 2 0.177** 34.94** 2350.04** 3.098** 241.28** 0.018 * gxs 4 0.187** 0.535** 5.49** 0.357** 2.136** 0.16** error 18 0.023 0.037 1.73 0.036 0.153 0.003 source of variance d.f ms protein (%) fat (%) carbohydrate (%) fiber % phytic acid t. antioxidant capacity genotype 2 4.76** 0.046 2.650** 0.135 0.049** 1536.37** statue 2 4.38** 3.440** 2.374** 1.895** 0.056** 7600.31** gxs 4 0.36** 0.029 0.350 6.078 0.012** 293.96** error 18 0.02 0.024 0.321 0.045 0.02 0.675 table 2. anova analysis of characters under study performance. main effect amylose content % gelatinization temperature (gt) elongation % hardness n (kg·m/s) water uptake % cultivars sakha 104 giza 178 giza 182 17.32!0.208a 17.73!0.327a 17.53!0.302a 4.40!0.577a 4.21!0.552a 4.33!0.601a 28.43!4.931 b 31.42!4.720 a 27.47!4.404 b 4.10!0.142 b 4.14!0.096 b 4.46!40.293a 13.05! 1.603 a 12.35! 1.374b 13.13! 1.550a statue of rice white rice brown rice germinated brown rice 18.39! 0.096a 17.26! 0.042b 15.58!0.076 c 6.17! 0.067a 2.29! 0.094c 4.53! 0.124b 47.18! 0.647a 16.08! 0.375 c 24.56! 0.082b 3.64!0.091c 4.82! 0.164a 4.24!0.082 b 18.59! 1.82a 8.56! 0.189c 11.37! 0.264b table 3. effect of rice cultivars and statuses on some cooking and eating characters during 2020 season. data represent means ± se. values with the same letter in a column of the same cultivar are not significantly different (p < 0.05). 745 indicated some cooking and eating characters. the amylose content affected on the stickiness of cooked rice was similar in the three types under study and classifying it as low-amylose varieties. the amylose content was highly significant between the statuses under the study. germinated brown rice showed the lowest amylose content (15.58%) among all, followed by brown rice (17.26%) and white rice (18.39%). germinated brown rice had a reduction of amylose content % but gradually increased in brown rice then increase in white rice. gelatinization temperature and amylose content of germinated brown rice starch, when compared with brown starch, indicated that the germination process can indeed reduce the amylose content. (kaneko and morohashi,2003). similarly, charoenthaikij et al. (2009) had reported that germination increased not only the activity of amylose but also increased in reducing sugars in brown rice, supporting our findings of lower amylose content in germinated brown rice when compared with brown rice. similar findings have also been reported by other researchers (mohan et al., 2010). the amylose content in rice affects the softness and palatability of steamed rice (feng et al., 2017) and so its reduction in brown rice will bring about a softer texture. additionally, amylose content was reported to have a positive correlation with hardness and a negative correlation with stickiness after cooking (musa et al., 2011). it means that germination reduces the hardness of brown rice, while it increases its stickiness, indicating that germinated brown rice would be more acceptable than brown rice taking into consideration its amylose content. gelatinization temperature (gt): the gelatinization temperature of the rice samples has been classified as high, intermediate, and low which means the temperature required for normal cooking time is below 70-74°c.and no significant difference with the three rice varieties under study. it is evident from data shown in table 3 that there were significant differences in gelatinization temperature among the three rice statuses. brown rice gave the highest temperature followed by germinated brown rice then milled rice.the amylose content % and gelatinization temperature among types and statuses of rice were evaluated (fig. 3). elongation (%): in this study elongation of rice kernel% (table 3), maximum elongation ratio was observed in giza178 ( japonica indica) (31.42) followed by sakha 104 (28.43). no significant was found among sakha104 ( japonica), and giza182 (indica). (bassuony and el abed 2016). also, results in table (3) showed significant differences were detected for elongation ratio among statuses. white rice gave the maximum elongation %( 47.18). whereas brown rice recorded the minimum value (16.08 %). while germinated brown rice gave a medium value between them (24.56 %). hardness: japonica rice exhibited lower hardness than indica rice (table 3). these results suggest that the ultrastructure of rice affects the texture of the cooked product (kang et al 2006). there were significant differences among statuses for hardness. the a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) fig. 3. effect of rice cultivars and statuses under study on (a) amylose content% and (b) gelatinization temperature. ! " 746 strongest (4.82) was recorded at brown rice. while the weakest value (3.64) was found in white rice. the elongation% and hardness among types and statuses of rice were evaluated (fig. 4). water uptake: the water uptake ratio is a good indication of the volume expansion of rice (table 3). high significant was observed in the water uptake ratio, which ranged from 12.35 to 13.13. giza 182 variety produced the maximum water uptake (13.13), while giza 178 gave the minimum value (12.35). there were significant differences in water uptake due to the statuses of the study. white rice gave the maximum number value (18.59). while brown rice recorded the minimum value (8.56). while germinated brown rice gave a medium value between them (11.37). water uptake among types and statuses of rice was evaluated (fig. 5). chemical composition the indica rice variety giza181 had the highest protein and fat% in compared the other types under study (6.98) and 2.46 respectively), while the japonica rice variety sakha 104 gave (5.57 and 2.32) (table 4). ( kang et al 2006) recorded the same results, while japonica -indica giza 178 gave the medium value between them in protein content %. whereas no significant differences were detected between the japonica rice variety sakha 104 and japonica indica giza 178 in fat % table 4. data showed that (table 4) the germinated brown rice had the highest value of protein, crude fiber, and fat (7.27, 1.98, and 2.87%), respectively, compared with brown rice and white rice. brown rice had the highest value of protein, fat, ash, and crude fiber content compared with white rice elhissewy et al., (2002) reported that increasing milling caused a significant decrease in oil, protein, and ash may be due to most of the nutrients present in the outer layer of the brown rice grain which were removed during the milling process. it may be the metabolic activity of dry seed increases as soon as it is hydrated during soaking. complex biochemical changes occur in different parts of the seed during germination. because no external nutrients are added during the germination process, only water and oxygen are consumed by the germinating seed, a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) ! " fig. 4. effect of rice cultivars and statuses under study on (c) elongation% and (d) hardness. ! fig. 5. effect of rice cultivars and statuses under study on (e) water uptake. 747 desirable nutritional changes mainly stem from the decomposition of complex compounds to more simple forms, and their transformation into basic constituents (chavan and kadam, 1989). zheng et al.(2007) found that the bioavailability of proteins was improved by the changes in the storage proteins of brown rice during germination. during germination, the content of amino acids increased significantly. the germination conditions exhibited an effect on the changes in amino acid content, and that treatment was useful to the accumulation of a higher concentration of amino acids in germinated brown rice (fengfeng et al., 2013). concerning carbohydrate content, the data observed that white rice had the highest value (89.03 %), compared with germinated brown rice (88.22%). while the lowest value (87.83 %) was found by brown rice. this means that the germination process is caused by the decrease in carbohydrate values compared with white rice. the chemical composition among the types and statuses of rice was evaluated (fig 6). phytic acid and total antioxidants capacity phytic acid: data shown in table 5 illustrate phytic acid (%) and total antioxidant capacity (mg/100g) of cultivars under study. no significant difference was between white rice and germinated brown rice. data revealed that white rice had lower phytic acid (%) comparing with the other two statues under study followed by germinated brown rice, then brown rice, liang et al. (2008) pointed out that germination could decrease phytic acid levels in brown rice also they found that hydrolysis of a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) main effect protein (%) carbohydrate (%) fat (%) fiber % ash (%) cultivars (v) sakha 104 giza 178 giza 182 5.57!0.199c 6.54!0.358b 6.98!0.251a 88.88!0.332a 88.53!0.224a 87.75!0.091b 2.32!0.212 2.43!0.172 2.47!0.170 1.78!0.172a 1.54!0.137a 1.67!0.141a 1.033!0.024a 1.08!0.032a 1.06!0.024a statue of rice white rice brown rice germinated brown rice 5.47!0.204c 6.35!0.171b 7.27!0.292a 89.03!0.294a 87.83!0.257b 88.22! 0.131b 1.70! 0.656c 2.65! 0.067b 2.87!0.032a 1.15!0.053b 1.87!0.060a 1.98 !0.82a 1.011!0.015 a 1.05! 0.26ab 0.88!0.033a table 4. chemical composition of cultivars and statues under study in 2020 seasons. data represent means ± sd. values with the same letter in a column of the same cultivar are not significantly different (p < 0.05). cultivars (v) phytic acid (%) total antioxidant capacity (mg/100 g) cultivars (v) sakha 104 giza 178 giza 182 0.706!0.026a 0.598!0.048b 0.565!0.005 b 193.260!10.67a 165.158!9.92b 111.908!5.68c status of rice white rice brown rice germinated brown rice 0.549! 10.07b 0.705! 14.82a 0.615! 11.06ab 133.359!0.026b 189.298!0.033a 147.669!0.023b table 5. effect of some rice cultivars on phytic acid and total antioxidant capacity. data represent means ± se. values with the same letter in a column of the same cultivar are not significantly different (p < 0.05). 748 phytate can be obtained by activation of the endogenous phytase during soaking germination. (shallan et al., 2010) stated that the phytate content is lower in milled rice because of the separation of the bran layers throw the polishing process. more than 80% of the phytate in the rice grain is found in the bran and aleurone layers. total antioxidants capacity: japonica rice cultivar (sakha 104) has a significantly higher antioxidants level than indica rice cultivar (giza 182) (ding et al., 2018). no significant difference have been observed between indica rice (giza 182) and japonica a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) ! " # $ % fig. 6. effect of rice cultivars and statuses under study on the chemical composition (f) protein (%) (g) carbohydrate (%) (h) fat (%) (i) fiber % ( j) ash (%). 749 indica rice cultivars (giza 178) (table 5). the antioxidants can protect cells against oxidative damage, thereby reducing the risk of diseases associated with oxidative damage (shao and bao2015). brown rice contains the highest value of antioxidant followed by germinated brown rice, while the lowest level of antioxidant was recorded by white rice. brown rice grains are harder to chew and have fewer taste qualities. thus, pre-germinated rice is favored. it is also shown that pre-germinated brown rice increases mental health and immunity (ravichanthiran et.al., 2018). the phytic acid and total antioxidants capacity among types and statuses of rice were evaluated (fig 7). panel test data in (table 6) showed the palatability characters fig. 7. effect of rice cultivars and statuses under study on (k) phytic acid and (l) total antioxidant capacity. t ab le 6 . p al at ab il it y ch ar ac te rs o f w h it e, b ro w n , a n d g er m in at ed b ro w n r ic e u n d er s tu d y. t ot al 10 0 t as te s ti ck in es s h ar dn es s e xp an si on w hi te n es s o de r c oo ki n g t im e (m in ) b ef or e co ok in g r ic e/ w at er ra ti o r ic e cu lt iv ar s t ra ns . g ra in s ha pe g ra in le ng th s ak ha 1 04 c ul ti va r 78 g oo d (9 ) f lu ff y ( 9 ) b ro ke n (7 ) h al f ( 6 ) w hi te (6 ) n o (1 0) 20 -2 5 (6 ) t ra ns (8 ) m ed iu m (8 ) sh or t ( 8 ) 1: 1. 25 w hi te 57 a cc ep te d (5 ) f lu ff y (8 ) u nb ro ke n (1 0) q ua rt er (2 ) d ar k (2 ) m ed iu m (5 ) m or e th an 30 (3 ) t ra ns (6 ) m ed iu m (8 ) sh or t ( 8 ) 1: 1. 25 b ro w n 63 a cc ep te d (5 ) f lu ff y ( 9 ) u nb ro ke n (1 0 t hi rd (4 ) d ar k h al f ( 3) m ed iu m (5 ) 20 -3 0 (5 ) t ra ns (7 ) m ed iu m (8 ) sh or t( 8 ) 1: 1. 25 g er m in at ed b ro w n g iz a 17 8 cu lt iv ar 75 g oo d (9 ) f lu ff y ( 9 ) b ro ke n (7 ) h al f ( 6) w hi te (6 ) n o (1 0) 20 -2 5 (6 ) t ra ns (8 ) m ed iu m (7 ) sh or t 7) 1: 1. 25 w hi te 55 a cc ep te d (5 ) f lu ff y ( 8 ) u nb ro ke n( 1 0) q ua rt er (2 ) d ar k (2 ) m ed iu m (5 ) m or e th an 30 (3 ) t ra ns (6 ) m ed iu m (7 ) sh or t ( 7 ) 1: 1. 25 b ro w n 62 a cc ep te d (5 ) f lu ff y ( 9 ) u nb ro ke n( 1 0) t hi rd (4 ) d ar k h al f (3 ) m ed iu m (5 ) 20 -3 0 (5 ) t ra ns (7 ) m ed iu m (7 ) sh or t( 8 ) 1: 1. 25 g er m in at ed b ro w n g iz a 18 2 cu lt iv ar 73 g oo d (9 ) f lu ff y ( 9 ) b ro ke n (7 ) h al f (6 ) w hi te (6 ) n o (1 0) 20 -2 5 (6 ) t ra ns (8 ) sl en de r (6 ) l on g ( 6) 1: 1. 25 w hi te 53 a cc ep te d (5 ) f lu ff y ( 8 ) u nb ro ke n( 1 0) q ua rt er (2 ) d ar k (2 ) m ed iu m (5 ) m or e th an 30 (3 ) t ra ns (6 ) sl en de r (6 ) l on g ( 6) 1: 1. 25 b ro w n 61 a cc ep te d (5 ) f lu ff y ( 9 ) u nb ro ke n( 1 0) t hi rd (4 ) d ar k h al f ( 3) m ed iu m (5 ) 20 -3 0 (5 ) t ra ns (7 ) sl en de r (6 ) l on g ( 6) 1: 1. 25 g er m in at ed b ro w n ! " a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) of three rice cultivars under study and the three statuses to increase the nutritional value of the white rice. rice to water ratios was constant to all statuses at 1:1.25. translucency before cooking for a white status of all cultivars was the best then germinated brown rice, while brown rice was the lowest one due to having dark color resulted in bran. white rice recorded the lowest cooking time followed by germinated brown rice then brown rice for all cultivars. brown rice had a strong odor than germinated brown rice and white rice for all cultivars under study. the whiteness of rice after cooking is affected directly by brown rice and germinated brown rice, it's clear from data shown in table 6. volume expansion of white rice status gave the highest value as shown in table 6. followed by germinated brown rice and brown rice gave the lowest one for all cultivars under study. germinated brown rice and brown rice gave the highest values for hardness for all cultivars under study, white rice status was more stickiness than germinated brown rice and brown rice. germinated brown rice recorded the second two good taste (table 6) after white rice. conclusion germinated brown rice recorded the second two good tastes after white rice. so, this study recommended that we can increase the nutritional value of rice by germinated brown rice with little change in taste. references association of official analytical chemists (a o.a.c ) (1990) official methods of analysis association of official analytical chemists. washington. d.c., usa. azeez mh and m shafi (1966) quality in rice.teck . bull. no.13 dep. agric. govt west pakistan, p.so. banerjee a, n dasgupta, and b de (2005) in vitro study of antioxidant activity of syzygium cumini fruit. fd chem 90: 727– 733. bassuony nn and m a el abed (2016) cooking qualities evaluation for white and black rice mixture. plant production, mansoura university, 7 (2): 197-202. doi: 10.21608/jpp.2016.45253. charoenthaikij p, k jangchud, a jangchud, k piyachomkwan, p tungtrakulnd, and w prinyawiwatkul (2009) germination conditions affect the physicochemical properties of germinated brown rice flour. j. food sci. 74 (9): c658-c665. chavan jk and ss kadam (1989) nutritional improvement of cereals by sprouting. crit. rev. food sci. 28: 401–437. cho dh and st lim (2016) germinated brown rice and its biofunctional compounds. food chemistry 196: 259 –271. cornejo f, pj caceres., m villaluenga, c rosell, and j frias (2015) effects of germination on the nutritive value and bioactive compounds of brown rice breads. food chemistry 173: 298 – 304. ding c, q liu, p li, y pei, t tao, y wang, w yan, g yang, and x shao (2018) distribution and quantitative analysis of phenolic compounds in fractions of japonica and indica rice. food chem 15 (274): 384-391. doi: 10.1016/j.foodchem. duncan db (1955) multiple ranges and multiple f. test. biometrics 11: 1-24. el-hissewy aa, fr laila, and ad hanaa (2002) effect of degree of milling on the chemical composition and nutritional value of the milled rice. egypt j. agric. res. 80 (1): 341-353. feng, f, y li1, x qin , y liao and khm siddique (2017) changes in rice grain quality of indica and japonica type varieties were released in china from 2000 to 2014. frontiers in plant science j. 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lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) investigating soil microbiota for antimicrobial activity against safe relatives of eskape pathogens eunha kim, amanda o’besso, danielle e. graham, and justin w. graham* department of biological and forensic sciences, fayetteville state university, fayetteville, nc 28301, usa received: may 5, 2025 / accepted: july 16, 2025 __________________________________________________ * corresponding author: jwgraham01@uncfsu.edu 856 abstract according to the cdc, there are more than 2.8 million antibiotic resistant infections occurring in the united states each year, and more than 35,000 people die as a result (cdc 2019). furthermore, the cdc classifies a group of bacteria known as eskape pathogens as six emerging antibiotic-resistant pathogens that are difficult to eradicate with current antibiotics. our study aims to identify and characterize soil-derived microorganisms with the potential to produce antimicrobial compounds effective against safe relatives of eskape pathogens, with the goal of translating these findings to combat their pathogenic counterparts. we hypothesize that bacteria identified from the soil will inhibit the growth of the following nosocomial associated safe relatives bacillus subtilis for e. faecium, staphylococcus epidermidis for s. aureus, escherichia coli for klebsiella pneumoniae, acinetobacter baylyi for a. baumannii, pseudomonas putida for p. aeruginosa, and enterobacter aerogenes for enterobacter species. to test our hypothesis, soil samples were collected from fayetteville state university (fsu) campus and serially diluted onto lb agar plates. sixty-three distinct colonies were isolated and screened against non-pathogenic eskape safe relatives. of the 63 fayetteville state university soil isolates (fsis) screened, 12 (19%) exhibited antimicrobial activity against at least one of the six eskape safe relatives, with all 12 inhibiting acinetobacter baylyi and only fsi 15 demonstrating broad-spectrum inhibition. characterization assays revealed that 11 of the 12 isolates were gram-negative, catalase-positive, and motile; the single gram-positive isolate (fsi 4) was catalase-negative and non-motile. all isolates displayed resistance to penicillin, while most remained susceptible to tetracycline and ciprofloxacin. these findings support our hypothesis that soil-derived bacteria can produce putative antimicrobial compounds effective against non-pathogenic eskape safe relatives. this study underscores the potential of soil microbiota on the campus of fayetteville state university as a source of novel antimicrobial agents capable of inhibiting antibiotic resistant eskape pathogens and warrant further investigation into their therapeutic potential. keywords: microbiology; antibiotic resistance; eskape pathogens; soil. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. published in the united states of america https://www.atlas-publishing.org 1. introduction in 1943, dr. selman waksman a prominent microbiologist and biochemist, made groundbreaking discoveries in antibiotic development by exploring soil samples, leading to the identification of antibiotics like streptomycin which was a significant breakthrough in the treatment of tuberculosis. this was groundbreaking work that revolutionized the field of microbiology and pharmacology, particularly in the discovery of antibiotics derived from soil microorganisms (opimakh 2023 and baltz 2006). dr. waksman innovative techniques and insights into microbial competition allowed him to postulate that soil bacteria could secrete substances that inhibit other bacteria, thus opening a new avenue for antibiotic discovery (ribeiro da cunha et al., 2019; hughes and karlén, 2014). dr. wakman’s platform for screening bacterial extracts set a precedent that dominated the domain of antibiotic research for decades, leading to significant breakthroughs during the 1940s and 1950s when many of the major antibiotic classes were identified (hughes and karlén, 2014; lewis, 2013). however, as antibiotic resistance began to emerge, the novelty of discovering new antibiotics waned, leading to a significant decline in pharmaceutical interest in exploring soil-derived compounds. this indicated that although waksman’s discoveries were monumental, they also painted a complex picture of antibiotic development that continues to evolve today (lewis, 2013). currently, antibiotic discovery has stagnated, and antibiotic resistance continues to rise. globally in 2019, this issue is estimated to have claimed 1.27 million lives by antibiotic-resistant infections. to better understand the ecological and physiological traits of bacteria that may influence their antimicrobial capabilities, it is important to consider their fundamental cellular structures. bacteria can be broadly categorized as either gram-positive or gram-negative, each exhibiting unique characteristics that enable them to thrive in their respective environments. gram-positive bacteria are characterized by their thick peptidoglycan cell walls, which provide robust protection against environmental threats (wang et al., 2023; hessle et al., 2000). in contrast, gram-negative bacteria lack a substantial peptidoglycan layer but compensate with various virulence factors that enhance their adaptability (wang et al., 2023; tsuchido et al., 2019). the rise of antibiotic-resistant bacteria is a pressing global health crisis attributed to several connected factors (overuse and misuse of antibiotics in human medicine, veterinary practices, and agricultural settings) that exacerbate effective antimicrobial treatment challenges. eskape pathogens, which consist of enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter species, represent a significant challenge in modern medicine due to their capacity to evade conventional antibiotic treatments, leading to severe healthcare-associated infections (hais) with high morbidity and mortality rates (ramsamy et al., 2018; dinesh and karthick, 2018; li et al., 2022). the term “eskape” was first introduced in 2008 by rice, highlighting a group of multidrug-resistant (mdr) bacteria that are prevalent in clinical settings (alsharedeh et al., 2023; santajit and indrawattana, 2016). these organisms are identified as critical priority pathogens by the world health organization, underscoring the urgent need for effective therapeutic strategies and accounting for approximately two-thirds of all hais in the united states (ramsamy et al., 2018; dinesh and karthick, 2018). the emergence of resistance mechanisms in eskape pathogens is facilitated by biofilm formation, significantly enhancing their resilience against standard antimicrobial therapies (rizki et al., 2024). studies have demonstrated that biofilm-producing strains can remain viable on various surfaces for extended periods, contributing to increased transmission rates within healthcare environments (rizki et al., 2024; motiwala et al., 2022). moreover, their virulence factors further complicate treatment, as these pathogens often cause more severe outcomes in infected patients, including prolonged hospital stays and increased healthcare costs (marturano and lowery, 2019; li et al., 2022). the implications of their prevalence necessitate ongoing research and global collaboration to develop targeted interventions that can effectively mitigate their impact on healthcare systems. in this context, leveraging soil microbes presents a promising strategy for combating these formidable pathogens. soil microorganisms, particularly those residing in the rhizosphere, have been shown to exert significant antagonistic effects against plant pathogens, potentially providing insights into new biocontrol strategies against human pathogens. for instance, studies reveal that specific groups of soil bacteria such as oxalobacteraceae and burkholderiaceae can be particularly effective in suppressing the growth of pathogenic fungi in their environment (tian and gao, 2021). these findings suggest that the microbial communities in soil can be harnessed to develop natural products or biological agents capable of disrupting the growth and biofilm formation of eskape pathogens, which are notoriously resilient to conventional antibiotics due to their biofilm-associated resistance mechanisms (artini et al., 2023). recent research has identified antimicrobial compounds produced by environmental bacteria, including those derived from unique habitats, which exhibit effectiveness against eskape pathogens (artini et al., 2023; postich and kiser, 2018). this indicates that not only can soil microbes provide a reservoir for potential new antibiotics, but they can also contribute to biofilm disruption, which is critical for improving treatment outcomes against these pathogens (artini et al., 2023). furthermore, the utilization of microbial consortia that include beneficial soil bacteria may enhance their efficacy when applied to clinical settings, promoting a synergistic action against infections involving eskape bacteria (mulani et al., 2019). the integration of soil microbes into clinical therapies could also stem from their natural adaptive mechanisms, which facilitate the development of resistance against environmental challenges, making them potentially valuable in overcoming the multidrug resistance exhibited by eskape pathogens (yi et al., 2021). moreover, soil microbes could complement existing treatment modalities by offering novel mechanisms of action such as the production of bioactive compounds and metabolites that target the unique molecular pathways of these bacterial pathogens (pliego et al., 2008). soil bacteria have emerged as a novel frontier in the battle against eskape pathogens, which are notorious for their multidrug resistance and virulence. this strategy is based on the unique capabilities that certain soil bacteria exhibit, including the production of antimicrobial compounds, competition with pathogenic bacteria, and the potential to disrupt biofilm formation. bacteria residing in soil, particularly those with plant growth-promoting properties, can synthesize a variety of antimicrobial agents. for instance, pseudomonas species, known for their ecological versatility, produce secondary metabolites such as phenazines and antibiotics, which have been shown to exert inhibitory effects on eskape pathogens like pseudomonas aeruginosa and staphylococcus aureus (artini et al., 2023). these natural products can inhibit bacterial growth and may also interfere with cellular processes critical for pathogenicity, thus mitigating the virulence of these pathogens (artini et al., 2023). moreover, soil bacteria actively engage in microbial antagonism, a phenomenon wherein beneficial microbes compete with pathogens for resources and habitats. this competitive dynamic can suppress the growth of eskape pathogens in contaminated environments. for example, the introduction of specific soil microbial communities into clinical settings might enhance biocontrol measures against persistent pathogens through direct competition and the production of inhibitory substances (mulani et al., 2019). mining natural genetic diversity among soil bacteria has unlocked potential new antimicrobial compounds to combat eskape pathogens. advances in genomics and metagenomics allow for the identification and characterization of unique biosynthetic gene clusters in soil microbes, which may lead to the discovery of new drugs or bioactive molecules that specifically target eskape pathogens (marie et al., 2024). these bioactive compounds could include novel antibiotics or inhibitors of critical resistance determinants, offering new avenues for therapeutic intervention. to our knowledge, this is the first study conducted on the fayetteville state university campus aimed at isolating soil-derived bacteria as a potential source of novel antimicrobial compounds. through this work we hope to identify microorganisms capable of producing antimicrobial agents effective against non-pathogenic safe relatives of eskape pathogens, including bacillus subtilis (safe relative of e. faecium), staphylococcus epidermidis (s. aureus), escherichia coli (k. pneumoniae), acinetobacter baylyi (a. baumannii), pseudomonas putida (p. aeruginosa), and enterobacter aerogenes (enterobacter spp.). these non-pathogenic surrogates provide a safe and effective initial screening platform, serving as a starting point for identifying antimicrobial-producing isolates prior to testing against clinically relevant eskape pathogens. this study contributes to the growing body of undergraduate-driven research focused on local microbiomes as untapped reservoirs of antibiotic discovery. 857 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) 2. materials and methods 2.1. strains and growth media soil samples were obtained from the campus of fayetteville state university on june 22, 2023, from topsoil. one gram of soil was resuspended in 9 ml distilled h2o (dh2o), homogenized, and serially diluted in dh2o. 100 µl was spread plated onto luria broth (lb) solid media (bd difco) with 1.5% w/v agar (bd difco) and 25µg/ml cycloheximide (fisher scientific). single colonies were picked and streaked for isolation. the following safe relatives of the eskape pathogens were used for this study: bacillus subtilis (atcc 6051), staphylococcus epidermidis (14990), escherichia coli (atcc 11775), acinetobacter baylyi (atcc 33305), pseudomonas putida (atcc 23467), and enterobacter aerogenes (atcc 51697). all strains were cultured at 28°c on lb agar. incubating at 28°c instead of 37°c reduces the risk of growing potential pathogens and helps promote greater microbial diversity by preventing fast-growing bacteria from outcompeting slower-growing soil species. lb agar is used in experiments to maintain consistency across samples, support the growth of control strains, and provide a standardized baseline for enumerating culturable bacteria. 2.2. soil collection all soil samples were collected from the campus of fayetteville state university. sampling sites were selected near vegetated and/or moist areas to maximize microbial diversity, and included coordinates such as 35.0742° n, 78.8925° w; 35°04’33.4” n, 78°53’19.4” w; and 35.069141° n, 78.891485° w. all soil samples had a ph range between 6.0 and 7.0. soil samples were collected using a sterile metal scoop to dig no more than 6 inches below the surface. this depth corresponds to the topsoil, which is the most fertile layer and contains most of the soil’s nutrients. to maximize microbial diversity, dark soil rich in organic material was targeted, particularly near plant roots and areas with moisture. approximately 5–10 grams of soil were transferred into sterile 50 ml conical tubes. samples were stored at 4°c to preserve microbial viability. all samples in this study were processed within 24 hours of collection. 2.3. selection of soil isolates the soil harbors a diverse collection of microbial biodiversity that is rich and dynamic. to best capture a representative sample of this diversity, 63 isolates were selected in this study based on macroscopic characteristics, including size, color, form, margin, elevation, and surface. table 1 contains characteristic information for the 12 fayetteville state isolates (fsi) that were used throughout the study. 2.4. macconkey agar test macconkey agar plates were prepared according to the manufacturer’s instructions (mp biomedicals™). using sterile flat toothpicks, isolates were patched onto the agar surface in a square grid pattern and incubated overnight at 28°c. macconkey agar is a selective and differential medium that inhibits the growth of gram-positive bacteria through the presence of crystal violet and bile salts, which disrupt their cell walls. it selectively supports the growth of gram-negative bacteria and differentiates them based on their ability to ferment lactose. a ph indicator in the medium detects acid production from fermentation, causing lactose-fermenting colonies to appear pink/red. the resulting colony color and growth pattern were recorded the following day to determine gram identity and fermentation capability. 2.5. gram stain test isolates were grown overnight on lb agar plates at 28°c. a single pure colony was selected and transferred to a clean microscope slide containing a drop of dh₂o. the resulting bacterial smear was air-dried and heat-fixed by passing the slide briefly through the flame of a heat source. the smear was then stained with crystal violet for one minute, followed by rinsing with dh₂o. gram’s iodine was applied for one minute to fix the primary stain and subsequently rinsed off with dh₂o. decolorization was performed using 95% ethanol for 10 seconds, after which the slide was immediately rinsed with dh₂o. a counterstain of safranin was applied for one minute and rinsed off with dh₂o. the stained slides were blotted with bibulous paper and examined under a light microscope using oil immersion. cell color and morphology were observed and recorded to determine gram reaction and cellular characteristics. 2.6. catalase test to assess catalase activity, isolates were grown overnight on lb agar at 28°c. a single pure colony was selected and emulsified in a drop of dh₂o on a clean microscope slide. a drop of 3% hydrogen peroxide (h₂o₂) was then applied directly to the bacterial suspension using a sterile dropper. the presence of catalase was indicated by immediate bubble formation, resulting from the enzymatic breakdown of h₂o₂ into water and oxygen gas. this assay indirectly screens the presence of catalase; a key enzyme associated with the ability of bacteria to detoxify reactive oxygen species. 2.7. motility test motility agar plates (0.5% agar; carolina biological) were prepared according to the manufacturer’s instructions and poured into sterile petri dishes, then allowed to cool to a semi-solid consistency. using a sterile inoculation wire, a single pure bacterial colony from each isolate was aseptically transferred and stab-inoculated into the center of the motility agar. plates were incubated at 28°c for 24 hours. a qualitative analysis of bacterial motility was documented based on the presence or absence of diffuse growth radiating from the stab site. in addition, a quantitative assessment was performed by measuring the diameter of outward growth in millimeters, conducted in triplicate, and reported as the mean ± standard deviation. 2.8. antibiotic resistance test antibiotic susceptibility of bacterial isolates was assessed using the kirbybauer disk diffusion method. briefly, 1 ml of lb broth was transferred into a sterile 2 ml microcentrifuge tube. a sterile cotton swab was used to collect a single bacterial isolate colony from an overnight culture plate and was then submerged into the lb broth to create a turbid suspension. the same swab was used to uniformly streak the suspension across the entire surface of an lb agar plate to establish a bacterial lawn. using sterile tweezers, antibiotic-impregnated disks were placed gently onto the surface of the agar and lightly pressed to ensure contact. antibiotics tested included ciprofloxacin (5 µg), neomycin (30 µg), chloramphenicol (30 µg), penicillin (10 µg), streptomycin (10 µg), and tetracycline (30 µg). a blank disk was included on each plate as a negative control. this procedure was repeated in triplicate for each isolate. plates were incubated at 28°c for 24 hours. zones of inhibition were measured in millimeters (mm) and recorded. interpretations of susceptibility were based on the clinical and laboratory standards institute (clsi) guidelines, where isolates were categorized as sensitive (≥ 18 mm), intermediate (14 – 17 mm), or resistant (<13 mm). 858 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) surface textureelevationmarginform colorsize isolate smoothraisedentirecircularlight yellowmediumfsi 2 smoothraised entirecircular yellowsmallfsi 3 smoothraisedentirecircularwhitemediumfsi 4 dryflatentireirregularwhitemediumfsi 7 smoothconvexentirecirculardark yellowsmallfsi 8 wrinkledraised entirecircularwhitelargefsi 9 smoothraised entireirregularwhitelargefsi 10 smoothraised entirecircularyellowlargefsi 11 smoothconvexentirecircularyellowsmallfsi 12 wrinkledraised entirecircularlight yellowsmall fsi 13 smoothflatentirecircularwhitemediumfsi 14 dryraisedentirecircularwhitelargefsi 15 table 1. colony morphology characteristics of fsis. colony morphology of antimicrobial-producing fsis was assessed based on visual characteristics observed after incubation on lb agar. parameters recorded include colony size, color, form, margin, elevation, and surface texture. observations were made following 24-hour incubation at 28 °c. 859 3. results 3.1. soil isolate activity against eskape safe relative strains soil samples were collected from the campus of fayetteville state university, a historically black college & university (hbcu) located in fayetteville, north carolina. founded in 1867, the university sits on a 156-acre campus that is rich in biodiversity, with an abundance of trees and a small river that runs immediately adjacent to the grounds. these natural features contribute to a variety of microhabitats, increasing diversity of soil microorganisms present. we predict that this environment serves as a reservoir of potentially beneficial soil bacteria that can be utilized to inhibit the safe eskape relatives tested in these experiments. to investigate the potential of antimicrobial molecules producing soil microbiota, soil samples were collected from different locations around fayetteville state university’s campus using 50 ml conical tubes. from each sample, one gram of soil was measured, and serial dilutions from 10-1 to 10-7 were a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) performed to obtain countable and retrievable bacteria colonies. the dilutions are spread onto lb agar plates. to reduce fungal contamination and enrich for bacterial growth, 25 µg/ml of cycloheximide, an antifungal agent, was added to the media. a total of 63 colonies were selected from the serial dilution plates based on morphological characteristics and patched onto fresh plates to allow for individual growth before further testing. after overnight incubation, the 63 fayetteville state isolates (fsis) were screened for antimicrobial activity against the six safe relatives of eskape pathogens to determine their ability to inhibit bacterial growth. this screening was performed in triplicate to ensure reproducibility. for each assay, the safe relative was spread as a lawn across the surface of an agar plate, and fsis were patched on top in a square grid using a sterile flat toothpick. plates were incubated overnight at 28°c and examined the following day. inhibition was indicated by the presence of a clearing zone (zone of inhibition) surrounding the patched colony (figure 1a). of the 63 fsis tested, 12 isolates (19%) exhibited antimicrobial activity against one or more of the six eskape safe relatives, as shown in the stacked bar chart (figure 1b). zone of figure 2. cell wall characterization of fsis using macconkey agar and gram staining. (a) growth of fayetteville state isolates (fsis) on macconkey agar. growth indicates gram-negative bacteria; lack of growth suggests gram-positive identity. all isolates that grew were non-lactose fermenters, as evidenced by the yellowish coloration of colonies. (b) gram staining results for each fsi. samples were stained using standard procedures. microscopy was performed at 100× magnification under oil immersion. pink-stained cells indicate gram-negative bacteria, while purple-stained cells confirm gram-positive status (fsi 4). scale bar in fsi 9 represents 100.00px and applies to all images. figure 1. inhibition of eskape safe relatives by fsis. (a) representative images of zone of inhibition assays showing antimicrobial activity of fsi strains against safe relatives of eskape pathogens. clear halos indicate bacterial growth inhibition. (b) stacked bar chart displaying the number of safe relatives inhibited by each of the 12 active fsis. (c) table presenting the mean diameter of inhibition zones (in millimeters ± standard deviation) for each fsi against six eskape safe relatives. all assays were conducted in triplicate and incubated at 28 °c for 24 hours. nz = no zone of clearance observed. 860 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) inhibition measurements for each isolate, recorded in triplicate, are reported as mean ± standard deviation in figure 1c. notably, acinetobacter baylyi was inhibited by all 12 active isolates, suggesting it may be particularly susceptible to antimicrobial compounds produced by these soil bacteria. fsi 15 was the only isolate to exhibit broad-spectrum activity, producing zones of inhibition against all six safe relatives. additionally, fsi 3, fsi 9, fsi 12, and fsi 14 demonstrated inhibitory effects against five out of six safe strains, indicating strong and diverse antimicrobial potential. these results not only highlight the variability in antimicrobial profiles among soil-derived isolates but also emphasize the promising potential of certain isolates, such as fsi 15. 3.2. phenotypic characterization of soil isolates after identifying fsi strains that inhibited the eskape pathogen safe relatives, a series of phenotypic characterization assays were performed to further analyze each individual isolate. to determine cell wall differentiation and lactose fermentation capability, macconkey agar was used as a selective and differential medium for streaking each isolate. eleven of the twelve fsi isolates were gram-negative and non-lactose fermenting. fsi 4 did not grow on macconkey agar, indicating that it is likely gram-positive (figure 2a). to confirm the results obtained from macconkey agar, gram staining was performed on all isolates. the staining results were consistent with observations from the selective media. microscopic examination revealed that isolates were either rodor cocci-shaped and exhibited gram-negative staining characteristics. only one isolate, fsi 4, was identified as gram-positive (figure 2b). catalase activity was assessed by applying hydrogen peroxide (h2o2) to bacterial cultures to detect the presence of the catalase enzyme, which breaks down hydrogen peroxide into water and oxygen; the release of oxygen gas results in visible bubble formation, indicating a catalase-positive reaction. eleven isolates were catalase positive, and fsi 4 was catalase negative (figure 3a). motility was assessed using soft agar plates containing 0.5% agar, a lower concentration than standard solid media, which creates a semi-solid consistency that allows motile bacteria to move through the medium. motile isolates exhibited diffuse, outward growth from the point of inoculation, while non-motile isolates remained confined to the original inoculation site (figure 3b). average motility distances, measured in millimeters, indicated that 11 of the 12 isolates exhibited motility, as evidenced by outward diffusion from the inoculation site. only fsi 4 was non-motile (figure 3b). figure 3. catalase activity and motility assays of fsis. catalase test results. representative images show catalase-negative (left; no bubbling) and catalase-positive (right; oxygen bubble formation) reactions upon exposure to hydrogen peroxide. catalase activity for each fsi is denoted as (+) for positive and (–) for negative. (b) motility assay results. representative images depict non-motile (left; confined growth) and motile (right; diffuse outward growth) phenotypes in soft agar (0.5%). the table below shows motility diameters measured in millimeters (mean ± standard deviation) for each fsi. nm = non-motile. all assays were performed in triplicate and incubated at 28 °c for 24 hours. figure 4. antibiotic susceptibility profiles of fsis. representative image of the kirby-bauer disk diffusion assay showing inhibition zones around antibiotic-impregnated disks. (b) zone of inhibition diameters (mean ± standard deviation, in millimeters) for each fsi tested against six antibiotics. interpretive breakpoints are based on clinical and laboratory standards institute (clsi) guidelines: sensitive (s) ≥ 18 mm, intermediate (i) = 14–17 mm, resistant (r) < 14 mm. all tests were conducted in triplicate and incubated at 28 °c for 24 hours. 861 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) 3.3. evaluation of antibiotic resistance in soil isolates testing antibiotic susceptibility is critical in contemporary research due to the increasing prevalence of antibiotic-resistant bacterial infections. antibiotic susceptibility testing (ast) plays a vital role in determining the effectiveness of specific antibiotics against bacterial pathogens. this is especially important in the context of the global health crisis posed by multi-drug resistant bacteria, which complicate treatment strategies and exacerbate morbidity and mortality rates associated with bacterial infections (shafrin et al. 2022 and tuite et al. 2017). to evaluate the antibiotic susceptibility profiles of the fsis, each isolate was tested against six antibiotics: ciprofloxacin, neomycin, penicillin, streptomycin, tetracycline, and chloramphenicol. for this assay, each fsi was streaked as a lawn on agar plates, followed by placement of antibiotic-impregnated disks. susceptibility was determined by the presence of a zone of inhibition surrounding the disk, indicating bacterial sensitivity; the absence of a zone indicated resistance (figure 4a). the average zone of clearance of antibiotic susceptibility for each isolate, in triplicate, is shown in figure 4b. several key findings emerged from this analysis. all fsis were resistant to penicillin, consistent with widespread natural resistance to this antibiotic among environmental bacteria. in contrast, all fsis were susceptible to tetracycline and ciprofloxacin. tetracycline targets protein synthesis, while ciprofloxacin inhibits bacterial dna gyrase; both antibiotics are commonly effective against gram-positive and gram-negative bacteria. notably, only fsi 15 was resistant to neomycin. streptomycin showed no inhibitory effect on fsi 2, 3, and 15. additionally, resistance to chloramphenicol was observed in fsi 2 and fsi 3 (figure 4b). 4. discussion in this study, 12 soil isolates from the campus of fayetteville state university (fsu) were found to inhibit at least one eskape safe relative, out of a total of 63 soil isolates tested, for a rate of 19%. previous studies have reported rates of about 8% to 12% of soil samples containing antimicrobial-producing organisms (valderrama et al, 2018). this rate is slightly higher than the average reported in previous studies, suggesting that the fsu landscape, which includes diverse plant life, moist soils, and relatively undisturbed microhabitats, may serve as an ideal environment for the discovery of putative antimicrobial-producing soil microbiota. fsu sits on a 156-acre campus and features a rich diversity of native trees and is bordered by a small river, creating a variety of microhabitats. this diverse and relatively undisturbed landscape likely supports a wide range of microbial communities. as such, educational campuses like fsu may represent underexplored reservoirs of antimicrobial-producing microorganisms, offering unique opportunities for bioprospecting in non-traditional environments. several fsis inhibited more than one safe relative, suggesting that these isolates may produce multiple antimicrobial compounds and/or a broad-spectrum agent. most of the fsis were gram-negative, consistent with reports that soil ecosystems enriched by organic matter, land use, and nutrient availability often support gram-negative bacterial populations. literature suggests that gramnegative bacteria tend to flourish in certain nutrient-rich contexts characterized by their metabolic efficiency in utilizing simpler substrates, often proliferating in nutrient-rich environments due to physiological adaptations, such as the presence of an outer membrane and periplasmic space, which aid in their survival against antimicrobial agents (shirakawa et al 2019 and kumar et al 2023). a correlation was observed among gram-negative identity, catalase activity, and motility. specifically, 11 of the 12 isolates demonstrated all three of these phenotypic traits. catalase activity indicates the presence of the enzyme catalase, which catalyzes the decomposition of hydrogen peroxide (h2o2) into water and oxygen. this may reflect an adaptive advantage conferred by catalase activity, enabling resistance to oxidative stress. this trait is especially beneficial in environments with high levels of reactive oxygen species (ros), such as those encountered in microbial competition or host immune responses (masued et al 2024). motility was observed in the same 11 isolates that were both gram-negative and catalase positive. motility contributes to survival in dynamic or unstable soil environments. this trait enables bacteria to migrate away from hostile conditions or toward more favorable microenvironments, further supporting their adaptability and ecological success. given this pattern, a future hypothesis to test is whether environmental gram-negative bacteria that are catalase positive and motile are more likely to exhibit antimicrobial activity due to enhanced stress tolerance and competitive fitness. prior studies have reported that motility and catalase production often co-occur in soil and rhizosphere-associated gram-negative bacteria, particularly those involved in plant-microbe interactions or secondary metabolite production (zhang et al., 2018; lugtenberg and kamilova, 2009). testing this hypothesis could help clarify whether this shared phenotype is predictive of antimicrobial potential in soil bacterial communities. despite their activity against safe relative of eskape pathogens, majority fsis remained susceptible to several commercially available antibiotics, with the exception of universal resistance to penicillin which is consistent with natural resistance commonly seen among environmental bacteria. this intrinsic resistance raises important considerations for future therapeutic applications, as there is potential risk for horizontal gene transfer of resistance determinants to pathogenic bacteria, which could complicate their use in drug development. fsi 2 and fsi 3 showed resistance profiles that suggest they may be useful in future studies exploring synergistic interactions with streptomycin and/or chloramphenicol. fsi 15 also exhibited patterns of interest for potential combination with neomycin and/or streptomycin. while these findings are preliminary, they offer a foundation for further investigation into how soil-derived isolates might enhance or complement antibiotic therapies. this study is not without limitations. antimicrobial activity was assessed only against non-pathogenic safe relatives of eskape pathogens, which do not fully replicate the resistance mechanisms or virulence of clinical isolates. as such, these results should be interpreted cautiously and not assumed to indicate therapeutic potential against true eskape pathogens. therefore, it is premature to conclude that these isolates produce novel antimicrobial agents, as the bioactive compounds responsible for the observed inhibitor activity have not yet been isolated, identified, or chemically characterized. additionally, molecular identification of the isolates using 16s rrna gene sequencing has not yet been conducted, which limits taxonomic classification at this stage. future work will involve testing the most promising fsis against clinically relevant, multidrug-resistant eskape pathogens to assess whether the observed inhibitory activity extends to pathogenic strains. additionally, studies will focus on the extraction, purification, and chemical characterization of the active compounds, followed by mechanism of action analysis. 16s rrna gene sequencing will also be conducted to identify the antimicrobial-producing isolates at the genus level. lastly, evaluating potential additive or synergistic interactions between bacterial isolates and commercially available antibiotics may provide insight into how such combinations could be leveraged in the fight against antibiotic resistant infections. acknowledgements we thank fayetteville state university for granting permission to survey and collect soil samples on campus. we also extend our appreciation to our colleagues for their valuable feedback and assistance in proofreading the manuscript. this work was supported by the burroughs wellcome fund student stem enrichment program (ssep) award (to jwg) and national science foundation award 2000260 (to deg). we are grateful to the students who contributed to data collection and analysis, and we acknowledge the support of the department of biological and forensic sciences. references alsharedeh rh, a yehya, ob yonis, o alameri, and n alshraiedeh (2023). characterization of eskape pathogens in urinary tract infections among jordanian patients. the journal of infection in developing countries 17(03): 374-380. artini m, r papa, g vrenna, m trecca, i paris, c d’angelo, et al. 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(2021). nanostructured copper surface kills eskape pathogens and viruses in minutes. chemmedchem 16(23): 3553-3558. zhang q, x gao, y ren, x ding, j qiu, n li, f zeng, and z chu (2018). improvement of verticillium wilt resistance by applying arbuscular mycorrhizal fungi to a cotton variety with high symbiotic efficiency under field conditions. int. j. mol. sci. 2018, 19, 241. atlas journal of biology 2016, pp. 236–248 doi: 10.5147/ajb.2016.0138 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) history of mirabilis expansa (ruiz and pav.) standl.; growth and use in the andes miriam kritzer van zant department of plant, soil and agricultural systems, southern illinois university, carbondale, il 62901, usa received: april 9, 2016 / accepted: may 2, 2016 __________________________________________________ * corresponding author: mkvzant@siu.edu 236 abstract several documents concerning the history and growth of mirabilis expansa (ruiz and pav.) standl. (nyctaginaceae), were found to be available by 2012 on international library and agency websites from south america. these documents, written in spanish, had previously been difficult for this author to find in north america. information in these documents and from additional papers and reports referenced in them, made it possible to summarize here what had been known historically about m. expansa. modern research in andean universities and agencies, completed but not seen by us prior to doing our research on m. expansa in southern illinois, is included in the summary. several points are covered in this material, including ideas on the origin of the crop, a list of indigenous names for the crop, and general descriptions of the appearance of several unnamed crop morphotypes grown in peru, ecuador and bolivia. this information is considered relative to the appearance of and growth data from two crop morphotypes grown in southern illinois. additionally, there is information from the south american documents on ecological considerations for m. expansa in latin america, germplasm information, how the crop has been grown traditionally, preparation of roots and stems for consumption including reduction of astringency and use in traditional dishes and for animal feed, and use of the leaves for fodder. brief consideration is given here to invasive and rare mirabilis spp. and how this relates to introduction of m. expansa into new locations, along with information on disease and pests known for the crop. discussion includes our considerations for conservation of the crop, and comparison of andean crop yields with published yield data for comparable crops grown in the united states. introduction m. expansa (ruiz and pav.) standl. is a rare andean crop plant grown for food and fodder in the highlands of peru, bolivia, and ecuador. however, it is disappearing from cultivation in the andes. the goal of this paper, is to present information that preceded our own research on the crop, and which may prove valuable for utilizing m. expansa in alternative cropping systems in the future. there were four objectives met in this paper. first was presentation of all information that we could find published on the crop, prior to our own research with the crop in southern illinois (kritzer van zant, 2016a,b), in one document and in english. our second objective was to draw comparisons with two morphotypes of the crop used in our research in southern illinois, with the m. expansa morphotypes described though unnamed in the previous literature. morphotypes were not specified for most of the information presented in this paper. information here is given for the crop at the species level except where otherwise stated. descriptions of the morphotypes used in latin america and in southern illinois are based on the language used to described morphotypes in the earlier literature. for the third objective, information is added on whether or not there is a potential for the crop to become invasive, based on what is known about it and other mirabilis spp. (kritzer van zant, 2016a,b). the fourth objective is to show how the crop’s root and forage yields compare with published information for similar uses of other crops and rangeland in the united states. specifically, m. expansa root yields per hectare in the andes (rea, 1994) are compared with yields for potatoes grown in idaho (olsen, 2004). in addition, forage yields per hectare for m. expansa in the andes (seminario, 2000c, 2004b) are compared to forage yields from improved alfalfa in california (putnam et al., 2007), and from unimproved rangeland in utah (perry, 2012). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 237 this crop is considered by us to be a subspecies of the wild type of m. expansa, based on kritzer van zant’s monograph of andean mirabilis, in preparation for publication. detailed descriptions and precise latin names for the two morphotypes that we have worked with in southern illinois will be given in the monograph on the andean species. for the present, the first initial of each intended name is utilized at the level of variety, var. ‘l’ and var. ‘t’. upon beginning field work on growing this endangered indigenous crop, little information was available to us, even after an extended period of taxonomic research on andean mirabilis. several attempts were made by us earlier, to recover information about research in the andes. some of this work had been mentioned to us by jaime estrella (estrella, personal communication, 1999), an andean researcher associated with centro internacional de la papa (cip), based in lima, peru. carlos ochoa had also mentioned to us unpublished reports on m. expansa at cip. now it is clear that in the 1980’s, renewed interest in preserving and understanding a number of indigenous andean root crops resulted in research on m. expansa (tapia, 1990; seminario, 1993, 2000a, 2000b, 2000c, 2004a, 2004b; cip, 1998; lizárraga, 2000, 2004), primarily under the auspices of cip. these documents, and certain other south american documents addressing research on m. expansa, have only recently become available in north america. this increase in availability is thanks to the practice of international university and research libraries digitizing much of their holdings and placing them on the internet. this has made in-house agency documents and others with limited circulation, easier to acquire by other libraries and individuals. availability of these particular documents on the internet came to our attention in 2012. further, these documents also brought to our attention, through their bibliographies, applicable papers by others. some of these secondary sources were perhaps available earlier, but their meaning for research described in kritzer van zant (2016a) had previously been obscured. therefore, results applicable to m. expansa, from cip researchers and their collaborators are incorporated here along with information from the limited number of publications on these topics earlier known to us. we also include some of our observations from our own research in the results, and offer our opinions on the implications of some of this research in discussion. methods most of the documents on the growth and use of m. expansa were written in spanish. to summarize them required translation into english by this author (rea and león, 1965; rea, 1982; tapia, 1990; seminario, 1993, 2000a, 2000b, 2000c, 2004a, 2004b; cip, 1998; seminario and valderrama, 2004; túpac yupanqui, 2000; lizárraga, 2000, 2004; valderrama et al., 2004; vásquez et al., 2004). though few of the internet-posted documents concerning m. expansa obtained by us from 2012 on, were published in peer reviewed academic journals, they include bibliographies which led us to other documents (rea and león, 1965; rea, 1982; izquierdo and roca, 1998; tapia, 1990, 2007) on m. expansa. these include papers published in academic journals. this review article examines this combined content and some of its implications. results origin of m. expansa julio rea studied the crop in peru, bolivia, and ecuador. rea concluded that the cajamarca region of peru is the center of distribution and the origin source of mauka for bolivia and ecuador, spread by human migration during the incan empire (rea and león, 1965; rea, 1982). this origin theory had been supported by the national research council of the united states since 1989 (popenoe et al., 1989; seminario, 1993). after studies of several traditional root crops during the 1980s, the origin theory for m. expansa was expanded. the place of origin of mauka in peru may incorporate a region including ancash, la libertad, cajamarca and amazonas (seminario, 2000a). venezuela and chile also have wild populations of m. expansa, but little exploration has occurred to find the crop in either nation, perhaps because botanical explorers there were interested in wild plants, while what is known about the crop for bolivia, ecuador and peru was discovered by persons looking for agronomic information (seminario, 1993). indigenous names for m. expansa m. expansa is known by numerous indigenous names including: miso and tazo in ecuador; mauka in bolivia and peru; and achagu, chago, chagos, chaco, shago, cushpe, cushpín, cushpenes, siguas, arricón, arracacha de toro, yuca inca, yuquilla, camotillo, pega-pega (beat-beat), rabanito, shalca yuca and yuca de jalca, in a variety of indigenous languages and dialects (popenoe et al., 1989; tapia, 1990; sperling and king, 1990; seminario, 1993, 2000a, 2000c, 2004b; flores et al., 2003; ugent and ochoa, 2006). rabanito is also an indigenous name for cultivated radish, raphanus sativus l. (brassicaceae) (seminario, 1994, 2000c). adjectives for color are often part of common names for varieties of the crop, added to the local name for the crop as a whole (seminario, 1994, 2000c, 2004b). a long list of common names for this crop indicates a long-term relationship between m. expansa and humans (seminario, 1994, 2000c, 2004b). apichu, the quichua name for the m. expansa crop, is more often applied to camote (ipomea batatas (l.) lam. (convolvulaceae) (seminario, 2000c, 2004b). duel use of names like rabanito and apichu reflect how m. expansa can be confused or lumped with other roots, and like it’s cousin, m. jalapa l., mistaken as a member of the family convolvulaceae (kritzer van zant, 2016b). wild mauka in bolivia can be called: llaki llaki, mauka, and sacha mauka (seminario, 2000c, 2004b). in northern peru, wild mauka is known as camotillo, pega pega, pegajera, or papelilla (seminario, 2000c, 2004b). in ecuador, the wild type is called moradilla or pega pega (seminario, 2000c, 2004b). names for m. expansa vary with particular ethnic groups and populations (seminario, 2000c, 2004b). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 238 appearance of m. expansa in the andes and of andean material grown in southern illinois bolivian roots have been reported as being salmon-colored (rea and león, 1965; popenoe et al., 1989; tapia, 1990). yellow and white roots are also reported from bolivia, growing at altitudes of 3200-3500 m (rea, 2000). roots can also be yellow to cream-colored (seminario, 1993). new collection of mauka from cajamarca, peru, was reported by seminario (1993) from 1984 along with donations of a form of white chago by campesinos. campesinos are primarily subsistence farmers, and frequently culturally indigenous people. these campesinos reported that their grandparents had grown the plant (seminario, 1993). in comparison, var. ‘l’ sent for research purposes to southern illinois from peru (kritzer van zant, 2016a) produces a root with either a turmeric or salmon-colored epidermis, and an off-white starchy interior. var. ‘t’, brought to southern illinois from ecuador by miriam kritzer van zant, has a light-tan epidermis and a yellowish to off-white starchy interior. three morphotypes were described for the crop for northern peru from the conserved collection of chago roots in cajamarca (seminario, 2000b). the three morphotypes all have decumbent habits, though the purple stemmed morphotype is the most decumbent (seminario, 2000b). varieties ‘l’ and ‘t’ were both decumbent to the same degree at the end of the growing season outdoors in southern illinois. however, var. ‘l’ spread out more quickly than var. ‘t’ as they grew in southern illinois. also, plants were not field grown in southern illinois for as many months as in the andes. other morphotypes have stems described as dark green and yellowish-green, respectively (seminario, 2000b). the purple stemmed morphotype has lilac-colored conical flowers, with dark green cordate leaves with purple blotches or spots, a yellowish cream-colored root epidermis and cream-white root interiors, and is ready for harvest between the other two (seminario, 2000b). the dark green stemmed morphotypes have green to dark green ovate leaves, fusiform-conical flowers which are white with lilac-colored pigmentation, and a yellowish-cream colored root epidermis and cream-white root interiors (seminario, 2000b). the dark green morphotype is ready for harvest later than the other two morphotypes described by seminario (2000b). the morphotypes with yellowish-green stems have cordate clear green leaves, conical white flowers, roots with white interiors, and are the earliest of the three (seminario, 2000b). each of these descriptions contains slightly different combinations of characters than would apply to var. ‘l’. var. ‘l’ has purple flowers, splotchy dark green stems and leaves, and turmeric-colored root exteriors. var. ‘l’ seed was sent to us by cip and is probably a different accession than those described in seminario (2000b). var. ‘t’ appears close to the description of the morphotype with yellowish-green stems. however, var. ‘t’ has pale, splotchy, slightly yellowish green leaves and stems, which seems to be different than “clear green leaves”, so it may also be a different accession than the ones above. var. ‘t’ was collected by us from an ecuadorian farmer with permission from the government of ecuador. germplasm of m. expansa there were up to 35 cultivars in live germ plasm banks in peru as of 1994, three of those accessions were kept separately from the others, all in cajamarca (rea, 1994). valderrama and seminario (2004) state that there were 56 chago collections in 2000 at unc (universidad nacional de cajamarca). this was after a search was made for germplasm in 22 provinces and 56 districts in northern peru, and before ten cultivars of chago were lost during the 1997-1998 el niño event (seminario and valderrama, 2004; valderrama and seminario, 2004). this el niño event caused soils to be more damp than usual in cajamarca (seminario and valderrama, 2004; valderrama and seminario, 2004). in 1991, two chago cultivars were returned to a farmer growing a third variety in huamachuco, peru (seminario and valderrama, 2004). in 1994 and 1995 chago was returned along with three other species to the farm communities of pariamarca and chetilla (seminario and valderrama, 2004). these communities later provided duplicate material to replace some entries lost in the 1997-98 el niño event (seminario and valderrama, 2004). in total 16 locations, 56 entries, 13 farmers, three morphotypes and 36 kilos of seed were involved in the repatriations for chago, though the emphasis of the program was switched to arracacha, which had greater familiarity for the farmers (seminario and valderrama, 2004). in addition, three collections of chago were found in peru in 2000. all three were wild types (valderrama and seminario, 2004), and are described in the section above. dna work, however, indicates there may be many fewer cultivars (chia et al. 2006). chia et al. (2006) looked at 37 peruvian accessions for the m. expansa crop, using 31 rapd markers, and concluded there may be only eight genetic variants among the 37 accessions examined. rapd cannot distinguish epigenetic variants. though accession numbers are given (chia et al., 2006) neither describes the visual or other kinds of distinctions among their morphotypes. chia et al. (2006) also do not say whose morphological concepts of taxa were initially followed, though they claim to have improved on these unspecified concepts for identification of m. expansa varieties. cytogenetic studies indicate that somatic chromosomes in m. expansa number 58, with n = x = 29 chromosomes (seminario, 2000b), however there has been much discussion of ploidy variation in mirabilis in the literature (kritzer van zant, 2016a). ecological information relevant to the growth of m. expansa lomas are patches of built up soil, from succeeding generations of mist-adapted vegetation where there is little rainfall. m. expansa cultivated varieties were grown in south america in the tough growing conditions of lomas patches within hot sand desert, found on the peruvian coastline. m. expansa is also known to grow in very high altitude cold deserts, known for their volcanic soils, in particular the paramo regions in the andes. known climates and locations for growth of m. expansa in the andes are given in several sources (rea, 1982, 1994; cip, 1998). extensive root systems and long duration of growth make m. expansa a good erosion control species (rea, 1982; cip, 1998). 239 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) use of m. expansa for erosion control makes sense for mirabilis, not in terms of root systems but of rhizomes, which send runners far into the ground. when m. expansa roots were dug up at harvest time in southern illinois, they came up fairly easily from the sand with a pitchfork. however underground rhizomes always broke off, and from their diameter and tendency to taper gradually, it appeared that they could easily extend down to the water table a few feet below. m. macfarlanei constance and rollins (oda, 2011), a rare endemic in the u.s. limited to idaho and oregon, has been reported to have roots typically under 2 m in length which can extend down as much as 10 m. observation of m. expansa and m. jalapa, growing outside in southern illinois, leads us to believe that the longer “roots” on m. macfarlanei, mentioned in oda (2011), are actually underground rhizomes. m. expansa is extremely drought resistant and can tolerate heavy rain. this has been observed in southern illinois. however, both varieties utilized in southern illinois research are intolerant of standing water. in southern illinois it was observed that standing water invariably caused roots to quickly disintegrate, both in the greenhouse and outside. var. ‘t’ in southern illinois is slightly more vulnerable to water damage than var. ‘l’. var. ‘t’ may also be more drought resistant than var. ‘l’. root disintegration in m. expansa, in standing water, occurs in spite of known resistance to some soil fungi. this resistance is due to class i ribosome inhibiting proteins which have been extracted from the roots (vivanco et al., 1997, 1999a,b; vivanco and flores, 2000). in cajamarca, conditions where the crop grows best are between 2300-3500 m in altitude, with average annual temperatures between 11-16˚c (seminario, 1993). m. expansa tolerates highest temperatures between 22-29˚c, and lowest temperatures between 4-7˚c, in deep soils with moderate texture and plentiful organic matter (seminario, 1993). experiments took place in the valley of cajamarca with an average annual temperature of 14˚c, average precipitation of 680 mm, 73% rh, open clay-lime soils of ph 6.8, at 2536 m, and 7˚ 10’ s lat (seminario, 1993). the crop also grows in black loose soils and sandy reddish soils in cajamarca, both having plenty of organic matter (seminario, 1993, 2004b). the plants are thought to have little sensitivity to day length (seminario, 2004b), and this has not been contradicted in southern illinois. native clay soils in southern illinois are too wet and heavy to grow the crop. a typically high water table in southern illinois may also be a factor. mauka is grown between 2400 to 4300 meters according to flores et al. (2003). izquierdo and roca (1998) place the crop at 2500 m. tapia (1990, 2007) puts altitudes for m. expansa between 1000 and 2500 m in altitude. seminario (1994, 2004b) both expands the range of altitudes known for the crop and breaks it down by country. in ecuador, the plant has been found between 2700-3100 m. var. ‘t’ was found by kritzer van zant in an agricultural field in pichincha province of northern ecuador, near the road to the tocacachi ruins, at 2760m +/-140m [garmin gis]. in bolivia, seminario (1994, 2004b) places m. expansa between 2900-3500 m for cultivars, and 2100-2500 m for wild types. in peru, altitudes are between 2900 and 3300 m, while in northern peru plants occur between 2500-3400 m (seminario, 1994, 2004b). plants are also said to prefer neutral ph soils, and have been found growing at temperatures typically at 13-14˚c, between extremes of 5-25ºc (seminario, 1994, 2004b). in southern illinois july and august temperatures can reach 43ºc, and frequently remain near 37ºc for more than a week at a time. a sowing experiment in texcoco, mexico resulted in plants that persisted without flowering for 1.5 years at an altitude of 2250 m, causing researchers to suspect that the crop can only complete its life cycle in the central andes (seminario, 1993, 2004b). plants flowered but did not seed in the greenhouse in southern illinois. at least six years passed before seed grown plants of var. ‘l’ and its subsequent clones, commenced flowering in southern illinois. rhizome grown material of var. ‘t’ began flowering in the greenhouse within a year of arriving in southern illinois. however, flowering has been very irregular in the greenhouse since, sometimes not occurring at all for more than one year, then re-occurring, with little seasonal correlation. we’ve never seen flowering in the field for either variety in southern illinois. mirabilis spp. can be rare or invasive neither cultivar has successfully wintered over outside in southern illinois. no special efforts were made there to cover or otherwise protect them. they do survive freezing temperatures in the andes. perhaps this has to do with the size of the roots, degree of establishment, and depth of the water table. neighbors, living in the town where the owners of the rural property where var. ‘t’ was collected also live, told kritzer van zant that there were dormant roots of the crop growing underground in the dirt path alleyway which ran behind their homes, though there was no evidence of the plants above ground. this level of dormancy is similar to what established storage roots of north american prairie plants can do during droughts, particularly in winter. rare endemic from the western us m. macfarlanei, invasive tropical m. jalapa, and sometimes invasive midwestern m. nyctaginea (michx.) macmill. are discussed in kritzer van zant (2016b). this information indicates that no blanket statement can be issued concerning invasiveness for mirabilis. farmers in humachuco are said to not still grow m. expansa much because it becomes very aggressive (seminario, 1993). in addition, m. expansa is thought to be competitive with maize, diminishing its growth, and possibly a reason that it has become less popular for cultivation in modern times (seminario, 1994, 2004b). however, other problems with growing m. expansa may indicate that aggressiveness is not the major reason that the crop is disappearing in the andes. additional problems for growing m. expansa some farmers say they only grow a small amount of m. expansa for themselves because of the lack of a market for the crop (seminario, 1993). seeds are also in short supply (seminario, 1993). problems with chago, as well as with a number of other indigenous food crops, include weight loss during processa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 240 ing and handling. chago is also at risk for damage including breakage or cutting of the roots during harvest (túpac, 2000), and stems during cultivation (seminario, 2000c). vines of both varieties grown in southern illinois became fragile as they became longer and as stems became thicker and denser in number. stems broke easily when disturbed by late summer (kritzer van zant, 2016a). though natural conditions do not allow for long term storage of chago, these roots store better than those of yucca or arracacha (túpac, 2000; seminario, 2004b). long term storage of fresh roots is difficult (seminario, 2000c). known agricultural companions for m. expansa m. expansa is thought to have been grown traditionally with corn (seminario, 1993). m. expansa is frequently grown with other crops, including corn (zea mays l.), beans (phaseolus vulgaris l.), squash (cucurbita ficifolia wall. or bouché), and other cultivars of beans and squash, as well as being associated with other ancient crops including arracacha (arracacia xanthorrhiza bancr.), llacón (smallanthus sonchifolius (poepp. & endl.) h.rob.), potatoes (solanum tuberosum l.), tarwi (lupinus mutabilis sweet or lindl.), and occasionally appearing spontaneously with wheat (triticum vulgare vill.) (seminario, 2000c, 2004b). ipni (2012) show the author of c. ficifolia both as wall. 1832, and bouché 1837. so, they may not be the exact same squash species. ipni (2012) gives the author of lupinus mutabilis as sweet 1830, and lindl. for both 1539 and 1832, so either one is a synonym of the other, or they are also not the exact same species. m. expansa is also occasionally mono-cropped, and can be found with diverse weeds on fallow land (seminario, 2000c, 2004b). agricultural research on m. expansa in the andes agriculture developed in the andes in dry inter-andean valleys, when the techniques for irrigation were discovered, predating agriculture in humid areas of the same region (tapia, 1990). initially, cultivation of m. expansa was described only for bolivia and ecuador (rea and león, 1965; rea, 1982; popenoe et al.,1989; tapia, 1990). cultivation of m. expansa is thought to be a very old practice, though there is no published archeological record of this (seminario, 2000a). in ecuador, bolivia and peru, farmers with small operations incorporate m. expansa into systems of mixed farming and polycultures (seminario, 2004b). in the andes, propagation is usually done utilizing stem pieces (rea, 1994). traditional propagation of mauka in bolivia was done clonally from basal buds or stem or shoot pieces (rea and león, 1965). however, seed growth is possible (rea 1994; rea and león, 1965). plants grown from basal buds or seed require about one year before harvest, while plants grown from shoots take a little longer (rea and león, 1965). var. ‘l’ was propagated from seed in southern illinois, and since propagated from stem cuttings of two surviving f1 plants and their vegetatively cloned descendants. var. ‘t’ has been propagated vegetatively since it arrived in illinois from ecuador. in the andes plants are typically grown a year or longer, with plants started in furrows in 1 x 1 m holes (rea, 1994). production typically takes seven to nine months (rea, 1994). this is a longer growing season than what is typically available in temperate north america. seed grown plants of m. expansa in the andes emerge in 4-6 days, first leaves emerge between 6-9 days, and tuberization occurs 25-30 days after first leaves emerge (seminario, 1993). seed to harvest is about 225 days, and next generation seeds appear 250-280 days after sowing (seminario, 1993). floral buds of this andean grown m. expansa appear 7080 days after tuberization, another 25-30 days are needed to achieve anthesis, and from anthesis to the commercial harvest stage is 90-100 more days (seminario, 1993). timing was also broken down separately for the three northern peruvian morphotypes. the purple stemmed morphotype took 200-215 days from sowing to production of floral buds, and 19-25 days from production of flower buds to anthesis (seminario, 2000b). the dark green stemmed morphotypes took 225-230 days from sowing to production of floral buds and an additional 2227 days to anthesis (seminario, 2000b). the yellowish-green stemmed morphotypes took 225-228 days from sowing to production of floral buds and 20-28 more days to anthesis (seminario, 2000b). plants were said to be propagated with ease in the andes, from underground stems, stem cuttings, and lateral shoots (seminario, 1993, 2000c, 2004b). franco and rodriguez (1988*) reported shoot stems gave 71-97% of the expected productivity in evaluation trials (seminario, 2004b). in a bed made from reeds (arundo donax) (poaceae) and filled with sand, beginning with stem cuttings and lateral shoots, roots appeared in 22 days, plants were ready for transplant 23 days after rooting, 96% of cuttings rooted, cuttings each produced 9-37 roots, and roots had reached lengths of 3-12 cm by the time of transplant 45 days after starting (seminario, 1993, 2000c, 2004b). ipni (2012) has a. donax l. 1753, and a. donax georgi 1780, it is unclear to us if they are the same species. ninety-six percent of cuttings root in the andes, and 23 roots typically develop from each cutting (rea, 1994). it is not clear to us whether rea (1994) meant 23 root segments per root mass, or whether runners establish separate root clusters. in indigenous plots, most plants produce two to five roots (rea, 1994). seven month old plants were evaluated in peru, revealing an average of 266 stems per plant, each with four nodes or eight buds (seminario, 2000c, 2004b). plants are arranged about 80-100 cm between furrows and 40-60 cm between holes dug for individual plants in fields (seminario, 1993). plants can also be given a head start to the bud stage in the greenhouse (seminario, 1993). another method is to harvest the roots, and then put the crowns back into the same ground, also sometimes wells are dug in the ground, crowns set in them, and soil is used to cover the crowns (seminario, 2004b). crown no doubt refers to the transition zone between root and shoot. the precise meaning of ‘wells’ was unclear in translation. perhaps wells just means holes, or perhaps this refers to areas of loosened or otherwise prepared soil. natural sowing is done by allowing seed to fall to the ground to germinate with the first rains, the beds being thinned out later (seminario, 2004b). evaluations were completed for ten entries of chago, maintained in one location for two years each at unc (valderrama and seminario, 2004). plants of m. expansa that do not get harvested, generally drop their leaves and go dormant in winter, reappearing as fresh shoots from surviving roots the following spring (seminario, 1993; valderrama and seminario, 2004). though above ground growth is considered cold intolerant, m. expansa is more resistant to cold than achira or yacón (seminario, 2004b). anthesis means floral opening. flowers, apparently for all three northern peruvian morphotypes, open only once in cajamarca, beginning to open at 5:30 pm, and beginning to close at 10:30 am (seminario, 2000b). these plants are fully open at 9:30 am and fully closed by 12:15 pm, for a total anthesis of six hours and 45 minutes (seminario, 2000b). wild type flower anthesis is one or two hours longer (seminario, 2000b). thus, the cultivars are true four o’clocks. the three morphotypes are thought to mostly self-pollinate, with several insects acting as secondary pollinators (seminario, 2000b). tuberization begins early whether plants are started from seed or are vegetative clones, with primary roots thickening within 30 days from sowing (seminario, 2000b). flowering typically occurs after 100 days with another 100 days to harvest (rea, 1994). however, harvest has also been reported to occur when plants are seven to eight months old, when flowers begin to wilt (túpac, 2000). none of the plants grown in southern illinois flowered outside though after several years annual flowering did commence in the greenhouse and continued for several years, even occurring in var. ‘l’ plants started in the same year from cuttings that had already flowered. rhizomes from ecuador of var. ‘t’ flowered in the greenhouse their first year in southern illinois. flowering eventually stopped occurring with any regularity, until the plants died a few years later (kritzer van zant, 2016a). based on 90% survival, and sowing distance of 0.80 x 0.50 m, it was concluded that 103 seven-month old mother plants, would be needed to plant one hectare of chago (seminario, 2000c, 2004b). in the andes weeding and cultivation are usually done 2-2.5 months after sowing and again after four months, with care needed not to break the fragile stems (seminario, 2004b). in the same series of papers, a table describing the handling of germplasm in the andes for four indigenous crops appears twice. information in the chart for chago (m. expansa) addresses use of shoots or underground stems for vegetative propagation, seed propagation and planting in furrows. distance between furrows and plants, is given as 0.70/0.60 m. information is presented in a chart for ten entries, on three weedings, one cultivation, three irrigations, one pruning, the cutting of roots and vines, pests present, the lack of diseases, the lack of applied organic fertilizer, and growth of a crop grown 9-12 months (vasquez et al., 2004; valderrama and seminario, 2004). in southern illinois, better harvest weights were obtained with bi-monthly weeding than with monthly weeding, and better results were obtained from monthly weeding than from only weeding at planting and mid-season. plants grown for two years must survive winter conditions at extremely high altitudes, and typically yield 0.5-2 kg roots, with a yield per hectare of 12-52 tons of roots while also typically producing 7 kg each of fodder (rea, 1994). in the unc trials, root yields for m. expansa spanned from 1.2-4.2 kg (valderrama and seminario, 2004). in the previously described experiments in the valley of cajamarca, in fertile soil, at 25,000 plants/hectare, harvest weights of roots were 1.8-5.5 kg/plant or 45,000-137,000 kg/hectare (seminario, 2000c, 2004b). this much more densely planted cajamarca experiment yielded about 1/3 to 2/3 the kg/hectare of roots compared with the less dense plantings reported above. harvest in the andes is usually timed to wilting of flowers (seminario, 2004b). allowing plants to grow two years increases harvest yield with little loss of quality (seminario, 2004b). it was not clear from this document if this referred to total or annual yield. it may mean both. in ecuador, plants of var. ‘t’ were seen by us in the field, reaching about one meter around and one meter high, with roots that penetrated the soil by about one meter deep. rea (1982) notes this is the typical size of the plants in ecuador and bolivia, which are usually harvested after one year of growth for seed grown plants, though some stems may be left in the ground to create a crop for later harvest. yet rea (1994) has also said the crop needs 7-9 months to mature. seminario (1994) clarified that plants derived from basal shoots require one year to mature, though plants can take longer if started from suckers, and 7-9 months is typical for production. they are biologically perennial but considered annual in terms of their use (seminario, 2004b). seed of m. expansa stores for several years without much loss of viability, and seeds stored at 18˚c lose viability at about 4% per year according to peruvian studies (seminario, 1993, 2000c, 2004b). preparation of m. expansa for food including traditional methods for mediating astringency there are reports of indigenous people curing m. expansa roots before eating them (rea, 1982) and restricting human consumption to very young leaves. chago is both said to be usually consumed fresh, and also after sun drying for a few days, to eliminate certain principal astringents (túpac, 2000). andean root crops, including oca (oxalis tuberosa (oxalidaceae) molina), have been briefly sun-treated to sweeten them (arbizu and tapia, 1994, 1998). astringency is a problem for marketing m. expansa, though less astringent varieties, such as those from ecuador, and indigenous processing methods, may also increase palatability (popenoe et al., 1989). popenoe et al. (1989) stated that tubers of m. expansa are cured to sweeten them, giving them a similar sweetness to that of sweet potatoes. curing the roots is a simple process. plants are either hung in a tree or buried in straw for eight days, and then roots are consumed like other root vegetables (popenoe et al., 1989; sperling and king, 1990, seminario, 2000c). pits have been dug for sweetening, containing layers of m. expansa roots, interlaid with layers of barley straw (popenoe et al., 1989; rea, 1982). if the roots depicted in these sources are full-grown, the pit could easily be 3-4 m deep. as mentioned above, var. ‘l’ can produce a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 241 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a turmeric-colored crystalline powder on the outside of the epidermis. the turmeric color and crystalline appearance are lost from roots on plants hung in a barn to dry, by the end of one week. this color change may correlate with another chemical change, making them safer to eat or more palatable. though rea (1994) indicates they are cured for sweetening, no experimental data is referenced by rea to back this. var. ‘t’ roots did not have turmeric colored powder on the epidermis, nor any similar substance which underwent an obvious color change, though the epidermes of all roots of both varieties were observed to darken slightly while drying in the barn. mauka can be parboiled and eaten like sweet potato and cassava in bolivia, where roots also could be sweetened by sun drying to neutralize astringent components (rea and león, 1965; popenoe et al., 1989). in peru, roots are said to be left out sometimes during the sunny hours (seminario, 2004b). this perhaps means that they are not left out overnight, though this is unclear to us. perhaps this wording means they are left outside and covered at night. in bolivia, stems and enlarged roots cured to eliminate astringent flavors can be cooked with sweeteners (rea, 1994). water left over is then consumed as a soft drink (rea, 1994). other sources include more specifics about preparation of this soft drink and also describe additional ways to use the plant. in more detailed reports, corn syrup, chancaca [brown sugar], or sugar, have been used to make roots more palatable to humans, the water in which the roots are boiled is served as a soft drink, while pigs are fattened with stems (rea and león, 1965; popenoe et al., 1989; seminario, 2000c, 2004b). this may refer to enlarged stems. they have amino acid profiles similar to roots (kritzer van zant, 2016a). roots were fed to domestic birds (tapia, 1966* reported by seminario, 2004b). m. expansa is used less frequently to fatten pigs, usually stems and roots are mixed with other crops for this purpose, though sometimes pigs are allowed to feed at will on post-harvest remnants in the field (seminario, 2004b). in ecuador, lard from pigs fed chago was said to be of good quality (tapia 1966* reported by seminario, 2004b). when feeding stems to animals, preference was seen for basal and terminal portions, probably due to the wealth of carbohydrates in the basal sections, which are more likely to be enlarged with starch, and for the smoother less fibrous texture of the upper parts relative to the middle sections (seminario, 2004b). cavies, also called cuyes, are andean domestic guinea pigs. whole plants are fed to pigs, cavies/cuyes, vicuna, sheep and cattle, which are partial to it (rea, 1994, seminario, 2000c, 2004b). bolivian mauka has been shown to contain 4-7% protein (rea, 1994). it seems this percentage is only for roots, based on research on protein content in collaboration with william banz of southern illinois university (kritzer van zant, 2016a). this analysis of amino acids in the southern illinois grown m. expansa (kritzer van zant, 2016a), gives the first look at the amino acid components of m. expansa protein. the amino acid profiling work was presented along with results from preliminary testing for micro molecule toxicity by hee-byung chai (kritzer van zant, 2016a) in douglas kinghorn’s laboratory at ohio state university. laboratory methods and data are not presented for most of the reports of protein content for m. expansa in the andean literature, so those measurements may be of total nitrogen, not total protein. chago leaves can be used in a traditional technique, along with other greens, herbs and roots, to stuff a pig for roasting (seminario, 2004a). in ecuador, leaves are added to salads and chili sauces, and buried roots are allowed to sweeten in pits to concentrate sugars, producing one of the two basic edible forms, “sal” [salt] and dulce “sweet” (rea, 1982, 1994; popenoe et al., 1989; seminario, 1994, 2000c, 2004b). roots can also be served with fish and tomatoes (seminario, 2000c, 2004b). in ecuador, tuberized roots and stems are skinned immediately after harvesting, whether preparation continues for the “sal” or “dulce” form (seminario, 2004b). chago is also said to be usually consumed fresh, and also after sun drying for 2-3 days to eliminate certain principal astringents (túpac, 2000). though it was a little unclear in translation, this probably means the plants could be eaten after this short drying period. after peeling, roots are parboiled (túpac, 2000). in peru, a carnival dish called sancochado containing large pieces of seasoned roots and meat, including yucca and sweet potato, can include mauka as well as chicken or pork and cabbage (seminario, 2004a, 2004b). sancochado is usually made at the time that cultivation and weeding of chago occurs (seminario, 2004a). roots can also be parboiled, prepared like yucca, cooked in a stew with peppers, prepared in soups and broths with rice and/or cabbage, or ground with salt to consume with coffee (seminario, 2004b). one way to prepare roots is to finely grate or ferment them, and then boil them twice to remove the astringent ingredients (seminario, 2004b). some people prefer to eat it cold since the astringent ingredients give it a heat of its own (seminario, 2004b); however, it was not clear to us if this applied to the leaves in salads or the roots. taste tests in cajamarca, serving roots with the fish dish ceviche and sweet potato or yucca, met with general approval (seminario, 2004b). peeled roots were broken up and air dried, making them suitable to add to flour (seminario, 2004b). roots could also be peeled then used fresh, described with the phrase “leave them in a milky liquid,” and then washed to remove the astringents (seminario, 2004b). this apparently means to leach the astringents from fresh roots out in water prior to cooking, though this did not necessarily preclude changing the cooking water. use of m. expansa for fodder mature leaves were used as fodder for livestock (popenoe et al. 1989; cip 1998). leaves of m. expansa have been reported to contain up to 17% protein (flores et al., 2003; kritzer van zant, 2016a), and this protein is considered among the more digestible from plant sources which grow in the andean highlands (flores et al. 2003). leaves have been used for rabbit forage in peru, giving superior weight gain when compared with a mixture of rye grass (lolium multiflorum) and clover (trifolium repens), and the same is seen with whole plants fed to vicuna, reported by (rea, 1992*; seminario, 2004b). by 6.5 months, plants grown two years at high altitudes typically produce 7 kg each of fodder, these were the same plants a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 242 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) that produced 0.5 to 2 kg of roots (rea, 1994). seminario (2004b) also reports yields of 7 kg of fodder per plant. forage was estimated at about 5.5 kg per plant in the densely planted experiment in the valley of cajamarca (seminario, 2000c, 2004b). marketing of m. expansa in peru, m. expansa roots were only found for sale in the market in huamachuco, under the name “yuquitas” (seminario, 2004b). most are grown for private consumption though they may also be traded or exchanged (seminario, 2004b). in bolivia roots are found in rural markets but not in cities (seminario, 2004b). additional nutritional data for m. expansa peruvian mauka has a phosphorous content of 117 mg/gram yet the plants are low in sodium and iron (rea, 1994). there are no methods presented for assessing sodium and iron content reported by rea. calcium levels in the plant, though reported as very high, are considered mostly unavailable as most are in the form of calcium oxalate raphides (flores et al. 2003). our observations indicate that this is true, as mature plants are packed with raphides in all tissues, seen in cross sections of roots, leaves and stems under a light microscope. there are two summary tables (tables 1 and 2) of previously published nutritional information for m. expansa in the supplemental material for this paper. both compilations are original to kritzer van zant (2016a). again, this information was only given for the crop at the species level. we standardized units in these tables when possible to do so. table 1 examines reports of humidity/moisture, ash, protein , fat, carbohydrates and fiber, distinguishing between wet and dry material and plant part when possible, from several sources. table 2 similarly compares reports of mineral content, including as categories calcium, phosphorous, iron and sodium also reported from several sources. though m. expansa does not have outstanding numbers for any single amino acid, it does contain all essential amino acids, most other amino acids as well, and is high for total protein relative to other plant protein sources considered (kritzer van zant, 2016a). it should be noted that peeled roots may not be as nutritious as whole roots. all of the amino acid tests in kritzer van zant (2016a) combined root epidermises with root interiors. none of the information on nutrition from the prior literature described the presence or absence of the root epidermes when tests were performed, even though it appears that roots were peeled at least prior to boiling and before traditional drying. pests, disease, and resistance in the crop mauka roots are prone to spoil in storage unless refrigerated or desiccated (seminario, 1993, 2004b). a fungus rot is known to attack roots after harvest, thought to enter wounds in roots and stems resulting from harvesting (seminario, 1993). a fusarium sp. is known to attack the root in soils with excess humidity, spreading upward from root tips to the base of the plant in keeping with rising water tables, and affecting the dark green morphotypes most heavily, as seen in the mid 1990s (seminario, 2000c, 2004b). no viruses were known for m. expansa before 1993, when phase i of the collaborative program for biodiversity of andean roots and tubercles (programa colaborativo de biodiversidad de raíces y tubérculos andinos) began (lizárraga, 2000). m. expansa was found to be host to the mosaic virus mirabilis potyvirus 1 (mir-1), transmitted by aphids, which systematically deforms leaves of c. quinoa, upon evaluation in the laboratory with ncm-elisa (lizárraga, 2000, 2004). c. quinoa is presumably chenopodium quinoa willd. (chenopodiaceae). mirabilis mosaic caulimovirus (mimv), also vectored by aphids, was found to cause disease in two mirabilis species, including mosaic and chlorosis in m. jalapa l., and intervenal infection, rings, and necrosis in m. nyctaginea (michx.) macmill. (lizárraga, 2000, 2004). however, serology and affected crops were not listed for mimv (lizárraga, 2000, 2004). in contrast, in laboratory studies conducted by rolando estrada at universidad de san marcos in lima, peru, chago ribosome inhibiting protein has been shown to also offer potential as a low cost biological control against potato viruses including pvx, pvy, and pstvd (seminario, 2004b). discussion of m. expansa protein research (kritzer van zant, 2016a), includes a reference by seminario (2004b) to an anonymous 1966 document which seems to be about research on m. expansa and m. multiflora type i ribosome inhibiting proteins (rips). rips were discovered by flores, vivanco and their coauthors. these proteins were also shown by flores and colleagues to be in m. jalapa, though each species has its own specific rips. in february 2012 in the hrc (horticultural research center) greenhouse at southern illinois university, stem cuttings of both varieties ‘t’ and ‘l’, grown under mist, developed black rot at the tips of emerging leaves about seven days after starting the cuttings, which then spread and destroyed many of the plants. this had not been seen by us before, and may have originated from un-sterilized vermifuge in the starting mix. plants were quickly removed from the mist bench, yet continued to deteriorate. a few days later, the plants were sprayed heavily and the medium they were growing in drenched with a mild hydrogen peroxide solution at about 0.5%. a few days later still, many plants continued to die but some had improved. a decision was made to wait a few more days and if the rot was still present, destroy all of the plants. when the plants were destroyed, it was realized that most of the recovering plants were var. ‘t’, the majority of var. ‘t’ plants were recovering, only a small number of var. ‘l’ plants had improved, and nearly all var. ‘l’ plants were dead. all of the plants were destroyed rather than risk transferring the infection to other soils. no pests were considered to have serious economic repercussions after ten years of continuous cultivation of m. expansa in peruvian field trials, though the purple flowered morphotypes are most susceptible to problems (seminario, 2004b). adult moths of the family cosmopterigidae feed on m. expansa buds (seminario, 1993, 2000c, 2004b). unspecified lepidoptera spp. larvae attack stems, and suck the sap from leaves (seminario, 1993, 2004b). a butterfly, thought to be in the siphingidae group, eats the leaves (seminario, 1993, 2004b). rootworm, 243 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) thrips and aphids are minor pests (seminario, 1993, 2004b). damage from lepidoptera was observed by us on both varieties ‘t’ and ‘l’ in the field in southern illinois. occasionally there have been problems with both aphids and thrips in the greenhouse in southern illinois. unidentified pests have consumed tender leaves of young plants in the field during the growth trials in southern illinois. discussion chia et al. (2006)’s concept of eight genetic variants are difficult to compare with the morphotypes in the collections described by seminario, and colleagues above, or with varieties ‘l’ and ‘t’. in light of m. expansa’s propensity for morphological variation for the same kinds of structures, seen even on individual herbarium specimens, and after growing varieties ‘l’ and ‘t’, it is very easy to believe that some varieties of the crop may be epigenetic variants. there is a great deal of disagreement in keys and descriptions by taxonomists, concerning the limits of mirabilis species for purposes of identification, and andean mirabilis is no exception. this includes keys by paul clark standley (1931b), antoine heimerl (1934), and bittrich and kühn (1993). disagreement is also common among publications by the same authors, including heimerl (1889, 1897, 1934) and standley (1909, 1918, 1931a, 1931b). bittrich and kühn did not produce an additional key for the nyctaginaceae. this does not mean chia et al. (2006) are incorrect in their rapd analysis, but that there should be other considerations besides their data in protecting biodiversity for this crop. mirabilis spp. are notorious for reported variation in chromosome numbers so chromosome counts reported in seminario, (200b) may not hold up for all varieties. ploidy may be a major factor in distinguishing some varieties of the crop, as is well known for several other crops including wheat and cotton. perhaps variation in flowering cycles is a question of the age of the plants, or need for a companion organism such as a mycorrhizal fungus or bacterium to be present to trigger the flowering response. in the greenhouse this could also have been a response in part to lights on some groups of neighboring plants, a situation which was frequently in flux in southern illinois. all of this is speculation, awaiting future research. high susceptibility to water should limit m. expansa’s ability to be invasive in many habitats, though aggressiveness may be a problem in some agricultural settings. usda does not include m. expansa in its listings for invasive species which is why it could be legally brought into the u.s. our acquisition of seeds of var. ‘l’ from cip in peru was greatly delayed by the need to get an official letter, graciously sent by a usda official directly to cip, saying that there were no restrictions for bringing m. expansa into the united states. documentation was necessary for the cooperating governments and agencies in peru to release plant material for export. cip finally accepted the letter in lieu of a usda approved permit which cip requires to release seeds to the united states. usda officials were initially reluctant to produce a letter that they deemed unnecessary, delaying import of the seed for an additional year. since that time, david a. lightfoot of southern illinois university-carbondale requested seeds of other varieties from cip. however, cip did not send them. perhaps this is due to biodiversity treaty concerns, which have become an issue amongst andean countries as well as other developing nations. also, perhaps seeds were not sent because peruvian researchers had started to publish their own research on m. expansa, since the var. ‘l’ seeds were first received in southern illinois. possibly, the southern illinois research has sparked more intense interest in these plants in their home range. hopefully this is so. difficulty in storing fresh roots may also be another reason why indigenous people typically dry them. correlation between harvest weights and more frequent weeding may have been due to the greater humidity and intensive weed growth in the southern illinois climate versus the arid regions in the andes. class i ribosome inhibitors (vivanco et al., 1999) are thought to be safe for humans and other mammals to eat (vivanco, personal communication, 2009). however, to our knowledge, there is no published tests addressing the safety of any mirabilis ribosome inhibiting proteins for consumption. roots of both cultivars, grown in the southern illinois plots, and then dried at least eight days, were cooked until they fell apart with other vegetables and quinoa, resulting in a pleasant flavored soup not much different in flavor from similar soup made without the mauka. our first batch of soup made with an m. expansa root included the root epidermis, and was completely palatable and digestible. it should be added that only one small dried root was used in a large pot of soup. a second batch of similar soup, kept on a stove top in warm weather, developed a distinct fermented-lemony flavor after a couple of days of insufficient reheating. at that point the second batch had to be disposed of as it caused extreme indigestion. freeze dried roots of a greenhouse-grown var. ‘l’ plant, while being ground in preparation for methanol extraction (kritzer van zant, 2016a), gave off a pleasant fragrance reminiscent of pancake or cake batter with vanilla and spices included. all other batches ground to an innocuous odor, more typical of plain flours or starches. perhaps mauka or at least some of its varieties will prove most useful in today’s world cured, ground, dried and then added to flour to enrich the protein and mineral content of baked goods and pasta. also, it may be that peeling the roots before drying them will help with reducing the astringent flavor, though this would not be the case if the astringent taste is due to the presence of calcium oxalate in the form of raphides. our observations of thin root sections mounted on microscope slides, showed raphides are present in large numbers in the starchy interior of the roots as well as on and in all structures of these plants. interestingly, the m. expansa starch gave our soup a silky texture and did not separate from the water even when left out overnight. most likely the small size of m. expansa starch molecules allowed them to remain in suspension. this property may be of industrial use. m. expansa starch samples sent for evaluation to thailand were considered comparable to cassava starch (triwitayakorn, personal communication, 2016). however, removing or otherwise mitigating calcium oxalate will again be an issue for production. lack of an archeological record for m. expansa may be due to the limits of archeological methods for 244 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) finding minute starch grains (kritzer van zant, 2016a,b). seed grown potato yields measured from 1999 to 2004 in idaho were reported by olsen (2004), and include the lowest reported yield of 8.4 tons/hectare for variety shepody, to 42.4 tons per hectare for the high yielding variety yukon gold. these numbers for potatoes are within range of and much higher than for shepody, though somewhat lower than for yukon gold, than the 12-52 tons per hectare reported by rea (1994) for an unspecified variety or varieties of m. expansa. most likely, m. expansa was grown with considerably less water and fertilizer than is needed for potatoes grown anywhere. however, rea’s yields for m. expansa roots may be the result of two years of growth compared with one season for the potatoes, so the comparability of the yields and costs of production remain to be seen. also, the yields for the same varieties of potatoes could be lower or higher had they been grown in the same location and conditions as the m. expansa described by rea (1994). however, even if m. expansa did gave a lower annual yield for its roots than yukon gold, unlike potatoes, it can produce a fodder crop at the same time. using seminario’s (2000c, 2004b) estimate of 103 plants per hectare would give a potential yield of 721 kg of fodder per hectare over a single extended growing season, in addition to a root yield of 6-26 tons of roots per hectare averaged per year based on rea (1994). putnam, summers and orloff (2007), evaluated improved fodder yields for alfalfa grown in california’s central valley, low and high desert, and coastal and intermountain regions. they reported forage yields for alfalfa ranging from about five to approaching eight tons per acre in 2004 and 2005 (putnam et al., 2007). using the usda forest service units conversion calculator (provisional) (usda forest service, 2004), this becomes 0.32 tons/acre for m. expansa, a considerably lower yield than for improved alfalfa. however, alfalfa is a heavy feeder which prefers rich soils, and requires considerable fertilizer and 2.6 to 6 acre feet of water per year plus often more water to deal with salinity, and is limited by the availability of water for irrigation (putnam et al., 2007). m. expansa requires almost no assistance with obtaining water, and appears to prefer soils generally considered less fertile (kritzer van zant, 2016a). in addition, the fodder crop is a bonus to the root crop or visa-versa. unimproved western u.s. range lands in utah typically yield 384.83 lbs/acre of forage in the fall/winter late october to january season (perry 2012). converting pounds to tons gives .19 tons/acre of forage, only 59% of the projected six month yield for m. expansa grown at a paramo location, which is high altitude cold desert. it must be conceded that this is for a considerably shorter growing season than for the fall/winter utah range land, and details of fertilizer and watering are not specified. still the point is made that m. expansa, even when planted for a root harvest, has the potential to be a valuable addition to fodder resources on marginal land, and based on kritzer van zant (2016a) with a minimal input of water and resources, simultaneously producing quantities of fodder at least comparable to unimproved range land in utah. yields from the densely planted cajamarca experiment (seminario, 2000c, 2004b) multiply to 137.5 kg/hectare of fodder from m. expansa. converting this 137.5 kg/hectare with usda forest service (2004), gives 61.3 tons/acre of fodder from m. expansa, more than 7.5-12 times the 5-8 tons/acre reported for improved alfalfa in california (putnam et al., 2007). this is also more than 320 times perry’s (2012) estimate of 0.19 tons/acre for fodder from winter season unimproved utah range land. these numbers are more than enough to be impressive over a possibly shorter growing season. we must also consider both the high protein quality and quantity in m. expansa fodder as well as how to mitigate the high amounts of calcium oxalate. this should be examined even though there does not appear to be a history of harm to a wide variety of andean livestock, from eating mature m. expansa leaves. perhaps the discrepancy in cured vs. fresh consumption depends on whether or not the variety is sweet, or at least less astringent than varieties which are always cured prior to eating. perhaps, processing for m. expansa developed alongside or from methods applied to other andean crops. possibly varieties ‘l’ and ‘t’, originating as far as we know separately from peru and ecuador respectively, also correlate to bitter and sweet versions of the crop in the same manner well known for crops such as cassava. cassava is also known as manioc (manihot esculenta (euphorbiaceae) crantz). however, it should be considered that m. expansa var. ‘t’ was collected by us in ecuador from a person who cured the roots prior to consumption, seemingly in contradiction to the concept of ecuadorian roots being sweet instead of astringent. interior tissue is difficult to separate from the thin epidermes on dry roots, making them as difficult to peel as dried ginger. had we known that roots are typically peeled prior to consumption, before doing our research, we would have peeled them prior to drying and testing, or tested both ways. var. ‘t’’s ability to recover more readily than var. ‘l’ in the greenhouse in southern illinois, makes the point that specific resistance to disease may be different in different varieties of m. expansa and that this is worth further investigation. conversely it may be an indirect effect, as the var. ‘t’ material may root faster. yet, rapid growth may have been due all or in part to the maturity and amount of stored resources in the stem material utilized for propagation. this could further be interpreted two ways. if the starting material for one variety was more mature than most of what was used for starting the other variety, did it need time to dedifferentiate before rooting, or would more stored reserves cause it to root faster? one thing is certain, none of the published research to date, on the effect of m. expansa proteins on disease causing organisms, distinguished between varieties of m. expansa. conclusion as a result of examining these documents a clearer image emerges of use, consumption and interest in m. expansa in its home range. comparisons could therefore be drawn with our observations and data from southern illinois. in addition, published information on other root and forage crop yields, when compared to results from andean researchers with mauka, point to the reasons why it makes sense to continue examining m. ex245 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 246 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) pansa’s potential as a modern food source and cash crop, both in and outside the andes. consideration should be given to m. expansa for more extensive genetic, nutrition and agricultural studies. greater attention should also be given to conserving both wild and domesticated germplasm of this crop. we recommend utilizing the broadest reasonable taxon concepts for distinguishing horticultural varieties of mauka. currently, those would be the traditional indigenous crop variety concepts. finally, we believe there are great opportunities to develop new varieties of m. expansa for use throughout the world in places where soils drain rapidly. as for all introduced crops, m. expansa should only be grown where it is unlikely to become a threat to endangered species and ecosystems. m. expansa’s extreme susceptibility to standing water will assist with keeping it in check, making it even more desirable for development as a modern crop. mirabilis expansa history review author contributions all conceptualization, research on source material, interpretation of information and writing for this paper are by miriam kritzer van zant. this includes translation from sources published in spanish, and any interpretation and updating of latin names. references arbizu c, and m tapia (1994, 1998) andean tubers. in: je hernándo bermejo, j león (eds.) 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protein isolated from the andean root crop species mirabilis expansa. in: he flores, jp lynch, d eissenstat (eds.) radical biology: advances and perspectives on the function of plant roots. american society of plant physiologists, pp. 454-457. vivanco jm, bj savary, and he flores (1999a) characterization of two novel type i ribosome-inactivating proteins from the storage roots of the andean crop species mirabilis expansa. plant physiol 119:1447-1456. vivanco jm, lf salazar, and m querci (1999b) antiviral and antiviroid activity of map-containing extracts from mirabilis jalapa roots. plant dis 83:1116-1121. vivanco jm, and he flores (2000) biosynthesis of ribosome inactivating proteins from callus and cell suspension cultures from mirabilis expansa. plant cell reports 19: 1033-1039. 247 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) van zant, 2016 supplementary data material (wet / dry) humidity / moisture ash protein fat carbohydrates fiber source roots (100 g) 59.3 % 2.2 g 4.4 g 0.1 g 33.1 g ** 1.0 mg tapia (1990) montenegro & pebe (1988*) roots 59.3 % -4.4 g 0.1 % 33.1 mg ** 1.0 mg seminario cunya (2004a) subterranean parts dry 7 months --6.9 0.7 g 87.0 g 1.3 seminario cunya (1993) eealm (1972*) subterranean parts wet 7 months 65 -2.4 6.3 g 30.5 g 0.4 seminario cunya (1993) eealm (1972*) subterranean parts wet 7 months --2.5 -35.0 g 6.5 seminario cunya (1993) unc* subterranean parts dry -4.20 (% wt.) 6.90 (% wt.) 0.72 (% wt.) 86.90 (% wt.) 1.25 (% wt.) seminario cunya (2004b) eealm (1972*) subterranean parts-wet -1.47 (% wt.) 2.40 (% wt.) 0.25 (% wt.) 30.50 (% wt.) 0.43 (% wt.) seminario cunya (2004b) eealm (1972*) subterranean parts-wet -2.18 (% wt.) 4.30 (% wt.) 0.09 (% wt.) 33.08 (% wt.) 0.96 (% wt.) seminario cunya (2004b) montenegro & franco (1988*) stems dry --17.2 5.0 g 43.3 g 20.8 seminario cunya (1993) eealm (1972*) stems wet 75 -4.3 1.3 g 10.8 g 5.2 seminario cunya (1993) eealm (1972*) foliage dry 59.39 % 2.2 % 4.0 % -10.0 % 0.9 % seminario cunya (1993) unc foliage dry -13.57 % 17.2 % 5.04 % 43.33 % 20.84 % seminario cunya (2004b) eealm (1972*) edible parts -2.2 % 4.4 % 0.1 % 33.1 % 1.0 % seminario cunya (1993) montenegro & franco (1988) unspecified compared w/ 9 andean crops --4.3 % range: 0.7-4.3 1.3 % range: 0.11.3 10.8 % range: 9.8-32.8 -seminario cunya (1993) eealm (1972*) edible part compared w/ 7 (6 for fats, 4 for fiber) andean crops --4.30 % range: 0.7-4.3 0.09 % 0.09-0.51 33.08 % range: 9.8-33.08 0.96 % range: 0.54-1.10 seminario cunya (2004b) ** there is disagreement between tapia (1990) and seminario cunya (2004a) as to whether carbohydrates were given in g or mg, the numerical values are the same. units are as given. table 1. nutrition values for roots, stems, foliage and unspecified parts of m. expansa from various authors and their sources. table 2. mineral values for roots and stems of m. expansa from various authors and their sources. units are all in mg. *** by comparison, must be per 100 g of roots. material (wet/dry) calcium phosphorous iron sodium source roots (100 g) 283 mg 111 mg 0.1 mg -tapia (1990) montenegro & pebe (1988*) roots*** 283 mg 111 mg 0.1 mg -seminario cunya (2004a) stems dry 2726.0 mg 590.0 mg --seminario cunya (1993) eealm (1972*) stems wet 680.0 mg 150.0 mg --seminario cunya (1993) eealm (1972*) unspecified (100 g) *** 283 mg 111 mg -< 0.01 mg seminario cunya (2004b) 248 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) atlas journal of biology 2020, pp. 699–705 doi: 10.5147/ajb.v0i0.211 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) nitrate reducing and denitrifying bacteria in oil / brine contaminated soils samer m. abubakr department of biological and forensic sciences, fayetteville state university, fayetteville, nc 28301, usa received: august 29, 2019 / accepted: january 19, 2020 __________________________________________________ * corresponding author: sabubakr@uncfsu.edu 699 abstract soil microorganisms are a fundamental part of biogeochemical cycling of nitrogen. nitrate reducing bacteria and denitrifying bacteria are diverse groups of bacteria involved in nitrogen transformations in soils. current research has shown that soil denitrification releases nitrogen from the soil ecosystem to the atmosphere. as a result, the balance between denitrification and n-fixation can determine the biologically available nitrogen for soils. in fact, nitrogen deficiency in soil is a common limiting factor for plant growth and productivity. nitrate reduction and denitrification could be affected by some environmental factors (e.g. oxygen levels) and could also be affected by various contaminants such as crude oil and brine as they may alter the abundance and species composition of nitrate reducing and denitrifying bacteria. on the other hand, different environmental factors (e.g., oxygen and humic substances) affect the degradation of petroleum hydrocarbons in soil by soil microorganisms. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction soil microorganisms are a fundamental part of biogeochemical cycles in general and biogeochemical cycling of nitrogen in particular. denitrification is an important component of nitrogen cycling in soils, in which some microorganisms (e.g. denitrifying bacteria) use nitrate or nitrite as alternative electron acceptors. multiple studies have focused on various aspects of nitrogen cycling. philippot et al. (2009) linked the distribution of the fraction of bacteria with the genetic capacity to reduce n2o to n2 to areas with low potential n2o emissions in a pasture. in addition, it was shown that a map of denitrification activity across a whole farm was reflected by maps displaying the community size and structure of a specific fraction of the denitrifiers at the site (enwall et al., 2010). since denitrification releases mineralized nitrogen from the soil ecosystem to the atmosphere, the balance between denitrification and n-fixation can determine the biologically available nitrogen for soils (ollivier et al., 2011). denitrification could be affected by soil ecosystem contaminants such as crude oil and brine as they may alter the abundance and species composition of denitrifying bacteria in predictable ways. for example, γ-proteobacteria are known to increase in crude-oil contaminated sites (shim and yang, 1999; lee et al., 2002), and a wide diversity of γ-proteobacteria including pseudomonas and vibrio species were shown to degrade hydrocarbons under nitrate reducing (nr) conditions (rockne et al., 2000). also, it was suggested that n2o production activity review article a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 700 could be sensitive to heavy metal pollution (liu et al., 2016). in addition, other studies (vosswinkel et al., 1991; song et al., 2000, green et al., 2010) showed that strains for several genera of γ-proteobacteria have the ability to denitrify. it was shown that nitrate and nitrite reduction rates were increasingly inhibited at increasing nacl concentrations when comparing treatment of fishery wastewaters (mariángel et al., 2008). bacterial diversity in brine-contaminated sites is expected to be less because of selection for salt-tolerant genera such as bacillus (boch et al., 1997) and pseudomonas (egamberdiyeva, 2005). in this review, i summarize the role nitrate reducing and denitrifying bacteria play in biogeochemical cycling of nitrogen in soils, discuss some factors that affect their abundance and their species composition, and highlight the necessity of investigating communities of denitrifying bacteria using an approach based on the enzymes responsible for key steps in denitrification pathway rather than 16s rrna gene since closely related species vary in their ability to denitrify and denitrification is common among phylogenetically unrelated microbial groups. biogeochemical cycling of nitrogen nitrogen is an abundant element, with the volume of nitrogen gas (n2) forms 78% of the atmospheric total volume. although n2 forms the highest percentage, nitrogen deficiency in soil is a common limiting factor for plant growth and productivity because vascular plants cannot combine n2 directly into organic compounds (nie et al., 2011). nitrate (no3 -) or ammonium ions (nh4 +) are the main nitrogen forms absorbed by plants from the soil. nitrogen cycles through the environment and living organisms including bacteria. fallen leaves, animal feces, and dead plants or animals provide the soil with nh4 + a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) by the soil microorganisms that break down organic nitrogen in a process called ammonification. on the other hand, other bacteria obtain energy by oxidizing nh4 + to produce no3 in a process called nitrification (zumft, 1997). however, not all the nitrate produced is absorbed by plants. some bacteria convert nitrate to nitrite and then to ammonia in a process called ammonifying nitrite reduction (zumft, 1997). other bacteria use nitrate and nitrite (no2 -) as alternative electron acceptors during respiration and convert them to the end products nitric oxide (no), nitrous oxide (n2o) and n2 for energy production when oxygen is limiting, generally under anaerobic conditions. these end products are released to the atmosphere (figure 1). this latter process is the traditionally defined pathway of denitrification (braker et al., 2000). physiology, taxonomic, and distribution patterns of nitrate reducing (nr) / denitrifying (dn) bacteria nitrate reducing (nr) bacteria use nitrate as an alternative electron acceptor to obtain energy from dissimilatory reduction of nitrate into nitrite by nitrate reductase enzymes (zumft, 1997). nitrate reducing bacteria are facultative anaerobes that can use oxygen as their terminal electron acceptor (chèneby et al., 2010). nitrate reducing bacteria represent a diverse group with members among α, β, and γ-proteobacteria, some members of firmicutes, and even archaea (philippot, 2005). nitrate reducing bacteria are abundant in various environments such as human digestive tract (bru et al., 2007), earthworm guts (drake and horn, 2007), and rhizosphere (brunel et al., 1992). nitrate reduction is a facultative process, it depends primarily on the figure 1. the nitrogen cycle. atmospheric nitrogen (n2) denitrifying bacteriaassimilation 701 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) presence of nitrate, oxygen limitation, and electron donor availability (tiedje, 1988). in low o2 environments, no3 and no2 are used as electron acceptors by some bacteria that perform a denitrification-like respiration (bock et al., 1995; braker et al., 2000). a series of enzymes direct the denitrification route in denitrifying bacteria: nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase. in anaerobic respiration, these enzymes consume no3 -, no2 -, no, and n2o, respectively, as terminal electron acceptors (zumft, 1997). after oxygen consumption (1), denitrification (2) is the highest energy-yielding process as shown in equations 1 and 2 (strohm et al., 2007). c6h12o6 + 6o2 → 6co2 + 6h2o (δg°ʹ = -2,870 kj per mol glucose) (1) 5c6h12o6 + 24no3 + 24h+ → 30co2 + 12n2 + 42h2o (δg°ʹ = -2,670 kj per mol glucose) (2) denitrification is estimated to remove 40-50% of external inputs of dissolved inorganic nitrogen in marine coastal sediments (seitzinger, 1990), causing an unbalance of nitrogen quantities in the ocean (devol, 1991; codispoti, 1995). the production and accumulation of no and n2o contributes to global warming and the destruction of the ozone layer (dickinson and cicerone, 1986; conrad, 1996). since denitrification is common among phylogenetically unrelated microbial groups and closely related species vary in their ability to denitrify, it is very unsuitable to investigate communities of dn bacteria by using an approach based on 16s rrna gene sequences (zumft, 1992). so, to detect and to analyze denitrifying bacteria, gene sequences coding for the enzymes responsible for key steps in the denitrification pathway have been used (braker et al., 1998; scala and kerkhof, 1998; scala and kerkhof, 1999). denitrification consists of four reaction steps in which nitrate is reduced to dinitrogen gas (table 1). the first step where nitrate is reduced to nitrite is common step to a taxonomically diverse group of bacteria (nr bacteria) and it is catalyzed by two different types of nitrate reductases, either membrane bound encoded by the narghji operon or periplasmic encoded by the napabc operon (kandeler et al., 2006). nitrite reductase is a significant enzyme in the denitrification pathway because it produces no, the first gaseous product (ye et al., 1994; casciotti and ward, 2001), thus nitrogen loss from soil. this enzyme is found as two different forms. the first one contains copper and is encoded by nirk, while the second contains cytochromes (hemes) c and d1 and is encoded by nirs (zumft, 1997). these two forms are functionally similar (casciotti and ward, 2001; avrahami et al., 2002) although structurally different (adman et al., 1995; casciotti and ward, 2001; avrahami et al., 2002). many different bacteria contain nirs, including paracoccus denitrificans atcc 19367, pseudomonas stutzeri atcc 14405, and roseobacter denitrificans atcc 33942t. some bacteria that possess nirk include hyphomicrobium zavarzinii ifam zv-622 t atcc 27496, alcaligenes sp. strain dsm 30128, and alcaligenes xylosoxidans subsp. denitrificans dsm 30026 (braker et al., 1998). nirs appears to be more abundant in nature while nirk is found to be more widespread in different taxonomic groups (coyne et al., 1989; gruntzig et al., 2001). nirk was discovered in several ammonia oxidizing bacteria (aob) such as nitrosomonas europaea (casciotti and ward, 2001). physiological evidence suggests that nirk might be employed by aob as a protection against no2 -, the toxic product of ammonia oxidation (poth and focht, 1985; stein and arp, 1998; beaumont et al., 2004). the reduction of nitric oxide is catalyzed by nitric oxide reductase small and large subunits encoded by norc and norb, respectively (braker and tiedje, 2003). norb includes 2 classes: the first class encodes cytochrome bc-type complex (cnorb) while the second class encodes the quinol-oxidizing single-subunit class (qnorb) (braker and tiedje, 2003). the last step in the denitrification pathway is the reduction of nitrous oxide and is catalyzed by nitrous oxide reductase genes that are arranged in three transcriptional units consisting of the nosz gene that encodes the catalytic subunit plus the nosr gene and the nosdfyl genes (philippot, 2002). environmental factors influencing nitrate reduction / denitrification denitrification is an environmentally regulated process with respect to oxygen supply, the presence and nature of a nitrogen oxide, and possibly additional external factors such as metal ions (philippot, 2002). it was shown that some bacterial strains demonstrated nitrite reduction under strict anaerobic conditions, but not in the presence of oxygen (abubakr and duncan, 2015). also, the diversity of nr bacteria in a waste water table 1. denitrification steps in bacteria. step no. step catalyzed by (enzyme) encoded by (operon/gene) reference 1 nitrate to nitrite membrane bound nitrate reductase narghji kandeler et al., 2006 periplasmic nitrate reductase napabc 2 nitrite to nitric oxide nitrite reductase (contains copper) nirk zumft, 1997 nitrite reductase (contains cytochromes (hemes) c and d) nirs 3 nitric oxide to nitrous oxide nitric oxide reductase small subunit norc braker and tiedje, 2003 nitric oxide reductase large subunit norb 4 nitrous oxide to dinitrogen gas nitrous oxide reductase nosz philippot, 2002 nosr nosdfyl treatment system was affected by salinity where the highest diversity of nr bacteria was observed at low salinity (yoshie et al., 2004; santoro et al., 2006). moreover, previous studies identified soil moisture and surface hydrology as important factors for supporting high denitrification activity (groffman and tiedje, 1989; hunter et al., 2008). however, it was suggested that factors other than levels of soil moisture controlled the % nr and dn bacteria since the relative abundance of nr and dn bacteria had a wide range (e.g. 0% to 100%) in different samples with the same moisture level (abubakr et al., 2019). degradation of petroleum hydrocarbons by facultative anaerobic bacteria petroleum hydrocarbons (hc) consists mainly of saturated hc (e.g. alkanes), usaturated hc (e.g. alkenes and alkynes), cycloalkanes, mono-aromatic and polycyclic aromatic hc (zhang et al., 2011). low-molecular-weight molecules, such as straight, branched, cyclic alkanes and aromatic hc, have been shown to be readily degraded by many microorganisms, while long-chain alkanes and polycyclic aromatic hydrocarbons are generally considered to be not as easily biodegraded due to their higher hydrophobicity (zhang et al., 2011). many different bacterial genera including pseudomonas, acinetobacter, and rhodococcus were shown to degrade alkanes (atlas, 1981; rojo, 2009). on the other hand, stenotrophomonas and pseudomonas species are among those shown to degrade toluene, benzene, ethylbenzene, and xylene (shim and yang, 1999; lee et al., 2002). also, a wide diversity of γ-proteobacteria such as pseudomonas and vibrio species were shown to degrade naphthalene (rockne et al., 2000). therefore, the presence of crude oil might either positively or negatively affect the abundance of nr and dn bacteria and their ability to perform nitrogen transformations. different environmental factors (e.g., oxygen, temperature, ph, and nutrient levels) affect the degradation of petroleum hydrocarbons in soil. in addition, several physicochemical factors such as the number and types of microbial species present; the nature, amount, and bioavailability of contaminants (macnaughton et al., 1999; röling et al., 2002; and smith et al., 2008) also play an important role in degradation of petroleum hydrocarbons. the presence or absence of oxygen determines the pathway of biodegradation of hydrocarbons. fuchs et al. (2011) summarizes 4 major pathways of aromatic hydrocarbon biodegradation. the first pathway comprises an attack by oxygenases that hydroxylate and finally cleave the ring with the help of activated molecular oxygen. the second pathway is an anaerobic process that converts benzoyl-coa to cyclic 1,5-dienoyl-coa. the third pathway also occurs under anaerobic conditions in which fumarate can be added to toluene with the subsequent β-oxidation of the intermediate benzylsuccinate to benzoylcoa. and fourth is the anaerobic hydroxylation of ethylbenzene to 1-phenylethanol, and the atp-dependent carboxylation of acetophenone that is involved in the conversion to benzoylcoa. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 702 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) humic substances: interaction with hydrocarbon degradation humic substances also affect hydrocarbon degradation. humic substances comprise about 60 to 80% of the soil organic matter and consist of three chemical groups based on solubility in water adjusted to different acid alkaline (ph levels) conditions: humic acids, fulvic acids, and humin (brady and weil, 2002). dissolved organic matter (dom) plays a critical role in determining the chemical and biological fate of organic contaminants in soils and sediments (johnson and amy, 1995). sequestration and irreversible binding of dom has been thought to shield organic contaminants from degradation (ragle et al., 1997; engebretson and wandruszka, 1999). some agricultural soil microorganisms were found to be nitrate-dependent humic acid (ha)-oxidizers. these microorganisms are phylogenetically diverse and included members of α-proteobacteria, β-proteobacteria, and γ-proteobacteria (van trump et al., 2011). also, it was shown that microbial reduction of humic substances (hs) may play an essential role during the anaerobic oxidation of organic pollutants in anaerobic environments (cervantes et al., 2008). some nitrate-reducing organisms also reduced hydroquinones within humic acids (ha) (lovley et al., 1999; coates et al., 2002). this reduction of ha by nitrate reducing bacteria may make hydrocarbons more accessible to this group of bacteria. it was illustrated that naphthalene 1,2-dioxygenase (ndo) is a humic-modifying enzyme that alters the bioavailability of organic contaminants associated with dissolved organic matter under aerobic conditions (abubakr et al., 2008). nitrogen as a limiting nutrient in the tallgrass prairie nitrogen limitation is an important regulator of plant growth (aber et al., 1997; shaver et al., 2001; lebauer and treseder, 2008). the effect of nitrogen on photosynthesis is simulated by most ecosystem models by using a relationship between leaf nitrogen content and photosynthetic capacity (aber et al., 1997; thornton et al., 2002). however, this relationship, in reality, may vary with different light, nitrogen availability, temperature, and co2 conditions (friend, 1991; reich et al., 1995; ripullone et al., 2003). photosynthesis and respiration are main biological processes in plants in which nitrogen is a major constituent of proteins for these processes (marschner, 1995). however, nitrogen is a limiting factor for plant growth (aber et al., 1997; shaver et al., 2001; lebauer and treseder, 2008). therefore, denitrification may decrease soil nitrogen levels available for plants, thus affecting the growth and survival under specific environmental conditions (friend, 1991; verkroost and wassen, 2005). atmospheric nitrogen is converted into ammonium that is available to organisms by biological nitrogen fixation. this process is an important natural input of available nitrogen in many terrestrial habitats (zehr et al., 2003). sublette et al. (2007) showed that nitrogen is a critical nutrient in the tallgrass prairie a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 703 soil where the addition of n-containing fertilizer was used to bioremediate a terrestrial crude oil spill, re-establish pre-spill n cycling and microbial diversity in order to accelerate the subsequent restoration. this microbial diversity is assumed to help in re-establishing the wide range of biogeochemical functions that are responsible for the recycling of soil nutrients and, as a result, supporting life in that area (sublette et al., 2007). a different study on the fate of the applied nh4 through five growing seasons illustrated that the flow of nitrogen within native tallgrass prairie soils was controlled by the incorporation of nitrogen into soil organic matter (som). however, plants appeared to maintain productivity by firmly conserving immobilized nitrogen (dell et al., 2005). burning of grasslands has long been recognized to maintain their plant diversity. burning also affects the nitrogen pools. although burning of grasslands causes loss of n from tallgrass prairie systems, burning can increase the total recovery of applied nitrogen due to greater n immobilization in the som where n is not available for plant uptake and needs to be mineralized first. the mechanism for increasing nitrogen immobilization was likely due to the greater microbial nitrogen demand in response to larger organic matter inputs with wider c:n ratios that is typical of burned prairie. this n immobilization increase microbial activity and aid rapid decomposition and turnover of organic matter, resulting in more available nutrients for plants over time (dell et al., 2005). a different study showed that unburned prairie was wetter and had higher concentrations of no3 in soil solution than annually burned sites. also, although the rate of denitrification varied seasonally, denitrification was significantly higher (p < 0.05) in unburned sites than that in annually burned, annually burned and grazed, and cultivated sites. in fact, the denitrification enzyme activity (e.g. n flux rate) was highest in the unburned sites (e.g. 327 ± 69 μg. kg-1. h-1) and lowest in the cultivated sites (e.g. 30 ± 5 μg. kg-1. h-1) (groffman et al., 1993).these results indicate that nitrate was released to the atmosphere and did not accumulate in soils in the annually burned sites. references aber jd, sv ollinger, and ct driscoll (1997) modeling nitrogen saturation in forest ecosystems in response to land use and atmospheric deposition. ecological modelling 101: 61–78. abubakr sm, ke duncan, gj thoma, and k sublette (2019) the effect of environmental factors on the abundance of culturable nitrate reducing/denitrifying bacteria from contaminated and uncontaminated tallgrass prairie soil. atlas journal of biology pp. 592–602. abubakr s, sl macmil, ma nanny, and ke duncan (2008) enzymatic transformation of humic substances by ndo. soil biology and biochemistry 40: 2055-2062. abubakr sm, and ke duncan (2015) the effect of oxygen supply on nitrite reduction by tallgrass prairie soil bacteria. proceedings of the oklahoma academy of science 95: 147-160. adman et, jw godden, and s turley (1995) the structure of coppernitrite reductase from achromobacter cycloclastes at five ph values, with no2 bound and with type ii copper depleted. journal of biological chemistry 270: 27458-27474. atlas rm (1981) microbial degradation of petroleum hydrocarbons: an environmental perspective. microbiological reviews 45: 180209. avrahami s, r conrad, and g braker (2002) effect of soil ammonium concentration on n2o release and on the community structure of ammonia oxidizers and denitrifiers. applied and environmental microbiology 68: 5685-5692. beaumont hj, si lens, wn reijnders, hv westerhoff, and rj van spanning (2004) expression of nitrite reductase in nitrosomonas europaea involves nsrr, a novel nitrite-sensitive transcription repressor. molecular microbiology 54: 148-158. boch j, g nau-wagner, s kneip, and e bremer (1997) glycine betaine aldehyde dehydrogenase from bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine. archives of microbiology 168: 282-289. bock e, i schmidt, r stuven, and d zart (1995) nitrogen loss caused by denitrifying nitrosomonas cells using ammonium or hydrogen as electron-donors and nitrite as electron-acceptor. archives of microbiology 163: 16–20. brady n, and rr weil (2002) soil organic matter. in: brady, n., weil, r.r. 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2 medical imaging department, faculty of health professions, alquds university, palestine received: december 23, 2019 / accepted: february 1, 2020 __________________________________________________ * corresponding author: mhjouj@hotmail.com 706 abstract understanding the impact of radiation on occupational workers has been a huge concern; particularly when it comes to cancer, which is considered a stochastic effect from radiation. this paper aims at investigating the effects of low radiation doses on tumor markers tests among radiation workers (rws). it also aims at demonstrating the effect of independent variables, such exposed dose, smoking status, and the type of work on tumor markers. the researchers reviewed the collected database of tumor marker tests in four governmental hospitals between the period (20132019) in order to trace the patterns of tumor marker over the years. the exposed dose record was taken from the energy department. additionally, a questionnaire was distributed to acquire correlated independent variables with tumor markers records and 78 rws participated in this study. results indicated that after several years of tracing tumor markers, they all tend to increase in a normal range. significantly, the carcinoembryonic antigen (cea) has increased by approximately 57% in rws. moreover, the smoking workers have a statistically significant change in cea. finally, the study has not shown any relationship between the radiation doses and tumor markers. in conclusion, to the best of the researchers’ knowledge, this is the first study to investigate the correlated tumor markers with low radiation doses among occupational worker. the researchers believe that these findings will contribute to ‘gap-filling’ in low dose effects, and demonstrate the importance of laboratory medical tests in prediction of low doses effect. however, further investigations are needed in order to achieve more accurate results. keywords: low dose, radiation workers, tumor markers, cancer, stochastic effects, retrospective. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction occupational radiation workers (rws) are more likely to receive low doses of radiation over a long period. the basic of low doses radiation effect is non-lethal mutations, with the biggest concern being the induction of cancer. this concern comes from the fact that some radiation risks are associated with dose by a linear, no-threshold model. in brief, linear means a proportional relationship between the dose and the risk. additionally, any dose -no matter how small this does is-, which may cause some risks is called no-threshold. (calabrese and o’connor, 2014). furthermore, the study reported that cancer in rws has significantly increased over the years (choi et al., 2013). several recent studies have shown evidence of cancer among occupational worker. one study used human’s peripheral blood lymphocytes to assess the dna damage. antioxidant status shows that the chronic low dose exposure is an unavoidable hazard, which would lead to an oxidative stress, increased genomic instability, and a rise in cancer among rws (siama et al., 2019). moreover, the incidence of thyroid cancer rates is higher among korean rws than the general population (lee et al., 2018; lee et al., 2019). another study on health risks shows an increase in all of skin lesions, orthopedic illness, cataract, hypertension, and hypercholesterolemia in cardiac catheterization workers (andreassiet al., 2016). one study, which focused on reliable cancer mortality risks from a low-dose in a nuclear industry, showed an evidence of stochastic effects and statistical significance for lung cancers (qu et al., 2018). based on that, it is crucial to provide the population with more intensive and protective measures, safe development tools, and acceptable tests for the early detection of cancer (schiffman et al., 2015). tumor biomarker is one of the most important tools, referred as a secreted molecule in the blood arising out of a tumor in case of cancer presence. although the field of radiation detection shows that, an assessment of absorbed dose using a protein with gene expression, will be considered as a good biomarker a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 707 of radiation, the insufficient sensitivity and specificity of tumor marker tests generate positive results, yet this does not necessarily indicate the presence of cancer. this means that additional procedures, such as biopsy, x-rays, or etc., are needed in conjunction with these tests. (sahoo, 2018; kayaba, 2003; rana et al., 2010). some tumor markers, such as cea glycoprotein, are used for screening the elevated levels found in colorectal, breast, lung, or pancreatic cancer and, in smokers (bhatt et al., 2010). however, it has special sensitivity among markers for colorectal cancer (gao et al. 2018). another marker is cancer antigen 19-9, commonly used for pancreatic cancer (scarà, 2015). while the regular ca19.9 measurements show an improvement in early detection of biliary tract cancer, the last one will included in this study is a carbohydrate antigen 125, a tumor marker for ovarian cancer. however, study shows that a combination of ca125 and human epididymis protein 4 (he4) improves the sensitivity and specificity of ovarian cancer (wannhoff, 2019). on the other hand, several studies have reported that multiple markers are more useful in the detection of some cancers. some other studies reported that the combination of ca 19-9, cea, and other markers will increase sensitivity and specificity in gastric cancer. moreover, other studies have shown that the combination contributes to diagnoses and prediction of pancreatic cancer. the combination of ca19-9 and ca125 has shown to encourage sensitivity for pancreatic cancer (kotzevet al., 2018; li et al., 2013; meng et al., 2017; o’brien et al., 2015). similarly, the impact of occupational radiation on some tumor markers, and the effect of high background radiation on them have been studied. a study in 2019 in the field of biomarkers indicates that, levels of serum 8-ohdg which is a biomarker resulted in oxidative stress, were found to be significantly higher in interventional rws (gao et al., 2019). likewise, a study in ramsar (iran) has shown that there is significant alteration in cyfra 21, cea, and tag 72 levels due chronic exposure to high background radiation (taeb et al., 2014). the researchers believe that studying and investigating tumor markers might help highlight low radiation effects, especially cancer, among the workers are exposed to chronic low doses. materials and methods study participants for the study approval, al-quds university sent letters including the purpose of the study to the ministry of health (moh). official permission was obtained to visit the hospitals and distribute the questionnaires in addition to facilitate data collection procedures. a one-page participants’ consent form was attached to the questionnaire. the researchers gave total freedom to accept or reject participation in this research. (78) rws participated. workers with diseases, such as (hepatic disorders, diabetes or any other kind of disease), were excluded for the accuracy of the results. a worker with diagnostic modalities that did not utilize ionizing radiation (like mri, u/s) was also excluded. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) data collection after institutional review board approval, the researchers retrospectively reviewed a collected database of tumor marker tests in four governmental hospitals between 2013 and 2019 to trace the patterns of tm tests over the years. commercial architect kits had been used to measure the serum levels of ca125, cea, and ca19.9 tumor markers. the test records are distributed as follows, 42 records of cea, 45 records of ca 19.9, and 35 records of ca125 conducted over the years of the study. independent variables, such as smoking status and the type of work, were reached and obtained by the distributed questionnaire, while the dose recorded was taken from a database of the energy department. statistical data analysis collected data were analyzed by using spss, version 22. student’s t-test was used for testing the equality of the means in the groups. p-value <0.05 was considered significant. descriptive statistical analysis was used to determine the tumor marker behavior along several years. results and discussion assessment of tumor markers test through years at the beginning, the researchers tried to study whether the exposed dose had an effect on tms by dividing rws into two groups according to the exposed dose. one group had a registered radiation dose of about 1msv or less, while the second had over 1 msv. then, the researchers compared their average means to each tumor markers. as noted, the average doses for each year from 2019, 2018, 2017, 2016, and 2015 with an annual average dose found to be at 0.50, 0.67, 0.77, 0.5, and 0.72 msv, respectively. the maximum doses were found to be 2.021, 4.519, 4.266, 2.679, 4.875 msv, respectively. this means that the average dose for most rws did not exceed the annual dose limit of 20 msv for occupational workers. as shown in table 1, the independent-sample t-test is used to investigate whether the exposed dose had an effect on tms. the result shows no significant mean difference according to the exposed dose. actually, the irregular readings of the exposed doses make the results inaccurate. therefore, the researchers decided to explore what is going to happen to the tms through years of exposure to the radiation. accordingly, one of the most important questions in this study is: what happens to tms test over several years of work in the radiation department? according to table 2 which represents the percentages of how tm tests of rws behave over the years (from 2013 to 2019), the tm test pattern is expressed by scale levels (decrease, increase, and stable). however, the unstable state is ignored due to an unclear behavior. in addition the average number of each behavior according to specific year is demonstrated in (figures 1, 2, and 3), for each type of tumor markers separately. these figures show how the average value was for each tumor marker and its behavior from the first year 708 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 1. statistically significant relationship between tumor markers according to an exposed dose (independent t-test). table 2. percentages of the behavior patterns of tumor markers during the years. tumor markers annual exposure dose mean std. deviation t sig cea less than 1msv 1.38 0.82 -0.59 0.56 more than 1msv 7.42 6.40 ca199 less than 1msv 11.34 5.11 -0.40 0.69 more than 1msv 1.38 0.82 ca125 less than 1msv 7.42 6.40 1.03 0.31 more than 1msv 11.34 5.11 tumor markers *decrease * increase *stable cea normal range (0-5 ng/ml) percent% 9% 57% 34% ca199 normal range( 0-37 ng/ml) percent% 31% 46% 23% ca125 normal range (0-35 ng/ml) percent% 37% 54% 9% **note that the increase, decrease and stable value was within normal range to all type of tm. figure 1. demonstration of the average value for cea behaviors by the year. the average cea value was (0.75 ng/ml) in the first year (2013), which illustrates the average of the rws who have an increase (which represent about 57% of the rws, see table 2) and the average of increase became (2.0 ng/ml) in the last year (2019). this shows the rate of increase over the years. it is worth mention that this example applies to all behaviors. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 709 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 2. demonstration of the average value for ca 19.9 behaviors by the year. the average value was (2 ng/ml) in the first year (2013), which illustrates the average of the rws who have an increase (which represent about 46% of the rws, see table 2), then the average of increase became (12.0 ng/ml) in the last year (2019). this shows the rate of increase over the years. it is worth mention that this example applies to all behaviors. figure 3. demonstration of the average value for ca 125 behaviors by year. the average value was (6.0 ng/ml) in the first year (2014), which illustrates the average of the rws who have an increase (which represent about 54% of the rws, see table 2), then the average of increase became (14.0 ng/ml) in the last year (2019). this shows the rate of increase over the years. it is worth mention that this example applies to all behaviors. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 710 to the last year. as shown in table 2, we found a 57% increase in cea tms but within the normal range of 0-5 ng/ml (table 2, figure 1). only one rw had abnormal results in the cea. this reminds us of the alteration occurrence in cea levels in a high background radiation (taeb et al., 2014). these findings may highlight cea as the most sensitive tm to radiation, due to the highest percentage of increase among tms, being more stable and having the lowest percentage of decrease over the years. the elevated cea may appear in case of lung cancer as mentioned by bhatt et al. (bhatt et al., 2010). in fact, lungs are considered as one of the sensitive organs to radiation with a risk factor at 2.0 *10 -3 sv-1 (till and grogan, 2008). moreover, it is important to mention the effect of smoking in cea (as will be discussed below), which may help in the interpretation of this increase. in any case, more investigation is needed to accurate the findings. however, ca-19.9 and ca125 have an increase of (46%) and (54%) within the normal range (figures 2 and 3) respectively. additionally, there is a tendency for a decrease of (31%) and (37%), respectively, with a very low percentage of stability in ca-19.9 and ca125 as tms. overall, the increased pattern was the most behavior tms have followed. this is despite the fact that, as mentioned in more than one study, the combination of tms has shown to be an encouraging sensitivity, for detecting several types of cancer, such as colorectal cancer (gao et al., 2018 ), gastric cancer (kotzev et al., 2018; li et al., 2013), and pancreatic cancer. (meng et al., 2017; o’brien et al., 2015). lastly, the effect of low radiation on tms behavior may have shown a little increase over the years, but a lot of investigation is needed to obtain results that are more accurate. it should be taken into account that this study is retrospective; therefore, the researchers have neither considered the systematic errors of equipment nor used any different types of equipment over the years, which, in fact, can affect the accuracy of the results. the effect of tumor markers by independent variables the determination of how different variables can affect tms is vital. it can help to understand how tms are affected by radiation, after isolating the effects of other variables. the following tables illustrate significant differences at the level of (p ≤ 0.05) due to tms effects according to the independent variables, such as smoking status, and type of work. the researchers tested the hypotheses for each variable separately as follows: smoking & tms the researchers compared the average means to each tumor marker for the two groups of rws, smokers and non-smokers. the independent-sample t-test is used (table 3),the results show that only the means of cea concentration between other markers have significant differences (p=0.01). the significant differences were in favor of the rws who are smokers, thus, coinciding with other studies that proved that smoking affect cea markers (bhatt et al., 2010; nan et al., 2017). according to that, smoking may cause the effects of radiation on rws to be worse. on the other hand; smoking does not affect the ca19-9 & ca125. this was suspected because smoking is not considered as an important factor in these markers, according to previous studies. work type & tms the researchers tried to find if there was a relationship between the two groups of workers who work on different devices, the x-ray / ct group and the interventional group. they conduct this by comparing the mean averages to each tumor marker for each group. based on the results of the independentsample t-test (table 4), the means of the two groups were not significantly different. this may be attributed to the rotation system in the radiation departments (there is no specific rw table 3. statistically significant relationship between tumor markers according to smoking (independent t-test). tumor markers smoking status mean std. deviation t sig cea smoking 1.71 0.84 2.88 0.01 non-smoking 1.21 0.59 ca199 smoking 5.99 6.07 0.39 0.70 non-smoking 5.47 4.73 ca125 smoking 11.14 4.54 0.77 0.45 non-smoking 10.12 5.37 table 4. statistically significant relationship between tumor markers according to type of work (independent t-test). tumor markers type of work mean std. deviation t sig cea ct , x-ray 1.35 0.72 -1.05 0.30 interventional 1.55 0.80 ca199 ct , x-ray 6.51 6.48 1.39 0.17 interventional 4.68 3.22 ca125 ct , x-ray 10.29 4.81 -0.50 0.62 interventional 10.96 5.29 711 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) to any type of equipment). it benefits to distribute a dose while working with high dose procedures (like interventional) among rws. world health organization (who) reported, “where higher doses are possible, careful use of worker rotations will reduce the risks” (world health organization, 2003). conclusion the researchers believe that these findings will contribute to ‘gap-filling’ in low dose effects, and demonstrate the importance of laboratory medical tests in the prediction of low doses effect. the effect of radiation may be presented as little increase from years to years, as the increase is the most pattern that tms is behaved. however, further investigation is still needed. it is important to repeat this study after few years in order to understand how radiation affects our bodies. smoking workers have a significant difference in cea tumor marker. a recommendation must be issued to reduce smoking when working with radiation because it might make the effects of the radiation worse for rws. the rotation system does not only aim to train rws to work with different types of procedures, but also helps distribute high doses to all workers, thus, making everyone in a safer environment. surely this does not prevent tests from being inaccurate for the early detection of ca as mentioned earlier in previous studies. according to that, workers in this field may need more accurate tests, or we may consider radiation to be safer than what we really think. in light of this, studying tumor markers helps policymakers decide if they should approve policies to decrease the irradiation of occupational radiation work, or recommended better laboratory tests that monitor the chronic low doses effect on rws. finally, the researchers state that due to chronic low doses of radiation received by occupational rws, an effective remedial action program to protect this population should be of the highest concern. conflict of interest the authors declare no conflict of interest. funding this work was not funded by any grant from public, commercial, or not-for-profit agencies. acknowledgments tha authors thank mr. marwan zuhd for his help in statistical data analysis. references andreassi mg, e piccaluga, g guagliumi, m del greco, f gaita, and e picano (2016) occupational health risks in cardiac catheterization laboratory workers. circ cardio vasc interv 9(4) : e003273. bhatt an, r mathur, a farooque, a verma, and bs dwarakanath (2010) cancer biomarkers-current perspectives. indian j med res 132 (2): 129-149. calabrese ej, and mk o’connor (2014) estimating risk of low radiation doses–a critical review of the beir vii report and its use of the linear no-threshold (lnt) hypothesis. radiat res 182 (5): 463-474. choi kh, m ha, w lee, ss hwang, 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african americans and west africans: a cultural exploration of agreement and divergence in attitudes in food environments and food responses catrina johnson1,2*, robert corruccini3, daniel becque4, wanki moon5, kola ajuwon6, and david lightfoot1,2 1 genomics core facility, southern illinois university carbondale, il 62901,usa; 2 department of plant, soil & ag systems, southern illinois university carbondale, il 62901, usa; 3 center for health, nutrition & biomedicine p.o. box 1062, park forest, il 60466, usa; 4 department of anthropology , southern illinois university carbondale, il 62901, usa; 5 department of kinesiology, southern illinois university carbondale, il 62901, usa; 6 department of physiology, southern illinois university carbondale, il 62901, usa; 7 department of agribusiness,southern illinois university carbondale, il 62901, usa; 8 department of animal sciences & nutrition, purdue university-elkhart, in 49707, usa. received: january 16, 2019 / accepted: december 14, 2019 __________________________________________________ * corresponding author: johnsoncatrina@gmail.com 661 abstract african americans, currently over-represented among low ses groups, have been found to be among the most at risk groups for obesity and type 2 diabetes. phenotypic expression, such as obesity, result from the combined effect of genetic inheritance and environmental influences. environmental dynamics interact with individuals on micro, meso and ecto levels (bronfrenbrenner, 1989), are diverse and cultural (vygotsky, 1993) and are learned by individuals through modeling and observation (bandura, 1989). environmental factors such as parental influence and modeling, availability and accessibility of produce, sweetened drinks and fast foods, can contribute to or minimize the onset of obesity and type 2 diabetes. consumption of fruits, vegetables and water has been shown to militate against the onset of obesity and type 2 diabetes. in the study ‘genetic bottlenecks impacting genes governing food responses in african americans’ the author considers the genetic and environmental responses to the onset of obesity and type 2 diabetes among african americans. the author uses a culturally competent model for repeated traits *(p=µ+g+ep+et) to evaluate the genotype/phenotype of african americans. this model incorporates the contribution of temporary and permanent cultural environmental factors of historical origin, related to food responses, predisposition, metabolism and ethnic adjusted mean values of physical assessment this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. to examine if the environment/genotype variables are informative towards the type 2 diabetes expression among african americans, and to assess the validity of bmi as a predictor of adiposity and/or obesity prevalence among non-european populations. the main goal was to evaluate the relationship between bmi, fat distribution/prediction, variation among phenotypes and assess their relationships to the obesity genotype and environmental influences affecting metabolism and food choice. an independent study confirmed bmi to be an ineffective predictor of obesity in african and african american populations. there was found to be different correlates and determinants of bmi and diabetes in african and african americans. some aspects differed between american-born and african-born african americans. barriers existed on the level of community affecting african americans. changing u.s. food environment exposed african and african americans to the obeseogenic effects of fast food, gmos and growth hormones. diabetes will continue to affect aa’s in response to historical pressures of the past and additive pressures of subjugation pressure and poverty. keywords: african, african-americans, ses, type 2 diabetes, environment, modeling, observation, parental influence, produce consumption, affordability. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) introduction man, is said to be a product of his/her environment. similarly, man is a product of his/her food environment. environment, the stage upon which genes act, is a dynamic that influences the phenotype, and consequently the genotype of all individuals, in complex ways, encompassing temporary and permanent affects and playing out on macro and micro levels. this study unpacks the dynamics of environment towards a revelations of food preferences for an african and african american cohort, across 131 variables and 20 categories that included: food availability and affordability, ses, parental modeling, proximity, and bmi. studies have shown there to be a direct association with income and food choice (turrell et al., 2003). healthy consumption of fruits and vegetables, low or nonfat milk and whole grains is a privileged behavior (gregson, 2009). affordability has been shown to present a barrier to fruit and vegetable consumption among low income households (cassady et al., 2007). in order to reach national dietary benchmarks, low income households would have to spend as much as 70% of their income on fruits and vegetables (cassady et al., 2007). the higher cost of whole grains and lean meats causes poor families to make poor food choices that are affordable and unhealthy (jetter and cassady, 2006). in general, low ses also causes high environmental stressors, increases cortisol levels in children and negatively impacts health and well-being (lupien et al., 2000). feeding restriction and food control is the same among class regardless of income. studies show that shared family meals gives parents a good chance to impact nutrition, produce consumption and prevent obesity causing factors like high fat/ sugar consumption and eating while watching t.v. (utter et al., 2008). parents can model a positive food environment for their children. parental consumption of milk and produce influences that of their children (arcan, 2007). good nutrition among african american children can be influenced by observed eating habits of their parents (tibbs et al., 2001). observed produce consumption in parents and the child’s food preferences, influences the produce intake of the that child (wardle et al., 2005); (nanney, 2007). produce availability at mealtimes influence consumption in adolescents (arcan, 2007). children raised in households with authoritative styles (highly strict and highly involved) showed the most favorable consumption of fruits and vegetables (kremers et al., 2003). “experiences such as eating fresh-picked fruits and vegetables while growing up or vegetable gardening as an adult may enhance vegetable consumption among members of some ethnic groups” (devine et al., 1999).”households where produce consumption is encouraged and observed in parents impacts produce consumption in children favorably (young, 2004).homes with produce gardens have been shown to have increased produce availability (nanney, 2007). parents can model a negative food environment in the home for their children that can set the stage for adult obesity. fat intake in children is related to parental behaviors. children who grow up in environments having parents who are high fat con662 sumers are likely to model this behavior (rossow, 1994). studies have shown that consumption of sweetened drinks from childhood to adulthood causes overweight in women (nissinen et al., 2009). parents have been shown to influence consumption of sweetened drinks in children (van der horst & et al., 2007). the choice to breast feed or not to breast feed can also set the stage for adult obesity. breast feeding prevents obesity in children. lack of breast feeding among the poor leads to fat children (martorell et al., 2001). and there are additive factors in childhood that will compound issues of future obesity. fat cells formed in children during adolescence will stay the same during adulthood. a parent’s failure to control a child’s calories during the slow growth period leads to adult obesity (spalding, 2008). the food preparation/consumption environment of the home can also be obesogenic. consumption of fried foods and low consumption of vegetables have been associated with obesity (bmi) in african americans (weinrich et al., 2007). fried foods and lack of vegetable consumption have been associated with obesity in studies involving black men (weinrich et al., 2007). studies show that equidistance to agricultural products, like gardening, can positively influence fruit and vegetable consumption (devine et al., 1999). among black youth produce consumption is influenced more by convenience than home availability (befort, 2006). but there exists a problematic reality to consumption. there are separate but unequal food environments for black and white, wealthy and poor (gregson, 2009). grocery stores with adequate produce supply are often out of reach for disadvantaged african americans (zenk et al., 2005). only canned and frozen vegetables are available in neighborhoods that are all black (morland et al., 2007), thereby putting blacks at risk for bpa exposure and its adverse effects on health. local grocery stores with inadequate supplies of healthy foods contribute to a nutritionally poor food environment with regards to consumption of whole grains and lean meats (jetter and cassady, 2006). no health clubs or grocery stores with produce sections cause more obese neighborhoods. it is a function of wealth and race (black, 2008). aside from parental/home influences, food preparation, availability, ses status and environmental estrogens that act upon the phenotype, proximity of fast food restaurants to residential areas increases the risk of community obesity. equidistance to fast food outlets can have a negative nutritional impact because fat consumption is influenced by fast food visits (befort c, 2006). obesity, high fat, low produce consumption and poor health is associated with fast food consumption among african americans (satia et al., 2004). proximity of produce markets to residential areas compared to fast food vendors has been shown to be an obesogen, or an environmental factor, shown to have a direct relationship to obesity among local populations (spence & et al., 2009). structural inequality translates to local inequality of place and poverty has an effect on bmi (gregson, 2009). the school environment can play a role in student food choice and potentially impact the home/parent environment. targeting student self efficacy could improve fruit and vegetaa tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 663 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) ble consumption (young, 2004). in urban populations, education and cultural upbringing can influence fruit and vegetable consumption (devine et al., 1999).“educators might focus on improving home fruit and vegetable availability, student self efficacy, as well as parent support and modeling. the level of availability might indicate where efforts should focus for enhancing parent behaviors (young em, 2004).”educational programs promoting awareness of local produce sources and facilitating the development of garden programs may be a worthwhile investment (nanney ms, 2007).” on a macro level, the growth of fast food in our local and worldwide consumption environment is exacerbating obesity. modernizing countries are experiencing obesity due to americanized fast foods (astrup a, 2008). there is a global variation of transfat levels among fast food chains (stender & et al., 2007). abdominal obesity is related to transfat consumption of fast food (astrup a, 2008). in general, large portion sizes, high trans fat levels and frequent consumption leads to obesity and related diseases worldwide (stender et al., 2007). on a micro level of ethnic-cultural environment, variation can be significant. there is cultural variation in obesity attributable to environment. the change from walking to driving in society has been shown to impact obesity in some ethnic groups. increases in african american obesity, beginning in 1940 might be attributable to automobiles (komlos et al., 2009). the food preferences survey the food preferences survey is a tool that was designed to understand where food preferences came from. are they individual, learned, environmental or cultural? it also examined an individual’s relationship to food in their environment and if this relationship is a contributing factor to obesity or leanness? the questions, by design, provide insight into what kinds of foods are in the participant’s pantry, freezer, and refrigerator. how they prepare food and if they prepare foods like their parents did? the survey looked at barriers to nutrition like affordability, socio-economic status, and availability due to factors like proximity of grocery stores. was the individual raised and/or do they currently reside in a food desert where grocery stores containing fresh fruits vegetables and meats are more than 5 miles away? overall 131 variables were included in questions asked that spanned across 20 categories of: affordability, availability, body mass index, bpa (environmental estrogen) exposure, consumption, cultural influence, diabetes affected, education, exercise, food preferences, food preparation, gardening, geography, meal planning, meals w/ family, obesity, obesity prevention, parental facilitation, socio-economic status and time spent in the u.s. the categories and variables represent a micro perspective that captures the actions of the actor. the food preferences survey also pans out to include a macro perspective of the actor in their context of environment and challenges the observer to imagine how the particulars of environment could impact food choices and preferences? for instance, could rural, suburban or urban geography act upon food availability? could the diets particular to tribe and culture impact consumption? what cultural norms may have impacted the parents and the maternal and paternal grandparents that could have been passed down to participant? methods environment, according to vtgoksky (vygotsky, 1993) is diverse and cultural. the participants included 3 groups in two categories: african american and their west african reference population. the african participants represent those who had been in the u.s. for 10 yrs or more and 10 yrs or less. the study concluded with a diverse african population that included a majority of west africans. the greater diaspora of african countries are reflected that capture west african cultures not just where they traditionally reside but cultures and generations in migration. the survey captures the geography of not just the participants but their parents and maternal and paternal grandparents. in all, 16 countries are represented that include: zimbabwe, ethiopia, zambia, nigeria, togo, eritrea, ghana, the republic of congo, trinidad, benin, cote d ivoire (ivory coast), jamaica, ireland, the european union and the united states of america. the regions of west africa, east africa and south africa are represented as well as the u.s. southern, northern, central, midwest and eastern regions and rural, suburban and urban lifestyles. 24 african tribes are represented that include: shona, edo, oromo, akan, urhobo, tigrinya, twi, benin, afro-trinidad, luba, ijaw, ibo, ashanti, etuno, amhara, yoruba, kuric, efik, ewe, tchokossi, dominican, zamunda, puerto rican, lakota, cherokee and african american. eighteen languages were spoken among our participants and their parents in addition to english: yoruba, izon, efik, hausa, edo, benin, ibo, baoule/agni, ewe, etuno, urhobo, benin, oromo, tigrinya, amharic, shona, french, spanish and patois. lineage. self reported maternal and paternal lineage data was collected from participants include: age, tribe, birthplace and rural vs. city migratory origin. this data was used to establish participant’s admixture status. this study required participants have both parents that are of african american, west african or gullah origin. food preferences. participants were required to complete a survey that measures food preferences and produce availability and food preparation methods. data was used to establish a potential point of origin for food preferences. food journal. participants were also required to fill out a 3 day journal recording food intake. data was evaluated by a registered dietitian who will analyze the nutritive content of food intake. results and discussion how the responses were examined the responses in this survey were examined in terms of all group and individual responses. for instance, if a question was asked like: “do you like meat?” if 67% of all participants asked says “yes” or “no”, this percentage is discussed here as “the response of all groups.” next, the percentage of responses within each of the two groups of african or african american is discussed. the african group represented a combination of africans who have been in the u.s. 10 yrs or more and ten yrs or less. of the 76 total participants, the african group consisted of 43 individuals and the african-american group consisted of 33 u.s. born blacks of slave descent, who had never been to africa. the percentages of each groups’ response were responses in reference to a part of the whole. for instance, all participants might respond “yes” or “no” to a particular question. using the above stated example, if 67% of all responses said this or that, the group responses looked at what percentage of that 67% was african or african-american. if 20% of the total group response was african and 47% was african-american, the group response asked of the 20% who responded this way, what percentage of the response of all africans would that represent. using our example, 20% of total group response by african american could mean that 87% of everyone in that group comprised the response of “yes” or “no” to that particular question. the responses of both groups were then compared in terms of similarities and differences between the two groups and this was discussed and placed in context of the larger question of if food preferences are shared within a group of the same origin like african and african americans-though separated by geography by hundreds of years? the non-responses were not indicated where they occurred for the sake of simplicity. affordability fruit, vegetables and meat were affordable to africans and african americans. there were subgroups within each that couldn’t afford these items (table 1). the indication here was, that if meat were free, these groups would over consume this product, therefore, the removal of cost as a barrier to meat consumption may lead to overconsumption. affordability of fruits and vegetables were correlated among africans and african american groups and may pose a barrier to produce consumption (nord, 2002; alaimo, 2001). it is indicated that that affordability of meat was not a barrier and did not put these groups at risk for poor food choice with regard to meat. availability both groups had fruit, vegetables and meat in their kitchen in some form. what was problematic was that african-americans derived their fruits and vegetables from cans and then from frozen (table 1). this indicated that the participants were from neighborhoods where fresh fruit and vegetables are only available in canned and frozen forms. this was confirmed by principal component analysis that showed a highly significant interaction between “vegetables in the pantry awaiting meal preparation” and,” if there were vegetables in the freezer awaiting meal preparation (p=<.0001).” there was a highly significant positive correlation with “if there were fruit in the refrigerator” and “if there were vegetables in the freezer (p=<.0001).” the location of a grocery store within 2-5 miles of the participant was a highly significant interaction with, “if there were fruits in the refrigerator (p=<.0001).” there was an interaction between” raw meat in the refrigerator awaiting meal preparation” and, “if there was raw meat in the freezer awaiting meal preparation (p=<.0001).” there was a highly significant interaction between “if there were vegetables in the freezer” and “if there were vegetables in the pantry awaiting meal preparation (p=<.0001).” and,” if there was a fast food outlet where the participant lived’ and “how many times they ate there,” were also highly significant interactions (p=<.0001).these factors indicated that african americans were under the effects of disadvantaged community (black, 2008; zenk et al., 2005; moreland, 2007; gregson, 2009), the effects of bpa (poole, 2004; hyoung et al., 2007) and spatial access to grocery stores or food deserts (moreland, 2007; zenk et al., 2005) and these barriers may have effects on the bmi of both groups (spence et al., 2009; ruel et al., 2010). bmi 1. bmi falsely assumed the presence of fat. it was assumed that the resultant values for mass in this population correlated with fat (table 1). it did not (kok et al., 2004; leading toxicology laboratories, 2015, 2017; kennedy et al., 2009; johnson, 2019). 2. bmi cutoffs were not found to be accurate across ethnicity (evans et., 2006; mills et al., 2007; kok et al., 2004). according to the bmi standard, 48% of both of these groups would be classified as overweight and obese, with african americans comprising 41% of this category and africans comprising even more at 45%. there was stern disagreement in the data that this population was 48% overweight and obese. when applied within the comparison of african americans and their reference population-west africans, in the within group comparison, showed their bmi value to be within normal range w/ 70% of body mass to be explained by fat free muscle compositionwhich is in agreement with the cultural standard for africans and african american somatotype of mesomorphy, having a large component of body mass comprised of muscle mass (heath and carter). therefore the bmi standard could not be used accurately to assess adiposity among all cultural groups, only within groups (satija, 2016; wagner and heyward, 2000; smalley et al., 1990; johnson, 2019). statistics multiple regression analysis using the fat free mass value of muscle weight in lbs as the dependent variable showed it to have a highly significant (p=<.0001) positive correlation with bmi. muscle percentage had a highly significant (p=<.0001) positive correlation with bmi. the fat free mass value of bones, when used a dependent variable in multiple regression analysis had a highly significant (p=<.0001) positive correlation with bmi (table 2). linear regression analysis examining bone density (db) as a variable against bmi found it to be a highly significant (p=<.0001) positive correlation. linear regression analysis examining the interaction between bmi and fat resulted in a a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 664 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 665 highly significant (p=<.0001) positive correlation. a multiple regression analysis examining the same interaction found it to be a significant positive correlation (p=.0060) (kennedy et al., 2009; smalley et al., 1990) (table 2). principle component analysis (pca) of the interaction of height and weight revealed that weight accounted for 54% of the variation in bmi among africans and african americans (table 1). bpa africans and african americans were found to be of a generation that grew up in the u.s. under plastics and microwaving. microwaving was a preferred method of cooking for african americans vs. africans. african americans were found to derive almost all of their vegetables and fruits from cans and they liked vegetables heated from cans. african americans were found to be in the low income group below $25,000 where mean bpa concentrations were found to be the highest (lang et al., 2008). africans were found to be in a demographic age group between 18-25 yrs where mean bpa concentrations have been shown to be the highest (lang et al., 2008). african americans were found to be in the ethnic demographic group having the highest mean concentrations present in urine samples (lang et al., 2008). additionally, because african americans weren’t breast fed as their african components were, they were found to have likely derived further exposure to bpa from plastic baby bottles as babies and formula (union, 1999; cao, 2008; newbold et al., 2007) and may have been at risk of underdevelopment of their metabolic systems as babies (janssen s. in kotz, 2008). therefore, african americans were found to be at great risk for the effects of bpa which include obesity (takeuchi et al., 2004; lang et al., 2008) adverse effects in the brain, immune system and reproductive system (vom saal and hughes, 2005); heart disease (newbold et al., 2007) ; increased disposition towards breast cancer, infertility and reproductive malfunction (brotons, et al., 1995), adverse effects on cell function (welshons et al., 2006); ovarian cyst disease in women (vom saal, 2005) advanced sexual maturation (howdeshell et al., 1999) and diabetes, per standard deviation of exposure (lang et al., 2008). consumption beverages with respect to beverages, africans and african americans were neutral in their preferences for milk and they deviated from their parent’s preference for whole milk by preferring low fat milk; they both liked sweet tea, soda, koolaid and water. sweetened drinks were a part of the nutritional belief system of both african and african american parents and they facilitated consumption to their children accordingly. africans liked the sweetened drinks more than their african american counterparts. female africans and african americans were found to be at risk for energy overweight in adulthood due to their exposure of consumption of sweetened drinks from childhood (nissinen et al., 2009), with the risk among the african group to be greater. both africans and african americans were found to be at risk for obesity and type 2 diabetes, due to exposure of high fructose corn syrup (hcfs) in soda (goran et al., 2012). both african and african american parents were found to possess poor nutritional beliefs with regards to feeding their children sweetened drinks (anderson and blackburn, 2008). africans were found to be at risk for transition away from traditional diets towards westernized eating habits and obesity and obesity related diseases, due to their consumption of sweetened drinks (astrup, 2008). fast food both groups liked to eat fast food and attributed their love of fast food to themselves. african americans were more likely though to have exposure to fast foods in the region where they grew up and lived than africans. the parents of african americans raised them eating fast foods, while the parents of africans raised their children on home cooked meals. more african american parents ate fast foods in front of their children, thereby modeling fat consumption, than african parents. both africans and african americans were at risk of fat consumption from fast food outlets (befort, 2006); abdominal obesity due to trans fat consumption from fast foods (astrup, 2008; weinrich et al., 2007) and poor health from fast food consumption (satia et al., 2004). african americans were found to be at risk for the effects of disadvantaged community due to promixity and access to fast foods in their community, making their neighborhoods obesogenic, or an environmental factor shown to have a direct relationship to obesity among local populations (spence et al., 2009). africans were found to be at risk for transition away from traditional diets towards westernized eating habits and obesity and obesity related diseases, due to their consumption of fast foods (astrup, 2008). because their parents raised them eating fast foods, this was found to be in the nutritional belief system of african american parents (anderson and blackburn, 2008); african americans were found to be at risk for acquisition of fat cells in childhood due to exposure of fast food; not being raised in calorie controlled environments in childhood and therefore gaining permanent fat cells in adulthood that contribute to the obese phenotype (spalding, 2008). african americans were found to be at risk for modeling high fat consumption before their children because their parents modeled this behavior toward them (rossow, 1994). fish, fruit, meat and poultry both groups liked to eat fish, fruit and meat. they both liked their vegetables steamed and had parents who modeled facilitated consumption of vegetables to them during dinner and lunch time. more african american parents facilitated vegetable consumption during dinner to their children than african and more african facilitated consumption of vegetables to their children during lunchtime than african american. only african 666 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 1. fp survey category tables pca analysis of significant interactions. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) fp survey category tables principal component analysis significant interactions variables significance / variation promotion 1. affordability 22freefruit 10vegprice <.0001 .50593 2. availability 8vegpantry 6vegfreeze <.0001 .64378 18fruitfridge 6vegfreeze <.0001 .43281 61grcwhere 18fruitfridge <.0001 .45626 39rmtfrdg 38rmtfrz <.0001 .43543 6vegfreeze 8vegpantry <.0001 .64378 18fruitfridge 6vegfreeze <.0001 .43281 18fruitfridge 61grcwhere <.0001 .45626 76bffregetx 76ffreg <.0001 .53297 3.cultural 89ht 70rorurb <.0001 .50465 62awtrl1eat_m 62awtrl1eat_c <.0001 .455776 62awtrl1eat_f 62awtrl1eat_m <.0001 .68400 69regbrn tribe <.0001 .51477 4. geography cntry, regbrn, tribe (all values for ptribe, m,f,mg,mgf,pgm,pgflines) <.0001 5. obesity 92owc 90owself <.0001 .49914 92owc 91nwt <.0001 -0.45198 90owself 91nwt <.0001 -0.77344 90owself fat_p <.0001 .43528 91nwt fat <.-0001 .46351 91nwt age <.0001 -0.43424 fat_p bmi <.0001 .68101 fat_p 89wt <.0001 .64505 bmi 89wt <.0001 .84076 6. parental preferences 64pvegeat 67parmlk <.0001 .46009 66pareatfrt 64parvegeat <.0001 .66855 66pareatfrt 67parmlk <.0001 .49054 7.ses 82pses 84pared <.0001 .46025 8.time in the u.s. 88wt 4cfat <.0001 .64505 91nwt 90owself <.0001 -0.77344 91nwt 4cfat <.0001 -0.46351 90owself 4cfat <.0001 .43528 4cbones 88wt <.0001 .73468 fp survey category tables principal component analysis significant interactions variables significance / variation promotion 667 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) americans preferred vegetables heated from a can, while their african counterparts were raised to and consumed vegetables from the garden. both groups liked vegetables, ate on the run, liked sugar and ate while watching television. african american preference for fried foods can put them at risk for obesity (weinrich et al., 2007) and male prostate cancer development via the consumption of grilled red meat (tang et al., 2007). both africans and especially african americans were found to be susceptible to colon cancer, due to their heavy red meat consumption and preferences for well-done meat in particular, due to the tendency of overcooked red meat releasing enzymes that metabolizes carcinogens (cotterchio et al., 2008). african american women may benefit from protection against colon cancer due to consumption of poultry (tang et al., 2007; cotterchio et al., 2008); giovanni et al., 1994; sharma, 2007). africans were found to be at risk of obesity and related diseases due to their tendency to like their poultry and fish fried. both african and african americans were found to have parents who influenced vegetable consumption positively and influenced good nutrition in them (nanney, 2007; tibbs et al., 2001; young, 2004; anderson et al., 2009; anderson and blackburn, 2008; kremers et al., 2003) that could result in their future consumption (arcan, 2007). both africans and african americans were found to be at risk for obesity and poor nutrition because they liked to eat on the run (utter, 2008). both africans and african americans were found to be at risk of obesity due to sugar consumption (goran et al., 2012; nissinen et al., 2009). both africans and african americans were found to be at risk of fat consumption influenced by television viewing (befort, 2006). african americans were found to be at risk for exposure to the obesogenic environmental estrogen bisphenol a (bpa) due to their consumption of fruits and vegetables from cans (brotons et al., 1995). african parents were found to facilitate the cultural upbringing of eating produce from the garden that may influence consumption in their children for garden produce in adulthood (devine, 1999). african and african american parents were found to provide home cook meals, african parents did this more frequently than african americans (anderson and blackburn, 2008). cultural influences african americans were from midwest and africans were from west africa. no parents in either group grew up or currently lived on a farm. african parents were from an urban setting, african american parents were from a rural setting. participants in both groups were from urban settings. [the african american participants were primarily from the midwest. this region has the distinction of being the bread basket to the world and though they are from chicago, this metropolis is surrounded by a rural setting (illinois is a prairie biome), therefore the participants responded “rural setting” vs. “urban”]. with regards to diets africans had more of a tendency to be food restricted than african americans, from pork. principal component analysis table 2. fp survey significant questions multiple regression (bf%). fp survey significant questions multiple regression dependent variable =body fat % [code] / questions secondary variables p/f value 1. [brorbtl] were you breast or bottle fed? n/a <.0001 2. [2friedfs] do you eat fried foods? n/a <.0001 3. [2a2x] how often (do you eat fried foods? n/a <.0001 4. [63fsitdown] were you raised having family sit down meals? n/a <.0001 5. [92owc] if you describe yourself as overweight, were you overweight as a child? n/a <.0001 6. [26sodalike] do you like soda? weight <.0001 <.0001 7. [26asodaxwk] if so, how often do you consume soda per week? weight <.0001 <.0001 8. [13tveat] do you eat while watching t.v. weight <.0001 <.0001 9. [yrs] how long have you been in the u.s. weight <.0001 <.0001 10. [24waterdrink] do you drink water regularly? weight <.0001 <.0001 11. [1ahowmswts] do you eat sweets more than 5 times a week? n/a .02804 12. [57rsetgff] were you raised eating fast foods? group #1 <.0001 .0092 668 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) (pca) correlated the eight of participants with the fact of “if they were from rural or an urban setting (p=<.0001);” the “religious restriction of the consumption of meat” with “religious restriction of chicken (p=<.0001);” “religious restriction of the consumption of fish” was correlated with the “religious restriction of the consumption of meat (p=<.0001);” and the “region born” had a highly significant interaction with the “tribe” of participants (p=<.0001) (table 1). diabetes affected principle component analysis examined the relationship between responses to the questions of “if type 2 diabetes ran in the participants families” and “if the participants themselves reported a diagnosis of type 2 diabetes.” the diagnosis of type 2 diabetes among participants accounted for 52% of the variation of the diabetes, affected category (table 1). the majority of the participants did not report a diagnosis of type 2 diabetes, however there were two participants who did, both were african american. one was from the caribbean west indies and having a very remarkable family history with the disease (i.e. amputations, blindness) on her maternal side. the other was the oldest female in the study (48yrs old) who reported adult onset type 2 diabetes. a regional pattern of fat distribution used to predict risk for onset of type 2 diabetes was ascertained from physiology data. 9% of african americans were in the at-risk category. all other participants were in normal range 1.0 for men and .8 for women and therefore not in the at-risk category for type 2 diabetes. education africans had more graduate school experience than the african american students in the study because more graduate students responded to participate among the african groups. more undergraduates responded among african americans (table 1). both african and african american parents had some college, african parents had more exposure to college than african american parents did. in principle component analysis, parents’ education drove the education of the participants. the education of the parents accounted for 51% of the variation of the education category. also the parents’ education was found to be a highly significant driver of parents’ income (p=<.0001) in the category of socio-economic status. african american parents therefore may be at risk for chronic stress increases in cortisol levels and bmi among populations without a college degree (daniel et al., 2006). exercise both africans and african americans were exercising for at least 1.5 hours a day. africans were more likely to ride bikes and walk to school or work than african americans. african americans were more likely to drive to school or work than africans. african americans were from neighborhoods (midwest, chicago) where structural violence was an issue, an effect that results in residents of bad neighborhoods not being able to exercise (walking) outdoors (lillioja et al., 1988). there was also an effect of poverty of place and disadvantaged community acting upon african american neighborhoods, where there are no health clubs access to fresh produce and where fast food outlets are plenteous (spence et al., 2009; black, 2008; gregson, 2009; ruel et al., 2010). african americans were getting 30 minutes of exercise, they just were walking to other places besides school or work. “walking vs. driving” and “driving to school or work” were found to be highly significant negative interactions in principle component analysis .physical activity may be able to help both groups diminish any additive effects, if they exist, of bmi heritability and waist circumference (mustelin et al., 2009). food preferences both african and african americans admitted to being selfmotivated fast food consumers from outlets like mcdonalds and red lobster. both groups consumed steamed vegetables, fruit, water and low fat milk, deviating from their parental preference for whole milk. african and african americans had a common taste preference for chicken. when african americans were asked to list “what their favorite foods were,” what they didn’t list was a strong preference for cultural soul food staples like collard greens and sweet potato pie. this could represent an equistance from cultural preferences in foods, either attributable to the fact the parents of the african american participants aren’t preparing foods like their parents did, or the fact that the taste preferences of african american youth are becoming less cultural and more assimilationist. both africans and african americans were found to be at risk for poor health associated with fast food consumption (satia, 2004). the parents of both groups modeled good nutritional influence before them with regards to milk consumption (arcan, 2007). african americans may be reflecting the modeling of their parents’ poor behavior of high fat consumption though their consumption of fast food (rossow, 1994). africans may be demonstrating transition away from traditional diets towards westernized eating habits and may be putting themselves at risk for obesity and obesity related diseases due to consumption of americanized fast food (astrup, 2008). both groups may reflect the modeling of positive nutritional influences of facilitated consumption of fruit, vegetables and milk from their parents (young, 2004). both groups were participating in privileged behavior with regards to their preferences for low fat milk (gregson, 2009). food preparation african and african americans were cooking their own meals and were cooking like their parents. the preferences among african americans to prepare their foods fried most often may reflect negative nutritional influences of their parents in the home (rossow, 1994). although the african participants may have had more positive nutritional modeling from their parents, they were not prea tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 669 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) serving these parents’ cooking methods at a faster rate than african americans, thereby opening the possibly of the gain of poorer and unhealthy habits (satia, 2004). the fact that both groups like to try new recipes, may facilitate the acquisition of new preferences, independent of parental cultural methods that may not be unhealthier (larson, 2006). african americans liked fried meats more than africans but africans liked frying their chicken more than african americans, who preferred baking it. in cooking their foods african americans preferred to first use the frying pan, then the microwave; for their meat. they preferred frying primarily and then grilling, and chose to bake their chicken. africans preferred to grill and then slow cook their meats and to use baking and microwaving to prepare the food in general and a rice cooker. it was problematic that both groups used the microwave to prepare their foods because this exposes each to plastics and heat and the possibility of exposure to bpa and its effects (vom saal, 2005). both groups on average took at least an hour to prepare their meals. therefore, in preparing their own meals both african and african americans were participating in better diet quality (larson, 2006). the preferences for fried foods among african americans could lead to obesity in black men (weinrich et al., 2007). gardening neither group gardened. geography this research study did not assume that cultures were static. a person born in one place may not have been raised in another. among africans participants who could have been of west african heritage, they could have moved within the continent of africa, the same is true for african americans within the u.s. principle component analysis of the category of geography correlates placement of “tribes” with “region” and “country (p=<.0001).” the main country of influence among african participants was nigeria with regional birthplaces in west and south africa. the main tribes represented in the study were yoruba and shona. african americans were more staticly influenced by the usa with regional birthplaces of the midwest and south. both groups were born in urban settings. african mothers were from nigeria, zimbabwe and ethiopia and represented the east and south african regions and the shona and yoruba tribes. african american mothers were from the midwest, usa. african fathers were born in nigeria, ethopia, zimbabwe and ghana, represented the west, south and east regions and the yoruba and shona tribe. african american fathers were born in the midwest and southern usa. both african and african american parents were from urban regions (even though african american parents listed rural as “region of exposure,” they are from the midwest which is an entirely rural setting even for residents of chicago. neither african nor african american parents were exposed to farms. maternal african grandmothers were born in nigeria, represented the west and south african regions and were of the yoruba and shona tribes. african american grandmothers were from midwestern and southern usa. maternal african grandfathers were born in nigeria, ethiopia, zimbabwe and ghana, represented the west, south and east african regions and were of the yoruba, shona and ewe tribes. african american grandfathers were from the usa. paternal african grandmothers were from nigeria, represented the west, south and east african regions and were of the shona and yoruba tribes. paternal african grandfathers were from nigeria, zimbabwe and ethiopia, represented the west, east and south african regions and were of the yoruba, shona and akan tribes. african american paternal grandmothers were from southern and midwestern usa. paternal african american grandfathers were from usa representing the southern and the midwestern regions. height africans were slightly taller and leaner than their african american counterparts. this may be attributable to geographic location of origin in proximity to the equator that influences taller leaner phenotypes due to warm climate. meal planning both africans and african american groups like to grocery shop, took the time to plan meals, liked to try new recipes and make grocery lists. africans had a stronger tendency to try new recipes and plan meals than african americans and this may explain why this group is not preparing meals like their parents at a faster rate than african americans. meal planning has the potential to improve self-efficacy among both groups and have a favorable impact on consumption and better diet quality (young, 2004; larson, 2006). obesity obesity encompasses four categories: heritability, cultural consumption, future prevalence and a combination of the three. 18% of african americans compared to 10% of african parents were reported to be overweight, therefore the heritability component of bmi (ludwig et al., 2001) isn’t strongly supported in the data. but the nutritional beliefs of both african and african american parents to feed their children sweetened drinks in childhood, could have had implications on control of calorie consumption during the slow growth period prior to adolescence, this combined with the obeseogenic side effect of sugar (nissinen et al., 2009) could have contributed to the presence of fat cell formation in adults today (spence & et al., 2009). and the fact that both groups were heavy consumers of meat, soda, koolaid, liked sweets and fast foods; could result in a future tendency towards obesity (ludwig et al., 2001; goran et al., 2012; astrup, 2008; satia, 2004; befort, 2006). additionally, in african americans the tendency of their parents to model fast food consumption, the lessened tendency towards sit down meals and 670 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) the fact that their parents did not breast fed them in considerable numbers, eliminating obesity prevention benefits of breast feeding, and exposure to the endocrine disruptor bpa through plastic baby bottles, infant formula, plastics, microwaving and consumption of fruits and vegetables through cans-could put them more at risk than africans of obesity (rossow, 1994; anderson and blackburn, 2008; martorell et al., 2001; vom saal, 2005). though the majority of both groups did not self report as overweight, there were responses that were given that could set the stage for overweight. in multiple regression analysis “whether the participant were breast or bottle-fed”; “ate fried foods”;the “frequency of fried food consumption;”” the tendency to have family sit down meals”; ‘whether one was raised eating fried foods;” “were overweight as a child;” “liked soda” and “how often they drank it;” “whether participants ate while watching television”; “time spent in the u.s.;” and “whether or not they drank water”-were all highly significant to fat percentage (p=<.0001). whether participants “described themselves as overweight” and “time spent in the u.s.” were highly significant interactions in principal component analysis (p=<.0001). “overweight in childhood” and “self description of overweight” or “normal weight”; “self description of overweight” and “body fat percentage;” “normal weight” description and “body fat percentage;” “normal weight” description and “age;” “bmi” and “body fat percentage”; “self description of overweight;” and “body fat percentage” and “weight” were all highly correlated interaction in principle component analysis (p=<.0001) (table 1). parental preferences the contribution of parental preferences was both positive and negative. the parents of both groups modeled and facilitated consumption of fruit, vegetables and milk in the home (nanney, 2007; tibbs, 2001; young, 2004; arcan, 2007). principle component analysis (table 1) confirmed that parents were “eating vegetables” and “drinking milk;” parents “eating fruit” and “eating vegetables” and parents “eating fruit” and “drinking milk”-were all highly significant interactions (p=<.0001). the tendency to impact good nutrition at mealtimes and the practice of family sit down meals was stronger in africans than african americans (utter, 2008). yet, both parents held the consumption of sweetened drinks in their nutritional belief system and fed this to their children thereby putting them at risk for adult obesity (anderson et al., 2009; anderson and blackburn, 2008; ludwig et al., 2001). and african american parents modeled fast food consumption to their children, increasing their likelihood for high fat consumption in adulthood (rossow, 1994). ses the “socio-economic status of the parents or ses,” was correlated with their “education” in principle component anaylsis (p=<.0001) (table 1). african parents were slightly more affluent than african american and had more college exposure. african american parents were more at risk for chronic stress increases to cortisol levels and bmi among populations w/o a college degree (daniel et al., 2006) and poor food choice due to income (turrell, 2003). time in the us “time spent in the u.s.” was highly correlated with “body fat percentage” and “weight” in multiple regression analysis (p=<.0001) (table 2). the longer participants stayed in the u.s. the more likely their tendency to increase body fat percentage. time spent in the u.s. for africans varied between 4 months and 33 yrs. african americans were in the u.s. all their lives and had a higher tendency to be affected by increase in body fat percentage than africans, yet both groups are in this risk category. conclusions fruit, vegetables and meat were generally affordable and available to all groups. there were subgroups within each that couldn’t afford these items (table 1). the indication here was that if meat were free, these groups would over consume this product, therefore the removal of cost as a barrier to meat consumption may lead to overconsumption. both groups had fruit, vegetables and meat in their kitchen in some form. however, african americans derived their fruits and vegetables from cans and frozen, which presents another factor of food exposure to an undesirable chemical in preservatives, such as bpa. africans and african americans were found to be of a generation that grew up in the u.s. under plastics and microwaving. microwaving was a preferred method of cooking for african americans vs. africans. african americans were found to derive almost all of their vegetables and fruits from cans and they liked vegetables heated from cans. both groups fell within income groups with high mean bpa concentrations according to lang et al. (2008). exposure to bisphenol a (bpa) from plastic baby bottles as babies from formula (consumers union, 1995; cao, 2008; newbold, 2007) and may have been at risk of underdevelopment of their metabolic systems as babies (janssen s. in kotz, 2008). therefore, african americans were found to be at great risk for the effects of bpa. the study revealed that “cultural influences” played an important role in food preferences (tables 1 and 2). both groups liked to eat fast food and attributed their love of fast food to themselves. african americans were more likely though to have exposure to fast foods in their childhood than africans. the parents of african americans raised them eating fast foods, while the parents of africans raised their children on home cooked meals. thus, all groups were at risk of fat consumption from fast food outlets, in agreement with earlier studies (befort, 2006). with respect to beverages, africans and african americans were neutral in their preferences for milk and they deviated from their parents’ preference for whole milk by preferring low fat milk; they both liked sweet tea, soda, koolaid and water. sweetened drinks were a part of the nutritional belief system of both african and african american parents and they facilitated consumption to their children accordingly. physical 671 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) activity may be able to help both groups diminish any additive effects, if they exist, of bmi heritability and waist circumference (mustein, 2009). this study showed that both africans and african americans were exercising for at least 1.5 hours a day. africans tended to ride bikes and walk to school or work than african americans (tables 1 and 2). both africans and african americans were found to be at risk for obesity and type 2 diabetes due to exposure of high fructose corn syrup (hcfs) in soda (goran et al., 2012). examination of the relationship between responses to the questions of “ if type 2 diabetes ran in the participants’ family” and ”if the participants themselves reported a diagnosis of type 2 diabetes?” the diagnosis of “type 2 diabetes” among participants accounted for 52% of the variation of the diabetes, affected category. the majority of the participants did not report a diagnosis of “type 2 diabetes.” a regional pattern of fat distribution used to predict risk for onset of type 2 diabetes was ascertained from physiology data (johnson, 2019). 9% of african americans were in the at-risk category. all other participants were in normal range 1.0 for men and .8 for women, and therefore not in the at-risk category for type 2 diabetes. obesity encompassed four categories: heritability, cultural consumption, future prevalence and a combination of the three. more african americans were reported to be overweight than africans. therefore, the heritability component of bmi (ludwig, 2001) was not supported in the data. • there are different correlates and determinants of bmi and diabetes in african americans than european americanssome aspects differ between american-born and african-born african americans • there exists barriers on the level of community affecting african americans. • the food environment in the u.s. has changed from agrarian to convenience to fast foods and now gmos. our food supply is toxic and the public is unprotected from the effects of gmos and growth hormones • diabetes will continue to effect aa’s in response to historical pressures of the past and additive pressures of subjugation pressure and poverty. note: proximity to produce does not necessarily impact consumption of produce in populations where healthier food consumption like produce and water are not a part of their nutritional beliefs or has been a part of parental modeling. the success of efforts to increase consumption of healthier foods may benefit from a protocol of facilitated consumption, like food preparation guidance and creative campaigns that encourage consumption; 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2 yousef bin abdul latif and sons agriculture co. ltd. research and development department. al-shihiyah 52758, gassim, saudi arabia. atlas journal of biology, 2020, pp. 713-723 doi: 10.5147/ajb.vi.223 abstract as many studies on other crops showed an acropetal decline of fruit-set and fruit size in the inflorescence, this paper investigates the effect of flower position in spikelets in the ‘sukkary’ variety of date palm on fruit-set and final fruit weight. also studied was the best time to apply strand-cut (bunch cut) as an alternative to hand thinning, to increase final fruit weight. hand thinning is a labor cost operation and worker productivity operation is one adult tree per day maximum. to study fruit-set in relation to fruit position, three types of pollen were used to pollinate female trees: new pollen harvested in march 2018, pollen stored for one year at room temperature (25-35° c), and pollen stored for two years at room temperature. four weeks after pollination, fruits aborted and those non-aborted in every bunch were counted. recording was done in every spikelet from proximal to distal. to study the effect of fruit position in spikelet on fruit weight, fresh pollen was used to pollinate ‘sukkary’ females. every two days, fruit weight was measured from pollination to harvest, and fruit growth pattern was drawn. an analysis of variance (anova) was carried out for the final fruit weight in every spikelet part. this investigation showed no pattern in fruit-set or in final fruit weight when using new pollen. when pollinated with old pollen that was one and two years old, the proximal ‘sukkary’ flowers of the spikelets showed less fruit-set compared to those of the middle and distal part. this basipetal decline is explained by low viability of old pollen, combined with the basipetal opening of the spathes in date palm. for strand-cut timing, the highest fruit weight was obtained when the strands were cut earlier at time of pollination (cut0: 15.82 g), 15 days after pollination (15 dap or cut1: 15.36 g) or 30 dap (cut2: 14.89 g). cutting strands later generated lower fruit weight respectively equal to 13.86 g for cut3 (45 dap), 13.61 g for cut4 (60 dap), and 13.75 g for cut5 (75 dap); all not significantly different from the 13.20 g obtained in the control where the strands were kept without cut (non-cut). keywords: bunch cut, acropetal, date palm, fruit, thinning. received: november 17, 2020 / accepted: december 6, 2020 ________________________________________ *corresponding author: a.eljiati@gmail.com this is an open access article distributed under the terms of the creative commons attribution license (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 713 a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) introduction date palm (phoenix dactylifera l.) has been cultivated in the middle east and north africa (mena region) for millennia; however, the exact origin of the date palm has not been verified. the date palm can be cultivated in all five continents of the world, largely between 39 degrees northernmost and 20 degrees southernmost latitudes. the main region of production is the middle east and north africa, where 89% of dates are produced (manickavasagan et al., 2012). current world date production is 8.68 million tons. the kingdom of saudi arabia (ksa ) is the fourth largest producer of dates. with about 25 million date palm trees, covering approximately 72% of the total area under permanent crops, ksa produces 1.3 million tons of dates annually, which represents 15% of the world date production (faostat, 2018). the date palm is one of the major crops that need to be improved from the technical side to optimize production expenses and decrease dependence on manpower. the ‘sukkary’ date is one of the most popular varieties consumed in saudi arabia due to its appealing taste. therefore, the authors’ objective for this study is to investigate the best time to apply bunch cutting and keeping 12 fruits on each spikelet as an alternative method to hand thinning, which is a high-cost labor operation. whatever method of thinning used in the crops they studied, many authors agreed on the fact that the time of thinning had a great influence on fruit size, and that early thinning generated good cell division and, therefore, larger fruit size (goffine et al., 1995; sutton et al., 2020; szot, 2010). in their investigation of the ‘sukkary’ date palm variety, eljiati et al. (2020) concluded that cutting the bunch at the time of pollination, and keeping 12 fruits on each spikelet, was a better alternative to hand thinning in increasing fruit size without affecting the yield per bunch and yield per tree. when two other methods of thinning were applied by moustafa et al. (2019), two months after pollination on ‘khadrawi’ date palms, and soliman and harhash (2012), applied thinning three months after pollination on the ‘sukkary,’ date palm they both obtained an increase in fruit size that negatively affected the yield per bunch and yield per tree compared to un-thinned trees (control). the two methods consisted of removing 15% or 30% of the total number of strands from the bunch center. another objective of this study is to examine the effect of flower position in spikelets on fruit-set and on fruit weight in the ‘sukkary’ date palm variety, as many studies on other crops showed an acropetal decline of fruit-set and fruit size in the inflorescence (bawa and webb, 1984; berry and calvo, 1991; diggle, 1995; obeso, 1993). fruit-set is considered to be higher in the base of the inflorescence than in the top. this is because basal flowers open and pollinate first and as a fruit exert a competition on water and nutrients to apical delayed ones (diggle, 1995; lee, 1989; stephenson, 1981). 714 materials and methods this study was carried out on the nafeesa farm of yousef bin abdul latif and sons agriculture co. ltd. (yala) in shihiyah, saudi arabia. the first part of the experiment was undertaken during the year 2018, and the second part during the year 2019. effect of bunch cut at different times after pollination on fruit weight nine date palm trees aged nine years old of the variety ‘sukkary’ were used to study the effect on final fruit size of cutting the strands (spikelets) at different intervals of time after pollination. to evaluate the best time to apply bunch cut as an alternative method to hand thinning, the authors selected nine trees of the ‘sukkary’ variety, aged nine years old, and in each tree, one bunch per treatment was chosen in such a way that they all opened at the same time. they were then pollinated with fresh pollen and tagged. the bunch cut was applied by cutting the strands in such a way that it kept 12 fruits per strand and removed the rest of the strand. the times of bunch cut evaluated (treatments) were: at the time of pollination (cut0), and 15 days after pollination (15 dap or cut1), 30 dap (cut2), 45 dap (cut3), 60 dap (cut4), and 75 dap (cut5), compared to the control where no bunch cut was applied (non-cut). every three days, one strand per treatment, per tree, was collected periodically and repeatedly, and their fruits were weighted from pollination until fruit maturation and harvest. in every measurement the authors had nine strands per treatment to measure their fruits and calculate their average fruit weight that would be used to draw their fruit pattern curve. fruit fresh weight was measured using a top load balance. effect of flower position in the spikelet on fruit-set and fruit weight to study fruit-set in relation to fruit position, three types of pollen was used to pollinate female trees used in this investigation: new pollen harvested in march 2018, pollen stored for one year at room temperature (25-35 °c), and pollen stored for two years at room temperature (25-35 °c). the three types of pollen were all extracted from the same male genotype. their viability was assessed before pollination. to do so, pollen staining was done by preparing agar medium, where 10% sucrose was added to 1% agar and the medium was poured into a petri dish. after cooled, the authors placed pollen on the agar medium and added a droplet of 1.0% mtt (2,5-diphenyl mono tetrazolium bromide) solution to pollen. after incubation at 28° c for one hour, the pollen activity was examined under an optical microscope. the pollen activity percentage was calculated after counting ap a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) 715 proximately 1,000 pollen (khatum and flowers, 1995; rodriguez-riano and dafni, 2000). non-viable pollen grains, which remain light colored, were distinguished from viable ones, which turned to a dark color (violet-purple), indicating the presence of dehydrogenases (sheffield et al., 2005). to study the effect of fruit position in the spikelet on fruit weight, one type of fresh pollen extracted from a male genotype was used. the pollen grain was prepared by cutting male flower strands and spreading them onto a paper sheet for two days to dry at room temperature. the pollen grain was extracted manually from the flowers by shaking the male strands and sieving them to remove impurities. each of the three types of pollen was used to pollinate one bunch in each of the three nine-year-old trees of the ‘sukkary’ variety used in this experiment. all bunches pollinated were chosen so that they were homogenous in size and would open at the same time. to avoid pollen contamination, bunches were covered before and after their pollination with paper craft bags (figure 1). the pollination was made with pure fresh pollen (100% pollen) without diluting it in any carrier, and by using a bulb duster (figure 2). every bunch was dusted with 10 grams of pollen powder. three powder squirts were applied from the top to the bottom of the female bunch. effect of flower position in the spikelet on fruit-set three weeks after pollination, the paper bags were removed and replaced by mesh bags (figure 3). four weeks after pollination, after making sure that fruit-set occurred; the authors recorded whether every fruit was aborted or non-aborted. during the recording, the authors respected the order of the fruits in their spikelet from proximal to distal in every bunch of the three trees. aborted fruits, called “shiss’’ by farmers, have three carpels (figure 4.a). the fruit-set was counted in every third of the spikelet and abortion percentage was calculated. effect of flower position in the spikelet on fruit weight to study the effect of fruit position in the spikelet on its weight, three nine-year-old female trees of the ‘sukkary’ variety were used. one bunch from each tree was pollinated and tagged using fresh pollen extracted from a male genotype employing the same above mentioned methods for both extraction and pollination. from the day of pollination until maturation and harvest, a destructive method was carried out using a top load balance to measure fruit fresh weight periodically and repeatedly. wherefore, every three days, three strands were collected from three of the nine bunches to measure their weight. the data were recorded in such a way that the order of the fruits in their spikelets from proximal to distal was respected in every measure. the data were collected and the fruit growth pattern of every part of the spikelet was drawn. an analysis of variance (anova) was also carried out to assess the final fruit weight in every spikelet part. results and discussion effect of bunch cut at different times after pollination on fruit weight hierarchical cluster analysis: hierarchical clustering using the average linkage clustering method with the euclidean distance measurement method was performed to the 49 measurements of fruit weight taken from pollination to harvest for all bunch cut methods (figure 5). the dendrogram obtained grouped the bunch cut methods into two clusters with additional subgroups in each group. group i was composed of the methods of cutting the bunch respectively, at the time of pollination (cut0), 15 days after pollination (cut1), and 30 days after pollination (cut2). group ii included the control where the bunch was kept without cut (non cut) plus the methods of cutting the bunch respectively 45 dap (cut3), 60 dap (cut4), and 75 dap (cut5). both groups were further divided into two subgroups each: in group i, the subgroup 1 contained the two methods of bunch cut: at time of pollination (cut0) and 15 dap (cut1). subgroup 2 is composed of solely the method of bunch cut at 30 dap (cut2). in group ii, the subgroup 1 is composed of only the method of bunch cut at 45 dap(cut3), and in subgroup 2, the methods of bunch cut were at 60 dap (cut4), at 75 dap (cut5), in addition to the control where no bunch cut was applied (non-cut) additionally, the heat map compared the value of the weight taken during every measurement time period. each data cell was colored based on the highest and lowest value, where the yellow color was the highest, the white was the medium and the blue was the lowest. the bunch cut methods respectively, at the time of pollination (cut0), at 15 dap (cut1), and at 30 dap (cut2), kept the highest weight value during all the periods of fruit growth. they all belonged to group i, in comparison to the rest of bunch cut methods belonging to group ii, where only bunch cut method 45 dap (cut3) that had a high fruit weight in the beginning of the fruit growth and finished with a lowest weight value. an anova was also run to analyze whether any of the final fruit weight mean differences between the treatments were significant. accordingly, the results showed significant (p<0.01) differences between all bunch-cut methods (table 1). this confirmed the results obtained by the heat map analysis applied to data of fruit weight from pollination to harvest. the four subgroups of treatments are clearly shown in figure 5. the first subgroup with the highest fruit weight composed of cutting strands at time of pollination (cut0) and 15 dap (cut1), is followed by subgroup 2 where strands were cut at 30 a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) 716 dap (cut2). both subgroups were placed into the same group (a) by the heat map analysis (figure 5). while cut3 (45 dap), cut4 (60 dap), and cut5 (75 dap) were placed into the same subgroup (b). non-cut, however, was in an isolated subgroup with the lowest fruit weight. fruit weight, despite following the same growth pattern in all treatments (figure 6), reached “tamr,’’ or mature stage, maintaining the same ranking as the one the authors obtained using the anova and heat map. fruit growth of the ‘sukkary’ date palm variety followed a sigmoidal pattern in accordance with the findings of rouhani and bassiri (1976), torahi and arzani (2010), and awad et al. (2011) for other date palm varieties, and eljiati et al. (2020) for ‘sukkary’ and ‘khlass.’ three main growth phases were identified accordingly (figure 6). lag phase: starting from pollination until 35 dap and where there is a very slight increase in fruit weight. exponential phase: starts from 35 dap and ends at 130 dap. this phase is characterized by maximum fruit growth. stationary and diminishing growth phase: increase in fruit size became stationary from 129 dap, and fruit developed into mature stage, or “tamr,’’ where the fruit started losing humidity gradually until reaching around 20% of moisture at harvest. the operation of bunch cut as an alternative to hand thinning, when done in the lag phase (cut0, cut1, and cut2), generates an increase in final fruit weight (figures 5 and 6, and table 1). in most crops, the lag phase, known as phase s1, plays a relevant role in defining the final fruit size. farinati et al. (2017) reported that in rosaceae fruits, the length of the early stage of development (s1), known as the cell division stage, plays a relevant role in determining the final fruit size at harvest, and that this stage is very short or absent in small fruits (i.e., in raspberry), while it lasts for 7–25 and 21–35 days in peach and apple fruits, depending on the variety. in peach fruit, cell division is restricted to the early part of the growth cycle. four weeks after full bloom, the final cell number in the mesocarp and endocarp appears to be fixed (masia et al. 1992). when they studied different varieties of peach, yamaguchi et al. (2002) concluded that the cultivar that ceased cell division early had a small final number of mesocarp cells, resulting in small fruit, and that the smallest size of wild peach is attributable to slow cell division and poor cell growth. in same species, wu et al. (2005) reported that early fruit size had a positive influence on subsequent fruit growth, while souza et al. (2019) and scorza et al. (1991) both stated that the number of cells had greater influence on the final peach fruit size than the cell area. sutton et al. (2020) considered that the majority of cell division during peach fruit growth occurs during the s1 stage at early fruit development. figure 1. sukkary bunches covered before and after pollination with paper craft bags. figure 2. a bulb duster is used to pollinate female bunches. a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) 717 the same finding was reported in melons. according to higashi et al. (1999), the size of melon fruit is defined by the cell number in the pericarp. in olives, a strong linear correlation, was found between the final cell number and the fruit size, indicating that fruit size increased linearly with cell number, but not with cell area, and that the rate of increase of cell number in period i was 5–11 times greater than in period ii (hammami et al., 2011). cell number has also been shown to be the major determining factor for fruit size differences among cultivars of japanese apricots (yamaguchi et al., 2004), japanese plums (nagashima et al., 2019), and sweet cherries (olmstead et al., 2007), as well as other fruits, such as apple (harada et al., 2005), blueberries ( johnson et al., 2011), and japanese pears (zhang et al., 2006). in an attempt to increase date fruit size, most date farmers are used to applying hand thinning starting from the “hababook’’ stage, when date fruit size reaches at least a chickpea grain, so that they can catch fruits between their fingers. this stage is equivalent to the end of lag phase and the beginning of the exponential phase (after 35 dap). this present investigation showed that this practice would have little impact on final fruit size. when the authors cut strands after the end of lag phase (35 dap) or later, it generated fruit weight respectively equal to 13.86 g for cut3 (45 dap), 13.61 g for cut4 (60 dap), and 13.75 g for cut5 (75 dap); all not different statistically from the 13.20 g, obtained in the control, where the strands were kept without cut (noncut). the authors only obtained high fruit weight when they cut the strands earlier during time of pollination (cut0: 15.82 g), 15 dap (cut1: 15.36 g), or at the latest, 30 dap (cut2: 14.89 g). in apples, szot (2010) reported that thinning done at different times has shown significant effects on the final fruit size closely associated with the differences in cell numbers in the cortex, and that smaller fruit size produced by heavily cropping trees can be attributed to low cell number. cell number is an important component in determining final fruit size. the time of thinning has a great influence on the mean fruit mass. he showed that there was a reduction in mean fruit mass when the time of hand thinning was delayed, and that flower thinning at the pink bud stages and towards the end of the flowering stage had significant effects in obtaining high fruit weight exactly similar to what the authors noted when strandcut was applied at time of pollination (cut0). demason et al. (1982) stated that in the endosperm of mejdool date fruit, at nine weeks after pollination (63 dap), figure 3. the bunches are covered with a mesh bag to make fruit-set counting easier. figure 4. (a) aborted flowers giving three carpels called “shiss,” and (b) flowers where the fruit-set was successful. 718 cell division was already completed and there was the beginning of cell wall thickening. few mitotic cells remained at 11 weeks (99 dap). depending on the layer, cells expand together with a thickening of cell walls, which lasts from 9-17 weeks (63 to 119 dap). at 17 weeks (119 dap), all cells had completed their elongation phase, resulting in the maximum fresh weight of fruits. in apples, fruits thinned at earlier stages were able to increase in weight and cell number, but fruit from trees thinned beyond 10 dap and fruit from un-thinned trees were not able to develop beyond a weight of 190 g or 50 million cortex cells. this is presumably because early thinning allows fruit to continue cell division in the later stages of fruit development (goffine et al., 1995). when the ‘sukkary’ fruits are at the end of stage 1 (lag phase), and the beginning of stage 2 (exponential phase), cell number is supposed to already be determined. early bunch cut (at pollination time) is, therefore, important to allow for a better fruit increase. the practical consequence of this study is that bunch cut as an alternative to hand thinning in the ‘sukkary’ date palm variety should be applied at time of pollination, or in the worst case, 15 days after pollination. the authors do not advise the last practice as it needs more time because, the workers will be asked to visit the tree two times (at time of pollination and at time of cut), the cut operation becomes difficult when the fruits are big, as the cut of the bunch needs two or three shots to be given, compared to one shot at time of pollination. if, for any reason, a farmer missed the strand cut after 30 days from pollination, it would be useless to do it later. this is because it will not generate any increase in the fruit size, as it will only decrease the number of fruits per bunch, and the yield. effect of flower position in the spikelet on fruit-set and fruit weight the spikelets were divided into three parts: the proximal third (down part), middle third (middle part), and distal third (upper part). at the level of each spikelet, the authors calculated the percentage of abortion in each of the three parts. table 2 shows the average abortion percentage per spikelet a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) figure 5. dendrogram of 6 bunch cut methods and the control based on 45 fruit weight measurements from pollination to harvest of the ‘sukkary’ date palm variety. part and per pollen age. when pollen is new (harvested in the same season), the position in the strand does not affect the fruit-set, as the authors noted no significant differences in abortion percentage between the three spikelet positions. whereas with old pollen (one-year-old and two-year–old), with very low viability (table 3), the proximal flowers of the spikelets are exposed to abortion more than those of the middle and distal parts (table 2). the spathe of date palm inflorescence always starts to open from the top of inflorescence to the bottom. only flowers exposed to sunlight open. demason et al. (1982) reported that flower primordia and their subtending bracts are formed first at the base of the rachillae, and are initiated in an acropetal sequence as the rachillae elongate. in later development, all flowers within an inflorescence are at approximately the same stage of development, although they may not be of the same size. however, the authors considered that a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) 719 treatment time of application of strand-cut average final fruit weight (g)(*) cut0 cut is applied at time of pollination 15.82 ± 0.75 a cut1 cut is applied 15 days after pollination (15 dap) 15.36 ± 0.84 a cut2 cut is applied 30 days after pollination (30 dap) 14.89 ± 0.71 a cut3 cut is applied 45 days after pollination (45 dap) 13.86 ± 0.74 b cut4 cut is applied 60 days after pollination (60 dap) 13.61 ± 0.54 b cut5 cut is applied 75 days after pollination (75 dap) 13.75 ± 0.66 b non-cut no strand cut is applied 13.20 ± 0.44 b ! table 1. average final ‘sukkary’ fruit weight when strand-cut was applied at different times after pollination (12 fruits per strand were kept). (*) averages final fruit weights that do not share the same letter are significantly different according to t-test anals. figure 6. fruit growth pattern (g) of ‘sukkary’ fruit where bunch was cut at different interval times. lag phase exponential phase stationar and diminishing growth phase 720 a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) maturation of date palm sexual organs seem to occur in a basipetal direction before anthesis. this is true as proximal flowers are always the last to open and, therefore, their receptivity to pollen is delayed compared to distal flowers. when the authors performed pollination, they covered the bunches with paper craft bags so as to not only avoid pollen contamination, but also keep pollen available as long as possible and minimize the negative effects of any of weather conditions on pollination (wind, rain, etc.). the basipetal decline pattern in the fruit-set obtained in the old pollen and the non-pattern, which was obtained with fresh pollen, differed completely from the acropetal decline trend found in many species where the fruit-set is higher at the base of the inflorescence than at the apex (bawa and webb, 1984; berry and calvo, 1991; diggle, 1995; obeso, 1993). many researchers explain the acropetal decline trend in fruit distribution by both, the earlier opening of the basal flowers whose transformation into fruit would limit later younger distal fruit-set, and by the position of proximal flowers/fruits with respect to resources compared to distal flowers and fruits located farther from the supply of resources (diggle, 1995; lee, 1989; stephenson, 1981). for date palms, the first assumption is not true, as flower anthesis starts from distal to proximal. the same finding has been reported by arista et al. (1999) in carob (ceratonia cilica) populations, a dioecious leguminosae tree. the authors found that racemes-setting fruit arrangement follows a definite basipetal decline pattern that remains constant between years and populations of carob trees: fruit production was significantly higher in the apical zone of the raceme and lower in the basal zone. they attributed this to pollinators’ activity (flies and wasps) that showed a clear preference for beginning their visits at the apex of a raceme. therefore, the pollen load deposited on the stigmas decreased from the apex to base of the raceme. they found out that in most of the flowers situated in the central and basal zones of the raceme, the number of pollen grains deposited on their stigmas was lower than the number of their ovules. to prove this, the authors applied hand pollination to the flowers of the carob, and obtained a contrasting acropetal decline pattern in both the number of fruits initiated and those ripening within the raceme (those closest to the resource origin). effect of flower position in the spikelet on fruit weight the fruit weight measured every three days from pollination to harvest in each of the three parts of the spikelets of the ‘sukkary,’ generated the information shown in figure 7. during all life cycles of fruits in every of the three parts of the strand, the three curves were almost similar, and no part of the spikelet showed a superiority compared to the other parts. to confirm this, an anova was used to analyze the average final fruit weight measured in the three spikelets to follow the fruit growth pattern. the analysis revealed no significant difference between fruit weight of the parts of the strand (table 4). this investigation showed no pattern of fruit growth related to fruit position in the spikelet. however, many studies on other crops demonstrated an acropetal decline in fruit size within inflorescences. diggle (1995) considered that declining fruit length may have been due to a decline in the number of ovules fertilized or to an increase in ovule or seed abortion, but the decline could also be caused by a decrease in ovary size and number of ovules per ovary with position. on other crops, distal flowers typically reach anthesis after proximal flowers and, thus, initiate fruit development at a later time. if sink strength of fruits increases with size and age, distal fruits will have lower competitive ability (with respect to resources) compared to early fruits within an inflorescence. in addition, distal flowers and fruits located farther from the supply of resources may, therefore, have less access to resources (diggle, 1995). however, the authors consider that competition for resources between distal and basal flowers and fruits, would not be the sole explanation for declining fruit-set acropetally. in date palms, the anthesis starts from distal to proximal flowers. when using fresh pollen, the authors did not obtain any pattern of fruit-set related to the position of the flowers in the spikelet. at the same time, no pattern was obtained in fruit weight (figure 7 and table 4). in tomatoes, bangerth and ho (1984) demonstrated that the final size of the tomatoes is determined by both the potential sink strength, which is determined to a great extent before fruit-set, and the competition for assimilate supply, determined by the sequence of fruit-set. the latter effect on fruit growth is also modified by the potential sink strength of the fruit and the time lapse between fruit-set. other studies, however, have identified opposite patterns in which the proximal positions are not associated with high fruit-set and maturation. that is the case of mckone (1985), who studied patterns of fruit-set in five bromus species (poaceae). he concluded that, except in bromus inermis, fruit production was not randomly distributed among florets with respect to spikelet position, and that in three of the five bromus species there was a peak in seed-set in the middle third florets, and then a decline in seed-set occurring in the upper (distal) florets; seed-set fell consistently with floret position acropetally in b. tectorum. stocklin and favre (1994) discovered that proximal and distal positions within the inflorescence of ovules within fruits showed higher abortion rates than intermediate positions in the two epilobium species they studied. they, however, concluded that proximity to resources and the absence of competing older fruits were not necessarily an advantage. berry and calvo (1991) studied the pollination method a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) 721 abortion percentage (%) proximal third of spikelet middle third of spikelet distal third of spikelet total new pollen 5.4 ± 1.3 % a 4.0 ± 1.2 % a 4.5 ± 1.9 % a 13.9% one-year-old pollen 25.0 ± 1.4 % a 19 ± 2.1 % b 11.6 ± 0.7 % c 56.0% two-year-old pollen 27.0 ± 2.7 % a 20.0 ± 2.5 % b 16.3 ± 1.0 % b 68.3% ! table 2. effect of the position in the spikelet in the ‘sukkary’ date palm on abortion percentage pollinated, with pollen having different storage times. means that do not share the same letter are significantly different according to t-test analysis. pollen viability % new pollen 89% one-year-old pollen 50% two-year-old pollen 19% table 3. viability of pollen with different storage times used to assess fruit-set, in relation to flower position in the ‘sukkary’ date palm variety. fruit position in spikelet average fruit weight (g) proximal fruits 10.7 ± 0.44 a middle fruits 10.1 ± 0.25 a distal fruits 10.0 ± 0.30 a table 4. fruit weight per spikelet part for ‘sukkary’ pollinated with fresh pollen. ! " # $ % &! &" &# &$ &% !" !# !$ #% ## #& '% '! '$ &% &# &$ $( $# $) )( )' )) *" *' ** (% " (% & (( % (( ! (( $ (" % (" ! (" $ (! % (! # (! $ (# ( (# # (# ) (' " (' ' (& ! (& $ !" #$ %&' ($ )* %&+ ), -./0&.1%("&2344$5.%$35 '()*+,-. /+00.1 2+34-. figure 7. fruit growth pattern (g) of the three parts of the spikelet of ‘sukkary’. means that do not share the same letter are significantly different, according to t-test analysis. 722 a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is s n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc ( w w w .a tla spu bl is hi ng .o rg ) and flower position in myrosmodes cochleare (orchidaceae). they found that when plants are self-pollinated, fruit-set decreased from 100% in proximal positions to 0% in distal positions, while fruit-set in open-pollinated plants was maximal in the middle third of the inflorescence and declined both basipetally and acropetally. this led the authors to conclude that the pattern observed in the open-pollination treatment reflected the activity of pollinators. a survey of the literature made by thomson (1989) revealed that the number of ovules per flower decline from proximal to distal position flowers in 13 of 15 species. he concluded that this decline was associated with a lower probability of fruit-set in later flowers. in aquilegia buergeriana var. oxysepala, itagaki and sakai (2006) expected that if they applied bud removal, this would reduce interflora competition, and then the number of seeds produced by the remaining flowers within inflorescences would increase. after conducting trials, they concluded that such removal had little impact on the number of seeds produced by the remaining flowers. this means that resource competition hypothesis does not always explain acropetal decline in fruit and/or seed-set within inflorescences. all above, research shows that the acropetal decline over flowers is not a universal biological model. conclusion at the opposite of many other crops, the ‘sukkary’ date palm variety did not show any pattern in fruit-set and final fruit weight regardless of the position of the fruit/flower in the strand. bunch cut as an alternative to hand thinning in the ‘sukkary’ date palm variety should be applied at time of pollination, as it is this generates the highest fruit weight, it is easy to apply due to the small size of the bunch. acknowledgements the authors would like to deeply thank mr youssef jameel the chairman of yousef bin abdul latif and sons agriculture co. ltd for his financial support and for allowing us to use the farm and laboratory facilities of his company during this research. references arista m, pl ortiz, and s talavera (1999) apical pattern of fruit production in the racemes of ceratonia siliqua (leguminosae: caesalpinioideae): role of pollinators. american journal of botany 86 (12): 1708-1716. http://doi.org/10.2307/2656669. awad ma, ad al-qurashi, and sa mohamed ( 2011) biochemical changes in fruit of an early and a late date palm cultivar during development and ripening, international journal of fruit science 11 (2): 167-183. http://doi.org/ 10.1080/15538362.2011.578520. bangerth f and lc ho (1984) fruit position and fruit set sequence in a truss as factors determining final fruit size of tomato fruit: annals of botany 53: 315-319. https://www.jstor.org/stable/42757182. bawa ks and cj webb (1984) flower, fruit and seed abortion in tropical forest trees. implications for the evolution of paternal and maternal reproductive patterns. american journal of botany 71: 736–751. http://doi.org/10.2307/2443371. berry pe and rn calvo (1991) pollinator limitation and position dependent fruit set in the high andean orchid myrosmodes cochleare (orchidaceae). plant syst. evol. 174:93-101. http://doi.org/10.1007/bf00937697. de mason da, ws kenneth, and b tisserat (1982) date. floral development in phoenix dactylifera. can. j . bot. 60: 1439-1446. https://doi.org/10.1139/b82-184. diggle pk (1995) architectural effects and the interpretation of patterns of fruit and seed development. annual review of ecology and systematics 26: 531-552. https://doi.org/10.1146/annurev.es.26.110195.002531. eljiati a, a rouifi, t m b silva, n terada , a sanada, and k koshio. 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98 (3): 537–543. https://doi.org/10.1093/aob/mcl144. atlas journal of biology 2024, pp. 830–838 https://doi.org/10.5147/ajb.vi.245 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) plant growth promoting rhizobacteria (pgpr) isolated from an plant growth promoting rhizobacteria (pgpr) isolated from an arid soil in saudi arabia improve maize growtharid soil in saudi arabia improve maize growth abdelazize eljiati1*, yassine elmaati2, and hammou ouchaou2 1 national centre for palms & dates, al dahi, hittin, riyadh 13512, saudi arabia; 2 r&d department, yala laboratory, yousef abdul latif and sons agriculture ltd. (yala) company, qassim, saudi arabia received: february 19, 2024 / accepted: april 29, 2024 __________________________________________________ * corresponding author: a.eljiati@gmail.com 830 abstractabstract the rhizosphere represents the main source of bacteria commonly referred to as rhizobacteria. such beneficial rhizobacteria with plant-beneficial activities are generally defined as plant growth promoting rhizobacteria (pgpr). the aim of this study was to investigate the ability of native rhizobacteria (pgpr) isolated from an arid soil of date palm in al-qassim region, saudi arabia, to enhance plants growth. maize (zea mays l.) was used as model crop for this research. maize seedlings roots were inoculated with bacillus and enterobacter bacteria. the seedlings showed significant increases in stem, leaf, and root growth. the maximal shoot lengths were obtained with strain (i2: bacillus cereus) (95.41 cm) with an increase of 33.45 % compared to uninoculated control seedlings. the three isolates i2: bacillus cereus, azs2: bacillus subtilis and commercial strain azb: azospirillum brasilense) caused a highly significant increase in the total number of leaves ranging from 10.9% to 12.7% compared to the uninoculated controls. seedlings inoculated with azs2: bacillus subtilis strain exhibited the highest aerial dry biomasses with an improvement of more than 85 % (30.76 g) compared with uninoculated control plants and more than 62 % compared to uninoculated nacl control plants. the inoculation treatment with i2: bacillus cereus strain induced an improvement of more than 65 % (27.44 g) over uninoculated control and more than 45 % over uninoculated nacl control. the strain this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. azs2: bacillus subtilis produced the highest root dry weights, in comparison to other isolates and induced an improvement of 30.17% (26.06 g) compared to uninoculated control plants and 24.09% compared to uninoculated plants (nacl control). the most effective rhizobacterial treatment in the dry biomasses of whole seedling (aerial dry biomass and root dry biomass) is azs2: bacillus subtilis strain which induced an improvement of 55% (56.83 g) compared to uninoculated plants (control) and 42% compared to uninoculated plants (nacl control). the most important production of kernels was recorded with azs2: bacillus subtilis strain. therefore, these findings suggested that the use of pgpr strains as inoculant biofertilizers might be beneficial for crop production cultivation especially in arid and semi-arid regions. keywords:keywords: plant growth promoting rhizobacteria (pgpr), bacillus subtilis, bacillus cereus, enterobacter ludwigii, inoculation, zea mays l. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) introductionintroduction bacteria that can aggressively colonize the rhizosphere or plant roots or both and promote growth and yield of plants are referred to as plant growth promoting rhizobacteria (pgpr) (kloepper et al., 1989). these rhizosphere bacteria enhance crop growth and yield directly, either by promoting nutrition, for example, by phosphate (hayat et al., 2010; sharma et al., 2007; das et al., 2003), and potassium solubilization (wang et al, 2020; han et al., 2006) and ammonia production (mukhtar et al., 2020; ahmad et al., 2008; joseph et al., 2007) or by synthesizing metabolites with great agricultural interest such as plant growth regulators and siderophores (tian et al., 2009; arkhipova et al., 2005; barazani et friedman, 2001). they can also promote growth indirectly, acting as bio-controlling agents for suppression of growth of soil borne phytopathogen microorganisms and as stimulator of other beneficial organisms for the plant (abbasi et al., 2011; bhattacharyya and jha, 2011). pgpr improve soil structure and bioremediate polluted soils by sequestering toxic heavy metal and degrading xenobiotic compounds (ahemad et al., 2012; braud et al., 2009). depending on their beneficial roles in the rhizosphere, pgpr have been classified as biofertilizers, phytostimulators, rhizoremediators and biopesticides (martínez-viveros et al., 2010). consequently, the application of these beneficial microorganisms as bioinoculants appears as an ecological friendly biotechnological tool (dimpka et al., 2009) to alleviate detrimental effects of intensive farming practices that are using synthetic fertilizers and pesticides without caring about environmental problems and soil health (elkoca et al., 2010). numerous laboratory, greenhouse and field studies are available on the screening of pgpr for their multiple plant growth promoting activities (wang et al, 2020; gouda et al., 2018; oteino et al., 2015; hayat et al., 2010; joseph et al., 2007) and utilization of pgpr-based products in agricultural crop production systems (yadav et al., 2017; cakmakci et al., 2006). these products are mainly applied as seed treatment, soil amendment, or soil drench at the time of sowing or immediately after transplantation, to facilitate better nutrient uptake, greater production of growth hormone and beneficial phytochemicals in crops leading to higher crops yield and quality (kloepper et al., 2004). pgpr activity has been reported in strains belonging to a several genera, such as pseudomonas, azospirillum, azotobacter, klebsiella, enterobacter, rhizobium, bradyrhizobium, alcaligens, arthobacter, burkholderia, bacillus, serratia and xanthomonas (verma et al., 2013; karnwal 2009; patten and glick 1996; glick, 1995; kloepper et al., 1989). pseudomonas and bacillus spp. have been the most studied bacteria for their plant growth promotion (pgp) activity and ability to produce beneficial substances (kejela et al., 2016; pham et al., 2017). isolation of native strains adapted to the arid environment may contribute to formulation of inoculants suitable for use in local crops, as they are adapted to the environment and can be, thereby more competent than imported microbial strains. the positive impact of pgpr has been studied in annual crops like wheat (bashan, 1986), soybeans (cattelan et al., 1999), beans (jarak et al., 2012) and corn (di salvo et al., 2018; ullah et al., 2014) in several ways. therefore, this study was designed to select effective strains from a series of of native rhizobacteria (pgpr) isolated in an arid area soil of date palm in al-qassim region, saudi arabia, by maize (zea mays l.) growth promotion assay under greenhouse conditions. these native strains are also compared with a commercial microbial strain, as a positive control. therefore, the application of the selected effective pgpr strains as microbial inoculants for crops would significantly promote their sustainable production in arid conditions and reduce the use of inorganic fertilizers and pesticides, which often pollute the environment. materials and methodsmaterials and methods bacterial inoculantsbacterial inoculants the rhizobacterial strains (i2: bacillus cereus, azs2: bacillus subtilis, aza2: enterobacter ludwigii and psa1: enterobacter ludwigii) used in this study were previously isolated from an arid area soil in qassim province, saudi arabia (elmaati et al., 2020). these bacterial strains were characterized and selected based on their plant growth promoting traits, comprising very good phosphate and potassium solubilization and ammonium production. a commercial strain (azb; azospirillum brasilense) was used as a positive control to compare it with these native strains. inoculation of maize plants by pgpr strainsinoculation of maize plants by pgpr strains to prepare the inoculum for each rhizobacterial strain, pure cultures were grown in nutrient agar (r2a agar medium). after 48 hours of incubation at 25 °c, a single colony from each strain was transferred into a 200 ml sterilized erlenmeyer flask, containing sterilized 102 medium broth (= lmg 1089 medium) with the following composition (g l-1): sucrose, 20.0; casein hydrolyzate, 16.0; yeast extract, 8.0; kh2po4, 4.0; mgso4 x 7 h2o, 0.30, and grown aerobically for 4-5 days on a rotating shaker (150 rpm) at 32 °c, to obtain a final concentration of 109 cfu ml-1. after incubation, bacterial growth is estimated by measuring the absorbance of the culture at 600 nm. to wash the bacteria, bacterial cells were centrifuged at 3000 rpm for 10 min in 15 ml tubes. the supernatant was then discarded and the pellet was washed once with 5 ml of sterile nacl solution (physiologic sterile water (0.85%)) and finally resuspended in 200 ml of the same solution. maize (zea mays l.) was used as the test plant for the inoculation in this experiment. seeds of a homogeneous variety were surface sterilized to eliminate all kinds of contamination according to the method of götz et al. (2006): the seeds were immersed for 1 min in ethanol (70%) with gentle agitation. they are then put back into 12% diluted sodium hypochlorite solution containing three drops of wetting agent (tween 20) for 15 minutes. to get rid of the chlorine, the seeds were rinsed several times with sterile distilled water. sterilized seeds were sown in alveolar plates containing 831 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) autoclaved (1: 1 v / v) mixture of peat and vermiculite respectively. they were then placed in a greenhouse at a natural photoperiod (at a temperature of 28 to 40 °c) and are regularly irrigated. twenty days after germination, the roots of maize seedlings were dipped into the inocula for 2 h at 25 °c. control seedlings were divided into two groups; the first is dipped into sterile nacl solution as the bacteria washing procedure containing this solution, while the second is dipped into sterile distilled water. each treated seedling was planted in a disinfected and labeled plastic pot (8 l) containing autoclaved (1/1: v / v) mixture of soil and vermiculite respectively. pots were then placed in a greenhouse at a natural photoperiod (at a temperature of 28 to 40 °c). the experiment was carried out in a completely randomized block design with 5 replications for each treatment. seedlings were irrigated with 300 ml of well water every two days to maintain at field capacity and received no fertilizers. estimation of some agro morphological parameters of plantsestimation of some agro morphological parameters of plants to evaluate the response to rhizobacterial inoculation, during the experiment, a daily monitoring of the evolution of the growth of the maize plants was done after the application of the inoculum for a period of 90 days. the height from the collar (size of the aerial part) and the number of leaves are the main growth parameters used in this study. these two growth parameters were measured at the start and every ten days during the 3 months of treatment. plant height was determined by measuring from the plant’s base to the top of the newest fully developed leaf. at the end of the experiment, the shoots and roots of each plant were put in paper bags and dried in an oven at 65°c for 72 hours (sfairi, 2013) to report the total dry biomass (shoot and root dry biomass). statistical data analysisstatistical data analysis the mean value of each treatment, as well as the corresponding standard deviation, were calculated using the data of all the replicates carried out. the data obtained was analyzed statistically using r 3.2.0 and multivariate analyses were performed using r language (dray & dufour, 2007; r development core team, 2011). resultsresults effect of rhizobacterial inoculation on shoot lengtheffect of rhizobacterial inoculation on shoot length the rhizobacterial isolates (azb, azs2, i2, psa1, and aza2) significantly affected the shoot length of maize seedlings. results reveal that pgpr promoted an increase in shoot length over un-inoculated (control) (figure 1 a and b). the maximal lengths of the maize seedlings were obtained with strain (i2) (95.41 cm) with an increase of 33.45 % compared to un-inoculated (controls). treatment of maize seedlings with strain (i2) showed a significant increase in shoot length improvement rates compared to other strains including the commercial strain (azb). there was no significant difference in shoot length between the two controls. effect of rhizobacterial inoculation on leaf numberseffect of rhizobacterial inoculation on leaf numbers from 1 to 38 days after emergence (figure 2a), the number of leaves of maize plants increased linearly in all treatments. these results were identical for all the rhizobacterial isolates and the controls. however, we noted the beginning of the stability of the number of leaves between the 40th and the last days of the cycle in some plants. the effects of different rhizobacterial isolates were significant on the total number of leaves of maize, compared with control. application of azs2 isolate to maize seedlings recorded non-significantly higher number of leaves, compared with azb and i2, and all these three isolates were comparatively more effective than rest of the isolates and the two uninoculated control. the three effective isolates caused a highly significant increase in the total number of leaves ranging from 10.9% to 12.7% compared to the un-inoculated control. no significant difference was observed between the two isolates (psa1 and aza2) and the control with nacl (figure 2b). 832 figure 1.figure 1. (a) effect of rhizobacterial inoculation on shoot length (b) height growth of maize seedlings after 50 days of cultivation under the effect of applied rhizobacterial strains. aa bb a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 833 effect of rhizobacterial inoculation on leaf desiccationeffect of rhizobacterial inoculation on leaf desiccation during the period of evolution of the different foliar levels of the maize seedlings, we noticed that the leaves of the base wither and turn brown, by a yellowing and drying which begins with the end of the leaves and extends thereafter, until they dry out completely. the number of dried older leaves was recorded for each plant. at the end of the experiment, the results of the analysis of variance relating to this parameter show that there is no statistically significant difference between the rhizobacteria and the controls without bacteria (figure 3). effect of rhizobacterial inoculation on aerial dry biomasseffect of rhizobacterial inoculation on aerial dry biomass the analysis of variance relative to the aerial dry biomasses of maize seedling shows that there is a highly significant difference between the different strains and the un-inoculated control and un-inoculated control nacl control (with a total aerial dry biomass which marked a rate of 16.59 g and 18.88 g respectively). seedlings inoculated with azs2 strain exhibited the highest aerial dry biomasses with an improvement of more than 85 % (30.76 g) compared with un-inoculated control plants and more than 62 % compared to un-inoculated nacl control plants. the inoculation treatment with i2 strain induced an improvement of more than 65 % (27.44g) over un-inoculated control and more than 45 % over un-inoculated nacl control. on the other hand, the commercial strain (azb) showed an improvement of 64.98% (27.37 g) compared to untreated control and 44.97% compared to untreated nacl control. the others two strains (psa1 and aza2) are characterized by their lowest significant effect on aerial dry biomass per comparison to the both control plants (control and nacl control) (up to 38 % increase) (figure 4). effect of rhizobacterial inoculation on root dry biomasseffect of rhizobacterial inoculation on root dry biomass the effect of the five rhizobacterial strains on the root dry biomasses of maize plants is illustrated in (figure 5a). all the tested strains were significantly improved the root dry biomasses in comparison with controls (control and nacl control), which scored a rate of 20.02 g and 21.00 g respectively. the strain azs2 produced the highest root dry weights, in comparison to other isolates and induced an improvement of 30.17% (26.06 g) compared to un-inoculated plants (control) and 24.09% compared to un-inoculated plants (nacl control). while, in comparison with controls, the inoculation with strains i2, psa1 and azb showed a significant difference with improvement rates ranging from 16.2% to 19.35%. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 0 2 4 6 8 10 12 0 20 40 60 80 le a ve s n u m b e r days number control nacl control aza2 (enterobacter ludwigii) azb (azospirillum brasilense) azs2 (bacillus subtilis) psa1 (enterobacter ludwigii) i2 (bacillus cereus) aa bb figure 2.figure 2. (a) evolutionary trend of the adjusted mean of the number of maize leaves (b) effect of rhizobacterial inoculation on leaf numbers. figure 3.figure 3. effect of rhizobacterial inoculation on leaf desiccation. figure 4.figure 4. effect of rhizobacterial inoculation on aerial dry biomass a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 834 it is observed from the results (figure 5b) that the rhizobacterial strains caused greater increase in root system of maize plants as compared with controls (control and nacl control). indeed, the highest root lengths was recorded with the inoculation of azs2 and i2 strains), in comparison to other strains. effect of rhizobacterial inoculation on whole seedling dry effect of rhizobacterial inoculation on whole seedling dry biomass biomass a very highly significant improvement rate in the dry biomasses of whole seedling (aerial dry biomass and root dry biomass) inoculated with the rhizobacterial strains is recorded compared to the un-inoculated plants (control and nacl control), which scored a level of 36.62 g and 39.94 g respectively. the most effective rhizobacterial treatment is azs2 strain which induced an improvement of 55% (56.83 g) compared to un-inoculated plants (control) and 42% compared to un-inoculated plants (nacl control). analysis of the variance applied to dry biomasses of whole seedling indicated that there was no significant difference between other strains (i2, aza2, psa1 and azb), but they induced an increase for this parameter compared to the un-inoculated control (figure 6). effect of rhizobacterial inoculation on kernel numbers effect of rhizobacterial inoculation on kernel numbers all the inoculated treatments proved statistically superior over un-inoculated control in improving number of kernels. no significant difference was observed between the four bacterial strains azb, azs2, i2 and psa1. however, the most important production of kernels was recorded with azs2 strain. while total kernels number was significantly increased with these four strains as compared to aza2 strain and compared to un-inoculated control (figure 7). discussiondiscussion plant growth promoting rhizobacteria (pgpr) are freeliving microbes that live on or around the roots (kloepper et al., 1989) and promote plant growth and yield (wu et al., 2005). native rhizobacteria (pgpr) isolated in arid area soil of date palm in al-qassim region, saudi arabia, were used in this study to constitute the rhizobacterial inoculum, which was then used to inoculate maize (zea mays l.) seedlings. these strains exhibited significant plant growth promoting attributes in vitro tests and selected from previous screening experiments (elmaati 2020). in addition to its nutritional and figure 5.figure 5. (a) effect of rhizobacterial inoculation on root dry biomass (b) effect of rhizobacterial inoculation on root length of maize seedlings aa bb figure 6.figure 6. effect of rhizobacterial inoculation on whole seedling dry biomass. figure 7.figure 7. effect of rhizobacterial inoculation on kernel numbers. economic importance, maize has been a keystone model organism for basic and applied research in plant biology (strable and scanlon, 2009). overall, the results of the study of the growth of maize seedlings treated with the various rhizobacterial strains enabled us to conclude that the efficacy of different strains for growth-promoting of maize was variable. indeed, the investigated pgpr strains in this work showed positive pgp traits. these potentialities seem playing an effective role for the plant in helping it to better absorb nutrients. pgpr have long been known to promote growth when added to seeds, roots or tubers in a wide range of plant species (kloepper et al., 1980), increasing both growth and yield (wu et al., 2005) by improving the concentration of nutrients in the host plant (canbolat et al., 2006). several studies have reported that inoculation of maize plants with pgpr strains caused significant increase in plant height, plant dry weight, stem diameter, root length and weight, yield, number of leaves and leaf area, and plant nutrient uptake of n, p, k, fe, zn, mn and cu (yazdani et al., 2009; jarak et al., 2012; gholami et al., 2012; calvo et al., 2017). according to our results maize seedlings inoculated by dipping the roots in bacterial suspensions showed a statistically significant improvement in the growth parameters studied as compared to treatments without inoculation. interestingly, the plant height, number of leaves, shoot dry weight, root dry weight and kernels number were significantly higher with plants treated with azs2 strain (bacillus subtilis) followed by i2 strain (bacillus cereus) in comparison with un-inoculated control and with other strains including the commercial strain (azb, azospirillum brasilense). this is consistent with previous studies, which demonstrated that plant growthpromoting activities of bacillus spp. are well characterized as evidenced by increased growth of roots, shoots, and leaves as well as enhanced yields. in this context, increased plant height and shoot biomass of arabidopsis, corn, and tomato under greenhouse conditions have been reported by inoculating with four isolated bacillus strains from rainforest soils (huang et al., 2015). results obtained by hassan (2017) report that b. cereus tp.1b and b. subtilis tp.6b strains significantly increased root length and root weight in maize compared to controls. co-inoculation of bacillus spp with other pgpr strains reduces phosphorus demand by 50% without affecting maize yield (yazdani et al., 2009). moreover, ferreira et al., 2018 reported that bacillus subtilis promotes positive influence on plant growth of maize plants under normal conditions (without salinity). bacillus subtilis strain was the most effective in promoting nitrogen accumulation and, therefore, increased chlorophyll content in maize (aquino et al., 2019; almaghrabi et al. al., 2014). when tomato seeds were treated with bacillus subtilis (epc016), a significant increase in seedling growth was observed relative to un-inoculated plants (ramyabharathi et al., 2013). in another study, tilak and reddy (2006) observed a significant increase in grain yield rate of 43.8% in maize plants inoculated with bacillus cereus. this last strain was found to exhibit the highest nitrogenase activity among 42 different strains of bacillus spp studied by ambrosini et al., (2016). in addition, b. cereus and b. megaterium have been reported as organic phosphorus mineralizing bacteria (guang can et al., 2008). the works of habib et al., (2015, 2016) on rhizobacteria isolated from saline soil and selected for their pgp activities revealed that they showed significant salt tolerance properties. these rhizobacteria were identified as enterobacter sp. and bacillus cereus. our research indicates that significantly lower values of different growth parameters were recorded in maize plants inoculated with enterobacter ludwigii in comparison to bacillus subtilis and bacillus cereus. on the other hand, and compared to the un-inoculated control plants, enterobacter ludwigii had significant positive effects on maize plant growth parameters. zaballa et al., (2020) found that barley plants inoculated with the enterobacter ludwigii strain showed improvement in growth and phosphate uptake compared to the un-inoculated control. tahir et al., (2013) reported that inoculation of wheat plants with phosphate-solubilizing and phytohormone-producing bacterial strains such as azospirillum, bacillus and enterobacter improved growth and yield. moreover, several studies have demonstrated the effectiveness of inoculating wheat grains with different rhizobacteria on plant growth (abbasi et al., 2011; rana et al., 2011; banerjee et al., 2010). numerous studies have highlighted the increase in dry matter weight of aerial parts in wheat (bashan, 1986) and maize (garcía de salamone and döbereiner, 1996; ullah et al., 2014; di salvo et al., 2018). the positive effects of pgpr on the yield and growth of crops such as wheat (ozturk et al., 2003; salanture et al., 2006) maize (egamberdiyeva, 2007; ullah s and b asghari, 2015; pereira et al., 2020) soybean (cattelan et al., 1999) and sugar beet (cakmakc et al., 2006) have been explained by the ability of these pgpr to fix n2, solubilize phosphate and produce phytohormones. thus, these rhizobacteria can be considered as an excellent tool for increasing the availability of phosphorus in plants by mineralization of soil organic phosphorus and by solubilization of phosphate precipitates (kucey et al., 1989; pradhan and sukla, 2006), production of aia (chaiharn and lumyong, 2011; swain et al., 2007), hcn (bakker and schippers, 1987), ammonia (nh3) (yadav et al., 2010) and siderophores (boopathi and rao, 1999). in general, our study clearly showed that the inoculation of maize plants with the rhizobacterial strains significantly promoted maize plants growth. these results suggest that these pgpr strains can be applied as biofertilizers for improving plants production. furthermore, their use can be an ecological alternative to reduce the dependence on chemical fertilizers. conclusionconclusion the rhizobacterial strains investigated in our study showed their plant growth ability. these native strains, which belong to the genera bacillus and enterobacter, significantly enhanced the growth of maize plants when compared with the un-inoculated control plants. the maximal lengths of the maize seedlings were obtained a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 835 with strain (i2: bacillus cereus) (95.41 cm) with an increase of 33.45 % compared to un-inoculated (controls). the three isolates i2: bacillus cereus, azs2: bacillus subtilis and commercial strain azb: azospirillum brasilense) caused a highly significant increase in the total number of leaves ranging from 10.9% to 12.7% compared to the un-inoculated control. seedlings inoculated with azs2: bacillus subtilis strain exhibited the highest aerial dry biomasses with an improvement of more than 85 % (30.76 g) compared with un-inoculated control plants and more than 62 % compared to un-inoculated nacl control plants. the inoculation treatment with i2: bacillus cereus strain induced an improvement of more than 65 % (27.44g) over un-inoculated control and more than 45 % over un-inoculated nacl control. the strain azs2: bacillus subtilis produced the highest root dry weights, in comparison to other isolates and induced an improvement of 30.17% (26.06 g) compared to un-inoculated plants (control) and 24.09% compared to un-inoculated plants (nacl control). the most effective rhizobacterial treatment in the dry biomasses of whole seedling (aerial dry biomass and root dry biomass) is azs2: bacillus subtilis strain which induced an improvement of 55% (56.83 g) compared to un-inoculated plants (control) and 42% compared to un-inoculated plants (nacl control). the most important production of kernels was recorded with azs2: bacillus subtilis strain. consequently, this finding suggests that these pgpr strains could be useful for the development of inoculants biofertilizers to improve the quality and the health of the soil and the plant species by increasing the nutrient availability for the soil and plants especially in arid and semi-arid regions. furthermore, using biofertilizers that contain these rhizobacterial strains will led to a decrease in the use of chemical fertilizers and will provide high quality products free of harmful agrochemicals for human and environment. acknowledgementsacknowledgements the authors would like to deeply thank mr youssef jameel the chairman of yousef bin abdul latif and sons agriculture co. ltd for his financial support and for allowing us to use the farm and laboratory facilities of his company during this research. referencesreferences abbasi mk, s sharif, m kazmi, t sultan and m aslam (2011) isolation of plant growth promoting rhizobacteria from wheat rhizosphere and their effect on improving growth, yield and nutrient uptake of plants. plant biosystems 145: 159-168. doi: https://doi.org/10.1080/11263504.2010.542318. ahemad m (2012) implications of bacterial resistance against heavy metals in bioremediation: a review. journal of institute of integrative omics and applied biotechnology (iioab) 3: 39-46. ahmad f, i ahmad and ms khan (2008) screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. microbiological 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(psm) and plant growth promoting rhizobacteria (pgpr) on yield and yield components of corn (zea mays l.). world acad. sci. eng. technol. 49: 90-92. doi: 10.5281/zenodo.1080014. zaballa ji, r golluscio and cm ribaudo (2020) effect of the phosphorus-solubilizing bacterium enterobacter ludwigii on barley growth promotion. american scientific research journal for engineering, technology, and sciences (asrjets) 63: 2313-4402. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 838 atlas journal of biology 2025, pp. 846–855 https://doi.org/10.5147/ajb.262 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) comparative genomics of foodborne pathogens: diversity, virulence, and epidemiological relevance bradford dreshawn1, khalid lodhi2, jiazheng yuan2, danielle graham2, justin graham2, mohamed maldani2, erin white2, afua arhin3, and my abdelmajid kassem1* 1 plant genomics and bioinformatics lab, department of biological and forensic sciences, fayetteville state university, fayetteville, nc 28301, usa; 2 department of biological and forensic sciences, fayetteville state university, fayetteville, nc 28301, usa; 3 school of nursing, fayetteville state university, fayetteville, nc 28301, usa received: june 6, 2025 / accepted: july 4, 2025 __________________________________________________ * corresponding author: mkassem@uncfsu.edu 846 abstract foodborne bacterial infections are a major global health concern, causing millions of illnesses and deaths annually. advances in microbial genomics have improved pathogen characterization, yet the relationship between genomic traits and public health outcomes remains unclear. this study investigates 50 foodborne bacterial species by analyzing genome size, gc content, virulence gene count, and antimicrobial resistance (amr) gene presence in relation to global infection rates and mortality. our findings reveal substantial genomic diversity, with genome sizes ranging from 1.2 mb to 9.0 mb and virulence gene counts from 2 to 312. genome size, gene number, and gc content are strongly correlated, but neither virulence nor amr gene counts consistently predict mortality or global case numbers. these weak associations suggest that host susceptibility, ecological adaptation, and gene expression contribute significantly to pathogenicity. this study also highlights the value of microbial forensics in foodborne outbreak investigations. integrating whole-genome sequencing (wgs), comparative genomics, and phylogenetic analysis allows for tracing pathogen origins during contamination events. bacteria such as salmonella enterica, escherichia coli, and listeria monocytogenes frequently feature in forensic cases due to their high public health impact. the use of machine learning (ml) and artificial intelligence (ai) enhanced genomic surveillance holds promise for improving pathogen source attribution and biosecurity. these results highlight the complexity of bacterial virulence and call for integrated approaches combining genomic, epidemiological, and forensic data. future work should emphasize functional genomics, host-pathogen interactions, and predictive modeling to enhance foodborne disease prevention and outbreak response strategies. keywords: microbial forensics, foodborne pathogens, genomic epidemiology, antimicrobial resistance, virulence factors, wholegenome sequencing. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. published in the united states of america https://www.atlas-publishing.org 1. introduction foodborne bacterial infections are a major global health concern, causing millions of illnesses and deaths annually. according to the world health organization (who), foodborne diseases affect 600 million people each year, leading to approximately 420,000 deaths, with the highest burden observed in lowand middle-income countries (who, 2025). these infections are commonly associated with contaminated food, water, improper hygiene practices, within children under five years old, immunocompromised individuals, and the elderly being the most vulnerable (kirk et al., 2015). the economic impact is also severe, with billions of dollars lost annually due to healthcare costs, loss of productivity, and food recalls (scallan et al., 2011). despite decades of research, foodborne bacterial infections remain a persistent challenge due to the emergence of antimicrobial resistance (amr), environmental adaptability, and complex pathogen-host interactions (rocourt et al., 2003). the field of microbial genomics has revolutionized our understanding of bacterial pathogens, providing valuable insights into the genetic basis of virulence, antimicrobial resistance, and epidemiological trends (didelot et al., 2017). the advent of whole-genome sequencing (wgs) and comparative genomics has enabled researchers to characterize the genetic diversity of bacterial species, facilitating the identification of key virulence determinants, mobile genetic elements, and resistance mechanisms (oniciuc et al., 2018; saini et al., 2024). however, despite these advances, the relationship between genomic features and disease severity remains poorly understood. while some pathogens exhibit large, complex genomes with numerous virulence factors, others have streamlined genomes yet cause severe disease, suggesting that genomic complexity alone may not fully explain pathogenic potential (merhej and raoult, 2011). several studies have explored genome size, gc content, and gene composition as potential indicators of bacterial adaptability, virulence, and resistance capacity (ochman and davalos, 2006; bobay and ochman, 2017). genome size has been linked to metabolic versatility and environmental adaptability, with larger genomes often associated with higher numbers of virulence and resistance genes (toft and andersson, 2010). however, this trend does not hold universally, as some highly virulent pathogens, such as clostridium botulinum and helicobacter pylori, have relatively small genomes, yet produce potent toxins or have evolved mechanisms to evade host immunity (rossetto et al., 2014). conversely, opportunistic pathogens like pseudomonas aeruginosa and burkholderia species have large genomes, allowing for adaptation to diverse environments but not necessarily higher virulence (stover et al., 2000). another important genomic feature is gc content, which varies widely among bacteria and may influence dna stability, mutation rates, and gene regulation (hildebrand et al., 2010). some studies suggest that higher gc content correlates with greater environmental persistence, as observed in soil-dwelling and free-living bacteria, whereas host-associated pathogens often exhibit lower gc content, possibly due to genome reduction and specialization (moran, 2002). however, the direct impact of gc content on bacterial virulence and epidemiological success remains debated (bentley and parkhill, 2004). in addition to genome size and gc content, virulence gene content and antimicrobial resistance (amr) gene presence are critical factors influencing pathogenic potential. bacteria encode a wide array of virulence factors, including toxins, adhesins, secretion systems, and immune evasion proteins, which collectively determine their ability to infect, survive, and proliferate within hosts (ribet and cossart, 2015). similarly, amr genes allow bacteria to withstand antibiotic treatment, complicating infection management and increasing mortality risks (martínez et al., 2009). the global rise of antibiotic-resistant foodborne pathogens, such as multidrug-resistant salmonella and extended-spectrum beta-lactamase (esbl)-producing escherichia coli, has been a growing concern in clinical and food safety settings (djordjevic et al., 2024). however, while amr genes contribute to treatment efficacy, their direct relationship with mortality rates is not well established, as some highly resistant bacteria cause mild infections, whereas others with few resistance genes can be highly lethal (baker et al., 2018). given these uncertainties, this study aims to systematically analyze the genomic features of 50 foodborne bacterial species and assess their correlation with public health metrics, including mortality rate and annual global infection cases. by evaluating genome size, gc content, virulence gene count, and amr gene presence, we seek to determine whether genomic complexity influences pathogenic severity. understanding these relationships could improve risk assessment, surveillance, and intervention strategies for foodborne diseases. the findings from this study may also contribute to genome-based predictive models, helping identify high-risk pathogens and guiding public health policies on food safety and infectious disease control. 2. materials and methods 2.1. data collection and selection criteria the genomic and epidemiological data for this study were obtained from well-established public databases. bacterial genome sequences were retrieved from ncbi and refseq databases (o’leary et al., 2016; sayers et al., 2024). in parallel, epidemiological data, including global infection cases and mortality rates, were collected from the global burden of disease (gbd) study and world health organization (who) reports. these sources were chosen for their comprehensive surveillance of foodborne diseases across different geographical regions and population groups. to ensure the reliability of our dataset, bacterial species were included only if they had well-documented genomic data and available epidemiological statistics related to human infections. species with incomplete genome sequences, low-quality assemblies, or insufficient epidemiological records were excluded from the study. 2.2. genomic feature analysis to explore the genetic diversity of foodborne bacteria, several key genomic features were extracted from assembled genome sequences. genome size (mb), gene number, and gc content (%) were directly obtained from refseq annotations. these fundamental metrics provide insights into bacterial genome organization, metabolic potential, and evolutionary adaptation. to assess virulence potential, the number of virulence genes in each bacterial species was identified using the virulence factor database (vfdb) (chen et al., 2016). this database contains curated information on known bacterial virulence determinants, including toxins, adhesion proteins, and immune evasion mechanisms. the presence of antimicrobial resistance (amr) genes was determined using resfinder and the comprehensive antibiotic resistance database (card) (alcock et al., 2020). these databases enable the detection of genetic determinants associated with antibiotic resistance, including genes encoding beta-lactamases, efflux pumps, and ribosomal modifications. to ensure data accuracy, we applied quality control filters during data extraction. only complete or high-quality draft genomes were included, and duplicate entries and plasmid-only assemblies were excluded. virulence and amr genes were identified using standardized thresholds of >90% nucleotide identity and >80% coverage to minimize false positives and ensure consistency across species. the wide ranges observed in virulence and amr gene counts reflect true biological variation among species and are not due to redundancy or assembly artifacts. cases with zero gene counts were verified as true negatives, typically occurring in species with reduced genomes (e.g., mycoplasma spp.), rather than data omissions. 2.3. statistical data analysis descriptive statistics were calculated to summarize the genomic and epidemiological characteristics of the dataset. for each genomic feature, mean, standard deviation, quartiles, and range were computed to assess the overall distribution and variability among bacterial species. all data analyses have been conducted using jupyter notebook (kluyver et al., 2016) with python (van rossum and drake, 2009) and its libraries: numpy (harris et al., 2020) for numerical operations, pandas (mckinney, 2010) for data handling, matplotlib (hunter, 2007) for plotting, and seaborn (waskom, 2021) for enhanced visualization. to evaluate potential relationships between genomic traits and public health impact, pearson and spearman correlation coefficients were used. pearson correlation was applied to assess linear relationships between continuous variables, while spearman correlation was used to capture potential non-linear associations. these analyses aimed to determine whether genome size, gene number, gc content, virulence gene count, or amr gene presence were predictive of mortality rate and global infection cases. to facilitate interpretation, multiple data visualization techniques were 847 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) employed. histograms and boxplots were generated to illustrate distribution patterns and outliers in genomic features, while heatmaps were used to depict correlation matrices. additionally, scatterplots were created to examine potential associations between genomic complexity and pathogenic severity. these visual representations provided an intuitive means of identifying trends, clusters, and potential outliers within the dataset. 3. results 3.1. genomic diversity among foodborne bacteria the comprehensive dataset summarizing the species, family, genome size (mb), gene number, gc content (%), virulence gene count, antimicrobial resistance (amr) gene count, annual global infection cases, and reported mortality rates (%) for 50 foodborne bacterial species analyzed in this study are shown in table 1. the summary statistics of the 50 foodborne bacteria (table 2) reveal substantial variability in genome size, gene number, gc content, virulence factors, amr genes, and their associated public health impact. the average genome size is 3.97 mb, with a wide range (1.2–9.0 mb), mirroring the diversity in gene number (0–18,000, mean 3,847). gc content varies from 27% to 68% (mean 44.28%), indicating different evolutionary adaptations. the number of virulence genes also shows high dispersion (2–312, mean 47.4), as does amr gene count (0–7,000, mean 152.4), suggesting that some pathogens possess extensive resistance mechanisms while others have none (table 1). the distribution of annual global cases (1–2.8 billion, median 141,000) and mortality rates (0.1–93%) highlights the uneven burden of these bacteria, with a few species causing massive outbreaks (table 1). the high standard deviations across most variables emphasize the heterogeneity in genome characteristics and public 848 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) table 1. . genomic, virulence, antimicrobial resistance, and epidemiological profiles of 50 foodborne bacterial species. comprehensive dataset summarizing the species, family, genome size (mb), gene number, gc content (%), virulence gene count, antimicrobial resistance (amr) gene count, annual global infection cases, and reported mortality rates (%) for 50 foodborne bacterial species analyzed in this study. this detailed table supports comparative analyses of genomic features and their potential associations with public health impact and microbial forensic investigations. mortality rate (%) annual cases worldwide amr genes vir genes number gc cont. (%) gene number genome size (mb) familyspecies 17800000010505246004.6enterobacteriaceaesalmonella enterica 30230000005303830003listeriaceaelisteria monocytogenes 5200000010405047005enterobacteriaceaeescherichia coli 0.1800000005355145004.8enterobacteriaceaeshigella_sonnei 7010003202836003.9clostridiaceaeclostridium botulinum 417900007605755005.5enterobacteriaceaeklebsiella pneumoniae 91,500,000393937004.1morganellaceaeproteus mirabilis 305,600,000104554,0005.6enterobacteriaceaeenterobacter cloacae 304,0001021585,0005enterobacteriaceaeserratia marcescens 10290,000712525,0005enterobacteriaceaecitrobacter freundii 34117,0001057474,0004.6yersiniaceaeyersinia enterocolitica 590,000867482604.6yersiniaceaeyersinia pseudotuberculosis 8.610,000916573,7003.8hafniaceaeedwardsiella tarda 2173,0001930513,5004morganellaceaemorganella morganii 181,000,000620675,0007pseudomonadaceaepseudomonas aeruginosa 917,000678,1009burkholderiaceaeburkholderia cepacia 10165,000100146686,5007.4burkholderiaceaeburkholderia pseudomallei 1018,0001300383,0004.8legionellaceaelegionella pneumophila 1540,00083126018,0005.5aeromonadaceaeaeromonas hydrophila 502,000,0004010473,9004vibrionaceaevibrio cholerae 18200,0005300534,4005.2vibrionaceaevibrio parahaemolyticus 331,0007160464,0007.5vibrionaceaevibrio_vulnificus 11,500,00030158301,7001.8campylobacteraceaecampylobacter jejuni 11,500,0001211312,0001.8campylobacteraceaecampylobacter coli 41,200,000426391,2001.7helicobacteraceaehelicobacter pylori 160,0001311446,8006.4bacillaceaebacillus cereus 20100,000102375,0005.3bacillaceaebacillus anthracis 93101008396,0005.5bacillaceaebacillus thuringiensis 61,500,00062294,0004.3clostridiaceaeclostridium difficile 301,000,000720302,5003.3clostridiaceaeclostridium perfringens 101,000,0003100282,7002.8clostridiaceaeclostridium tetani 501,00013273,4003.4clostridiaceaeclostridium septicum 101,600,000209332,0002.8staphylococcaceaestaphylococcus aureus 401,000863322,5002.5staphylococcaceaestaphylococcus epidermidis 11,500,00053332,5002.5staphylococcaceaestaphylococcus saprophyticus 2010014382,8002.9listeriaceaelisteria_ivanovii 1018,000,000646391,5001.9streptococcaceaestreptococcus pyogenes 828,000713352,0002streptococcaceaestreptococcus agalactiae 5500536392,5002streptococcaceaestreptococcus pneumoniae 52,800,000,00049362,0002streptococcaceaestreptococcus mutans 7240,00065408,0002streptococcaceaestreptococcus suis 10100,0001010373,0003.6enterococcaceaeenterococcus faecalis 1350,0001073803.2enterococcaceaeenterococcus faecium 509,0001015654,0004.4mycobacteriaceaemycobacterium tuberculosis 25200,00037684,0005mycobacteriaceaemycobacterium avium 520526321,8001.8francisellaceaefrancisella tularensis 110008574,2003.3brucellaceaebrucella abortus 2500,000410573,0003.3brucellaceaebrucella melitensis 11,00002422,0001.9coxiellaceaecoxiella burnetii 525005301,3001.2rickettsiaceaerickettsia rickettsii mortality rate (%) annual cases world amr genes virulence genes gc content (%) gene number genome size (mb) 50.0050.0050.0048.0050.0050.0050.00count 17.8360499803.32152.4047.4044.283847.203.97mean 19.40395649413.95988.3677.3012.062679.741.73std 0.101.000.002.0027.000.001.20min 5.005250.005.007.7535.252500.002.5825% 10.00141000.007.0014.0039.503650.003.9550% 28.751500000.0010.0047.0052.754575.005.0075% 93.002800000000.007000.00312.0068.0018000.009.00max table 2. summary statistics of genome size, gene number, gc content, virulence genes, amr genes, annual cases worldwide, and mortality rates among 50 foodborne bacteria. 849 health risks associated with these pathogens. the genomic features of the 50 foodborne bacterial species exhibit substantial variability, as seen in the distribution plots and boxplots. genome size ranges from 1.2 mb to 9.0 mb (mean: 3.97 mb), with most bacteria clustering around 2–5 mb (figure 1). the gene count follows a similar trend, varying from 0 to 18,000 genes (mean: 3,847), with a few species having exceptionally large genomes and gene content (figure 2). gc content shows a wide distribution (27%–68%) (figure 3), indicating diverse evolutionary strategies across species. boxplots reveal that some species exhibit outliers in genome size, gene number, and gc content (figure 4), suggesting the influence of horizontal gene transfer, environmental adaptation, or pathogenic specialization. a barplot ranking bacterial species by genome size (figure 5) highlights burkholderia cepacia, vibrio vulnificus, and burkholderia pseudomallei as the species with the largest genomes (>7 mb). in contrast, rickettsia rickettsii, helicobacter pylori, and campylobacter jejuni have the smallest genomes (<2 mb), suggesting a more specialized or host-dependent lifestyle. a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) 3.2. virulence and antimicrobial resistance (amr) gene distributions the number of virulence genes varies widely across species, ranging from 2 to 312 (mean: 47.4) (figure 6). most bacteria harbor relatively few virulence genes, but a few species possess over 100, suggesting enhanced pathogenic potential. the boxplot (figure 7) confirms this trend, with several outliers exhibiting exceptionally high virulence gene counts. similarly, amr gene distribution is highly skewed, with most bacteria containing few resistance genes (mean: 152.4), while a select few harbor thousands of amr genes (figure 8). the presence of extreme outliers in the boxplot (figure 9) suggests that some species have undergone extensive resistance acquisition, likely due to antibiotic pressure or horizontal gene transfer. 3.3. correlation between genomic features and public health impact a heatmap (figure 10) shows strong positive correlations between genome size, gene number (0.55), and gc content (0.67), indicating that larger genomes tend to have higher gc content and more genes. virulence gene count exhibits moderate correlations with genome size (0.32) and gene number (0.42), suggesting that more complex genomes may contain more virulence factors. amr gene count, annual cases, and mortality rates show weak or no correlation with genomic traits, implying that pathogenicity and public health burden are influenced by additional ecological or host factors. 3.4. scatterplot analysis of mortality rate vs. genomic traits scatterplots comparing mortality rates with genomic features (figure 11) indicate that genome size, gene number, and gc content do not strongly correlate with mortality, suggesting that larger genomes or higher gene counts do not predict increased lethality. virulence gene count shows a slight upward trend, with some species possessing >100 virulence genes exhibiting higher mortality; however, the overall pattern remains dispersed, indicating the influence of additional factors. similarly, amr gene counts and annual global case numbers do not show clear relationships with mortality, as bacteria with high resistance gene counts or widespread prevalence often exhibit low mortality rates. these patterns emphasize the complexity of bacterial pathogenicity and the role of host, ecological, and environmental factors beyond genomic content. 3.5. pairwise comparisons of genomic and epidemiological features a comprehensive pairplot (figure 12) visualizes relationships among numerical features across species. genome size, gene number, and gc content exhibit strong correlations, consistent with observations in the correlation heatmap, reflecting genome structural relationships across foodborne bacteria. in contrast, relationships between genomic features and public health metrics figure 1. distribution of genome size among 50 foodborne bacteria species. figure 2. distribution of gene number among 50 foodborne bacteria species. figure 3. distribution of gc content among 50 foodborne bacteria species. figure 4. boxplots of genome size, gene number, and gc content among 50 foodborne bacteria species. 850 figure 5. ranked barplot of genome size by species among 50 foodborne bacteria species. a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) figure 6. distribution of virulence gene count among 50 foodborne bacteria species. figure 7. boxplot of virulence gene count among 50 foodborne bacteria species. figure 8. distribution of amr gene count among 50 foodborne bacteria species. figure 9. boxplot of amr gene count among 50 foodborne bacteria species. 851 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) figure 10. correlation heatmap of genomic features among 50 foodborne bacteria species. figure 11. scatterplots of mortality rate vs. genomic traits among 50 foodborne bacteria species. 852 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) figure 12. pairplot of all numerical features among 50 foodborne bacteria species. (mortality rates, annual cases) remain weak or absent, highlighting that genome characteristics alone are insufficient to predict disease severity or burden. virulence and amr gene counts display skewed distributions, with a few species harboring high counts while most remain low, without clear clustering patterns based on pathogenic severity. these findings underscore the multifactorial nature of foodborne bacterial pathogenicity and the necessity of integrating genomic, epidemiological, and ecological data to improve risk assessment, surveillance, and disease prevention strategies. 4. discussion this study provides a comprehensive analysis of the genomic diversity, virulence potential, and antimicrobial resistance (amr) profiles of 50 foodborne bacterial species, linking these features to their public health impact. our findings reveal substantial variability in genome size, gene content, virulence factors, and resistance genes, but weak correlations between genomic traits and epidemiological severity. these results highlight the multifactorial nature of bacterial pathogenicity, emphasizing the need for integrated genomic, clinical, ecological, and host-related assessments to better predict and mitigate foodborne disease risks (pightling et al., 2021; hendriksen et al., 2019). 4.1. genomic complexity and its relationship to pathogenic potential our results confirm that genome size is strongly correlated with gene number (r = 0.55) and gc content (r = 0.67), consistent with previous findings that larger bacterial genomes often encode more genes, allowing for greater metabolic versatility and adaptability (ochman and davalos, 2006). however, genome size alone does not dictate pathogenicity, as some highly virulent bacteria, such as helicobacter pylori and campylobacter jejuni, have small genomes (<2 mb), while others with larger genomes, such as burkholderia species, are opportunistic rather than obligate pathogens. this suggests that pathogenicity is more reliant on specific virulence factors than on genome size alone. furthermore, virulence gene count exhibits only moderate correlations with genome size (r = 0.32) and gene number (r = 0.42), reinforcing that pathogenic potential is not solely determined by genomic expansion. some pathogens with relatively low gene counts, such as clostridium botulinum, produce highly potent toxins, making them lethal despite their small genomes (rossetto et al., 2014). conversely, some species with large genomes, such as bacillus cereus, are not always highly virulent, highlighting the role of regulatory mechanisms, environmental conditions, and host interactions in determining disease severity (ehling-schulz et al., 2019). 4.2. antimicrobial resistance and public health risk the distribution of amr genes across bacterial species is highly skewed, with some species possessing thousands of resistance genes, while others have none. this variability aligns with prior studies showing that horizontal gene transfer (hgt) plays a crucial role in the acquisition of resistance determinants, particularly in species frequently exposed to antibiotic pressure (frost et al., 2005; von wintersdorff et al., 2016). global surveillance efforts using metagenomics and wgs have highlighted the spread of amr determinants across environmental and clinical settings, underscoring the need for integrated monitoring frameworks (hendriksen et al., 2019; collignon and mcewen, 2019). despite the presence of high amr gene counts in some bacteria, no strong correlation was found between amr genes and mortality rate, suggesting that antibiotic resistance alone does not directly predict disease severity. while resistance may contribute to treatment failure and prolonged infections, it does not necessarily increase intrinsic virulence (martinez et al., 2009). additionally, some highly resistant bacteria, such as enterococcus faecium, typically cause chronic but non-lethal infections, whereas low-resistance pathogens like listeria monocytogenes can be fatal, particularly in immunocompromised individuals (radoshevich and cossart, 2018). 4.3. weak correlations between genomic features and mortality rate a key finding of this study is the lack of strong correlations between genome size, gene number, gc content, virulence genes, and mortality rate. scatterplots show that some highly lethal bacteria have relatively simple genomes, while others with large genomes and numerous virulence factors cause only mild infections. this reinforces the idea that mortality rate is shaped by a complex interplay of factors, including host susceptibility, transmission dynamics, and immune evasion strategies (casadevall and pirofski, 2018). for instance, pathogens such as vibrio vulnificus and yersinia pseudotuberculosis exhibit high fatality rates despite moderate genome sizes, whereas escherichia coli and salmonella enterica, which have relatively large genomes, cause a spectrum of diseases ranging from mild gastroenteritis to severe systemic infections (jolley and maiden, 2010; baker et al., 2018). this suggests that key virulence determinants, such as toxin production, immune evasion mechanisms, and host-pathogen interactions, may be better predictors of disease severity than genome size alone (pightling et al., 2021). 4.5. evolutionary and ecological influences on pathogenicity the pairwise comparison of genomic and epidemiological features indicates that certain bacterial species cluster together based on genome size and gc content, but not necessarily on virulence or resistance patterns. this aligns with previous research showing that bacterial genome evolution is driven by niche adaptation, environmental pressures, and host interactions rather than a simple accumulation of virulence factors (merhej and raoult, 2011). for example, intracellular pathogens like rickettsia and mycobacterium exhibit genome reduction, reflecting their reliance on host cellular machinery, while environmentally persistent bacteria like pseudomonas aeruginosa maintain large, flexible genomes to survive diverse conditions (toft and andersson, 2010). machine learning and ai approaches are increasingly being used to analyze these complex genomic and ecological patterns, enhancing our understanding of pathogen evolution and spread (jiang et al., 2022; danko et al., 2021; libbrecht and noble, 2015). this suggests that pathogen success is dictated by ecological fitness rather than genome complexity alone, underscoring the need for contextual analysis of bacterial pathogenicity. 4.6. implications for food safety and disease prevention these findings have important implications for foodborne disease surveillance and risk assessment. given the weak correlations between genomic traits and public health burden, a multifactorial approach incorporating genomic, epidemiological, and host-pathogen interaction data is essential for improving predictive models of foodborne disease risk (scallan et al., 2011; collignon and mcewen, 2019). genomic screening alone may not be sufficient to assess virulence risk; functional studies on toxin production, immune evasion, and transmission dynamics are critical. amr monitoring should focus on clinically relevant resistance genes and their impact on treatment outcomes rather than total gene counts. surveillance strategies should prioritize high-risk species with both high virulence and frequent foodborne transmission, such as listeria monocytogenes, vibrio vulnificus, and salmonella enterica. future research should explore machine learning approaches to integrate genomic, epidemiological, and clinical data for more accurate pathogenicity risk assessments (jiang et al., 2022; libbrecht and noble, 2015). additionally, functional genomics studies could help determine which virulence and resistance factors are most predictive of severe disease outcomes. 4.7. foodborne bacteria and their role in microbial forensics the genomic analysis of foodborne bacteria not only enhances our understanding of pathogenicity and public health risks but also plays a crucial role in microbial forensics—a field dedicated to identifying and tracking microbial agents in criminal, bioterrorism, and foodborne outbreak investigations. by leveraging whole-genome sequencing (wgs), phylogenetic analysis, and comparative genomics, microbial forensics can trace the origin, evolution, and transmission routes of foodborne pathogens, providing critical evidence in cases of food contamination, bioterrorism, and intentional adulteration (thirunavukkarasu et al., 2018). today, advancements in microbial forensics enable real-time genomic surveillance, allowing authorities to rapidly identify specific bacterial strains, their 853 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) virulence factors, and antimicrobial resistance genes, aiding in forensic investigations and outbreak control (oliveira et al., 2024; pightling et al., 2021). the integration of ml and ai models with wgs data can enhance source attribution, evolutionary tracking, and prediction of outbreak dynamics in microbial forensics (jiang et al., 2022; danko et al., 2021). the bacterial species analyzed in this study, including salmonella enterica, escherichia coli, listeria monocytogenes, and vibrio cholerae, are among the most common culprits in foodborne outbreaks and forensic investigations (todd, 2017; baliyan et al., 2025). as global food supply chains become increasingly complex, the combination of pathogen genomics and forensic tools will be essential for ensuring food safety, tracing sources of contamination, and mitigating biosecurity threats. 4.8. limitations and future directions while this study provides valuable insights into the genomic and epidemiological diversity of foodborne bacteria, several limitations remain. key factors such as host susceptibility, infection dose, and environmental conditions were not included, yet they play crucial roles in disease severity and transmission. future research should incorporate functional genomics approaches to analyze how gene expression influences virulence and antimicrobial resistance, rather than relying solely on gene presence. additionally, ml techniques could significantly improve predictive modeling by identifying genomic signatures linked to high-risk pathogens, enabling more precise risk assessment and targeted public health interventions. funding this research received no external funding. conflict of interest the authors declare no conflicts of interest. authors contributions conceptualization: mak data collection: db formal analysis: bd, mak writing—original draft preparation: mak, kl writing—review and editing: aa, dg, jg, jy, md, kl, ew supervision: mak project administration: mak references american academy of microbiology. 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(2021) seaborn: statistical data visualization, joss., vol. 6, no. 60, pp. 3021. https://doi. org/10.21105/joss.03021. 855 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) atlas journal of biology 2018, pp. 551–574 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) mirabilis expansa growth, yield, and quality traits: first us field trials for an ancient, endangered, andean crop miriam kritzer van zant*, ken diesburg, and david a. lightfoot department of plant, soil and agricultural systems, southern illinois university, carbondale, il 629014415, usa received: may 21, 2018 / accepted: august 8, 2018 __________________________________________________ * corresponding author: mkvzant@gmail.com 551 abstract crop introductions have historical importance. ancient, endangered, andean root and forage crop mirabilis expansa (ruiz and pav.) standl. (nyctaginaceae), has high quality starch and protein, is drought resistant, and exhibits high morphological variation on individuals. leaves are used for animal forage, roots for human food. processing of the calcium oxalate could make m. expansa a significant modern crop. cultivated varieties ‘l’ and ‘t’ were grown in constructed, lysimeter sand plots in southern illinois in 2008 and 2009. there was sufficient survival and growth data for var. ‘l’ for testing with repeated measures anovas. tables of mean averages of harvest data are included for each variety. var. ‘l’ plots had three levels of soil amendments plus all-sand as the control. plant height, longest and typical axial shoot length, emerging axial and lateral shoot numbers, lamina length with and without petiole included, lamina width, and estimated herbivory, all had significance at p<0.05, for tracking growth in at least some soil amendments. m. expansa cultivars were therefore shown as adapted to the southern illinois climate in the modified plots. m. expansa is unlikely to become invasive or a major crop where standing water can persist in the root zone. keywords: mirabilis expansa (nyctaginaceae); endangered; drought-resistant; growth; forage. abbreviations: avg, mean average; cip, centro internacional de la papa; coa, combined organic amendments; diam, diameter; qcne, herbario nacional; hrc, horticulture research center; h-f-l, hunyh-feldt-lecoutre epsilon correction; p, peat; control, 0.0% soil amendments; 1p, 1% peat; 2p, 2% peat; 3p, 3% peat; rcbd, randomized complete block design; sds, standard deviations; siu, southern illinois university; s, steer manure; 4-5s, 4% and 5% steer manure; 1p3s, 1% peat with 3% steer manure or combined organic amendments; var., variety. additional abbreviations are in each table’s notes. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction mirabilis expansa (ruiz and pav.) standl. (nyctaginaceae) is an ancient and endangered andean crop with high potential to serve as a new food and fodder crop for arid lands, including both hot desert and cool mountain ecosystems. it was cultivated in both for thousands of years (kritzer van zant, 2016a, 2016b, 2016c; popenoe et al., 1989). m. expansa roots have been used for human food in many parts of the andes (popenoe et al., 1989). m. expansa leaves, traditionally used for animal forage, were found to contain high-quality protein in greater amounts than in the roots (kritzer van zant, 2016a, 2016b, 2016c). m. expansa is rich in high quality protein and extremely fine starch. preliminary research on m. expansa starch indicates it is equal to or better in quality than that of cassava (kritzer van zant, 2016a, 2016b; thanyasiriwat et al., 2013). m. expansa appears to need porous soils (kritzer van zant, 2016a). m. expansa roots often have an astringent taste, though ecuadorian varieties may be less astringent (popenoe et al., 1989). astringency is probably due to the presence of needlelike raphides made of calcium oxalate crystals, which can contribute to kidney stone formation (korth et al., 2006; massey et al., 1993). raphides protect crops from chewing insects. indigenous processing methods increase palatability of the crop (popenoe et al., 1989). mechanical separation methods such as calcium binders and centrifuges, and/or enzyme digestion used for producing raw protein powders, might ameliorate the calcium oxalate (coe, 2016; ranganathan et al., 2010; turroni et al., 2007, 2010). biodiversity, crucial to agricultural production, may provide solutions for creating ecologically sound agricultural systems (di falco and perrings, 2005; partap, 1992). biodiversity is especially crucial to mountain agriculture’s highly variable soils, fragile ecosystems and gene pools, inaccessibility, and nichespecific adaptations of plants to conditions (partap, 1992; zimmerer, 1992). in situ conservation of native crops is needed to promote biodiversity and improve the lot of small farmers in a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) mountainous regions (camino, 1992; tapia, 1992). benefits of the green revolution were limited to flat and well irrigated areas (conway, 1992; evenson and gollin, 2003). resultant varieties and agronomic practices are of little use, even damaging, to arid mountain regions. in the early 1960s and 1970s, many aid and development organizations shifted their agricultural focus for arid mountains to emphasize human need and environmental conditions. indigenous crops have an important role in addressing these issues. most major crops underwent most of their evolutionary development in the highlyvaried conditions of mountain regions (zimmerer, 1992). tapia (1992) thoroughly summarizes variable conditions relevant for agriculture in the andes, which include the most varied conditions for a single mountain range in the world. in the andes, as in mountain regions generally, multiple factors are causing losses of indigenous crops and erosion of genetic variability (zimmerer, 1992). factors include seasonal labor shortages, trade issues, and loss of land. landslides and the world-wide demand for coca (erythroxylum sp.) also negatively impact indigenous farming practices and crops, particularly at altitudes up to 2000 m (camino, 1992; fjeldså et al., 2005). zones utilized in coca production overlap with where m. expansa was traditionally grown. we questioned whether m. expansa horticultural varieties could withstand the heavy rain and drought extremes common to southern illinois weather. also of interest, was whether m. expansa could withstand typical native southern illinois soils in better drained areas, such as hillsides and sandy soils. the question of whether there was growth through the summer was examined for two varieties of the crop. two hypotheses, detailed in methods below, address only var. ‘l’, as it was the only variety to produce sufficient data for repeated measures anova analysis both years. this paper addresses the successful growth of m. expansa, for a two-year period, in constructed plots containing sandy soils. there is also an evaluation of the growth characters measured for this morphologically variable crop. growth data was taken in the field while plants were growing. harvest data was taken from a small subset of plants of var. ‘l’ in 2008, and of var. ‘l’ and var. ‘t’ in 2009, which were grown on plots that received each of several different levels of soil amendments, after harvest. positive results for growth data are given from the repeated measures anovas (tables 1 and 2). there was insufficient harvested material of either variety for meaningful anova analysis, though a table of means is given for harvested material for each year (supplementary tables 1 and 2). harvest data is presented in tables as mean averages for each variety (supplementary tables 1 and 2); and in graphs based on the data in those tables, with error bars representing standard deviations (figures 4, 5, and 6; supplementary figures 1, 2, 3, 4, and 5). var. ‘t’ grew in plots with slightly different levels of steer manure and peat than those used for var. ‘l’. amounts of peat by itself, and steer manure by itself, were similar enough for non-statistical comparison of those two soil amendments between varieties. horticulturalists have been growing m. expansa in belgium and great britain, though to date, no papers have been published from these efforts (kritzer van zant, 2016a, 2016b). an 552 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) experiment with the crop in texcoco, mexico was considered unsuccessful, as the plants failed to flower, and roots are usually harvested during flowering in the andes (kritzer van zant, 2016a, 2016b; seminario cunya, 1993, 2004). however, these texcoco plants may have yielded useful forage and edible roots. m. expansa grown in the andes offered better forage yields than either improved alfalfa in california (putnam, 2007) or unimproved rangeland in utah (perry, 2012). this is based on forage yields in an andean trial with denser plantings than those typically used for growing roots (kritzer van zant, 2016a, 2016b; seminario cunya, 2000, 2004). in the denser planting trial, root yields were lower than from less dense plantings intended for root harvests. however, denser planting still produced enlarged roots simultaneously with forage. m. expansa planted for roots (rea, 1992) produced comparable yields to several potato varieties grown in idaho (olsen, 2004). in the root trial described by rea, m. expansa simultaneously produced forage. it should be possible to produce m. expansa roots and/or forage with considerably less water and fertilizer than needed for potatoes or alfalfa (kritzer van zant, 2016a, 2016b). unfortunately, few details presented in documents describing growth of the crop in the andes (seminario cunya, 1993, 2000, 2004), were directly comparable to results from our field data. materials and methods materials for the southern illinois field work, var. ‘l’ cuttings were grown from the two stock plants. var. ‘l’ stock plants were descendants from two sole survivors of plants started from seed and greenhouse grown in illinois. seeds of var. ‘l’ were sent to kritzer van zant by centro internacional de la papa (cip) in lima, peru (cip accession #208001 arb5395). variety ‘l’ was said by cip personnel to have originated in cajamarca, cajamarca, peru. however, as there were m. expansa accessions maintained there by personnel at universidad nacional de cajamarca, var. ‘l’ may have had a different place of origin. variety t’ was propagated from rhizomes, descendants of plants from the garden of eloisa de la cruz, an indigenous woman farmer in northern ecuador. her garden was on property in pinchincha province, near the road to the tocacachi ruins. collection of var. ‘t’ was made during an intensive search for cultivated m. expansa in northern ecuador by kritzer van zant. during that search, only var. ‘t’ was found, and only at that single location. variety ‘t’ was initially propagated in illinois from rhizomes. stem cuttings from both varieties were next propagated as stock plants. plants from stem cuttings were used for research in this paper, and for subsequent research to date on m. expansa amino acids and cytotoxicity (kritzer van zant, 2016a). permissions and protections upon request, seeds of var. ‘l’ were sent to kritzer van zant by common agreement between cip and usda. a few of these a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 553 table 1. the 2008 growth data repeat measure anovas positive results. abbre viations: axlatsht#, axial and lateral shoot number; coa, combined organic amendments; df1/df2, error; herb 0, 1, herbivory coded 0, 1 – 0 = absent, 1 = present; h-f-l, huynh-feldt-lecoutre epsilon correction, adjusted pr>f for time and the interaction; interaction, time*independent variable (specified); in/laxsht, internodes per longest axial shoot length in first 50.8 mm above the soil; lamina, lamina length without petiole (laminalg); lamina length with petiole; lamlpow, ratio of lamina with petiole length over lamina width; laminaw, lamina width; lamwolp, ratio of lamina width over lamina with petiole length; laxshtl, longest axial shoot length; plantht, plant height; p-value, at .05 level, pr>f for anova; steerm, steer manure; taxshtl, typical axial shoot length; *, significant; **, highly significant. test type interaction independent variable dependent variable f value df1, df2 p-value h-f-l adjusted h-f-l p-value at .05 level anova coa plantht 6.90 3, 4 0.0464 * time coa plantht 87.26 6, 24 <0.0001 0.3644 ** time*coa coa plantht 5.64 18, 24 0.0110 0.3644 * time peat plantht 27.76 6, 30 0.0006 0.2397 * time steerm plantht 42.69 6, 30 <0.0001 0.3555 ** time terrace plantht 25.48 6, 36 <0.0001 0.3208 ** time coa laxshtl 92.30 4, 16 <0.0001 0.4253 ** time peat laxshtl 28.46 4, 20 0.0014 0.3016 * time steerm laxshtl 44.89 4, 20 0.0002 0.3445 * time terrace laxshtl 40.57 4, 24 0.0002 0.3177 * time coa taxshtl 28.58 3, 12 0.0002 0.6644 * time peat taxshtl 20.08 3, 15 0.0004 0.6392 * time steerm taxshtl 29.63 3, 15 <0.0001 0.9844 ** time terrace taxshtl 24.69 3, 18 <0.0001 0.6191 ** time coa axlatsht# 10.73 3, 12 0.0025 0.8250 * time peat axlatsht# 11.40 3, 15 0.0019 0.7225 * time steerm axlatsht# 10.23 3, 15 0.0025 0.7460 * time terrace axlatsht# 17.10 3, 18 0.0002 0.7266 * time coa in/laxsht 29.47 4, 16 0.0001 0.5352 ** time peat in/laxsht 30.24 4, 20 <0.0001 0.5816 ** time steerm in/laxsht 28.68 4, 20 <0.0001 0.7118 ** time terrace in/laxsht 34.73 4, 24 <0.0001 0.7211 ** time coa laminalg 5.82 6, 18 0.0024 0.9160 * time peat laminalg 3.85 6, 24 0.0078 1.3577 * time steerm laminalg 4.94 6, 24 0.0023 0.9645 * time terrace laminalg 4.46 6, 30 0.0024 1.6054 * anova coa laminap 32.37 3, 3 0.0087 * time coa laminaw 7.22 6, 18 0.0005 1.0208 * time peat laminaw 6.86 6, 24 0.0002 1.1642 * time steerm laminaw 5.66 6, 24 0.0091 0.5500 * time terrace laminaw 6.58 6, 30 0.0014 0.6749 * time coa lamwolp 8.36 6, 18 0.0191 0.3282 * time peat lamwolp 8.17 6, 24 0.0146 0.3055 * time steerm lamwolp 6.03 6, 24 0.0022 0.7636 * time terrace lamwolp 6.16 6, 30 0.0025 0.6404 * time terrace lamlpow 4.13 6, 30 0.0171 0.6030 * anova peat herb 0, 1 20.89 2, 2 0.0457 * time peat herb 0, 1 17.33 6, 12 0.0312 0.2208 * time steerm herb 0, 1 5.97 6, 12 0.0301 0.5083 * time terrace herb 0, 1 13.75 6, 18 0.0005 0.5749 * time coa mass 57.00 6, 24 <.0001 0.3964 ** time*coa coa mass 4.74 18, 24 0.0151 0.3964 * time peat mass 21.37 6, 30 0.0010 0.2599 * time steerm mass 49.24 6, 30 <0.0001 0.3554 ** time*steerm steerm mass 4.38 12, 30 0.0230 0.3554 * time terrace mass 20.92 6, 36 <0.0001 0.4262 ** a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 554 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg )table 2. the 2009 growth data repeat measure anovas positive results. abbre viations: axlatsht#, axial and lateral shoot number; coa, combined organic amendments; df1/df2, error; herb 0, 1, herbivory coded 0, 1—absent = 0, present = 1; herb 0-4, herbivory coded with five levels—no noticeable herbivory = 0, noticeable though insignificant = 1, slight = 2, moderate = 3, and heavy = 4; lamina, lamina length with petiole; h-f-l, huynh-feldt-lecoutre epsilon correction, adjusted pr>f for time and the interaction; interaction, time*independent variable (specified); laminalg, lamina length without petiole; laminaw, lamina width; laxshtl, longest axial shoot length; plantht, plant height; p-value, at .05 level, pr>f for anova; steerm, steer manure; taxshtl, typical axial shoot length*, significant; **, highly significant. test type interaction independent variable dependent variable f value df1, df2 p-value h-f-l adjusted h-f-l p-value time peat plantht 6.81 4, 20 0.0378 0.2955 * time steerm plantht 5.63 4, 20 0.0452 0.3330 * time coa laxshtl 48.29 4, 16 <0.0001 0.7092 ** time peat laxshtl 40.41 4, 20 <0.0001 1.0162 ** time steerm laxshtl 35.54 4, 20 <0.0001 0.8876 ** time terrace laxshtl 33.61 4, 24 <0.0001 0.5571 ** time coa taxshtl 105.04 4, 16 <0.0001 1.3380 ** time*coa coa taxshtl 4.75 12, 16 0.0023 1.3380 * time peat taxshtl 61.99 4, 20 <0.0001 0.7635 ** time steerm taxshtl 49.04 4, 20 <0.0001 0.5995 ** time terrace taxshtl 35.10 4, 24 <0.0001 0.6579 ** time coa axlatsht# 212.57 4, 16 <0.0001 0.5761 ** time*coa coa axlatsht# 5.61 12, 16 0.0095 0.5761 * time peat axlatsht# 137.68 4, 20 <0.0001 0.4244 ** time*peat peat axlatsht# 5.30 8, 20 0.0222 0.4244 * time steerm axlatsht# 53.62 4, 20 0.0002 0.3111 * time terrace axlatsht# 62.60 4, 24 <0.0001 0.3184 ** time coa laminalg 25.55 4, 16 <0.0001 1.0377 ** time*coa coa laminalg 3.53 12, 16 0.0102 1.0377 * time peat laminalg 22.91 4, 20 <0.0001 1.0586 ** time*peat peat laminalg 4.02 8, 20 0.0055 1.0586 * time steerm laminalg 15.22 4, 20 0.0006 0.5387 * time terrace laminalg 12.13 4, 24 <0.0001 0.9150 ** anova coa laminap 6.97 3, 4 0.0457 * time coa laminap 15.88 4, 16 <0.0001 1.3054 ** time peat laminap 16.40 4, 20 <0.0001 0.9397 ** time*peat peat laminap 2.76 8, 20 0.0355 0.9397 * time steerm laminap 11.72 4, 20 0.0006 0.6794 ** time terrace laminap 11.15 4, 24 <0.0001 0.8512 ** time coa laminaw 13.19 4, 16 <0.0001 1.1839 ** time peat laminaw 12.59 4, 20 <0.0001 1.2686 ** time steerm laminaw 13.58 4, 20 <0.0001 1.0939 ** time terrace laminaw 13.21 4, 24 <0.0001 1.3674 ** time coa herb 0, 1 63.55 4, 16 <0.0001 0.9955 ** time peat herb 0, 1 65.75 4, 20 <0.0001 0.9495 ** time steerm herb 0, 1 63.12 4, 20 <0.0001 1.0712 ** anova terrace herb 0, 1 7.43 1, 6 0.0344 * time terrace herb 0, 1 75.11 4, 24 <0.0001 0.9900 ** time coa herb 0-4 189.49 4, 16 0.0002 0.2500 * time peat herb 0-4 200.44 4, 20 <0.0001 0.2500 ** time steerm herb 0-4 157.37 4, 20 <0.0001 0.2500 ** time terrace herb 0-4 273.24 4, 24 <0.0001 0.2500 ** anova coa mass 65.54 3, 4 0.0007 * time coa mass 97.57 4, 16 <.0001 0.5103 ** time*coa coa mass 21.16 12, 16 0.0001 0.5103 ** anova peat mass 37.48 2, 5 0.0010 * time peat mass 105.07 4, 20 <.0001 0.8146 ** time*peat peat mass 24.46 8, 20 <.0001 0.8146 ** anova steerm mass 6.59 2, 5 0.0360 * time steerm mass 35.76 4, 20 <.0001 0.05489 ** time*steerm steerm mass 8.22 8, 20 0.0023 0.05489 * time terrace mass 8.83 4, 24 0.0190 0.2882 * 555 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) seeds were shared with colorado researchers (vepachedu et al., 2003). variety ‘t’ was brought back to the united states with permission from the ecuadorian government, under ecuadorian permit #023-ic-flo-dnbap/ma, in conjunction with kritzer van zant’s then host institution, herbario nacional (qcne), in quito. guachuiun, puente and de la cruz signed human subjects forms protecting their rights and those of ecuador, along with the research rights of kritzer van zant. growth of m. expansa in the field m. expansa was grown outdoors in sand plots (supplementary figure 6; kritzer van zant 2016a appendix b-1). these plots had lain fallow for several years prior to 2008. these plots were originally set up for research on golf turf, 10-15 years prior to first use of them for growing m. expansa. plots were each 2 x 4 m, spaced 1m apart, and arranged in three consecutive terraces. terrace i, ii and iii slope gradually downward to the east, along the same hill, with terrace i at the top of the hill. plots were amended at the time of construction with varied percentages of peat and steer manure. eight levels of treatments, including allsand controls, were repeated on each terrace when constructed. as not all of the original plot types were used, plots used for m. expansa made up a modified replicated randomized complete block design (rcbd). constructed root zones in the plots were 30.5 cm deep. plots were made with 96-100% sand overlaying a 10.2 cm deep layer of 6.4 mm sized gravel. a drainage pipe was installed at the base of the gravel layer. lysimeters were incorporated into each plot at construction. each plot drained separately into a bottle, which overflowed into a channel alongside and 2 m below the plots, so little or no leaching occurred between plots and there was little standing water. not all lysimeters were still functioning at the time m. expansa was growing in the plots, so lysimeter measurements were not taken. however, each plot still drained well, and the lysimeter construction reduced overflow of water among plots. from each terrace, a single plot for each of three soil amendments, plus an all-sand control, were used for the var. ‘l’ experiments in 2008 (table 1 and supplementary table 1; figure 4 and supplementary figure 3). this made four treatments. these same plots were used again for experiment i with var. ‘l’ in 2009 (table 2 and supplementary table 2; figure 5, and supplementary figures 1, 3, and 4). thirty-two plants were measured each year for the repeated experiments with var. ‘l’. averaging was done separately for var. ‘l’ for each year. this helped to compensate for discrepancies in planting, measuring, and survivorship. experiment iv plots had var. ‘t’ plants exclusively in 2009 (supplementary table 2; figure 6 and supplementary figures 2 and 5). for growing var. ‘t’, two levels of treatments were used, different from those used for var. ‘l’, and excluding the all-sand controls. experiment iv plot types were 4% steer manure (4s) and 2% peat (2p), and were on terraces i (upper) and ii (middle). experiment iv plots were also planted with four plants each. additional plots on the same terraces were also planted with each variety in various combinations. however, results from plots containing both varieties, are only included as part of our general observations. the additional plots also provided material for nutrition and cytotoxicity research on the crop. four soil samples were taken in 2011 from plot treatments matching those which had been used for the var. ‘l’ statistically analyzed 2008 and 2009 experiments. these samples were tested for soil nitrogen concentrations in 2012 at the soil and plant testing laboratory at university of missouri extension (http:// soilplantlab.missouri.edu/soil/). each soil sample came from a different plot type on different terraces, so results were not averaged. nitrogen concentrations for different plots were low and did not vary in any pattern that correlated with the original plot amendments. they are only mentioned as a window into the overall condition of the plots. no additional samples from these plots have been subsequently tested for nitrogen. however, differences in weed cover among plot treatments indicated that there were still differences among plot types just prior to and during the mirabilis experiments. these plant community differences were probably due more to persistent changes in soil structure, than to levels of nitrogen present in the plots. each plant received a single pint of mushroom composted horse manure, at the time of planting. manure was covered with a small amount of sandy media from the plot being planted, beneath each plant. manure had been added to increase the possibility that subtle differences that might remain between plots with different levels of soil amendments, would be apparent in the results. manure was aged for the same length of time within each year for all plants. however, in 2009 the manure had aged an additional year over 2008. harvest data growth data had consisted of the measurements taken while the plants were still growing in the field and were analyzed in repeated measures anovas for var. ‘l’ (tables 1 and 2; figures 1, 2, and 3). harvest data (supplementary tables 1 and 2; figures 4, 5, and 6, supplementary figures 1, 2, 3, 4, and 5) were taken after plants were dug up at the end of each season. harvest data for 2008 and 2009 were insufficient in quantity for meaningful anova analysis for either variety, and therefore were considered separately from growth data. harvest data were compared as mean averages between the two varieties, from each plot type, for plots modified with similar amounts of peat or steer manure, 5s and 3p for var. ‘l’, to 4s and 2p for var. ‘t’. for var. ‘l’, within each year, the combined 1p3s and all-sand control plots were also levels of soil amendments available to compare to 5s and 3p. harvest data for var. ‘l’ was grown in 2008 (supplementary table 1; figure 4, supplementary figure 3) and 2009 (supplementary table 2; figure 5; supplementary figures 1 and 4). harvest data for var. ‘t’ came exclusively from the plants grown in experiment iv in 2009 (supplementary table 2; supplementary figures 2 and 5). measurements were taken before and after harvested specimens were dried. plants were dried hanging from ropes in a barn, to mimic traditional drying practices. drying time was shorter in 2008 than in 2009 though both periods of time exceeded the typical eight days minimum drying time reported a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 556 by indigenous people. whole dried plants were weighed, then herbage separated from below ground portions of the plants and re-weighed separately. harvest data for both years consisted of whole plant weights prior to and post drying, percentage change in dry weight, and above-ground and underground portions of the plant as dry weight. root size was averaged for each variety, for each soil amendment from which material was harvested (supplementary tables 1 and 2). results for root size from 2009 (supplementary table 2), from 4s and 2p plots from which var. ‘t’ roots were harvested (supplementary table 2; figure 6, supplementary figures 2 and 5), are again compared with results for var. ‘l’ taken from 5s and 3p plots (supplementary table 2; figure 5, supplementary figures 1 and 4), from the same year. two plants were taken for each kind of soil amendment, one each from separate plots on different terraces. remaining plants were left in the ground to see if they would winter over. however, var. ‘t’ was not available from 2p plots on the upper terrace, so only a single var. ‘t’ plant was harvested from a 2p plot on the middle terrace. thus no sds could be computed for 2p or upper terrace, on graphs for var. ‘t’ (supplementary figures 2 and 5). additional harvest variables were added in 2009 along with var. ‘t’ data (supplementary table 2; supplementary figures 1, 2, 3, 4, and 5). they are root length and root diameter in cms (supplementary table 2; supplementary figures 1 and 2), and the non-continuous character of position of the widest portion of the root mass (supplementary table 2). for this last character, each harvested root mass was visually divided into four zones or levels, from top to bottom as they had been oriented in the soil, and the widest of these zones was noted. harvest data results (supplementary tables 1 and 2) are otherwise self-explanatory and non-statistical, so harvest results are not much discussed in the body of the paper below. description is given for the harvest tables in kritzer van zant (2016a appendices b-4, b-5 and b-6). analyses and variables-field growth data results of the anova analyses, are divided into growth data from the var. ‘l’ experiments in 2008 (table 1; figure 1) and 2009 (table 2; figures 2 and 3). growth data for var. ‘l’ (tables 1 and 2; figures 1, 2, and 3) resulting from the anovas between independent variables, are labelled “anova”. anovas for the same data through time are labelled “time”, and anovas for the interaction of time with each independent variable, are labelled as “time*independent variable” (tables 1 and 2). an example is “time*peat”. only positive results of the statistical analyses are presented below. negative results are in extended tables in kritzer van zant (2016a appendix b-8). in addition, there are three figures of graphs given in this paper (figures 1, 2, and 3), illustrating the most compelling positive “anova” results from the repeated measures analyses, for the var. ‘l’ growth data. m. expansa var. ‘l’ field data was analyzed with repeated measures 2-way anovas in sas (cary, nc), separately for 2008 and 2009. the decision to use repeated measures anovas, sometimes used in ecological studies, resulted from consultation with biological statistician john d. reeve of siuc. repeated measures anovas take into account the lack of independence between measurements on the same individuals, by applying a correction to time and the interaction. regular anovas require an assumption of independence between measurements. the two independent variables in each repeated measures analysis, were treatment meaning a soil amendment or terrace, and time across the growing season in fixed intervals. the intervals for measurement were different for each year. repeated measurements may be correlated, so the tests can need adjustment. repeated measures analysis fixes the correlation issue, using correction factors. therefore, the h-f-l (huynh-feldt-lecoutre epsilon) value was used to alter the degrees of freedom for the time, and treatment x time interaction tests, making them more conservative. h-f-l is considered strict. when the h-f-l is 1, tests don’t need adjustment. since the treatment test is not affected by correlated observations, no correction is applied to it, making it similar to a 1-way anova. however, since the correction is applied to time, one of the two independent variables, 2-way repeated measures anovas are different from regular 2-way anovas. in this design, treatment, time, and the treatment x time interaction, are all fixed effects. t-tests and tables with means are more traditionally included in agricultural field research papers. repeated measures anovas made it possible to consider the subtle variations in plot treatments through time for each measured character, in spite of the lack of independence for measurements on the same individuals taken across the growing season. these analyses provided three kinds of results, labeled in tables 1 and 2 as anovas, time, and the interaction of time with each plot treatment as independent variables, ie: time x peat in the text, or time*peat in tables. the treatment results designated anova, use the mean for each subject over time, though they include no measurement of time itself. time and the interaction, include both time and repeated measurements, which sas refers to as “within subjects”. as these tests address measurements from each period of time in which growth measurements were taken, for each pair of variables, it is not practical to include means of individual measurements at particular points in time in tables. separate tables of means for each kind of growth measurement, at each time of measurement, would create confusion when interpreting this data set, as the purpose is to show the growth of m. expansa through time, and the usefulness of each measurement through the season. means considered separately at each point in time would not give a cohesive picture. three graphs produced by sas for the analyses are presented (figures 1, 2, and 3). they illustrate some of the strongest patterns in the data across the season from both years. graphs for each combination of variables are in kritzer van zant (2016a appendix b-9). growth rates were analyzed separately for several measured dependent variables. included dependent variables for 2008 were the continuous characters: axial and lateral shoot number counted on stems up to 5 cm above the soil surface; estimated mass; plant height; longest axial shoot; typical axial shoot length; internodes per longest axial shoot; lamina length with and without petioles; and lamina width. in 2008 the only nona tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) continuous character was estimated herbivory coded absent (0) or present (1). continuous characters were measured in cm or cm3 unless otherwise stated. some 2008 variables were excluded in 2009 including, counting internodes on the longest axial shoot, and ratios for lamina lengths and widths, as they had given little useful information the previous year. independent variable, starting size of the plant, had three levels—small, medium, or large. independent variable position in the subplots had four levels—nw, sw, ne, or nw. starting size and position in the subplots were both dropped in 2008, prior to the anova analysis, after preliminary t-tests showed they had no effect. herbivory 0, 1 coding had little meaning in the statistical analyses as all plants were subjected to at least some herbivory. therefore, in 2009 though herbivory was measured as present or absent again to compare to the 2008 data, it was also separately measured on a scale for degree of herbivory with five levels—no noticeable herbivory (0), noticeable though insignificant (1), slight (2), moderate (3) and, heavy (4). in addition, in 2009 the largest and most typical or averagesized leaves were measured for the continuous characters of length of the lamina alone, length of the lamina with petiole included, and width of the lamina at the widest point. the lamina measurements and herbivory estimates were taken for each plot in 2009, instead of for each plant as in 2008. lamina measurements were taken for both largest and average leaves, because m. expansa leaves appear to exhibit indefinite growth in the field, greenhouse, and herbarium. all lamina measurements taken for individual plants were averaged for each plot prior to running the anovas, to compensate for this difference in how measurements were taken. analyses and variables-harvest data means for harvest data (supplementary tables 1 and 2) were confirmed, and graphs with sds for that same data (figures 4, 5 and 6, supplementary figures 1, 2, 3, 4 and 5), were computed using microsoft excel (2016). means for the harvest data are presented for var. ‘l’ in 2008 (supplementary table 1; figure 4, supplementary figure 3), and for both varieties in 2009 (supplementary table 2; figure 5 and 6; supplementary figures 1, 2, 4 and 5). y axes for graphs, of the mean harvest weights are in kgs (figures 4, 5 and 6), lengths are in cms (supplementary figures 1 and 2), and percentage weight change for whole plants after drying are in percent (supplementary figures 3, 4, and 5). error bars in these graphs represent standard deviations (sds) of the means. hypotheses in addition to the general question, would m. expansa’ grow in the outdoor sand plots across the growing season, two hypotheses were addressed for var. ‘l’, prior to the analyses of the field growth data for m. expansa. var. ‘t’ could only be considered for the general question concerning growth. only var. ‘l’ gave sufficient data for a meaningful anova analysis of the characters measured, to see if there were significant differences among levels of soil amendments. both hypotheses were applied to var. ‘l’ grown in 2008 and 2009 in southern illinois. repeated measures anova results were applied separately to the hypotheses for var. ‘l’ for each year. for each analysis, p values of 5% or less indicated significance, and 0.01% or less indicated high significance. the hypotheses were: h0 = amounts of peat applied at the time of plot construction have no effect on growth of m. expansa horticultural varieties. h1 = amounts of peat applied at the time of plot construction have an effect on growth of m. expansa horticultural varieties; h0 = amounts of steer manure applied at the time of plot construction have no effect on growth of m. expansa horticultural varieties. h1 = amounts of steer manure applied at the time of plot construction have an effect on growth of m. expansa horticultural varieties. results and discussion positive results for analyses of variance, time, and interaction of time with independent variables there was a great deal of variation in means at different points in time and for different pairs of variables and levels, which the repeated measure anova results made sense of. these contradictions can be seen in 96 graphs which resulted from the repeated measures analyses of the field data, in kritzer van zant (2016a appendix b-9). the repeated measures anova analyses were particularly useful for finding significant results for the variable of time itself, which t-tests could not have addressed. anovas anovas for the 2008 growth data, were for treatment only (table 1), and did not include time as a separate variable. they were significant for the independent variable coa (combined organic amendments) for plant height (f = 06.9003,04, h-f-l adjusted p = 0.0464; figure 1), lamina with petiole length (f = 32.3703,03, h-f-l adjusted p = 0.0087), and for the independent variable peat (f = 20.8902,02, h-f-l adjusted p = 0.0457) for herbivory coded 0,1 (table 1; figure 1). anovas for the 2009 growth data (table 2) were significant for: the independent variable coa for lamina with petiole length (f = 06.9703,4, h-f-l adjusted p = 0.0457) and estimated mass (f = 65.5403,4, h-f-l adjusted p = 0.0007; figure 2); the independent variable peat for estimated mass (f = 37.4802,05, h-f-l adjusted p = 0.0010); the independent variable steer manure for estimated mass (f = 6.5902,05, h-f-l adjusted p = 0.0360; figure 3); and the independent variable terrace for herbivory coded 0, 1 (f = 07.4301,06, h-f-l adjusted p = 0.0344) (table 2). no one character gave consistent results both years for any of the soil amendments in the repeated measures anova for any treatment without time as a separate independent variable. increases in plant height for all the treatments over the control became more pronounced as the growing season continued in some plot types (figures 1 and 2). however, the 3% (v3/v3) steer manure amended plots, at the time of final measurement in a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 557 558 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 2. chart of effect of coa on var. ‘l’ estimated mass, grown in outdoor sand plots in 2009. x-axis: measurement dates: 1, 20jun; 2, 19jul; 3, 16aug; 4, 18sep; 5, 18oct. y-axis: estimated mass in cm3. abbreviations: amendm/coa, combined organic amendments; ppp, 3% peat; psss, 1% peat with 3% steer manure; sssss, 5% steer manure; cont, all-sand control. amendments consist of steer manure and/or peat added at time of plot construction. first measurement taken 4 wks post planting. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 1. chart of effect of coa on var. ‘l’ plant height, grown in outdoor sand plots in 2008. x-axis: measurement dates: 1, 19jul; 2, 2aug and 4aug; 3, 17aug and 19aug; 4, 2sep and 3sep; 5, 16sep and 17sep; 6, 2oct; 7, 14oct. y-axis: plant height in cm. abbreviations: amendm/coa, combined organic amendments; ppp, 3% peat; psss, 1% peat with 3% steer manure; sssss, 5% steer manure; cont, allsand control. amendments consist of steer manure and/or peat added at time of plot construction. first measurement taken 2 wks post planting. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 559 2009, gave about a 150% (cm3/cm3) increase in estimated mass over both the control and 5% steer manure amended plots (figure 3). this may show that too little or too much of the nutrients in steer manure limited growth. an alternative or additional explanation is that the intermediate 3% steer manure amended plots had the effect of retaining sufficient moisture to help m. expansa grow, though not enough to interfere with growth. herbivory results in the first set of anovas for treatment were also contradictory. herbivory results in anovas for treatment may have been affected by insect life cycles, as they were higher for all treatments in the middle of the growing season. the greatest difference for lamina with petiole length in the first set of anovas for combined organic amendments, was at mid-july in 2009, when 1% peat with 3% steer manure had roughly 75% greater length than the control, 5% steer manure was roughly 50% longer than the control, and 3% peat fell between the other two treatments, giving all treatments more length than the control (kritzer van zant 2016a appendix b-6 figure 81). similar patterns of all treatments producing greater growth than the control, can be seen for 1% peat and 3% steer manure on plant height in 2008 (figure 1) and on estimated mass in 2009 (figure 2). time: for the 2008 growth data was highly significant for coa, for plant height (f = 87.2606,24, h-f-l adjusted p = <0.0001; figure 1), longest axial shoot length (f = 92.304,16, h-f-l adjusted p = <0.0001), internodes per longest axial shoot length (f = 29.4704,16, h-f-l adjusted p = 0.0001) and estimated mass (f = 57.0006,24, h-f-l adjusted p = <0.0001) (table 1). time for the 2008 growth data was significant for combined organic amendments for typical axial shoot length (f = 25.5803.12, h-f-l adjusted p = 0.0002), axial and lateral shoot number (f = 10.7303.12, h-f-l adjusted p = 0.0025); lamina length (f = 05.826,18, h-f-l adjusted p = 0.0024); lamina width (f = 07.2206,18, h-f-l adjusted p = 0.0005); and lamina width over lamina with petiole length (f = 08.3606,18, h-f-l adjusted p = 0.0191) (table 1). time for the 2009 growth data was highly significant for coa for: longest axial shoot length (f = 48.2904,16, h-f-l adjusted p = <0.0001); typical axial shoot length (f = 105.0404,16, h-f-l adjusted p = <0.0001); axial and lateral shoot number (f = 212.5704,16, h-f-l adjusted p = <0.0001); lamina length (f = 25.5504,16, h-f-l adjusted p = <0.0001); lamina with petiole length (f = 15.8804,16, h-f-l adjusted p = <0.0001); lamina width (f = 13.1904,16, h-f-l adjusted p = <0.0001); herbivory coded 0, 1 (f = 63.5504,16, h-f-l adjusted p = <0.0001); and estimated mass (f = 97.5704,16, h-f-l adjusted p = <0.0001) (table 2). time for the 2009 growth data, was significant for coa for herbivory coded 0-4 (f = 189.4904,16, h-f-l adjusted p = 0.0002) (table 2). clearly, the analysis of the effect of time indicates multiple measurements are useful to track growth in var. ‘l’ throughout the growing season. however, which are best is less clear, when comparing results between 2008 and 2009, or between soil amendment within and between years, or for each level of soil amendment at different points in time. considering how varied expression is in the nyctaginaceae, this is not a surprise. we have witnessed extremely high variation within wild type individuals of m. expansa, on herbarium sheets. for combined figure 3. chart of effect of steer manure on var. ‘l’ estimated mass, grown in outdoor sand plots in 2009. x-axis: measurement dates: 1, 20jun, 2, 19jul, 3, 16aug, 4,18sep, 5, 18oct. y-axis: estimated mass in cm3. abbreviations: steer, steer manure; 0, 0% steer manure; 3, 3% steer manure; 5, 5% steer manure. steer manure added at time of plot construction. first measurement taken 4 wks post planting. 560 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) organic amendments, consistently significant results for growth through time, between years, were seen for longest axial shoot length, typical axial shoot length, axial and lateral shoot numbers counted on stems up to 5 cm above the soil surface, lamina length not including petiole length, lamina width, and estimated mass. time for the 2008 growth data, was highly significant for peat only for internodes per longest axial shoot length (f = 30.2404,20, h-f-l adjusted p = <0.0001) (table 1). however, time for the 2008 growth data, was significant for peat for: plant height (f = 27.7606,30, h-f-l adjusted p = 0.0006); longest axial shoot length (f = 28.4604,20, h-f-l adjusted p = 0.0014); typical axial shoot length (f = 20.0803,15, h-f-l adjusted p = 0.0004); axial and lateral shoot number (f = 11.4003,15, h-f-l adjusted p = 0.0019); lamina length (f = 03.8506,24, h-f-l adjusted p = 0.0078); lamina width (f = 06.8606,24, h-f-l adjusted p = 0.0002); lamina width over lamina with petiole length (f = 08.1706,24, h-f-l adjusted p = 0.0146); herbivory coded 0, 1 (f = 17.3306,12, h-f-l adjusted p = 0.0312); and estimated mass (f = 21.3706,30, h-f-l adjusted p = 0.0010) (table 1). time for the 2009 growth data, was highly significant for peat for: longest axial shoot length (f = 40.4104,20, h-f-l adjusted p = <0.0001); typical axial shoot length (f = 61.9904,20, h-f-l adjusted p = <0.0001); axial and lateral shoot number (f = 137.6804,20, h-f-l adjusted p = <0.0001); lamina length (f = 22.9104,20, h-f-l adjusted p = <0.0001); lamina with petiole length (f = 16.4004,20, h-f-l adjusted p = <0.0001); lamina width (f = 12.5904,20, h-f-l adjusted p = <0.0001); herbivory coded 0, 1 (f = 65.7504,20, h-f-l adjusted p = <0.0001); herbivory coded 0-4 (f = 200.4404,20, h-f-l adjusted p = <0.0001); and estimated mass (f = 105.0704,20, h-f-l adjusted p = <0.0001) (table 2). time for the 2009 growth data, was significant for peat for: plant height (f = 06.8104,20, h-f-l adjusted p = 0.0378) (table 2). for peat, consistently significant results for growth through time between years were seen for longest and typical axial shoot lengths, axial and lateral shoot numbers up to 5 cm above the soil surface, lamina length not including petiole length, lamina width, herbivory coded 0-1 and estimated mass. time for the 2008 growth data, was highly significant for steer manure for: plant height (f = 42.6906,30, h-f-l adjusted p = <0.0001); typical axial shoot length (f = 29.6303,15, h-f-l adjusted p = <0.0001); internodes per longest axial shoot (f = 28.6804,20, h-f-l adjusted p = <0.0001); and estimated mass (f = 49.2406,30, h-f-l adjusted p = <0.0001) (table 1). time for the 2008 growth data, was significant for steer manure for: longest axial shoot length (f = 44.8904,20, h-f-l adjusted p = 0.0002); axial and lateral shoot number (f = 10.2303,15, h-f-l adjusted p = 0.0025); lamina length (f = 04.9406,24, h-f-l adjusted p = 0.0023); lamina width (f = 05.6606,24, h-f-l adjusted p = 0.0091); lamina width over lamina with petiole length (f = 06.0306,24, h-f-l adjusted p = 0.0022); and herbivory coded 0, 1 (f = 05.9706,12, h-f-l adjusted p = 0.0301) (table 1). time for the 2009 growth data, was highly significant for steer manure for: longest axial shoot length (f = 35.5404,20, h-f-l adjusted p = <0.0001); typical axial shoot length (f = 49.0404,20, h-f-l adjusted p = <0.0001); lamina with petiole length (f = 11.7204,20, h-f-l adjusted p = 0.0006); lamina width (f = 13.5804,20, h-f-l adjusted p = <0.0001); herbivory coded both 0, 1 (f = 63.1204,20, h-f-l adjusted p = <0.0001) and 0-4 (f = 157.3704,20, h-f-l adjusted p = <0.0001); and estimated mass (f = 35.7604,20, h-f-l adjusted p = <0.0001) (table 2; figure 3). time for the 2009 growth data, was significant for steer manure for plant height (f = 05.6304,20, h-f-l adjusted p = <0.0452), axial and lateral shoot number (f = 53.6204,20, h-f-l adjusted p = <0.0002) and lamina length (f = 15.2204,20, h-f-l adjusted p = <0.0006) (table 2). for steer manure, consistently significant results for growth through time between years were seen for plant height, longest and typical axial shoot length, axial and lateral shoot numbers up to 5 cm above the soil surface, herbivory coded 0, 1, length not including petiole length, lamina width, and estimated mass (figure 3). lamina length excluding petiole length was more consistently significant than lamina length with the petiole length included. time for the 2008 growth data, was highly significant for terrace for: plant height (f = 25.4806,36, h-f-l adjusted p = <0.0001); typical axial shoot length (f = 24.6903,18, h-f-l adjusted p = <0.0001); internodes per longest axial shoot (f = 34.7304,24, h-f-l adjusted p = <0.0001); and estimated mass (f = 20.9206,36, h-f-l adjusted p = <0.0001) (table 1). time for the 2008 growth data, was significant for terrace for: longest axial shoot length (f = 40.5704,24, h-f-l adjusted p = 0.0002); axial and lateral shoot number (f = 17.1003,18, h-f-l adjusted p = 0.0002); lamina length (f = 04.4606,30, h-f-l adjusted p = 0.0024); lamina width (f = 06.5806,30, h-f-l adjusted p = 0.0014); lamina width over lamina with petiole length (f = 06.1606,30, h-f-l adjusted p = 0.0025); lamina with petiole length over lamina width (f = 04.1306,30, h-f-l adjusted p = 0.0171); and herbivory coded 0, 1 (f = 13.7506,18, h-f-l adjusted p = 0.0005) (table 1). time for the 2009 growth data, was highly significant for terrace for steer manure for: longest axial shoot length (f = 33.6104,24, h-f-l adjusted p = <0.0001); typical axial shoot length (f = 35.1004,24, h-f-l adjusted p = <0.0001); axial and lateral shoot number (f = 62.6004,24, h-f-l adjusted p = <0.0001); lamina length (f = 12.1304,24, h-f-l adjusted p = <0.0001); lamina with petiole length (f = 11.1504.24, h-f-l adjusted p = <0.0001); lamina width (f = 13.2104,24, h-f-l adjusted p = <0.0001); and herbivory coded both 0, 1 (f = 75.1104,24, h-f-l adjusted p = <0.0001) and 0-4 (f = 273.2404,24, h-f-l adjusted p = <0.0001) (table 2). time for the 2009 growth data was significant for terrace for estimated mass (f = 08.8304,24, h-f-l adjusted p = 0.0190) (table 2). for terrace, consistently significant results for growth through time between years were seen for longest and typical axial shoot length, axial and lateral shoot number up to 5 cm above the soil surface, lamina length without the petiole, lamina width, and herbivory coded 0, 1. time for the 2008 growth data for terrace was unusual for steer manure in that estimated mass was not significant. for all four of the independent measured variables, consistently significant results for growth through time between years was only seen for longest and typical axial shoot length, axial and lateral shoot numbers counted on stems up to 5 cm above the soil surface, lamina length not including petiole length and lamina width. each of these characters has both benefits and issues that might a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 561 limit their usefulness. axial shoot measurements and counts required handling of and/or stretching out of increasingly viney stems. as the season progressed, vines became more rigid and handling them often resulted in breakage, potentially throwing off subsequent measurement of length or shoot numbers. counting shoots in the overlapping vines within 5 cm of the soil surface became difficult as well, as the vines obscured each other, or worse became entangled, with rearrangement risking more breakage.’ on above and below lines. therefore, those characters are best used only in the first few weeks of growth, and at harvest. lamina length and width characters appear very useful. however, they may have little meaning individually or relative to plants at other sites, as leaves of both varieties appear capable of continuous growth, interspersed with plateaus which falsely appeared to be the maximum possible or typical size. several times we thought we knew the top of the range of leaf sizes for the plant, yet under different lighting or fertilizer conditions in the greenhouse, leaves would suddenly begin to grow again, exhibiting dramatic gains in size. some of these sudden and dramatic increases in leaf size only occurred after several years of consistent maximum leaf growth by an individual plant or group of plants in the greenhouse. also, new leaves are constantly emerging all season along the multiple vines that form the crop’s adult habit. for these reasons, leaves typical in size for a plot were measured. the next best character for tracking growth through time was estimated mass, which only lacked significance for terrace and only in 2008. estimated mass is often used as a measurement to track growth in agricultural studies. however, estimated mass can be a highly subjective measurement. even if individual researchers are consistent in how they measure estimated mass, other researchers may differ somewhat in method, making comparison between data sets from different workers less reliable for this character. though the herbivory coded 0, 1 character also gave positive results through time, it did not clearly show a pattern that correlated with the levels of soil amendments. in addition, herbivory coded 0, 1 did not give positive results for coa, even though it did so both years for peat and steer manure separately, as well as for terrace. monitoring herbivory was most useful for paying attention to what fed on the plants. several species of caterpillars voraciously consumed leaves, including hawk moth caterpillars which often clung tightly to m. expansa vines, resisting removal by hand. interaction of time with independent variables: interaction of time with coa for the 2008 growth data was significant for plant height (f = 05.6418,24, h-f-l adjusted p = 0.0110) and estimated mass (f = 04.7418,24, h-f-l adjusted p = 0.0151) (table 1). interaction of time with steer manure for the 2008 growth data was only significant and only for estimated mass (f = 04.3812,30, h-f-l adjusted p = 0.0230). interaction of time with peat and with terrace for the 2008 growth data each had no significance (table 1). interaction of time with coa for the 2009 growth data was only highly significant for estimated mass (f = 21.1612,16, h-f-l adjusted p = 0.0001) (table 2). interaction of time with coa for the 2009 growth data was significant for typical axial shoot length (f = 04.7512,16, h-f-l adjusted p = 0.0023), axial and lateral shoot number (f = 05.6112,16, h-f-l adjusted p = 0.0095) and lamina length (f = 03.5312,16, h-f-l adjusted p = 0.0102) (table 2). interaction of time with peat for the 2009 growth data was also highly significant only for estimated mass (f = 24.4608,20, h-f-l adjusted p = <.0001) (table 2). interaction of time with peat for the 2009 growth data was significant for axial and lateral shoot number (f = 05.3008,20, h-f-l adjusted p = 0.0222), lamina length (f = 04.0208,20, h-f-l adjusted p = 0.0055) and lamina with petiole length (f = 02.7608,20, h-f-l adjusted p = 0.0355) (table 2). interaction of time with steer manure growth data was only significant for estimated mass, for both 2008 (f = 04.3812,30, h-f-l adjusted p = 0.0230) (table 1) and 2009 (f = 08.228,20, h-f-l adjusted p = 0.0023) (table 2). interaction of time with coa, and with steer manure, were only consistently significant for both years for estimated mass. interaction of time with peat was not consistently significant for estimated mass for both years, nor for any measured character. interaction of time with terrace had no significant characters either year (tables 1 and 2). individual growth characters for m. expansa were more meaningful when analyzed through time than when examined in the anova results for treatment, or in the interactions between time and the independent variables. resolution of the general question and two hypotheses for the general question on growing m. expansa across the growing season, the expectation of failure was not met. for the hypothesis on the peat application at the time of construction influencing growth, the null of no effect was rejected. peat in 2008 had a slightly significant effect on herbivory, also a high significance for the interaction of peat with time for herbivory, and slight significance for the interaction of peat with time for the ratio of lamina width over the length of the lamina with the petiole included. peat in 2009 was slightly significant for the interaction of peat with time for both typical axial shoot length and axial shoot number. for estimated mass, peat and peat’s interaction with time were highly significant in 2009. lamina length somewhat, and lamina with petiole length slightly, were significant for the interaction of peat with time for var. ‘l’ in 2009. for the hypothesis that the original steer manure application had an effect on the growth of var. ‘l‘ in the sand plots, the null of no effect was again rejected. the same outcome was seen for coa, though there was no coa hypothesis. steer manure’s interaction with time for estimated mass in 2008, had a slightly significant effect on plant height and a highly significant interaction with time. steer manure in 2009 had a slightly significant effect on estimated mass, and a highly significant effect on the interaction of estimated mass with time. although no hypothesis was written for coa, that data was also analyzed. coa includes var. ‘l’ data from all plots containing only peat, or only steer manure, as well as from the 1p3s plots. coa in 2008 was slightly significant for plant height and extremely highly significant for its interaction with 562 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) time on plant height for var. ‘l’. coa interaction with time in 2009 was also somewhat significant for longest axial shoot length. coa interaction with time was highly significant for estimated mass in 2009. coa in 2009 was somewhat significant for lamina with petiole length. conclusions and recommendations varieties ‘l’ and ‘t’ of m. expansa were shown to have grown in the northern temperate climate of southern illinois even in very wet years. insight was also gleaned from our observations while growing the plants indoors and in the greenhouse, into plant requirements and differences between the ways the two varieties grow. some significant differences were seen for the impact of the original soil treatments, though there were inconsistencies between years, and within and among levels of soil amendment, for different measured characters. harvest data allowed for comparison of the two varieties for weight gain. harvest data also gave information on wet-to-dry ratios above and below ground for each variety in 2009, and between 2008 and 2009 for var. ‘l’. harvest data indicated that var. ‘l’ had larger roots than var. ‘t’. however, var. ‘l’ roots contained a higher percentage of water than those of var. ‘t’. additional differences between the two varieties were in sensitivity to moisture, as well as the size of plants early in the season. the volume of above-ground portions of var. ‘t’ appeared to have caught up with var. ‘l’ towards the end of 2009. var. ‘l’ seemed more tolerant to wet southern illinois conditions and produced more forage from early through mid-season, than var. ‘t’. var. ‘t’ seems to be more drought tolerant than var. ‘l’. these considerations concerning drought and flooding tolerance are based on multiple observations in both field and greenhouse, which varied in degree each year. in addition, by harvest time both years for var. ‘l’, fresh whole plant weights were several times heavier for the upper terrace over the middle terrace, in all-sand control plots. this resulted in very large sds in graphs of both growth and harvest data for var. ‘l’ controls (figures 4 and 5, supplementary figure 4). clones of each variety survived about ten years from the time they were first planted in the greenhouse in southern illinois. this was in a less than hospitable climate for the crop, due to combined extreme heat and humidity in the summers (angel, 2008, 2009, 2010a, 2010b; carbondale illinois airport/ noaa, 2008, 2009, 2010; changnon and black, 2009; daily republican, 2011; kritzer van zant, 2016a appendices b-2, b-3 and b-6). long survival times for serially grown clones are promising for several areas of long term research with the crop. this ability of m. expansa, to be clonally propagated in the field or greenhouse for extended periods, is similar to the ability of some cell types to withstand being passed to fresh media for generations, in that longevity and continuity are valuable traits for any model organism. m. expansa has a high possibility for being reclaimed as a highly valuable crop. this is especially true as the search intensifies for solutions to the impact of climate change upon agriculture. funding is required to help this to happen. four recommendations are given, which address the usefulness of m. expansa as a modern agricultural crop. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 4. chart of 2008 var. ‘l’ harvest weights fresh & >30 days dry with sds, grown in outdoor sand plots. x-axis: treatments: upper terrace and middle terrace, soil amended plot types 5s, 3p, 1p3s and control. y-axis: weights in kgs. abbreviations: 5s, 5% steer manure; 3p, 3% peat; 1p3s, combined organic amendments 1% peat and 3% steer manure; control was all sand. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 563 figure 5. chart of 2009 var. ‘l’ harvest weights fresh & nine days dry with sds, grown in outdoor sand plots. x-axis: treatments: upper terrace and middle terrace, soil amended plot types control, 3p, 1p3s and 5s. y-axis: weights in kgs. abbreviations: control was all sand; 3p, 3% peat; 1p3s, combined organic amendments 1% peat and 3% steer manure; 5s, 5% steer manure. figure 6. chart of 2009 var. ‘t’ harvest weights fresh & nine days dry with sds, grown in outdoor sand plots. x-axis: treatments: upper terrace and middle terrace, soil amended plot types 2p, 2% peat; 4s, 4% steer manure. y-axis: weights in kgs. first, more experiments are needed on a larger scale to address best methods for growing m. expansa. these experiments should additionally give a clearer picture of which growth characteristics should be measured to track growth in the crop. second, research on the crop’s complex genetics, epigenetics and possible ploidy issues should be funded. genetic research may add to the crop’s economic value. mirabilis can provide a fresh window into real time variation in expression on individuals, that is highly localized, and ongoing during the life of the plant. third, nutrition and food-safety research should be funded for use of both m. expansa roots for human food, and the leaves and stems for forage and dry feed. the plant is an excellent source of large amounts of amino acids, including all essential amino acids. the roots and enlarged rhizomes also contain large amounts of minute starch grains. though we will present currently completed research on these topics in future papers, additional investigation is needed. fourth, there are likely better places in north america and elsewhere to grow m. expansa than in southern illinois. areas with sandy and volcanic soils are probably best suited, though these results indicate soils can also be modified for these plants. anywhere the crop can obtain water through stems and leaves above ground or through rhizomes below ground, while the root zone remains dry, has potential for growing the crop. m. expansa is highly endangered, as well as highly nutritious and drought resistant. the crop should be grown at more sites as soon as possible, with monitoring to make sure it does not become invasive in specific ecosystems. m. expansa’s extreme sensitivity to standing water in soils, makes it unlikely to become widely, permanently, invasive in the united states. conflict of interest the authors do not have and do not declare any conflict of interests for the research in this paper. acknowledgements the following individuals and institutions are thanked by the authors for their assistance: s.k. chong; brian klubek; john miller; john preece; tod d. swanson; abraham kritzer; judy robins; andes and amazon field school and center for latin american studies at arizona state university; centro internacional de la papa (cip); herbario nacional quito (qcne); horticulture research center–siu-c; and the united states department of education. s.k. chong originally built the sand plots. john preece, brian klubek and ken diesburg gave advice on what field characters to measure. diesburg also gave permission for kritzer van zant to use the sand plots. john miller and john preece gave advice on growing m. expansa in the greenhouse. rich cole was first to try growing m. expansa from cuttings on a mist bench at siu. john d. reeve and david lightfoot gave advice on the statistical treatments. the lightfoot lab paid for testing of soil samples for their nitrogen content. tod d. swanson, the arizona state university field school in ecuador, as well as a united states department of education fellowship, made it possible for kritzer van zant to a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) travel to ecuador to attend kichwa, taxonomy and ethnobotany classes, the jumping off point for the research which led to finding var. ‘t’. there was no conflict of interest created by that funding. rhizomes were deposited in ecuador and later sent under agreement to siu-c. a set of voucher herbarium specimens for the var. ‘t’ plants, collected in the field in ecuador, were deposited at qcne [kvzant #s 318, 319, and 320]. variety ‘l’ seed was sent to kritzer van zant from centro internacional de la papa in lima, peru. herbario nacional in quito, equador was kritzer van zant’s host institution for collecting m. expansa var. ‘t’ samples. brigida guachuiun and lewis enrique puente transported kritzer van zant to their property to introduce her to eloisa de la cruz, who grew the crop. references angel j (2008, 2009, 2010a) illinois state water survey climate division data (division 8). 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transferase activity in lactobacillus acidophilus. j appl microbiol 103(5):1600-9. downloaded 17 aug 2015 from: http://www.ncbi. nlm.nih.gov/pubmed/17953571. turroni s, sc bendazzoli, scf dipalo, m candela, b vitali, r gotti, and p brigidi (2010) oxalate-degrading activity in bifidobacterium animalis subsp. lactis: impact of acidic conditions on the transcriptional levels of the oxalyl coenzyme a (coa) decarboxylase and formyl-coa transferase genes. appl environ microb 76(16):5609– 5620. downloaded abstract 17 aug 2015 from: http://www.ncbi. nlm.nih.gov/pmc/articles/pmc2918965/. vepachedu r, hp bais, and jm vivanco (2003) molecular characterization and post-transcriptional regulation of me1, a type i ribosome-inactivating protein from mirabilis expansa. planta 217 (3): 498-506. zimmerer ks (1992) the loss and maintenance of native crops in mountain agriculture. geoj 27 (1): 61-72. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 565 566 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) kritzer van zant et al., 2018 – supplementary data summary of supplementary materials supplementary material includes a description of the appearance of m. expansa varieties ‘l’ and ‘t’, details on how the crop was grown in the greenhouse, additional details on field plot conditions including precipitation, information on the lyophilization of harvested material for subsequent nutritional research, positive exploratory data that did not apply directly to hypotheses in this paper, consideration of patterns of negative data, and additional discussion including consideration of harvest data. information included in the supplementary material may be useful to future growers of the crop. description of m. expansa crop varieties crop varieties of m. expansa appear as dense masses of fine vines, cascading outward more or less in a circle. this cascading appearance was less pronounced for both varieties in the short growing season of southern illinois, in comparison with mature var. ‘t’ plants seen by kritzer van zant in ecuador. the wild type’s mature habit is reported as bush-like, in type descriptions, regional keys, and in descriptions on herbarium sheets. var. ‘l’ seed, var. ‘t’ rhizomes, and off-shoots from cuttings of both, in a greenhouse medium without extra fertilizer, emerged into a bush-like, branching habit. in the presence of additional fertilizer, new growth from greenhouse cuttings could emerge as a rosette, later developing the bush-like habit. stems normally develop into vines as the crop matures. both varieties ‘l’ and ‘t’ have ovate or nearly-ovate leaves, and are more stable for leaf shape than wild m. expansa, which frequently has varied leaf shapes. variety ‘l’ usually exhibits dark green foliage and stems, red-purple veins, flowers, and leaf and stem splotches, and its turmeric-colored root epidermis has a slightly sparkling crystalline appearance, at harvest. variety ‘t’ usually exhibits lighter green leaves and stems, with white or creamy light green veins, flowers, and leaf splotches, and a non-crystalline light tan-colored root epidermis. roots of both varieties have light-colored starchy flesh internally. the relatively young age at harvest of southern illinois field grown plants must be considered in matters of color. each variety can temporarily mimic the other for leaf and stem coloration, especially in the earliest stages of growth, no matter the method of propagation. however, red leaf veins on young var. ‘t’ plants are usually not as deeply colored as those on var. ‘l’ plants, and in most cases, red-veined var. ‘t’ plants soon revert to having lighter colored veins as they continue growing. m. expansa is unusual for having both enlarged storage roots and rhizomes. root here refers to true root tissue, free of nodes. rhizomes are enlarged underground stems and therefore have nodes. once enlarged with starch, roots and rhizomes become indistinguishable in appearance, even in cross-section, particularly where they meet in the crown region of mature plants. var. ‘l’ always gained size more quickly and exhibited greater harvest weights than var. ‘t’ in the southern illinois outdoor sand plots. size differences between varieties were less obvious at all stages of growth in the greenhouse. an extended description of m. expansa is in kritzer van zant (2016a), along with information on crop distribution, greenhouse culturing, and cropping rationale for these plants. additional information on growing m. expansa in the field and greenhouse propagation in greenhouses: basic greenhouse media for maintaining stock of m. expansa consisted of equal amounts of perlite, vermiculite, and sand, plus 5-15% peat. additions of 5-10% of the dense organic fertilizer vermifuge, increased moisture in the medium used to start cuttings. vermifuge is denser than mushroom composted horse manure. this may be why it took a larger volume of mushroom composted horse manure to get the same level of moisture retention as from using a smaller volume of vermifuge. with addition of either fertilizer, it was necessary to remove plants from the mist within two weeks of starting them or they rotted away. rotting usually occurred sooner for var. ‘t’ compared with var. ‘l’. root remnants, usually disappeared overnight, when more mature plants became too wet in the greenhouse. it appeared that rapid rotting of roots led to plant death. without using fertilizer, cuttings did not rot so easily but still did better if removed from the mist bench as soon as new leaf buds began to open. cuttings and planting: most stock plants were started from stem cuttings in a base of equal amounts of perlite, vermiculite, and sand, on a mist bench in the greenhouse, without heating pads. once plants had a few weeks off the mist bench, they were transplanted with 15% (v3/v3) peat, added to equal amounts of perlite, vermiculite, and sand. cuttings intended for field planting were started on the mist bench on 26 feb 2008, 10 jan 2009, and 10 apr 2009. the jan 2009 cuttings were started in a mixture of 80% 1:1:1 vermiculite: perlite: sand, with 20% (v3/v3) vermifuge [aka vermicompost], on the mist bench, without heating pads. red worms (eisenia fetida) fed food waste at the vermifuge center at siu-c (http://coas.siu.edu/research/university-farms-forest-research/ sustainability-center/vermicomposting.html), were the source of the vermifuge. for each batch of cuttings, once new leaves appeared, plants were transferred from the mist bench to a dry bench where they received two to three days of daily watering. after that they were watered infrequently, usually no more than twice a week. in 2008, young var. ‘l’ plants grown from cuttings, were planted outdoors on 2 july and no replacements were made through the season. var. ‘t’ plants were first planted outside in 2009. both varieties were planted into the outdoor sand plots in 2009. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) experiment i was planted outside on 14 june 2009. experiment iv was planted outside on 15 june 2009. experiment iv had only var. ‘t’ plants. experiments i and iv were exclusively planted with, and replaced with, apr 2009 cuttings. experiment iv plants were used to obtain all harvest data for var. ‘t’ considered in the body of the paper. april cuttings were started after losing most of the january cuttings, probably the result of overwatering in the greenhouse. fertilizers added to the january starting medium made it necessary to greatly reduce time on the mist bench. plants started in feb 2008 and apr 2009 did not receive vermifuge until they were removed from the mist bench. cuttings planted outside in 2008 had roots that were better established at planting, compared to those of the 2009 transplants. prior to planting the original outdoor plantings in june 2009, cuttings of var. ‘l’ were arranged into three size classes—small, medium, and large. distribution by size was randomized using microsoft excel (2007). april 2009 cuttings had not developed roots sufficiently well to hang on to media when removed from starter pots, during the original planting. this was unlike root development in cuttings for the 2008 season. either or both of two var. ‘t’ survivors from a mostly-failed january start for cuttings, and from april cuttings used as replacements in a failed experiment, may have been included in lyophilized material later submitted for out-sourced amino acid and cytotoxicity assays (kritzer van zant 2016a). details are available for the establishment of other cuttings in 2009 in kritzer van zant (2016a). because of planting discrepancies, statistical analyses were only made with var. ‘l’, considered separately by year, and averaged for each plot. replacement of plants in the field: the first month’s data from experiment i were taken from the original plants, planted on 20 june 2009. the second month, data were taken for surviving plants including 11 july replants on 19 july 2009. no plants of either variety that died after 11 july 2009 were replaced. fifteen of the 44 var. ‘l’ plants originally planted in the field in 2009 were dead by the time the second measurements were taken on 19 july. these numbers include the 32 var. ‘l’ plants in experiment i. the var. ‘l’ plants from experiment i which had died by 19 july 2009 are: 4l (tier i, 1p3s); 6l and 7l (tier ii, 1p3s); 9l, 11l, 12l (tier i, 3p); 15l (tier ii, 3p); 21l, 23l (tier ii, 5s); 25l, 27l, 28l (tier i, control); 30l, 31l, 32l (tier ii, control). by 16 august var. ‘l’ plant 17l (tier i, 5s) from experiment i was also dead. by 18 september the longest surviving var. ‘l’ plant in experiment i, 2l (tier i, 1p3s), was dead. data from the lost plants was excluded statistically, as imbalances would have been created had they been included.’ for line above and two below. first measurements in 2009 were taken 20 june for experiment i, and 21 june for experiment iv. no plants were replaced from the time of the second measurement in july 2009, though losses continued to occur. last measurements on surviving plants were taken in october both years. in 2008 there were no losses in either the upper or middle terrace and the lowest terrace was not planted with m. expansa. in experiment i with all var. ‘l’, by 19 july 2009, plants were discovered to be missing while taking measurements from: the upper terrace—two lost from the control plot, three each from 1p3s and 3s, and one was lost from 5s; the middle terrace—three were lost from the control, one from 3p, and two from 5s. no additional plants were lost from experiment i after 19 july 2009. in experiment iv with only var. ‘t’, in addition to losses discovered 8 july and a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 567 supplementary table 1. 2008 harvest data for var. ‘l’ with averages for controls, amendments, and terraces. terrace treatment whole wet whole dry whole dry/fresh lf/stem dry root dry terrace avg mid-all 2.9 kg 0.4 kg 15.4% 0.4 kg >0.1 kg terrace avg upr-all 4.2 kg 0.7 kg 16.7% 0.6 kg 0.2 kg upr control 3.1 kg 0.5 kg 16.8% 0.4 kg 0.1 kg mid control 0.6 kg 0.1 kg 23.3% 0.1 kg >0.1 kg treatment avg control 1.9 kg 0.3 kg 17.9% 0.3 kg >0.01 kg upr 3p 4.4 kg 0.8 kg 17.5% 0.7 kg 0.1 kg mid 3p 4.1 kg 0.6 kg 15.3% 0.6 kg 0.1 kg treatment avg 3p 4.2 kg 0.7 kg 16.4% 0.6 kg 0.1 kg upr 1p3s 3.6 kg 0.6 kg 15.8% 0.5 kg 0.2 kg mid 1p3s 2.9 kg 0.4 kg 15.1% 0.4 kg <0.1 kg treatment avg 1p3s 3.2 kg 0.5 kg 15.3% 0.4 kg <0.1 kg upr 5s 5.8 kg 1.0 kg 16.9% 0.9 kg 0.8 kg mid 5s 4.0 kg 0.6 kg 14.7% 0.5 kg <0.1 kg treatment avg 5s 4.9 kg 0.8 kg 16.0% 0.7 kg 0.4 kg notes: averaged by treatment, 2 plants averaged, one harvested per plot; averaged by terrace, 4 plants averaged, same individuals as for treatment; harvested roots were washed with sprayer hose, air dried 20 min, weighed, then hung with ropes to dry in barn; numbers = % soil amendment; root—obvious rhizome removed, transition zone retained; weights for wet were taken at time of harvest (16oct2008); weights for dry are from whole plants hung in barn (1dec2008). abbreviations in order of appearance: all, all treatments included; avg, mean average; dry%fresh, dry weight as a percentage of fresh weight; lf/stem, leaf/stem = part of plant growing above soil line; mid, middle terrace; p, peat; root, part of plant growing below soil line; s, steer manure; upr, upper terrace. soon replaced, one more plant was discovered dead on 16 aug from the 4s upper terrace plot. in addition, another plant that had been suspected to be dead in the same plot on 8 july was missing without any remnant by 19 july. that last plant was also not replaced. fertilizer: individual plants, planted outdoors 2 july 2008 and 1 july 2009, received one pint of mushroom composted horse manure at the time of planting. in 2009, the same batch of manure was used to fertilize the plants. however, by 2009 the manure pile had been exposed to the elements and resultant leaching for an additional year. mushroom composted horse manure was added to each plant by first digging a small hole, then stirring the manure into the sand or using a little more sand to cover the manure, then placing cuttings on top. chemical fertilizers were not used. m. expansa has potential as a superfood for the designer health product industry. if the plants can be grown organically, they will have broader appeal for this market. in 2008 leaves of the plants were treated at planting time with a natural latex-based commercial preparation, to reduce damage from pests. latex was applied because some of the young plants lost in 2007 in native soil had been badly damaged from herbivory, suspected to have been the work of small animals. as the latex clogged a sprayer, it was hand applied. as a result, the substance was probably applied too thickly. as applying latex seemed to do more harm than good, it was not used again. fortunately, most plants soon produced other leaves which were not treated, allowing them to survive. nitrogen plots were tested for nitrogen concentrations with samples taken from different plots and terraces in 2011. in 2012, a subset of these samples, from the first terrace, including the plot types for the repeated 2008 and 2009 experiments, were submitted for nitrogen testing to the soil and plant testing laboratory at university of missouri extension (http://soilplantlab.missouri.edu/soil). nitrogen concentrations for different plots varied, though not in any pattern that correlated with plot amendments at the time of construction. no additional samples from these plots were tested further for nitrogen levels. weeds and weeding weedy species inhabiting plots varied sufficiently to show effects, carried over from the original treatments. these differences in species communities, appeared to be independent of whether the original additions of steer manure and peat were still present. weed taxa present in each plot may indicate differences in aggregate structure and/or water-holding capacity of the plots. weeding was done, with hand tools, when data were collected. plants were weeded monthly in 2009 instead of bi-monthly as they had been in 2008. more detail on plot differences can be found in kritzer van zant (2016a) along with information on efforts to plant in native soils from 2007 to 2011. fencing fencing varied from year to year and made little 568 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) supplementary table 2. 2009 harvest data for varieties ‘l’ and ‘t’ with averages for controls, amendments, and terraces. var. terrace treatment whole plant wet whole plant dry dry % fresh herbage dry root dry root lgth dry root diam dry l terrace avg mid-all 0.8 kg 0.3 kg 40.7% 0.3 kg 0.1 kg 28.8 cm 10.4 cm l terrace avg upr-all 1.0 kg 0.4 kg 35.3% 0.3 kg <0.1 kg 31.3 cm 7.0 cm l upr control 0.8 kg <0.1 kg 03.8% <0.1 kg <0.1 kg 26.6 cm 5.6 cm l mid control 0.1 kg 0.1 kg 45.5% 0.03 kg <0.02 kg 21.5 cm 7.1 cm l treatment avg control 0.5 kg <0.1 kg 08.9% 0.02 kg <0.1 kg 24.1 cm 6.4 cm l upr 3p 1.0 kg 0.4 kg 42.3% 0.40 kg <0.1 kg 31.1 cm 7.0 cm l mid 3p 1.1 kg 0.4 kg 41.0% 0.39 kg <0.1 kg 36.7 cm 7.4 cm l treatment avg 3p 1.0 kg 0.4 kg 41.4% 0.39 kg <0.1 kg 33.9 cm 7.2 cm l upr 1p3s 1.6 kg 0.8 kg 47.2% 0.70 kg 0.1 kg 38.0 cm 8.4 cm l mid 1p3s 1.4 kg 0.5 kg 39.4% 0.47 kg 0.1 kg 40.0 cm 8.5 cm l treatment avg 1p3s 1.5 kg 0.7 kg 43.6% 0.58 kg 0.1 kg 39.0 cm 8.5 cm l upr 5s 0.6 kg 0.2 kg 36.5% 0.20 kg <0.1 kg 29.7 cm 6.9 cm l mid 5s 0.7 kg 0.3 kg 40.9% 0.24 kg 0.1 kg 17.0 cm 18.4 cm l treatment avg 5s 0.7 kg 0.3 kg 38.8% 0.22 kg <0.1 kg 23.3 cm 12.7 cm t terrace avg mid-all 0.3 kg 0.1 kg 50.0% 0.11 kg <0.1 kg 21.6 cm 7.0 cm t terrace avg upr-all 0.1 kg <0.1 kg 40.0% 0.02 kg <0.1 kg 15.1 cm 4.4 cm t upr 2p † † † † † † † t mid 2p 0.2 kg 0.1 kg 42.1% 0.07 kg <0.1 kg 21.2 cm 5.6 cm t treatment avg 2p 0.2 kg 0.1 kg 42.1% 0.07 kg <0.1 kg 21.2 cm 5.6 cm t upr 4s 0.1 kg <0.1 kg 40.0% 0.02 kg <0.1 kg 15.1 cm 4.4 cm t mid 4s 0.3 kg 0.2 kg 52.9% 0.15 kg <0.1 kg 21.9 cm 8.3 cm t treatment avg 4s 0.2 kg 0.1 kg 50.0% 0.08 kg <0.1 kg 18.5 cm 6.4 cm notes: averaged by terrace, 4 plants averaged for var. 'l', 2 for var. 't'†, same individuals as for treatment; averaged by treatment, 2 plants averaged†, one harvested per plot; †exception—no var. 't' plants survived past july 8, 2009, so no harvest weight was given for 2% peat upper terrace; both varieties were barn dried to the 9th day after harvest when weighed; harvested roots were washed with sprayer hose, air dried 20 min, weighed, then hung with ropes to dry in barn; lengths and diameters of roots were measured on dry material; numbers = % soil amendment; root—obvious rhizome removed, transition zone retained; var. 'l' harvest data is from experiment i in 2009. var. 't' harvest data is from experiment iv weights for wet taken at time of harvest (19oct2008); weights for dry from whole plants hung in barn (28oct2008). abbreviations in order of appearance: all, all treatments included; avg, mean average; dry%fresh, dry weight as a percentage of fresh weight; leaf/stem, part of plant growing above soil line (lf/stem); mid, middle terrace; p, peat; root, part of plant growing below soil line; s, steer manure; upr, upper terrace. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 569 supplementary figure 1. chart of 2009 var. ‘l’ harvest root size nine days dry with sds, grown in outdoor sand plots. x-axis: treatments: upper terrace and middle terrace, soil amended plot types control, 3p, 1p3s, 5s. y-axis: lengths in cms. abbreviations: control was all sand; 3p, 3% peat; 1p3s, coa 1% peat and 3% steer manure; 5s, 5% steer manure. supplementary figure 2. chart of 2009 var. ‘t’ harvest root size nine days dry with sds, grown in outdoor sand plots. x-axis: treatments: upper terrace and middle terrace, soil amended plot types 2p and 4s. y-axis: lengths in cms. abbreviations: 2p, 2% peat; 4s, 4% steer manure. 570 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) supplementary figure 3. chart of 2008 var. ‘l’ >30 days dry as a % of fresh weight with sds, grown in outdoor sand plots. x-axis: treatments: upper terrace and middle terrace, soil amended plot types control, 5s, 3p, 1p3s, control. y-axis: percent. abbreviations: 5s, 5% steer manure; 3p, 3% peat; 1p3s, combined organic amendments 1% peat and 3% steer manure; control was all sand. supplementary figure 4. chart of 2009 var. ‘l’ nine days dry as a % of fresh weight with sds, grown in outdoor sand plots in 2009. x-axis: treatments: upper terrace and middle terrace, soil amended plot types control, 3p, 1p3s, 5s. y-axis: percents. abbreviations: control was all sand; 3p, 3% peat; 1p3s, combined organic amendments 1% peat and 3% steer manure; 5s, 5% steer manure. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) supplementary figure 5. chart of 2009 var. ‘t’ nine days dry as a % of fresh weight with sds, grown in outdoor sand plots in 2009. x-axis: treatments: upper terrace and middle terrace, soil amended plot types control, 2p, 4s. y-axis: percent. abbreviations: 2p, 2% peat; 4s, 4% steer manure. difference within terraces. plots used in experiment i had been fenced early in the 2008 growing season and remained so in 2009. more details on fencing for these years, as well as information on native soil attempts from 2007 to 2011 are available (kritzer van zant 2016a and 2016a). precipitation both 2008 and 2009 were among the wettest years recorded for southern illinois as of 2009. precipitation data for southern illinois, used to interpret our data, were taken from the illinois state water survey prairie research institute (angel 2008, 2009, 2010a, 2010b). specific data for october from carbondale illinois airport/noaa (2008, 2009, 2010) were also examined. it should be considered that plants were no longer being measured after mid-october. the general precipitation for southern illinois averaged for each month in inches, as given for the 2008—2010 growing seasons from may to october shows: 2008—may 7.28, june 2.55, july 5.68, aug 2.33, sep 3.79, oct 2.22; total for season 23.85. 2009—may 6.81, june 4.57, july 5.59, aug 3.57, sep 3.32, oct 10.59; total for season 34.45. other than rain, water was given to plants outdoors only at the time of planting, if at all. more detailed information on precipitation is in kritzer van zant (2016a and 2016a appendix b-3). this includes summarization of published analyses of the significance of southern illinois weather in the years when m. expansa was planted there (angel 2010a, 2010b; carbondale illinois airport/noaa 2009; changnon and black 2009; daily republican 2011). most final data for 2009 were taken 18 oct. details are in kritzer van zant (2016a and 2016a appendix b-3). axial shoot data was taken 21 oct after that data point was discovered to be missing during computer entry. frost impacted above-ground parts of plants by 18 oct, partially wilting stems and leaves, and causing discoloration. despite the slight frost, final measurements for 2009 are thought to be accurate. lyophilization leaves with attached fine stems were lyophilized for additional testing (kritzer van zant 2016a and 2016a appendix b-3). roots lyophilized for additional testing had attached epidermi and retained connected enlarged stems. one var. ‘l’ plant from each of four of the eight original soil treatments, 5s, 3p 1p3s, and the control, were lyophilized in 2008. harvested and lyophilized var. ‘t’ roots were from 2p and 4s plots in 2009. plants of both varieties were brought into the greenhouses from field plots and transplanted into pots on 1 nov 2009. these plants continued growing until processed for lyophilization. they were repotted from the field at the hrc greenhouse, in media consisting of 40% vermiculite, 40% perlite, 15% peat and 5% vermifuge. at first these plants dropped most of their leaves in the greenhouse. soon, most of them leafed out and appeared to thrive indoors. var. ‘t’ plants numbered t3, t8, and t10, all died in the greenhouse prior to harvest for lyophilization, leaving an above-ground portion in the pot. these three plants were not discarded until checked for root remnants at the time of harvest for lyophilization. when m. expansa dies it is rare to find any remnant of the roots. of these three, only t8 (5s) had intact root remaining. this root remnant was stored in ethanol in a clear glass vial. some of the root epidermis fell away during handling, exposing possible fibers in the transition zone between root and stem. perhaps in the future t8 root remnants can be examined to identify which fungi and/or other micro571 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) supplementary figure 6. terrace plot map horticultural research center (hrc) siu-c, random blocks of 2 m x 4 m plots, repeating on three terraces. each plot received one of eight levels of soil amendment. tier 1 is the highest terrace, closest to the top of the hill, closest to the access road within hrc, and therefore closest to nearly daily human activity. tier 1 is the site of the var. ‘l’ repeat experiments in 2008 and 2009. tier 3 is the lowest terrace along the hillside, most isolated from human activity, though closest to the road adjacent to and outside of hrc. tier 3 is the site of the all var. ‘t’ experiment, from which harvest data for var. ‘t’ was taken. numbers indicate percentage of each soil amendment used for that plot at the time of construction. abbreviations: p, peat; s, steer manure; zero, all-sand control. organisms infected it, and/or whether there are possible fungal allies present. in late winter/spring 2010, specimens were harvested in batches for lyophilization from the plants moved from the field into the greenhouse at the end of the 2009 growing season, beginning 19 feb 2010. methods for lyophilization are given in greater detail in kritzer van zant (2016a). additional descriptions of positive data and strong trends in negative data additional descriptions—anovas 2008: figures of plant height and longest axial shoot length for coa reveal that percent increases for all treatments gradually increased over the all-sand controls (figures 1 and 5; kritzer van zant 2016a). the 1% peat combined with 3% steer manure treatment had the least effect on plant height until it increased to second place at the time of harvest in mid-october. however, 1% peat combined with 3% steer manure retained the lowest increase over the control for longest axial shoot length through the season. anovas for the 2008 growth data, for the independent variables steer manure and terrace were neither highly significant nor significant for any dependent variable (table 1). additional descriptions—anovas 2009: coa for lamina with petiole length exhibited a great deal of inconsistency through the growing season, though the control was lowest until the time of harvest in mid-october (kritzer van zant 2016a). anovas for the 2009 growth data, for the independent variables peat and steer manure, were neither highly significant nor significant for any dependent variable (table 2). the upper terrace consistently had more herbivory coded 0, 1 through the growing season, though only slightly so in midjuly and about 60% more than the middle terrace when differences were greatest. greatest differences occurred at the time of harvest in mid-october (figure 92 in kritzer van zant 2016a). additional descriptions—interaction of time with independent variables 2008: interaction of time with peat, steer manure, and terrace, for the 2008 growth data, had no significance for any dependent variable (table 1). additional descriptions—interaction of time with independent variables 2009: interaction of time with coa for the 2009 growth data was not highly significant for any dependent variable. interaction of time with steer manure, and with terrace, for the 2009 growth data, was neither highly significant nor significant for any independent variable (table 2). discussion and additional points discussion, additional points—cuttings: poorer root development of cuttings at the time of outdoor planting in 2009 compared to 2008 was unsurprising. this is because most of the 2009 transplants were younger at planting time than the 2008 transplants had been. adding vermifuge initially when starting 572 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) cuttings, was sometimes desirable when working with stock plants, as this often produced dense, bonsai-like habits in young plants, and faster initial growth. however, the ability to tolerate the richer and wetter media appears to be relative to overall heat and humidity inside the greenhouse. in 2009, starting stock plants by using vermifuge when initiating cuttings in 2009 seemed to have contributed to higher mortality in cuttings and less root development at the time of field planting, compared to 2008. though replacements plants in 2009 had less time in the field than originally planted individuals, this discrepancy was somewhat cancelled out by the replacements having stronger roots when planted outside than their predecessors earlier in the same year. var. ‘l’ plants in the repeated experiments were not replaced, and no replacements were measured for harvest data for either variety. therefore, replacements had no impact on the anovas, or interpretation of harvest data. discussion, additional points—field growth variables: at least some plants of each variety grew to maturity in at least one of each plot type utilized in both 2007 and 2008. lamina width over lamina with petiole length was significant for all four independent variables in 2008: coa; peat; steer manure; and terrace. the inverse relationship, lamina with petiole length over lamina width, was significant only for terrace, and only in 2008. no other ratio data had significance for any variable. lamina width over lamina with petiole length was not more informative than other characters. for these reasons, all ratios were dropped from analysis in 2009. discussion, additional points—anovas: anovas for growth data for the independent variables steer manure in 2008 and 2009, terrace in 2008, and peat in 2009, had no significance for any dependent variable. see discussion immediately above for consideration of terrace in ratio data. discussion, additional points—interaction of time with independent variables: interaction of time with any independent variable, for the growth data, was consistently not highly significant, and consistently not significant between years, for any combination of independent and dependent variables. interaction of time with the independent variable peat, for the growth data, had no significance for any dependent variable in 2008. interaction of time with either independent variable steer manure or terrace, for the growth data, were not significant for any dependent variable in 2008 or 2009. thus, there was a consistent lack of significance in 2008 and 2009 for the interaction of time with both independent variables steer manure and terrace. discussion, additional points—herbivory: herbivory coded 0, 1 was not significant in 2008, for coa for time. though herbivory coded 0, 1 in 2008 and herbivory coded 0-4 in 2009 were both significant for peat for time, herbivory coded 0, 1 in 2009 had no significance for peat. discussion, additional points—harvest: variety ‘l’ entire plants lost much more water weight during drying in 2008 than in 2009. this occurred, despite less rain across the 2008 growing season. this was true for entire plants from both the middle and upper terraces. there was an inverse relationship of water available in the final month of growth and water lost when var. ‘l’ was dried. extra rain in 2009 may have allowed var. ‘l’ to increase its solid mass even more than its water mass. looking at the individual terraces for entire plant wet and dry weights, upper terrace whole wet plants in 2008 were about 50% heavier than dried whole plants from the middle terrace for plots with the same soil amendments. these differences between 2008 and 2009 var. ‘l’ can be seen for every treatment for dry entire plant weights. for var. ‘l’ averaged entire dry plants, the control, 3p and 1p3s plots were about ¼ of a kilo heavier, and 5s plots were about ½ of a kilo heavier, in 2008 compared to 2009. for averaged entire plants in 2009, 2p plots of var. ‘t’ and 3p plots of var. ‘l’, retained nearly the same percentage of dryto-wet mass in 2009. for averaged entire plants in 2009, 4s plots of var. ‘t’ retained 11% more mass upon drying than 5s plots of var. ‘l’. comparing the all peat plots between varieties in 2009 for the averaged dry weights of the harvested entire plants, 3p grown var. ‘l’ weighed 0.4 kg, compared to 2p grown var. ’t’ which weighed less than 0.1 kg. comparing the all steer manure plots between varieties in 2009 for the averaged dry weights of the harvested entire plants, 5s grown var. ‘l’ weighed 0.3 kg, while 4s grown var. ‘t’ weighed less than ½ of that weight at 0.1 kg. averaged dry weights for leaves and stems for the upper terrace were nearly double those of the middle terrace in 2008. in 2009, averaged weights for dried leaves and stems of var. ‘l’ were again greater for the upper terrace though much less than for 2008. for var. ‘t’ in 2009, the averaged dried leaves and stems weighed less for the upper terrace. this is the opposite of var. ‘l’ in 2009. var. ‘t’ had much lower weights than var. ‘l’ that year. both varieties had considerable weight differences between terraces in 2009 for averaged dried roots. in 2008 and 2009 dry leaf and stem material of each variety weighed more than dry root material with what appears to be a single exception (supplementary table 2). this was true for both terraces and all plot types tested. the seeming exception was for var. ‘l’ for control plots in 2009, which had the same averaged weight for dried leaf and stem as for dried root. individual plots and terraces on average were also higher for dry weights of leaf and stem over root, for both varieties in 2009. in 2008 for var. ‘l’, the heaviest averaged, dried, entire plant, leaf and stem, and root portions, were in the upper terrace relative to the middle terrace. in 2009 var. ‘l’ averaged, dried, entire plant, and dried leaf and stem portions, were both heavier again in the upper terrace. however, the averaged dried root material of variety ‘l’ was heavier in the middle terrace as were the dried averaged entire plant, stem and leaf portion, and root portion, of var. ‘t’ in 2009. therefore, heaviest dry weights by terrace in 2009, were mostly inversed for the two varieties. for var. ‘l’ in 2009 root lengths were longer on the upper terrace though for var. ‘t’ root lengths were longer on the middle terrace. for both varieties in 2009 root diameters were wider on the middle terrace. averaged dried var. ‘l’ material in 2008, by plot type for both the dry entire plant and the dried leaf and stem portions, were heaviest in the 5s plots and lightest in the control plots. averaged dried var. ‘l’ material in 2008 by plot type for the dried root portions was again heaviest in the 5s plots though lightest in both the control plots and 1p3s plots. averaged dried a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 573 var. ‘l’ material in 2009 by plot type for the dry entire plant, leaf and stem portion, and root portion, was heaviest in the 1p3s plots and lightest in the control plots. thus the var. ‘l’ 2008 and 2009 material were somewhat similar in having the lightest dried root material primarily in the control plots. however, there was a lack of resolution for why 1p3s plots shared the lightest averaged weights with the control in 2008 yet had the heaviest averaged weights for the same variety in 2009. examining this in more detail, 1p3s plots in both the upper and middle terraces in 2008, had the lightest dry var. ‘l’ entire plants, leaf and stem portions, and root portions. for the 5s plots for var. ‘l’, whole dry plants, and dry leaf and stem portions, were lighter in both terraces in 2009. however, the 5s plots for var. ‘l’ had root portions which were lighter in the middle terrace in 2008, though lighter in the upper terrace in 2009. averaged dried var. ‘t’ material in 2008 by plot type was heavier for the entire plant, leaf and stem portion, and root portion, in the 4s plots over the 2p plots. this heaviest dry weight data for var. ‘t’ in 2009 was only similar to the var. ‘l’ harvest results for the all peat and all steer manure averaged plot types in 2008, which again contradicted the var. ‘l’ 2009 dry weight results. for var. ‘l’ in 2009 averaged root length was longest in the 1p3s plots and shortest in the 5s plots. for var. ‘l’ in 2009 averaged root diameter was greatest in the 5s plots and least in the control plots. thus, there was an inverse relationship for var. ‘l’ in 2009 between root length and diameter in the 5s plots. for var. ‘t’ in 2009 averaged root length was longest in the 2p plots and shortest in the 4s plots. for var. ‘t’ in 2009 averaged root diameters were greatest in the 4s plots and least in the 2p plots. as the relationship for var. ‘t’ at harvest in 2009 between root length and root diameter were in an inverse relationship between the 2p and 4s plots, and as 5s for var. ‘l’ and 4s for var. ‘t’ were the densest plot types in this study, it appeared that denser, richer soils favored a shorter broader root shape for m. expansa. this may indicate that the shape of m. expansa roots can change along with changes in soil density. harvest data complete tables and additional graphs harvest data tables: there were insufficient harvest data for statistical analysis beyond presenting means from the same plot types and graphs of the data in these charts with error bars representing standard deviations. most of this data are means from two plants unless only one plant was available for a particular plot type. eight figures are based on supplementary tables 1 and 2. three are in the body of the paper (figures 4, 5, and 6) show the relationships in kg between plot types and terraces with measurements taken at harvest time, respectively for var. ‘l’ in 2008 (figure 4), var. ‘l’ in 2009 (figure 5), and var. ‘t’ in 2009 (figure 6). additional figures based on these tables show the relationships for the same harvested plants measured in cm only in 2009, for variety ‘l’ (supplementary figure 1) and var. ‘t’ (supplementary figure 2). charts showing percentage the differences in fresh and dry whole plant weights from these tables, are for var. ‘l’ in 2008 (supplementary figure 3), var. ‘l’ in 2009 (supplementary figure 4) and var. ‘t’ in 2009 (supplementary figure 5). where only one plant was available for harvest from a particular plot type, standard deviations could not be computed in the charts. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 574 atlas journal of biology 2025, pp. 839–845 https://doi.org/10.5147/ajb.260 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) dissecting continental and intra-european genetic structure using chromosome 22 snps from the 1000 genomes project my abdelmajid kassem plant genomics and bioinformatics lab, department of biological and forensic sciences, fayetteville state university, fayetteville, nc 28301, usa. received: may 12, 2025 / accepted: june 21, 2025 __________________________________________________ * corresponding author: mkassem@uncfsu.edu 839 abstract understanding the population structure of global human groups remains fundamental to population genetics and medical genomics. in this study, i analyzed genomic variation from chromosome 22 using publicly available data from the 1000 genomes project, focusing on four populations: yoruba (yri), iberian (ibs), tuscan (tsi), and utah residents of northern/western european ancestry (ceu). using ~50,000 high-quality biallelic snps, i applied principal component analysis (pca), admixture, pairwise fst, multidimensional scaling (mds), and phylogenetic clustering to characterize inter-population relationships. pca revealed a strong continental split between african and european individuals, with minimal separation among the european subgroups. admixture analysis (k = 4) confirmed this pattern, showing consistent european ancestry clusters and distinct divergence from african ancestry. pairwise fst values highlighted low differentiation among the european groups (fst ≈ 0.0016–0.0030) and a much higher divergence from the yri population (fst > 0.137). the site frequency spectrum was dominated by rare variants, in line with recent population expansions. this study demonstrates that even a single chromosome’s snp subset can robustly capture major axes of genetic structure, offering a scalable model for population genomics education and exploratory research. keywords: human population structure, chromosome 22, 1000 genomes project, principal component analysis (pca), admixture, genetic differentiation (fst), population genomics, ancestry inference. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. published in the united states of america https://www.atlas-publishing.org 1. introduction understanding the genetic structure of human populations is central to population genomics, medical genetics, and anthropology. the spatial distribution of genetic variation among human groups reflects complex historical processes, including ancient migrations, geographic isolation, genetic drift, and admixture. continental-scale divergence – such as that between african and non-african populations – is a well-documented outcome of the “out of africa” expansion of anatomically modern humans (tishkoff et al., 2009; pagani et al., 2016). at a finer scale, intra-continental variation – particularly in europe – has revealed subtle but consistent population structure that often mirrors geography (novembre et al., 2008; lao et al., 2008). the availability of large-scale, publicly accessible genomic datasets such as the 1000 genomes project (1kgp) has significantly enhanced the ability to explore human genetic diversity (the 1000 genomes project consortium, 2015). these data sets provide dense, high-quality genotype information across hundreds of individuals from diverse populations, enabling robust analyses of genetic structure and ancestry patterns. such analyses are essential not only for understanding evolutionary and demographic history but also for improving the accuracy of disease association studies and personalized medicine in diverse populations (martin et al., 2017). in this study, i analyze data from four representative populations in the 1000 genomes project: the yoruba population from nigeria (yri), iberians from spain (ibs), tuscans from italy (tsi), and utah residents with northern and western european ancestry (ceu). this population set captures both broad continental divergence (africa vs. europe) and fine-scale european variation. previous research has consistently found a strong separation between african and non-african populations, attributed to the bottleneck effect and genetic drift following the migration out of africa ~60,000 years ago (reich et al., 2009; rosenberg et al., 2002). european populations, while more genetically homogeneous compared to africans, still exhibit discernible north-south and east-west clines in genetic variation (novembre et al., 2008; lao et al., 2008; capocasa et al., 2014). although most population genomic studies analyze the entire genome, chromosome-specific analyses provide a practical alternative for focused studies. chromosome 22 is one of the smallest human autosomes but is gene-rich and has been previously utilized to examine genetic diversity, recombination hotspots, and selective sweeps (mcvean et al., 2009; novembre and ramachandran, 2011). analyzing a representative autosome such as chromosome 22 allows researchers to reduce computational complexity while still capturing informative patterns of genetic structure, especially when using dense single nucleotide polymorphism (snp) data. in this work, i uzed a subset of ~50,000 biallelic snps randomly selected from chromosome 22 to assess population differentiation and structure among yri, ibs, tsi, and ceu samples. i applied a suite of complementary analytical methods including principal component analysis (pca), admixture clustering (alexander et al., 2009), pairwise fst statistics (weir and cockerham, 1984), multidimensional scaling (mds), and phylogenetic clustering. these tools are widely used in human genomics to visualize genetic relationships, infer ancestry proportions, and quantify genetic differentiation (patterson et al., 2006; rosenberg et al., 2002). pca and mds are powerful techniques for reducing high-dimensional genotype data into interpretable axes of variation, often revealing broad population clusters that correlate with geography (novembre et al., 2008). admixture, a maximum-likelihood model-based method, is used to infer the proportion of an individual’s genome originating from different ancestral populations. fst metrics, meanwhile, provide a quantitative measure of genetic differentiation between populations and can help interpret clustering and ancestry patterns in evolutionary terms. i also explored the site frequency spectrum (sfs), which summarizes the distribution of allele frequencies across populations. the sfs is shaped by demographic history and natural selection and often exhibits a strong skew toward rare variants in expanding populations (keinan and clark, 2012). additionally, i used hierarchical clustering to construct a simple phylogenetic representation of the genetic distances among individuals, providing an intuitive visualization of population relationships. this chromosome-focused study has both scientific and practical motivations. scientifically, it contributes to my understanding of continental and subcontinental genetic structure by confirming patterns observed in full-genome studies using a smaller genomic subset. practically, it demonstrates how publicly available data, and lightweight computational pipelines can be used to teach core concepts in population genetics and to explore hypothesis-driven questions in resource-constrained settings. by combining multiple lines of evidence, i aimed to (1) demonstrate clear population structure between african and european populations, (2) identify subtle genetic differentiation within europe, and (3) show how a single-chromosome approach can replicate major population genomic patterns. this work thus serves as a scalable and reproducible framework for both educational and exploratory research in human population genetics. 2. methods 2.1. data source and sample selection i used variant call format (vcf) files from chromosome 22 of the 1000 genomes project phase 3 release (danecek et al., 2011; the 1000 genomes project consortium, 2015), specifically the high-coverage phased genotypes: [all.chr22.phase3_shapeit2_mvncall_integrated_v5b.20130502.genotypes. vcf.gz] sample metadata were obtained from the corresponding 1000 genomes annotation file 20130606_sample_info.txt. from the full dataset, four populations were selected based on geographic and ancestral relevance: ceu (utah residents with northern and western european ancestry), tsi (toscani in italia), ibs (iberian population in spain), yri (yoruba in ibadan, nigeria). a total of 421 individuals were retained after filtering for completeness and ancestry. chromosome 22 was selected due to its relatively small size, high gene density, and frequent use in population genetic studies. its manageable size allows for efficient computational analysis while still capturing relevant evolutionary signals, making it ideal for both educational use and exploratory genomic work (mcvean et al., 2009; novembre and ramachandran, 2011). 2.2. vcf processing and genotype extraction i used bcftools v1.21 to subset the sample population and index the vcf file. the following command was used: [bcftools view -s sample_ids.txt -oz -o chr22_subset.vcf.gz all.chr22*.vcf.gz] this step extracted the four target populations from the full chromosome 22 dataset. the resulting vcf file was then parsed in python using the scikit-allel package (miles et al., 2019). only biallelic snps with no missing genotype calls were retained. to optimize computational performance, a filtered set of 50,050 snps was randomly sampled for downstream analyses including pca and admixture. no ld pruning or hardy-weinberg equilibrium filtering was applied in this analysis to preserve maximal variance for pca and admixture inference. all snps used were biallelic and had complete genotype calls. 2.3. principal component analysis (pca) genotype data were converted to alternate allele count matrices, transposed to a samples × variants format, and passed to scikit-learn’s pca module (pedregosa et al., 2011). the top two principal components were visualized using matplotlib and seaborn. additional pca was performed stratified by gender to explore sex-specific variation. 2.4. site frequency spectrum i computed the site frequency spectrum (sfs) by summing alternate allele counts across all individuals and plotting the distribution using 100 histogram bins. the sfs was used to infer overall patterns of allele frequency, highlighting common vs. rare variant contributions. 840 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) 2.5. admixture analysis the [chr22_subset.vcf.gz] file was converted to plink format using plink --vcf (purcell et al., 2007), followed by admixture analysis with k=4 using a dockerized admixture v1.3.0 container (alexander et al., 2009). crossvalidation error was recorded across k=2 to k=6 to assess optimal clustering resolution, with the minimum cv error observed at k = 4, supporting the choice to emphasize this value in the analysis. ancestry proportions (q matrix) were merged with population and gender metadata and visualized using stacked bar plots. gender-specific ancestry profiles were explored using grouped boxplots and mann–whitney u tests for significance. 2.6. fst calculation pairwise weir and cockerham’s fst statistics (weir and cockerham, 1984) were computed using scikit-allel. the weir_cockerham_fst function was applied across the filtered genotype array for each pair of populations, and results were aggregated to compute average divergence across chromosome 22. the resulting fst matrix was visualized as a heatmap using seaborn. 2.7. multidimensional scaling (mds) pairwise genetic distances between individuals were computed from the alternate allele count matrix using euclidean distance via scipy.spatial.distance. pdist. a 2d mds embedding was computed using scikit-learn’s mds implementation. 2.8. phylogenetic tree construction a hierarchical clustering dendrogram was generated using the same genetic distance matrix. ward’s linkage was used via scipy.cluster.hierarchy.linkage. the tree was visualized with individual sample ids. 2.9. software and environment all analyses were conducted in python 3.11 (https://www.python.org/), using anaconda on macos with m1 pro chip. major packages include: scikitlearn (pedregosa et al., 2011), scikit-allel (miles et al., 2019), numpy (harris et al., 2020), pandas (mckinney, 2010), matplotlib (hunter, 2007), seaborn (waskom, 2021), bcftools (danecek et al., 2011, 2021), plink (purcell et al., 2007), admixture (alexander et al., 2009), and docker for containerized execution (merkel, 2014). all analyses were performed on a standard personal computer (macbook pro) running mac os sonoma 14.4.1. all code and jupyter notebooks (kluyver et al., 2016) are available upon request. 3. results 3.1. principal component analysis (pca) figure 1 displays a principal component analysis of 421 individuals from four populations (ibs, ceu, tsi, yri), based on 50,050 randomly sampled snps from chromosome 22. the first two principal components explain 11.88% and 0.78% of the total variance, respectively. pc1 clearly separates the african (yri) population from the european clusters, while the three european groups—ibs, tsi, and ceu—exhibit substantial overlap, suggesting shared ancestry and limited substructure (patterson et al., 2006). a few outlier individuals may reflect cryptic population substructure or recent admixture. figure 2 overlays gender on the same pca coordinates. no visual evidence of sex-specific clustering was observed, indicating the absence of significant sexbiased allele frequency differences across these autosomal markers. 3.2. site frequency spectrum (sfs) figure 3 presents the site frequency spectrum, which shows a pronounced skew toward low-frequency variants—i.e., snps with rare alternate alleles. this pattern is consistent with expectations from recent population growth and purifying selection acting on deleterious alleles (keinan and clark, 2012). the steep decline from rare to common variants suggests that the sampled snps effectively capture demographic signatures across the studied populations. 841 a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) figure 1. pca of selected 1000 genomes populations (50k snps). figure 2. pca colored by gender. figure 3. a. site frequency spectrum. b. site frequency spectrum by population. 842 3.3. population structure via admixture figure 4 shows ancestry estimates inferred using admixture with k = 4. individuals from the yri population display nearly uniform membership in two distinct ancestry components (clusters 1 and 4), whereas individuals from ceu, ibs, and tsi are primarily composed of clusters 2 and 3. among europeans, tsi and ibs appear more similar to each other than to ceu, reflecting subtle intra-european differentiation. this pattern aligns with known geographic and historical relationships among these groups. figure 5 disaggregates ancestry proportions by gender for each cluster. minor differences between males and females are visible but fall within expected stochastic variation. no consistent or statistically significant sex-specific ancestry patterns were detected, as expected for autosomal loci. 3.4. pairwise genetic differentiation (fst matrix) figure 6 summarize pairwise fst estimates computed using the weir and cockerham method (weir and cockerham, 1984). genetic differentiation among the three european populations (ibs, tsi, ceu) is minimal, with fst values ranging from 0.0016 to 0.0030, indicating high genetic similarity and exa tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) figure 4. admixture ancestry proportions (k=4). figure 5. admixture ancestry proportions by gender (k=4). figure 6. weir & cockerham fst between populations. figure 7. mds of genetic distances. 843 figure 8. phylogenetic tree (hierarchical clustering). a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) a tla s j ou rn al o f bi ol og y i ss n 2 15 891 51 . p ub lis he d by a tla s p ub lis hi ng , l lc (w w w. at la s-p ub lis hi ng .o rg ) tensive gene flow. in contrast, comparisons involving the yri population yield substantially higher fst values (0.137–0.141), reflecting long-term geographic and evolutionary separation from the european groups (the 1000 genomes project consortium, 2015). these results corroborate pca and admixture findings, reinforcing the strong continental divergence between african and european populations and the fine-scale genetic continuity within europe. 3.5. multidimensional scaling (mds) figure 7 displays a two-dimensional embedding of pairwise genetic distances computed via multidimensional scaling. the mds results mirror the pca structure, with yri individuals forming a distinct cluster separated from the european samples. within europe, ceu appears slightly shifted relative to tsi and ibs, which again cluster more closely together. this convergence of results across dimensionality reduction methods enhances confidence in the robustness of the inferred structure. 3.6. phylogenetic tree figure 8 presents a hierarchical clustering dendrogram constructed from pairwise genetic distances. two major branches emerge: one composed entirely of yri individuals and another containing the three european groups. within the european clade, ibs and tsi cluster more closely, while ceu forms a distinct sub-branch. although this method is not strictly phylogenetic in a population-genetic sense, it provides a visual summary of genetic distances shaped by historical demographic processes and geographic separation (reich et al., 2009). 4. discussion this study leveraged publicly available whole-genome data from the 1000 genomes project to examine genetic structure and ancestry across four representative human populations—yri, ibs, tsi, and ceu—using a subset of approximately 50,000 snps from chromosome 22. despite being limited to a single chromosome, this targeted approach provided sufficient resolution to detect both broad continental divergence and subtle intra-european substructure, while also reducing computational demands. 4.1. continental and regional structure principal component analysis (pca) and multidimensional scaling (mds) consistently identified a dominant axis of genetic variation separating african and european populations, consistent with known human migration out of africa (tishkoff et al., 2009; pagani et al., 2016). within europe, populations clustered tightly with slight differentiation, reflecting historically documented gene flow across the continent. fst metrics and hierarchical clustering reinforced these patterns. genetic distances between european populations were minimal (fst > ≈ 0.0016– 0.0030), whereas fst values between yri and any european group were substantially higher (≈0.14), indicating deep evolutionary divergence (rosenberg et al., 2002; reich et al., 2009). notably, tsi and ibs showed the closest affinity (fst = 0.0016), likely reflecting their shared mediterranean ancestry and geographic proximity (capocasa et al., 2014). 4.2. site frequency spectrum and rare variants the site frequency spectrum (sfs) exhibited a characteristic skew toward low-frequency alleles, a hallmark of recent population expansion and purifying selection (keinan and clark, 2012; tennessen et al., 2012). this enrichment of rare variants supports the demographic history inferred from pca and fst metrics and illustrates the utility of random snp sampling in capturing global allele frequency distributions. 4.3. ancestry proportions and admixture admixture analysis at k = 4 revealed clear differentiation between african and european ancestries, with yri individuals displaying almost entirely distinct ancestry components from european populations. these clusters likely reflect deeper genetic structure within west africa (tishkoff and williams, 2002; gurdasani et al., 2015). european samples, particularly tsi and ibs, shared more mixed ancestry profiles, consistent with previously reported subcontinental structure (nelson et al., 2008). the similarity between tsi and ibs observed in both admixture and pca further supports historical gene flow across southern europe and the mediterranean basin (fiorito et al., 2016). the distinctiveness of ceu reflects its northern/western european background, in contrast to the more mediterranean-affiliated southern groups. 4.4. sex-specific analyses analysis of ancestry proportions by gender revealed no significant differences across male and female individuals, confirming the expected uniformity of autosomal inheritance (mathieson et al., 2015). while sex-biased admixture has been detected in studies using uniparentally inherited markers such as mtdna and y-chromosomes (goldberg et al., 2017), such effects were not evident in 844 this autosomal analysis, highlighting the importance of genomic context when interpreting sex-specific genetic patterns. 4.5. implications and limitations this study demonstrates the effectiveness of chromosome-specific snp analysis in identifying both global and regional population structure. the reduced computational burden of using a single autosome, coupled with robust analytical methods, makes this approach suitable for educational purposes, preliminary research, and scalable genomic studies. nonetheless, several limitations merit discussion. first, results derived from chromosome 22 may not fully represent genome-wide patterns, especially those influenced by linkage disequilibrium or selection on other chromosomes. second, while the sample sizes (~100 individuals per population) were sufficient to detect broad trends, more subtle signals (e.g., recent admixture or adaptive introgression) would require larger cohorts or whole-genome data. finally, the study’s focus on only four populations – though geographically informative – limits its applicability to broader global patterns of human genetic diversity. future work should consider expanding the population set, applying genome-wide snp data, and integrating functional annotations to investigate population-specific variants with biomedical relevance. 5. conclusion this study presents a focused analysis of population genetic structure and ancestry using a subset of chromosome 22 snps from the 1000 genomes project. by integrating principal component analysis (pca), admixture clustering, fst statistics, multidimensional scaling (mds), and hierarchical clustering, i uncover both broad continental divergence and fine-scale substructure among european populations. my key findings include: • a clear genetic separation between african (yri) and european (ibs, tsi, ceu) populations. • subtle yet consistent differences between southern (ibs, tsi) and northern/western european (ceu) groups. • a site frequency spectrum dominated by rare variants, consistent with recent population expansions. • no evidence of significant sex-specific differences in ancestry distribution at the autosomal level. these results demonstrate that even a single chromosome can yield robust insights into human population structure when paired with appropriate computational tools and population-genetic models. the analytical pipeline developed here is lightweight, reproducible, and adaptable, making it a valuable resource for exploratory research and population genomics education. future research may build upon this framework by incorporating genomewide data, expanding the number of studied populations, and exploring the functional and biomedical relevance of population-specific genetic variation. such efforts will be essential for improving our understanding of human evolutionary history and for ensuring that genomic research benefits diverse global populations. acknowledgments i gratefully acknowledge the developers and maintainers of the open-source tools and packages used in this study, including bcftools, plink, admixture, docker, and the python libraries scikit-allel, scikit-learn, numpy, pandas, matplotlib, and seaborn. their contributions to the scientific community have made this research possible. i also thank the 1000 genomes project consortium for generating and publicly releasing the high-quality genomic data used in this analysis. their work 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cockerham, c.c. estimating f-statistics for the analysis of population structure. evolution 1984, 38, 6, 1358–1370. https://doi.org/10.2307/2408641. atlas journal of biology 2019, pp. 674–698 doi: 10.5147/ajb.v0i0.196 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a culturally competent phenotypic evaluation/obesity assessment in african and african american populations: pilot study catrina johnson1,2,3*, robert corruccini4, daniel becque5,6, wanki moon7, kola ajuwon8, and david lightfoot1,2 1 genomics core facility, southern illinois university carbondale, il 62901,usa; 2 department of plant, soil & ag systems, southern illinois university carbondale, il 62901, usa; 3 center for health, nutrition & biomedicine p.o. box 1062, park forest, il 60466;usa; 4 department of anthropology, southern illinois university carbondale, il 62901, usa; 5 department of kinesiology, southern illinois university carbondale, il 62901, usa; 6 department of physiology, southern illinois university carbondale, il 62901, usa; 7 department of agribusiness,southern illinois university carbondale, il 62901, usa; 8 department of animal sciences & nutrition, purdue university-elkhart, in 49707, usa. received: january 16, 2019 / accepted: june 23, 2019 __________________________________________________ * corresponding author: johnsoncatrina@gmail.com 674 abstract best practice, movement towards individualized medicine and deployment of effective models that impact the diabetes epidemic and its related precursors like insulin resistance and the metabolic syndrome, requires terminal use of bmi, a biologically meaningless and crude indicator of obesity, in favor of effective and culturally-competent non-relative body composition evaluation of genetically determined adiposity, that untenably compares values among groups. african americans are among the increasingly affected groups for diabetes and possess unique composition variation requiring proper intra-cultural evaluation independent of inter-ethnic eurocentric assumptions that over assesses obesity risk. incorporating use of 4c models to evaluate adiposity and assess risk for diabetic predisposition and onset, provides an effective, unbiased assessment of the cultural components inherent within body composition variation among ethnicity, age and gender. obesity and type 2 diabetes onset and pre-disposition was assessed phenotypically, in creation of a body mass profile among african and african american groups, using 4c model, photography, anthropometry, somatotype and genetic evaluation. environmental, obeseogenic cultural factors were also explored. bmi was not found to be an accurate predictor of adiposity in africans and african americans. west africans and other african americans were found to be an accurate and cultur this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ally competent reference population for african american physiology vs. european. africans and african americans were found to be heavier and less fat and normal weight at higher bmi, attributable to cultural acceptance and more fat free mass. skeletal weights were heavy (6-7lbs) among africans and african americans. african americans had heavier bone density than africans but african bone weight increased the longer they stayed in the u.s. bmi falsely assumed the presence of fat in this population. 70% of body mass was attributable to muscle, confirming the mesomorphic phenotype in these groups. african american women were larger than their male counterparts vs. africans, a sexual dimorphic indicator that may be attributable to the absence of incarcerated black male phenotypic data in this study. keywords: african-americans, west-africans, physiology, obesity, bmi. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) introduction phenotypic evaluation/obesity assessment bmi, a ratio of weight over height, is a key tool used to measure obesity and diabetic predisposition by clinicians and government measurement standards like the nhanes survey. it emanated from 1959 ideal weight tables generated by metropolitan life insurance company that was exclusive to most of the population relative to white, upper class males (loos et al., 2008) and became a mandated shibboleth as a proper measure of adiposity by researchers and publishers by the 1985 nih consensus panel (kuczymarksi and flegal, 2000). what is problematic about this acceptance, is that a standard that fails to represent a proper relative sample or that excludes groups, cannot be used as a universal measure for all groups (harrison, 1985). and the notion of “ideal weight” has been found to be biased and inaccurate as a universal standard (knapp, 1983) across ethnicity, class, gender and age. adipose tissue contains hormones that can upset metabolic homestasis with regards to an increase in insulin release. however, it is not the general bodily presence of adipose tissue that increases susceptibility to diabetes, rather, the location specific adiposity(bjorntorp, 1985). fat patterning is not relatively assumed but genetically determined (wagner and heyward, 2000). adipose tissue located around the waist is correlated with metabolic syndrome, glucose intolerance-a precursor to diabetes, and diabetes onset (bjorntorp, 1985; fox, 2008). body composition is an acceptable and proven method of evaluation of adiposity, and ideal weight (wagner and heyward, 2000) and has a variation across cultures (harrison, 1985). african americans have been shown to have a higher percentage of lean, fat free masses that include heavier skeletal weights, muscle and bone mineral content (bmc), longer extremities, adipose concentrations in the trunk, subscapular, back and lateral areas and low waist to hip ratios (schutte et al., 1984; hortobagyi et al., 1990). the universal body composition model that evaluates body fat percentage, historically was exclusive to african americans relative to the evaluation of white, male cadavers and is therefore not an accurate representation for adiposity in african americans. the relationship between adiposity and weight is weakly correlated (harrison, 1985).adipose tissue, accepted to be the result of energy storage over expenditure, is not correlated with obesity in africans and african americans’ tissue (ebersole et al., 2008). body mass index represents an assumption of adiposity and its equal distribution and has a strong cultural component (kleerokoper et al., 1994) that is different among ethnicity, culture, gender and age (gallagher et al. 1996). bmi is not a useful tool to evaluate adiposity (kaarma et al., 2009; kennedy et al., 2009; smalley et al., 1990) among ethnicities. it is not comparable across ethnic groups (satija, 2016). it has been shown to over-estimate obesity among african americans (aloia et al., 1997; aloia et al., 1998).the 4 c model is a proper tool to evaluate body fat that eliminates bias across ethnicity (mott and et al., 1999; durenberg and durenberg, 2001). 675 genes and variants associated with obesity obesity has a genetic component. the following genes have been affiliated with obesity: 23hnf4a haplotypes in intron 3 7 region,23hnf4a haplotypes in p2 promoter region, adrb2, adrb3, agrp, ankrd26, apoe (snps), cart, cart (mutation), cdkal1 chromosome 6 snp-rs9350270, chromosome 3p26, chromosome 6, e2f3 chromosome-6 snp rs6939190, e2f3 chromosome 6 snp-rs6939190, enpp1, enpp1 (3 allele haplotype), esr 1 absence or variation, fetub, fto, fto (snp-rs11219800, fto intron 1, ghrl, gnpa2, gnpda2 (snp-rs10938397), il-6, iqgap1, kctd, mc4r, mc4r (mutation), mc4r (snp s17782313), mc4r (snp rs12970134), mc4r (snp-rs17782313), mc4r mutation, mc4r(snprs12970134), mc5r, mtch2, mtch2 (snp-rs4752856), multiple rare deleterous variants, myo18b, myo18b, negr1, negr1 (snp-rs2815752), nr0b2, nrob2 (mutation), pcski (variation in), pdss2, pomc, pomc (mutation), pparg, ppargc1b, ptprd, pyy, rela (1kbkb variants), rs2241766(adiponectin), rs23047595, rs2304795, rs2304795, rs2745367(resistin), rs8179071, rs894160, sdc3, sdc3 (snps), sh2b1, sh2b1 (snp-rs7498665), sim1, snp-rs6004901, snp-rs6870962, sstr2, tmem18, ucp1, ucp3 (ahituv et al., 2007; bagwell et al., 2005; barroso, 2005; bouchard et al., 1990; branson et al., 2003; calton and vaisse, 2009; chambers et al., 2008; dong et al., 2003; dong et al., 2005; doumatey, 2009; dubern et al., 2001; farooqi et al., 2003; gallagher et al., 2007; lucas et al., 2011); meyre et al., 2009; norman et al., 1997; paganini‐hill et al., 1981; proctor, 2009; sutton et al., 2005; willer et al., 2009; wing, 2010; zonta et al., 1987; nishigori et al., 2001). obesity genotypes affiliated with african americans hassanein et al. (2010) discovered an association between variants rs3751812 and rs9941349 with bmi in african americans. wing et al. (2010) found that genetic heterogeneity between african americans, hispanic americans and caucasian americans was affiliated with fto intron 1. he also found the ratio of visceral to subcutaneous fat (vsr) to be associated with myo18b, pdss2 and iqgap1. doumatey et al. (2009) found the il-6 was associated to body mass index (bmi), waist hip ratio (whr), and the homeostatic model assessment (homa_ ir) for insulin resistance in african americans. the environmental mode of action being telomere shortening (epel et al., 2004). it affects il-6 (lin et al., 2012). doumatey et al. (2009) also found rs2241766 (adiponectin) to be associated with waist hip ratios and rs2745367(resistin) associated with circulating resistin in african americans and west africans. these groups were also found to regulate adopokines differently. proctor et al. (2009) associated subcutaneous adipose tissue in african americans with e2f3 chromosome 6-snp rs4710930 and rs6939190. bagwell et al. (2005) found that 23hnf4a haplotypes in the p2 promoter region and 3-intron 7 region related significantly to measures of obesity in hispanics and african americans. barroso et al, 2005 found association with risk-raising waist a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 676 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) circumference, waist-hip-ratios, estimated percent body fat, body weight, and bmi>35 to be associated with gene variants rs2304795, rs894160 and rs230475. metabolic syndrome prevalence was associated with rs2304794.gallagher et al, 2004 associated the oprm1 gene to type 2 diabetes susceptibility among african americans. and sutton et al. (2005) discovered a link between chromosome 3p26 and obesity phenotypes in african americans. body mass index (bmi) is diabetesas predicted by body mass index (bmi) alone amongst african americansa problematic assessment? bmi is a key tool used to measure obesity and diabetic predisposition that uses the standards of americans of european descent as a normative. bmi is defined as: a ratio of weight over height squared. it is widely accepted as a modernized height and weight tables that standardizes the measure of body fat using a weight over height ratio. bmi and ethnicity there is variation in bmi among ethnicity. for the same bmi black women have 1% less body fat that white women (evans et al., 2006). currently accepted bmi, when adjusted for race, produced a low sensitivity to fatness (evans et al., 2006). it is different among whites and blacks (evans et al., 2006) and is an imprecise measure of fatness across ethnicity (mills et al., 2007). at the same bmi african american men had lower visceral/belly fat than white and hispanic men (carroll et al., 2008) and controlled for age, had less truncal fat and more skeletal muscle than hispanic men (aleman-mateo et al., 2009).bmi cut off points are necessary to determine metabolic risk among different ethnic groups (carroll et al., 2008). environmental factors like screen time, school commuting and consumption of calorie dense snacks and sweetened drinks is dependent upon bmi and ethnicity (singh et al., 2009). bmi and environmental factors in a study of african americans and west africans, bmi in african americans was associated with insulin resistance (doumatey, 2009).among middle aged and older women, weight gain was affiliated with age (ortega-alonso et al., 2009).variation in ponderosity-body weight relative to height as determined by bmi-23% was attributed to environmental or non-genetic factors (komlos et al., 2009). 48% of variation among rfpi, skinfold thickness was due to environmental effects (hasstedt et al., 1989). at all levels of bmi only 10% of fat cells die and are renewed annually. adult fat cells are established during the childhood environment (spalding, 2008). disadvantaged community has an effect on bmi. it reduces racial disparities in bmi but does not affect bmi over time (ruel et al., 2010). physical activity can diminish the additive effects of the heritability of bmi (mustelin et al., 2009). chronic stress increases cortisol levels and bmi among populations with no college degree (daniel et al., 2006). cultural differences in bmi may be explained by behaviors effecting energy expenditure like television viewing, commuting to school and consumption of fruit juices (singh et al., 2009). bmi was associated with smoking in aa males. bmi and inheritance a child with one or more parents overweight, inherits an increase risk for overweight (danielzik et al., 2002).the bmi of parents affect the offspring (li et al., 2009; robl et al., 2008). heritability of bmi from parents was found to be 79%. among males, physical activity reduced waist circumference and heritability to 78% and females reduced to 56% and 71% with physical activity (mustelin et al., 2009). in an international study bmi heritability was measured at 80-82% (hjelmborg et al., 2008). in an international study of 7 and 10yr olds, bmi heritability was found at 60-74%. the same environmental and genetic factors responsible for variation in bmi caused obesity (haworth et al., 2008). a study by hunt et al (n=38, 759) found evidence that increases in bmi act upon the genotype, increasing the allele frequency of the fto gene (hunt et al., 2008). in dutch families weight class (thin, median, overweight or obese) and bmi are inherited from mothers and fathers. different variants effected change and bmi levels. genetic influences related to bmi levels is 60%. genetic influences related to bmi change is 64%. (ortega-alonso et al., 2009). dong et al. (2005) located chromosomes responsible for genomic imprinting of obesity from parents among europeans. bmi imprinting from the father was on 12q24 and on 10p12. the additive and non-additive genetic effects in african americans on bmi are different from european americans (duncan et al., 2009). a small scale twins study (n=12) revealed genetic factors affecting the body’s tendency to store energy as fat or lean tissue and various determinants of resting energy expenditure. this tendency affected regional fat distribution and abdominal/visceral fat. hasstedt et al., in a study of 774 adults discovered 42.3% of variation in the relative fat pattern index (rfpi)-a ratio of subscapular skinfold thickness to the sum of subscapular and suprailac skinfold thickness, was due to recessive allele inheritance. 9.5% was polygenic and 48.2% attributable to random environmental effects(hasstedt et al. 1989). weight class (thin, median weight, overweight or obese) was found to be strongly related to the bmi of the mother (p=.0001) and the father (p=.02). bmi is not a valid indicator of regional fat distribution (kok et al., 2004).“controlling for bone size, there is considerable variation in density and thus weight of the skeleton in human adults and this variation is correlated with age, sex and race (harrison, 1985).” -bone density is associated with hormonal regulation. leptin levels have an inverse relationship to the regulation of era signaling. increased levels of leptin in animal models was shown to increase bone density (ohlsson, 2000).“an ideal weight cannot be identified at a point in time for a person or person differing from the group or groups on which the table was based (harrison, 1985).” it is not accurate across ethnicity (evans et al., 2006)”; (mills et al., 2007). many studies have disproven it as a reliable measure of adipoisity. bmi not a useful measure bmi cutoffs are not accurate across ethnicity (evans, 2006; mills, 2007). it is insensitive to the variation in body composition across ethnicity (kok, 2004).when compared with measures of skinfolds and body composition, bmi does not correlate with body fat, height or length of extremities (kaarma et al., 2009) and is inaccurate across levels of fatness (freedman and sherry, 2009). bmi is not a biological indicator of body fat distribution (kok,,2004) and should not be used to evaluate obesity prevalence (mcadams et al., 2007). it introduces bias and misclassification (rothman, 2008), overpredicts overweight and underpredicts obesity and should not be used in scientific or clinical research (kennedy et al., 2009). bmi is a poor predictor of fat mass in adolescents where ffm is attributed to variation in bmi (freedman et al., 2005).using dexa, bmi was found to be a measure of weight and not fatness or adiposity (freedman and sherry, 2009). height and weight as absolute values cannot be expressed by bmi because it represents part and not the whole body (kaarma et al., 2009). in meta-analysis it underpredicts excess body fat in half of its study participants(okorodudu et al., 2010). why is it still being used? given this history of quasi-breeding and phenotypic selection caused by slavery and its eight generations of african american commoditization, would the descendants of slaves thusly affected, present a bmi within the same normative range of a european culture that experienced no equivalent episodes of selection? could african americans posses a genetic tendency towards a larger bmi in response to historic selection pressures practiced in the era of slavery? this question is of general cultural significance and is an essential prerequisite towards validation of the “epidemic” of obesity/diabetes amongst blacks. african american physiology body composition and genetics waist circumference is inherited via parental bmi (mustelin et al., 2009). genes determine body fat percentage and leanness (ahituv et al., 2007). body composition and ethnicity body composition is variable across, ethnicity, age and sex and must be adjusted accordingly to determine health risks due to fatness (kok et al., 2004). race adds to prediction of body fat. for the same bmi black women have 1% less body fat that white women (evans et al., 2006). percent body fat is different between black and white (evans et al., 2006). caucasian males have higher body fat than african american men (mills et al., 2007). african american fat increases with age faster than asians and hispanics (mills et al., 2007). white women have higher percent body fat than other races (mills, 2007). at low bmi asian women have the highest percent body fat (mills et al., 2007). at the same bmi and waist circumference, african american men had lower visceral fat than white and hispanic men (carroll et al., 2008). whites and hispanics have more visceral fat than african american women (carroll et al., 2008). visceral fat (adipose) tissue defines metabolic risk in different populations (carroll et al., 2008). different waist circumference (wc) and bmi cut off points are necessary to determine metabolic risk among different ethnic groups (carroll et al., 2008). at the same bmi and age, mexicans have more truncal (derriere) fat and less total appendicular skeletal muscle than african americans (aleman-mateo et al., 2009). body composition and environment using a micro environmental analysis of phenotype shows that waist circumference inherited via parental bmi is subject to reduction by physical exercise (mustelin et al., 2009). a macro phenotypic analysis using environmental systems theory reveals that hot climates encourage tall and lean phenoytpes (walker and hamilton, 2008).cold climates encourage short and round phenotypes (walker and hamilton, 2008). dense populations (i.e. asia, india) encourage petite phenotypes via natural selection and small population density favors the large phenotype (walker and hamilton, 2008). genes and mutations have been associated with obese phenotypes of geographical regions of north america (feitosa et al., 2002), europe (bagwell et al., 2005; branson et al., 2003), japan (chambers et al., 2008), italy (dubern et al., 2001) and cultures like the pima indians (farooqi et al., 2003). genetics genes associated with bmi in the literature included: bmiq1, 7q31; bmiqi, bmiqi (near leptin gene) , 7q32.3;bmiq2, 13q14; bmiq3, 6q23-q25; bmiq4, (variation in ucp2), 11q24; bmiq5, 16p13; bmiq6, 20pterp11.2; bmiq7, 4p15-p14; bmiq8, 10p; bmiq9, (variation in mc3r), 20q; bmiq10; bmiq 11; bmiq12, (variation in pcsk1); bmiq13; bmiq14; bmiq15, (prkca); bmiq16; pparg2 (polymorphism); npc1; adipoq, 10q; xq24; 5q15q21; 2q14.1; 16q12.2; 17q23.2-q25.1; 16p11.2; 18q11; 3q27; mtmr9; npci (rs1805081); maf (rs1424233); gprc5b (proximity variant); mc4r (susceptibility loci); pomc; sh2b1; bdnf; fto; irs1; spry2; mc4r (rs17782313)higher bmi; mc4r (rs17782313); mc4r (rs17782313-c). increased risk of type 2 diabetes; mc4r (variant); fto; htr1b; htr1b; htr1b; ucp2; ucp3; vdr; igf1; il6r; ghsr; ppargc1a lep; cyp19a1; gldn; htr1b (polymorphism); cyp19a1 (polymorphism); htr1b (polymorphism); htr2c (aa, bmi x environment); adipor1 (aa, bmi x environment); igfbp3 (ca, bmi x environment); adipor (ca, bmi x environment); pparg (ca, bmi x environment); pparg (ca, bmi x environment), 8p23-p22; 18q22-q23; 16q22-q23; 16p12; rs243650 (effecting allele t); rs534870 (effecting allele a); rs17782313; c allele of rs17782313; c allele of rs17782313; rs12970134; 6 (rs13212041; 6 (rs6296); 6 (rs4140535); 11 (rs17132534); 11 (rs7110607); 12 (rs4334089); 12 (rs6214); 18rs (17066829); 1 (rs12083537); 1 (rs12083537); a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 677 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 678 3 (rs11929140); 3 (rs2948694); 4 (rs6821591); 7 (rs2278815); 15 (rs1902584); 15 (rs1961177); (rs4140535) for bmi <25, 2529, 30-34, >35)); rs1902584 for bmi, 25, 25-29, 30-34, >35); rs4140535 for bmi <25m 25-29, 30-34, >35); rs1902584 for bmi <25, 25-29, 30-34, >35); x (rs17095676xcigarette smoking) p=.001; 1 (rs6672643xcigarette smoking (current) p=.001; 7 (rs6670xsmoking (pack-years) p=.001; 1 (rs12045862 x physical activity) p=.001; 3 (rs709157 x time spent sitting) p=.001; 3 (rs1175540 x time spent sitting) p=.001; mtmr9; npci (rs1805081); maf (rs1424233); gprc5b (proximity variant); mc4r (susceptibility loci); pomc; sh2b1; bdnf; fto; irs1. new loci/decreasing body fat percentage; decreased irsi expression, impaired metabolic profile, increased visceral to subcutaneous fat ratio, insulin resistance, dyslipidemia, diabetes risk, and coronary artery may be discovered (edwards, 2012; feitosa et al., 2002; hsueh et al., 2001; kilpeläinen et al., 2011; loos et al., 2008; meyre et al., 2009; qi et al., 2008; speliotes et al., 2010; yanagiya et al., 2007). phenotype evaluations subjects signed consent from subjects and approval from our local irb preceded this study. up to 142 randomly selected africans and african american subjects, male and females-ages 18-45 from 4 populations (1-africans in us 10 yrs or more, 2-africans in u.s. to yrs or less, 3-african american, 4-african american gullah); of varying education level (no high school diploma, ged, college degree and graduate degree); socioeconomic status (working class, unemployed, undergraduate and professional students), having parents of west african lineage or both parents african american descent, were recruited from college campuses, community churches, mosques and sporting organizations via newspaper ads, posters, flyers and word of mouth. subjects filled out a questionnaire on lineage, a survey on food preferences and food frequency. phenotypic measurements body mass, bioimpedence analysis (bia) and anthropometrical data were collected by means of an examination in the sports medicine facility located in the student health centers of two college campuses. participants wore a hospital gown with underclothing (barefoot, w/underpants, sans the t-shirt for males, women retaining brassieres) a phenotypic profile was established by a trained clinician of six skin-fold, and nine girth and stature measurements. digital photographs were taken of the participants from the neck down in their underclothing. somatotype profiles were established (i.e. endo, ecto, mesomorphic) from the data collected. measurements were evaluated using an ethnic appropriate 4 component model (swan, ball, athena, 2006) of individual tissue composition that determined fat and fat free masses (water, bone mineral density and residual proteins) adjusted for ethnicity. bioimpedence analysis (aloia et al., 1997, 1998). mass, fat and ffm was measured by a digital bioimpedence analysis scale. output generated a value (+/-.003) for total body and muscle mass (g), total body water (tbw) and fat (%). girth measurements (9) (ross et al., 2003; carter, 2002). girth measurements (cm/mm) was taken using steel anthropometric (rosscraft, white rock, bc, canada) tape from the following positions: biepycondal humerus (relaxed and flexed), forearm, supine waist (abdominal), umbilicus, erect hip (hip/buttocks), biepycondal femur (thigh), flexed calf, foot width. skinfold thickness (6). regional body fat masses was evaluated by skinfold thickness using calipers (harpenden and lange) in the subscapular, suprasinale, abdominal, tricep, thigh and calf areas. stature (ozaslan et al., 2003). stature was evaluated by sliding calipers (campbell 10 & 20/rosscraft), a headblock and a segmometer 4 (rosscraft) in cm to measure standing, sitting and trochanteric heights, hand length, and lengths of the foot, leg (thigh and lower) and hand. somatotyping (1 of 2) (ross et al., 2003; carter, 2002). the heath-carter method was used to generate a somatotype profile ecto, endo or mesophoric) from 10 anthropometric positions: 1. body mass (ross et al., 2003; carter and heath, 1990). from a standing position body mass was recorded from a minimally clothed (hospital gown) subject using a bioimpedence analysis (bia) scale (scale and body composition analyzer, tanita, arlington heights, illinois, usa). values were estimated to nearest 0.1 kg and adjusted for clothing. 2. stretch stature (height) (ross et al., 2003; carter 2002). from a standing position height was estimated using a headsquare (rosscraft) and carpenters retractable tape (lufkin). subject was positioned against a wall, maximally erect with their back, heels and gluteals against the surface. the subjects’ head was oriented along the frankfort plane with the headsquare (rosscraft) resting w/ gentle pressure against the hair onto the vertex. measurements were recorded to the nearest (mm). 3. tricep skinfold (ross et al., 2003; carter 2002). from a standing position and arms at sides, triceps skinfold was taken from a raised vertical section of the back of the tricep between the acromion and olecranion using a skinfold caliper (harpenden). values were estimated to the nearest 0.1 mm. 4. subscapular skinfold (ross et al., 2003; carter 2002). from a standing position the subscapular skinfold was taken from the subject 45 degrees from the scapula, 2cm diagonal from the scapula using a skinfold caliper (harpenden). values were recorded to the nearest 0.1 mm. 5. supraspinale skinfold (ross et al., 2003; carter 2002). from a standing position supraspinale skinfold was taken from the top of the iliac spine on a medial 45 degree line along the anxillary border. a minimal (5-7 cm) skinfold amount was evaluated relative to the subject using a skinfold caliper (harpenden). values was estimated to the nearest 0.1 mm. 6. medial calf skinfold (ross et al., 2003; carter, 2002). 679 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) from a standing position, subject raised the right leg to a 90 degree bent knee position upon a stool. medial calf skinfold was obtained from the maximal girth site girth site on the medial side of the calf using a skinfold caliper (harpenden). values were recorded to the nearest 0.1 mm. 7. biepicondylar breadth of humerus (ross et al., 2003; carter, 2002). from a seated position, the subject raised the right humerus and bent it 90 degrees at the elbow. biepicondylar humerus breadth was recorded from the medial and lateral epicondyles using a sliding bone caliper (campbell 10). diameter values were recorded to the nearest 0.5 mm. 8. biepicondylar breadth of femur (ross et al., 2003; carter, 2002). from a seated position biepicondylar breadth of the femur was evaluated from the subject. the examiner located the medial and lateral epicondyles from a flexed femur, using a small bone caliper (campbell 10). the maximum epicondylar distance was taken and the diameter was recorded to the nearest 0.5 mm. values. 9. flexed arm girth (ross et al., 2003; carter, 2002). flexed arm girth was taken along the subject’s raised, flexed, right arm, bent to a 90 degree position using a flexible steel tape (rosscraft). the maximal flexed value was recorded at the highest peak of the tricep. values were estimated to the nearest mm. 10. tensed calf girth (ross et al., 2003; carter, 2002). from a standing position calf girth was taken from the right calf of the subject using a retractable steel tape (rosscraft). three to four circumference values was taken along the long axis of the lower leg and the highest circumference value recorded to the nearest mm. this profile was further utilized to evaluate the tendency of the participants towards mesomorphy using the heath carter somatoype method. data was plotted on a somatochart and a 2d somatochart was also produced. somatotyping (2 of 2). participants were photographed from the neck down (minimally clothed) with a digital camera (10mp) against a grid pattern to generate a photoscopic somatogram. this data was supplementary to the general somatoyping to further classify and accommodate the evaluation of a potential mixed proportioned participant. adjustments errant assumptions inherent in standardized lean density calculations was adjusted for bf% by ethnicity using, (schutte et al., 1984), for black women, (wagner and heyward, 2000) for black men the following calculations for higher proportions of lean body mass inherent in african/african americans: 4c model: (friedl et al., 1992) where: bf= body fat; db= body density; tbw= total body bone mineral; bm 2c model: (schutte et al., 1994) (black women) where: lean density (ld)=1.113g/ cm3 blacks (schutte et al., 1994) vs.1.100 whites (siri 1956) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 1. fp survey category tables pca analysis of significant interactions. fp survey category tables principal component analysis significant interactions variables signficiance/ variation proportion 17. obesity 92owc 90owself <.0001 .49914 92owc 91nwt <.0001 -0.45198 90owself 91nwt <.0001 -0.77344 90owself fat_p <.0001 .43528 91nwt fat <.-0001 .46351 91nwt age <.0001 -0.43424 fat_p bmi <.0001 .68101 fat_p 89wt <.0001 .64505 note: adapted from (johnson et al., 2019; in press). fp survey significant questions multiple regression dependent variable =body fat % [code]/questions secondar y variables p/f value [brorbtl] were you breast or bottle fed? n/a <.0001 [2friedfs] do you eat fried foods? n/a <.0001 [2a2x] how often (do you eat fried foods? n/a <.0001 [63fsitdown} were you raised having family sit down meals? n/a <.0001 [92owc] if you describe yourself as overweight, were you overweight as a child? n/a <.0001 [26sodalike] do you like soda? weight <.0001 <.0001 [26asodaxwk] if so, how often do you consume soda per week? weight <.0001 <.0001 [13tveat] do you eat while watching t.v. weight <.0001 <.0001 [yrs] how long have you been in the u.s. weight <.0001 <.0001 [24waterdrink] do you drink water regularly? weight <.0001 <.0001 [1ahowmswts] do you eat sweets more than 5 times a week? n/a .02804 [57rsetgff] were you raised eating fast foods? group #1 <.0001 .0092 note: adapted from (johnson et al., 2019; in press). table 2. fp survey significant questions multiple regression (bf%). 680 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) physiology data 4 component model-fat percentages id group 4c.fat (% ) age gender id group 4c.fat (% ) age gender p13 afr1a 23.4 20 m p32 aa 24 23 f p15 afr1 11.7 26 m p34 aa 11.6 21 m p18 afr1 21 32 f p300 aa 30 21 f p17 afr1 34.3 38 m p313 aa 60 20 f p19 afr1 12.7 32 f p325 aa 32 23 f p110 afr1 25.3 28 m p326 aa 29 21 m p111 afr1 17.9 34 m p328 aa 30 25 m p112 afr1 11.7 26 m p329 aa 31.2 19 f p114 afr1 24.2 36 m p332 aa 27.7 22 f p117 afr1 17 25 m p333 aa 25.7 25 f p121 afr1 23 27 m p334 aa 27.2 21 f p130 afr1 31 19 m p36 aa 39 33 f p131 afr1 12.1 24 m p338 aa 25 25 m p135 afr1 36.9 28 m p340 aa 27.8 20 f p136 afr1 23.8 24 m p344 aa 27 20 m p141 afr1 16.3 21 m p345 aa 26 27 f p144 afr1 22 23 m p347 aa 26.8 20 m p146 afr1 11.5 21 m p349 aa 27.8 22 f p148 afr1 17 28 m p350 aa 26.8 25 m p152 afr1 22.8 29 m p351 aa 32.2 45 f p154 afr1 25.1 33 m p356 aac 28 22 m p155 afr1 19.8 29 m p361 aa 16.6 21 m p157 afr1 22.3 37 m 1p3-12 aa 38.9 21 f p162 afr1 18.3 24 m 2p3a-12 aa 25.5 20 f p164 afr1 16.3 23 m 3p3-12 aa 27 43 f 1p1-12 afr1 28.6 37 m 4p3-12 aa 16 22 m 2p1-12 afr1 25.3 27 m 5p3-12 aa 25.4 19 f 3p1-12 afr1 48 27 f 6p3-12 aa 13.6 23 m 1p2-12 afr2b 18.8 18 m 7p3-12 aa 46.4 34 f 2p2-12 afr2 9.9 25 m 9p3-12 aa 17 21 m 3p2-12 afr2 10.6 18 m 10p3-12 aa 31.3 19 f 4p2-12 afr2 31 19 f 11p3-12 aa 41.3 29 m p215 afr2 16.7 21 f 12p3-12 aa 18 20 m p237 afr2 19.8 20 f aafr1= africans in the u.s. 10 years or less bafr2=africans in the u.s 10 years or more caa= non african affiliated black americans p239 afr2 17 19 m p242 afr2 21 28 f p243 afr2 18.8 20 m p253 afr2 24.2 18 m p260 afr2 18 33 f p263 afr2 23 19 f table 3. physiology data . 4 component model fat percentages african & african americans. figure 1. african & african american bone density (db) in (lbs). figure 2. african american bone density (db) in (lbs). 681 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) physiology 4-component model body weight (lbs) id group m.weig ht (lbs) age id group m.weig ht (lbs) age p13 afr1 173.4 20 p32 aa 142 23 p15 afr1 133 26 p34 aa 132.8 21 p18 afr1 166 32 p36 aa 313.8 33 p17 afr1 214.8 38 p300 aa 212 21 p19 afr1 129 32 p313 aa 486 20 p110 afr1 163.6 28 p325 aa 158 23 p111 afr1 153.4 34 p326 aa 200 21 p112 afr1 139.6 26 p328 aa 200 25 p114 afr1 174.2 36 p329 aa 136.8 19 p117 afr1 161 25 p332 aa 190 22 p121 afr1 200 27 p333 aa 165.6 25 p130 afr1 219 19 p334 aa 150.2 21 p131 afr1 145 24 p338 aa 162 25 p135 afr1 164.6 28 p340 aa 215 20 p136 afr1 155 24 p344 aa 176 20 p141 afr1 147.8 21 p345 aa 167 27 p144 afr1 165 23 p347 aa 175 20 p146 afr1 121.5 21 p349 aa 179.2 22 p148 afr1 220 28 p350 aa 170 25 p152 afr1 184 29 p351 aa 218.4 45 p154 afr1 169 33 p356 aa 165 22 p155 afr1 151 29 p361 aa 153.4 21 p157 afr1 120.2 37 1p3-12 aa 170.6 21 p162 afr1 154 24 2p3a-12 aa 143.2 20 p164 afr1 127 23 3p3-12 aa 157 43 1p1-12 afr1 223.8 37 4p3-12 aa 137 22 2p1-12 afr1 165 27 5p3-12 aa 137.8 19 3p1-12 afr1 188 27 6p3-12 aa 188.6 23 1p2-12 afr2 120.8 18 7p3-12 aa 213.4 34 2p2-12 afr2 180.8 25 9p3-12 aa 145 21 3p2-12 afr2 147.8 18 10p3-12 aa 151.4 19 4p2-12 afr2 160 19 11p3-12 aa 248.6 29 p215 afr2 132 21 12p3-12 aa 172 20 p227 afr2 219 19 afr1= africans in the u.s. 10 yr or less afr2= african in the u.s. 10 yrs or more aa= african americans p237 afr2 128.6 20 p239 afr2 130.4 19 p242 afr2 137 28 p243 afr2 160 20 p253 afr2 161.4 18 p260 afr2 136 33 p263 afr2 140 19 table 4. four component model physiology data. weight in (lbs) for africans and african americans. figure 3. african bone density in (lbs). figure 4. african & african american body weight in (lbs). data shows physiological agreement of african americans with reference population of west african. 682 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) physiology 4-component model muscle weight (lbs) & (%) id group muscle (lbs) m. weight (lbs) % id group 4c.muscle (lbs) m.weight (lbs) % p13 afr 126.2 173.4 0.727 p32 aa 101.9 142 0.717 p15 afr 111.6 133 0.839 p34 aa 112.2 132.8 0.844 p18 afr 124.7 166 0.751 p36 aa 181.8 313.8 0.579 p17 afr 134.1 214.8 0.624 p300 aa 117 212 0.55 p19 afr 107.5 129 0.833 p313 aa 184 486 0.378 p110 afr 116.2 163.6 0.710 p325 aa 99.4 158 0.629 p111 afr 119.7 153.4 0.780 p326 aa 135.5 200 0.677 p112 afr 117.3 139.6 0.840 p328 aa 132.8 200 0.664 p114 afr 125.6 174.2 0.721 p329 aa 89.5 136.8 0.654 p117 afr 127 161 0.788 p332 aa 131.4 190 0.691 p121 afr 147.8 200 0.739 p333 aa 117 165.6 0.706 p130 afr 144.1 219 0.657 p334 aa 103.9 150.2 0.691 p131 afr 122.5 145 0.844 p338 aa 114.5 162 0.706 p135 afr 98.9 164.6 0.600 p340 aa 147.5 215 0.686 p136 afr 112.3 155 0.724 p344 aa 130.5 176 0.741 p141 afr 117.7 147.8 0.796 p345 aa 116.9 167 0.7 p144 afr 122.5 165 0.742 p347 aa 121.1 175 0.692 p146 afr 102.5 121.5 0.843 p349 aa 123 179.2 0.686 p148 afr 175.6 220 0.798 p350 aa 118.4 170 0.696 p152 afr 135 184 0.733 p351 aa 140.9 218.4 0.645 p154 afr 120.4 169 0.712 p356 aa 107 165 0.648 p155 afr 115.1 151 0.762 p361 aa 121.7 153.4 0.793 p157 afr 88.8 120.2 0.738 1p3-12 aa 99 170.6 0.580 p162 afr 119.6 154 0.776 2p3a-12 aa 101.2 143.2 0.706 p164 afr 100.7 127 0.792 3p3-12 aa 109.2 157 0.695 1p1-12 afr 152 223.8 0.679 4p3-12 aa 108 137 0.788 2p1-12 afr 116.6 165 0.706 5p3-12 aa 97.6 137.8 0.708 3p1-12 afr 92.8 188 0.493 6p3-12 aa 155 188.6 0.821 1p2-12 afr 93.1 120.8 0.770 7p3-12 aa 109 213.4 0.510 2p2-12 afr 154.9 180.8 0.856 9p3-12 aa 114.2 145 0.787 3p2-12 afr 125.5 147.8 0.849 10p3-12 aa 98.8 151.4 0.652 4p2-12 afr 104.6 160 0.653 11p3-12 aa 138.8 248.6 0.558 p215 afr 104.7 132 0.793 12p3-12 aa 133.9 172 0.778 p216 afr 105 152 0.690 p222 afr 118.7 198 0.599 p227 afr 124.8 219 0.569 p237 afr 98.1 128.6 0.762 p239 afr 102.8 130.4 0.788 p242 afr 103.2 137 0.753 p243 afr 124.5 160 0.778 p253 afr 116.3 161.4 0.720 p260 afr 106.5 136 0.783 p263 afr 101.8 140 0.727 table 5. physiology data. 4 component model muscle weight (lbs) for africans and african americans. figure 5. african & african american body fat composition (%). data shows african american agreement with west african reference population. body fat comprises less than 30 percent of total body mass. figure 6. african & african american muscle compostion (%). both groups showed agreement with mesomorphic phenotype w/ muscle composition in excess of 60% of total body mass. 683 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 6. physiology data. group bmi (%) for africans and african americans. physiology data african and african american group bmi (% ) id group bmi gende r age id group bmi gende r age p13 afr1 21.9 m 20 p32 aa 22.1 f 23 p15 afr1 19.7 m 26 p34 aa 18.9 m 21 p18 afr1 32 f 32 p36 aa 62.8 f 33 p17 afr1 29.4 m 38 p300 aa 27 f 21 p19 afr1 24 f 32 p313 aa 75.4 f 20 p110 afr1 22.6 m 28 p325 aa 28.6 f 23 p111 afr1 22.6 m 34 p326 aa 28 m 21 p112 afr1 23 m 26 p328 aa 37.4 m 25 p114 afr1 25.7 m 36 p329 aa 21 f 19 p117 afr1 21 m 25 p332 aa 32.4 f 22 p121 afr1 27.1 m 27 p333 aa 27.3 f 25 p130 afr1 31.2 m 19 p334 aa 24.7 f 21 p131 afr1 21.8 m 24 p338 aa 22 m 25 p135 afr1 26.9 m 28 p340 aa 32.8 f 20 p136 afr1 42.9 m 24 p344 aa 27 m 20 p141 afr1 23.2 m 21 p345 aa 27 f 27 p144 afr1 24.8 m 23 p347 aa 23 m 20 p146 afr1 21.1 m 21 p349 aa 27.2 f 22 p148 afr1 29.9 m 28 p350 aa 31.9 m 25 p152 afr1 26.5 m 29 p351 aa 42 f 45 p154 afr1 25.6 m 33 p356 aa 28 m 22 p155 afr1 25.2 m 29 p361 aa 23.4 m 21 p157 afr1 22.3 m 37 1p3-12 aa 27.9 f 21 p162 afr1 24.2 m 24 2p3a-12 aa 23 f 20 p164 afr1 20.9 m 23 3p3-12 aa 26.3 f 43 1p1-12 afr1 32.2 m 37 4p3-12 aa 20.6 m 22 2p1-12 afr1 30.9 m 27 5p3-12 aa 20.4 f 19 3p1-12 afr1 30 f 27 6p3-12 aa 26 m 23 1p2-12 afr2 20.5 m 18 7p3-12 aa 39 f 34 2p2-12 afr2 27.7 m 25 9p3-12 aa 20.3 m 21 3p2-12 afr2 21.4 m 18 10p3-12 aa 23.3 f 19 4p2-12 afr2 28.9 f 19 11p3-12 aa 35.6 m 29 p215 afr2 20.4 f 21 12p3-12 aa 22.6 m 20 p216 afr2 16.5 f 18 p227 afr2 34.4 m 19 p237 afr2 22.2 f 20 p239 afr2 19.1 m 19 p242 afr2 20.6 f 28 p243 afr2 30 m 20 p253 afr2 25.3 m 18 p260 afr2 21.2 f 33 p263 afr2 22.6 f 19 figure 7. african body fat percentages. fat mass accounted for less than 30 percent of total body mass in west africans. figure 8. african american body fat percentage. body fat accounted for 30-40 percent of actual body mass in african americans. 684 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 7. significant variables for physiology data. table of s ignificant variables for physiology data one way anova (owa) =afr x aa linear regression (lr) = var1 x var 2 multiple regression (mr) =dependent var x v1, v2, v3, v4, v5…. variable dependent variable p/f value significance data bmi muscle_lbs <.0001 highly significant multiple regression bmi muscle % <.0001 highly significant multiple regression bmi <.0001 highly significant multiple regression bmi bones <.0001 highly significant multiple regression bmi x fat <.0001 highly significant linear regression bone density x bmi <.0001 highly significant linear regression bones muscle_lbs <.0001 highly significant multiple regression bones bmi <.0001 highly significant multiple regression bones intercept <.0001 highly significant multiple regression fat muscle % <.0001 highly significant multiple regression muscle % intercept <.0001 highly significant multiple regression muscle_lbs <.0001 highly significant multiple regression weight bmi <.0001 highly significant multiple regression fat 0.0002 very significant multiple regression fat 0.0003 very significant multiple regression fat 0.0006 very significant multiple regression group 1 (afr) fat 0.0002 very significant multiple regression muscle_lbs intercept 0.0027 significant multiple regression body fat % 0.0039 significant one way anova fat composition bmi 0.006 significant multiple regression group 2 bones 0.0061 significant multiple regression fat muscle_lbs 0.0071 significant multiple regression bmi intercept 0.0072 significant multiple regression bmi intercept 0.009 significant multiple regression bmi fat 0.0097 significant multiple regression fat bmi 0.01 significant multiple regression weight 0.0246 significant one way anova gender fat 0.0366 significant multiple regression group 1 (afr) bones 0.0367 significant multiple regression gender fat 0.0372 significant multiple regression bmi 0.0481 significant one way anova figure 9. african and african americans bmi (%). figure 10. african & african american muscle weights (lbs). table 8. physiology data . 4 component model fat percentages african & african americans. physiology data 4 component model-fat percentages id group 4c.fat (% ) age gender id group 4c.fat (% ) age gender p13 afr1a 23.4 20 m p32 aa 24 23 f p15 afr1 11.7 26 m p34 aa 11.6 21 m p18 afr1 21 32 f p300 aa 30 21 f p17 afr1 34.3 38 m p313 aa 60 20 f p19 afr1 12.7 32 f p325 aa 32 23 f p110 afr1 25.3 28 m p326 aa 29 21 m p111 afr1 17.9 34 m p328 aa 30 25 m p112 afr1 11.7 26 m p329 aa 31.2 19 f p114 afr1 24.2 36 m p332 aa 27.7 22 f p117 afr1 17 25 m p333 aa 25.7 25 f p121 afr1 23 27 m p334 aa 27.2 21 f p130 afr1 31 19 m p36 aa 39 33 f p131 afr1 12.1 24 m p338 aa 25 25 m p135 afr1 36.9 28 m p340 aa 27.8 20 f p136 afr1 23.8 24 m p344 aa 27 20 m p141 afr1 16.3 21 m p345 aa 26 27 f p144 afr1 22 23 m p347 aa 26.8 20 m p146 afr1 11.5 21 m p349 aa 27.8 22 f p148 afr1 17 28 m p350 aa 26.8 25 m p152 afr1 22.8 29 m p351 aa 32.2 45 f p154 afr1 25.1 33 m p356 aac 28 22 m p155 afr1 19.8 29 m p361 aa 16.6 21 m p157 afr1 22.3 37 m 1p3-12 aa 38.9 21 f p162 afr1 18.3 24 m 2p3a-12 aa 25.5 20 f p164 afr1 16.3 23 m 3p3-12 aa 27 43 f 1p1-12 afr1 28.6 37 m 4p3-12 aa 16 22 m 2p1-12 afr1 25.3 27 m 5p3-12 aa 25.4 19 f 3p1-12 afr1 48 27 f 6p3-12 aa 13.6 23 m 1p2-12 afr2b 18.8 18 m 7p3-12 aa 46.4 34 f 2p2-12 afr2 9.9 25 m 9p3-12 aa 17 21 m 3p2-12 afr2 10.6 18 m 10p3-12 aa 31.3 19 f 4p2-12 afr2 31 19 f 11p3-12 aa 41.3 29 m p215 afr2 16.7 21 f 12p3-12 aa 18 20 m p237 afr2 19.8 20 f aafr1= africans in the u.s. 10 years or less bafr2=africans in the u.s 10 years or more caa= non african affiliated black americans p239 afr2 17 19 m p242 afr2 21 28 f p243 afr2 18.8 20 m p253 afr2 24.2 18 m p260 afr2 18 33 f p263 afr2 23 19 f 2c model: wagner et al. {%bf=[(4.858/db)-4.394] x 100 (wagner and heyward, 2000) (black men) additionally, data was adjusted for age and gender. statistical analysis phenotypic data was evaluated among the three groups where: group 1: reference population of african americans-west africans in us less than 10 yrs group two: reference population for african americanswest african in the us 10 years or more group three: african americans statistical analysis was used to determine the significance of the variances among the groups. phenotypic data (mass, heights, breadths, girths, skinfold thicknesses) were used as variables (32). statistical significance was obtained among variables using anova (sas inc. cary, nc). significant variables shown on table 1 were ranked using principal component analysis (sas) and table 2 shows further analysis on a regression curve using multiple regression (sas). self-reported physiology survey response data non/normal weight (selfreported) the majority of africans and african americans described themselves as normal weight. a larger percentage of africans described themselves as normal weight than african americans. about 61% of african americans described themselves as normal weight, 39% did not (table 2). almost 79% of africans described themselves as normal weight, 21% did not. 685 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 9. four component model body measurement data for africans and african americans. african & african american 4-component model physiology data id group fat (% ) muscle (lbs) tbw (% ) bones (lbs) weight (lbs) bmi whr age (yrs) gender p13 afr_tenplus a 23.4 126.2 32 6.6 173.4 21.9 0.77 20 m p15 afr_tenplus 11.7 111.6 48 5.8 133 19.7 0.81 26 m p18 afr_tenplus 21 124.7 34.6 6.4 166 32 0.74 32 f p17 afr_tenplus 34.3 134.1 21 7 214.8 29.4 0.88 38 m p19 afr_tenplus 12.7 107.5 38.3 5.6 129 24 0.77 32 f p110 afr_tenplus 25.3 116.2 33.3 6 163.6 22.6 0.8 28 m p111 afr_tenplus 17.9 119.7 39 6.2 153.4 22.6 0.82 34 m p112 afr_tenplus 11.7 117.3 46.1 6 139.6 23 0.84 26 m p114 afr_tenplus 24.2 125.6 31.7 6.4 174.2 25.7 0.88 36 m p117 afr_tenplus 17 127 32 6 161 21 0.8 25 m p121 afr_tenplus 23 147.8 32 6.2 200 27.1 0.86 27 m p130 afr_tenplus 31 144.1 23.5 7 219 31.2 0.95 19 m p131 afr_tenplus 12.1 122.5 41.1 5 145 21.8 0.79 24 m p135 afr_tenplus 36.9 98.9 27.9 5 164.6 26.9 0.95 28 m p136 afr_tenplus 23.8 112.3 35.8 5 155 42.9 0.83 24 m p141 afr_tenplus 16.3 117.7 41.3 6 147.8 23.2 0.95 21 m p144 afr_tenplus 22 122.5 36.3 6.2 165 24.8 0.8 23 m p146 afr_tenplus 11.5 102.5 47.7 5 121.5 21.1 0.8 21 m p148 afr_tenplus 17 175.6 26.1 7 220 29.9 0.89 28 m p152 afr_tenplus 22.8 135 30.5 7 184 26.5 0.82 29 m p154 afr_tenplus 25.1 120.4 31.9 6.2 169 25.6 0.94 33 m p155 afr_tenplus 19.8 115.1 38.7 6 151 25.2 0.92 29 m p157 afr_tenplus 22.3 88.8 47.1 4.6 120.2 22.3 0.84 37 m p162 afr_tenplus 18.3 119.6 38.7 6.2 154 24.2 0.85 24 m p164 afr_tenplus 16.3 100.7 55 5.6 127 20.9 0.83 23 m 1p1-12 afr_tenplus 28.6 152 51.8 7.8 223.8 32.2 0.9 37 m 2p1-12 afr_tenplus 25.3 116.6 0.36 6.7 165 30.9 85 27 m 3p1-12 afr_tenplus 48 92.8 41.3 5 188 30 0.81 27 f 1p2-12 afr_tenlessb 18.8 93.1 61.1 5 120.8 20.5 0.89 18 m 2p2-12 afr_tenless 9.9 154.9 63 8 180.8 27.7 0.83 25 m 3p2-12 afr_tenless 10.6 125.5 62.5 6.6 147.8 21.4 0.79 18 m 4p2-12 afr_tenless 31 104.6 36.2 5.8 160 28.9 0.83 19 f p215 afr_tenless 16.7 104.7 45.9 5.4 132 20.4 0.73 21 f p216 afr_tenless 27 105 36.8 6 152 16.5 0.76 18 f p222 afr_tenless 37 118.7 32.8 6 198 37 0.79 20 f p227 afr_tenless 40 124.8 20.5 6.6 219 34.4 0.88 19 m p237 afr_tenless 19.8 98.1 45.4 5 128.6 22.2 0.84 20 f p239 afr_tenless 17 102.8 46.3 5.4 130.4 19.1 0.82 19 m p242 afr_tenless 21 103.2 32.1 5 137 20.6 0.78 28 f p243 afr_tenless 18.8 124.5 22.7 5.4 160 30 0.83 20 m p253 afr_tenless 24.2 116.3 34 6 161.4 25.3 0.81 18 m p260 afr_tenless 18 106.5 39.7 5 136 21.2 0.77 33 f p263 afr_tenless 23 101.8 45.7 6 140 22.6 0.78 19 f p32 aac 24 101.9 43.6 6 142 22.1 0.78 23 f p34 aa 11.6 112.2 48.4 5.8 132.8 18.9 0.82 21 m p36 aa 39 181.8 14 9.6 313.8 62.8 0.89 33 f p300 aa 30 117 31 8 212 27 0.8 21 f p313 aa 60 184 24 10.4 486 75.4 0.95 20 f p325 aa 32 99.4 34.1 8 158 28.6 0.93 23 f p326 aa 29 135.5 31.9 6.5 200 28 0.8 21 m p328 aa 30 132.8 48.9 7.2 200 37.4 0.96 25 m p329 aa 31.2 89.5 36.6 4.6 136.8 21 0.86 19 f p332 aa 27.7 131.4 32 6 190 32.4 0.81 22 f p333 aa 25.7 117 32.4 6 165.6 27.3 0.78 25 f p334 aa 27.2 103.9 35.2 5.4 150.2 24.7 0.81 21 f p338 aa 25 114.5 24.1 7 162 22 0.8 25 m p340 aa 27.8 147.5 24.5 7.6 215 32.8 0.85 20 f p344 aa 27 130.5 27 8 176 27 0.81 20 m p345 aa 26 116.9 42 6.7 167 27 0.83 27 f p347 aa 26.8 121.1 34.7 7 175 23 0.82 20 m p349 aa 27.8 123 29.4 6.4 179.2 27.2 0.75 22 f p350 aa 26.8 118.4 32.9 6 170 31.9 0.77 25 m p351 aa 32.2 140.9 22.6 7.2 218.4 42 0.77 45 f p356 aa 28 107 24 7 165 28 0.7 22 m p361 aa 16.6 121.7 39.4 6.2 153.4 23.4 0.89 21 m 1p3-12 aa 38.9 99 47 5.2 170.6 27.9 0.83 21 f 2p3a-12 aa 25.5 101.2 53.4 5.4 143.2 23 0.76 20 f 3p3-12 aa 27 109.2 32.7 5.4 157 26.3 0.76 43 f 4p3-12 aa 16 108 34.7 7.1 137 20.6 0.87 22 m 5p3-12 aa 25.4 97.6 53.1 5.2 137.8 20.4 0.81 19 f 6p3-12 aa 13.6 155 60 8 188.6 26 0.8 23 m 7p3-12 aa 46.4 109 51 5.8 213.4 39 1.06 34 f 9p3-12 aa 17 114.2 35.8 6.1 145 20.3 0.8 21 m 10p3-12 aa 31.3 98.8 50.7 5.2 151.4 23.3 0.75 19 f 11p3-12 aa 41.3 138.8 55 7.2 248.6 35.6 0.98 29 m 12p3-12 aa 18 133.9 38.3 7.1 172 22.6 0.83 20 m 686 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 687 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) overweight (self-reported) among african americans, 61% did not describe themselves as overweight, 39% did. about 81% of africans did not describe themselves as overweight, 16% did (table 1). childhood overweight (self-reported) the majority of african and african americans were not overweight as children. more african americans were overweight as children than africans. about 64% of african americans said they were not overweight as children, 21% couldn’t remember (table 2). just 18% of african americans said they were overweight as children. among african groups, 60% said they were not overweight as children, 32% didn’t remember. just 5% of africans said they were overweight as children (table 2). therefore, there may be a population at risk for adult obesity due to childhood obesity. parents participants were asked if either of their parents were overweight. about 59% of all groups responded that their parents were not overweight. almost 41% of all groups said their parents were overweight. the majority of africans and african americans said their parents were not overweight. african american parents were reported overweight at the same percentage as they were not reported overweight. more african americans reported their parents overweight than africans. among african africans, 52% said their parents were overweight, and the same percentage said their parents were not. about 65% of africans said their parents were not overweight. just 35% of african americans did not have parents who were overweight. therefore, because a majority population of african americans reported parents who were overweight, african americans are at risk of overweight due to parental inheritance of bmi (danielzik et al., 2002; robl et al., 2008). prevention/breast feeding almost all africans were breast fed and not even half of african americans were. among african americans 42% were not breast fed and 45% were. just 12% of african americans couldn’t remember. about 95% of africans were breast fed, 5% were not (table 2). therefore, african americans are were more at risk of childhood and adult obesity due to lack of breastfeeding than africans (dewey, 2003). bone density there was variation in bone density (db) between african participants in the u.s. ten years or more (tenplus) and those who had been in the u.s. 10 years or less (tenless). the former had lighter skeletal weights or bone density and the latter had heavier bone density. african americans had the heaviest bone density (table 3). it seems to suggest a relationship between time in the u.s. and bone density in africans and african americans (figures 1-3; suppl. figures a1 and a2). perhaps there are characteristics of foods grown in american soils that is related to this phenomenon. a future study might examine the relationship between u.s. soil nutrients and its effect on bone mineral density of immigrant populations like africans, over time. african american and african weights vs. gender african americans females weighed more on average than african american males and africans. african males weighed more than african females (tables 6-7). more than 60% of their body mass was attributed to muscle (figures 4-6; suppl. figuress a3-6, a7-14). fat percentage about 72% of african americans were found to be over-fat by the standard of hydrodensitometry (smalley et al) and 58% of africans. however, when adjusted for cultural acceptance, based on the participant’s response of themselves as overweight (39% of aas; 16% of afr), the total was corrected by respondents whose body fat exceeded 31%. the total number of overfat went from 72% african americans to 9% and 58% africans to 2% (figures 1-3; table 7). conclusion this study asked the question: “if bmi is a proper assessment tool to measure obesity among african and african american populations” and it is not. according to the bmi standard 48% of both of these groups would be classified as overweight and obese with african americans comprising 41% of this category and africans comprising even more at 45% (table 8, figures 5, 7-8). 1. bmi falsely assumed the presence of fat. it was assumed that the resultant values for mass in this population correlated with fat. it did not (tables 6-9). 2. a breakdown of values into a 4-component model (body fat percentage, body water, bone density and muscle mass) was more informative towards adiposity (tables 3-8, suppl. tables a1-a2; figures 1-10; suppl. figures a1-34). 3. body fat percentage among the groups was at 24.7% overall and 29.3% for females, 21.8% for males, 28.1 % for african americans and 22.1% for africans. according to the bmi tables these values are all within normal range. the hydrodensitometry standard for body fat percentage is 20% for male and 25% for female (smalley et al., 1990). therefore, 4.3% of aas in the study were over-fat and 1.8% of africans (table 7; figures 5, 7-8). 4. fat free mass value of bone density on average comprised 6lbs for africans and 7lbs for african americans of total body mass value (table 3). 5. fat free mass value of total body water (tbw) comprised 37.8% of body mass value in africans and afri688 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) can americans (suppl. table a1). 6. fat-free mass values of muscle composition were around 70% of body mass value for african and african americans, confirming a mesomorphic somatotype. muscle comprised 123lbs of weight in african americans and 118lbs for africans (tables 5, 7, and figure 10). the bmi standard cannot be used accurately to assess adiposity among all cultural groups only within groups. when applied within the comparison of african americans and their reference population-west africans, there is stern disagreement in the data that this population is 48% overweight and obese. the within group comparison showed the bmi value to be within normal range, w/ 70% of body mass to be explained by fat free muscle composition. this is in agreement with the cultural standard for africans and african american somatotype of mesomorphy, having a large component of body mass comprised of muscle mass (table 6; suppl. figures 18-25). ideal weight the notion of “ideal weight” is biologically meaningless and represents the efforts of persons well placed politically and well published academically. height and weight tables are popular, prevalent and standardized but not objective, biologically meaningless, and 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(2019) supplementary data physiology data 4 component model total body water (tbw) in (lbs) id group 4c.tbw (% ) gender id group 4c.tbw (% ) gender p13 afr1 32 m p32 aa 43.6 f p15 afr1 48 m p34 aa 48.4 m p18 afr1 34.6 f p300 aa 31 f p17 afr1 21 m p313 aa 24 f p19 afr1 38.3 f p325 aa 34.1 f p110 afr1 33.3 m p326 aa 31.9 f p111 afr1 39 m p328 aa 48.9 m p112 afr1 46.1 m p329 aa 36.6 m p114 afr1 31.7 m p332 aa 32 f p117 afr1 32 m p333 aa 32.4 f p121 afr1 32 m p334 aa 35.2 f p130 afr1 23.5 m p36 aa 41 f p131 afr1 41.1 m p338 aa 24.1 m p135 afr1 27.9 m p340 aa 24.5 f p136 afr1 35.8 m p344 aa 27 m p141 afr1 41.3 m p345 aa 42 f p144 afr1 36.3 m p347 aa 34.7 m p146 afr1 47.7 m p349 aa 29.4 f p148 afr1 26.1 m p350 aa 32.9 m p152 afr1 30.5 m p351 aa 22.6 f p154 afr1 31.9 m p356 aa 24 m p155 afr1 38.7 m p361 aa 39.4 m p157 afr1 47.1 m 1p3-12 aa 47 f p162 afr1 38.7 m 2p3a-12 aa 53.4 f p164 afr1 55 m 3p3-12 aa 32.7 f 1p1-12 afr1 51.8 m 4p3-12 aa 34.7 m 2p1-12 afr1 0.36 m 5p3-12 aa 53.1 f 3p1-12 afr1 41.3 f 6p3-12 aa 60 m 1p2-12 afr1 61.1 m 7p3-12 aa 51 f 2p2-12 afr2 63 m 9p3-12 aa 35.8 m 3p2-12 afr2 62.5 m 10p3-12 aa 50.7 f 4p2-12 afr2 36.2 f 11p3-12 aa 55 m p215 afr2 45.9 f 12p3-12 aa 38.3 m p216 afr2 36.8 f p222 afr2 32.8 f p227 afr2 20.5 m p237 afr2 45.4 f p239 afr2 46.3 m p242 afr2 32.1 f p243 afr2 22.7 m p253 afr2 34 m p260 afr2 39.7 f p263 afr2 45.7 f suppl. table a1. four component model total body water (tbw) data in (lbs) for africans and african americans. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 692 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) physiology data african & african american waist hip ratio (whr) in (% ) id group whr age gende r id group whr age gende r p13 afr 0.77 20 m p32 aa 0.78 23 f p15 afr 0.81 26 m p34 aa 0.82 21 m p18 afr 0.74 32 f p300 aa 0.8 33 f p17 afr 0.88 38 m p313 aa 0.95 21 f p19 afr 0.77 32 f p325 aa 0.93 20 f p110 afr 0.8 28 m p326 aa 0.8 23 f p111 afr 0.82 34 m p328 aa 0.96 21 m p112 afr 0.84 26 m p329 aa 0.86 25 m p114 afr 0.88 36 m p332 aa 0.81 19 f p117 afr 0.8 25 m p333 aa 0.78 22 f p121 afr 0.86 27 m p334 aa 0.81 25 f p130 afr 0.95 19 m p36 aa 0.89 21 f p131 afr 0.79 24 m p338 aa 0.8 25 m p135 afr 0.95 28 m p340 aa 0.85 20 f p136 afr 0.83 24 m p344 aa 0.81 20 m p141 afr 0.95 21 m p345 aa 0.83 27 f p144 afr 0.8 23 m p347 aa 0.82 20 m p146 afr 0.8 21 m p349 aa 0.75 22 f p148 afr 0.89 28 m p350 aa 0.77 25 m p152 afr 0.82 29 m p351 aa 0.77 45 f p154 afr 0.94 33 m p356 aa 0.7 22 m p155 afr 0.92 29 m p361 aa 0.89 21 m p157 afr 0.84 37 m 1p3-12 aa 0.83 21 f p162 afr 0.85 24 m 2p3a-12 aa 0.76 20 f p164 afr 0.83 23 m 3p3-12 aa 0.76 43 f 1p1-12 afr 0.9 37 m 4p3-12 aa 0.87 22 m 2p1-12 afr 0.85 27 m 5p3-12 aa 0.81 19 f 3p1-12 afr 0.81 27 f 6p3-12 aa 0.8 23 m 1p2-12 afr 0.89 18 m 7p3-12 aa 1.06 34 f 2p2-12 afr 0.83 25 m 9p3-12 aa 0.8 21 m 3p2-12 afr 0.79 18 m 10p3-12 aa 0.75 19 f 4p2-12 afr 0.83 19 f 11p3-12 aa 0.98 29 m p215 afr 0.73 21 f 12p3-12 aa 0.83 20 m p216 afr 0.76 18 f p222 afr 0.79 20 f p227 afr 0.88 19 m p237 afr 0.84 20 f p239 afr 0.82 19 m p242 afr 0.78 28 f p243 afr 0.83 20 m p253 afr 0.81 18 m p260 afr 0.77 33 f p263 afr 0.78 19 f suppl. table a2. african and african american waist hip ratio (whr). 693 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) suppl. figure a1. african american bone density (db) in (lbs) by gender. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) suppl. figure a2. african bone density (db) in (lbs) by gender. suppl. figure a3. african american body weight in (lbs). suppl. figure a4. african body weight in (lbs). suppl. figure a5. african american body weight (lbs) by gender. suppl. figure a6. african body weight (lbs) by gender. 694 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) suppl. figure a7. african american muscle composition (%). african americans showed agreement with mesomorphic phenotype, having muscle composition in excess of 60% of total body mass. suppl. figure a8. african muscle composition (%).african muscle composition was shown to be 60% and beyond, in agreement with mesomorphic phenotype. suppl. figure a9. african american male and female muscle composition (%). aa muscle composition in males and females were found to agree with the mesomorphic phenotype, accounting for 60% and beyond of total body mass. suppl. figure a10. african muscle composition (%). suppl. figure a11. african american muscle weight (lbs). suppl. figure a12. african american muscle composition (%) by gender. 695 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) suppl. figure a13. african muscle composition (lbs). suppl. figure a14. african muscle weight (lbs) by gender. suppl. figure a15. african and african american total body water (%). suppl. figure a16. african american total body water (%). suppl. figure a17. african total body water (tbw) by (%). suppl. figure a18. african american bmi (%). 696 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) suppl. figure a19. african bmi (%). suppl. figure a20. african & african american bmi (%) by age. suppl. figure a21. african american bmi (%) by gender. suppl. figure a22. african bmi by gender (%). suppl. figure a23. african & african american bmi (%) by age. suppl. figure a24. african american bmi (%) by age. 697 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) suppl. figure a25. african bmi (%) by age. suppl. figure a26. african & african american body weight (lbs) by age. suppl. figure a27. african american body weight (lbs) by age. suppl. figure a28. african american total body water (%) by gender. suppl. figure a29. african total body water (tbw) by gender. suppl. figure a30. african & african american waist hip ratio (whr). 698 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) suppl. figure a31. african american waist hip ratio whr (%). suppl. figure a32. african waist hip ratio (%). suppl. figure a33. african american waist hip ratio -whr (%) by gender. suppl. figure a34. african waist hip ratio (%) by gender. atlas journal of biology 2017, pp. 313–354 doi: 10.5147/ajb.2017.0148 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) proceedings of the third international american moroccan agricultural sciences conference amas conference iii, december 13-16, 2016, ouarzazate, morocco my abdelmajid kassem1*, alan walters2, karen midden2, and khalid meksem2 1 plant genetics, genomics, and biotechnology lab, dept. of biological sciences, fayetteville state university, fayetteville, nc 28301, usa; 2 dept. of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901-4415, usa received: december 16, 2016 / accepted: february 1, 2017 __________________________________________________ * corresponding author: mkassem@uncfsu.edu 313 abstract the international american moroccan agricultural sciences conference (amas conference; www.amas-conference.org) is an international conference organized by the high council of moroccan american scholars and academics (hc-masa; www.hc-masa.org) in collaboration with various universities and research institutes in morocco. the first edition (amas conference i) was organized on march 18-19, 2013 in rabat, morocco; amas conference ii was organized on october 18-20, 2014 in marrakech, morocco; and amas iii was organized on december 13-16, 2016 in ouarzazate, morocco. the current proceedings summarizes abstracts from 62 oral presentations and 100 posters that were presented during amas conference iii. keywords: amas conference, hc-masa, agricultural sciences. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i. session i. date palm i: ecosystem’s present and future, major diseases, and production systems co-chair: mohamed baaziz, professor, cadi ayyad university, marrakech, morocco co-chair: ikram blilou, professor, wageningen university & research, the netherlands 1. date palm adaptative strategies to desert conditions alejandro aragón raygoza, juan caballero, xiao, ting ting, yanming deng, ramona marasco, daniele daffonchio and ikram blilou*. abstract date palm cultivars are among the few plants adapted arid oral presentations abstracts wednesday & thursday december 14 & 15, 2016 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) conditions, however, the molecular mechanisms conferring date palm tolerance remain largely unknown. as the root system is an important agronomic trait, having the proper root system architecture in a given environment is critical to allow plants to survive periods of water and nutrient deficit, and compete effectively for resources. here we provide a detailed analysis of date palm root system architecture from embryogenesis to seedling state. we show that the described gene networks regulating stem cells in the model plants arabidopsis and rice are also present in date palm; we have mapped their expression domains at the cellular resolution and we are currently studying their function in the model plant arabidopsis. using rnaseq technologies, we identified date palm genes differentially expressed in roots and shoots. we also show that date palm uses several adaptive strategies to survive desert conditions, ranging from its mode of germination to traits acquired by different cultivars and to the microbial community colonizing the date palm rhizosphere. 2. date palm root microbiome: the ecological services involved in plant growth, survival and tolerance to abiotic stresses ramona marasco1*, maria mosquiera1, ikram blilou2, and daniele daffonchio1 1 bese, biological and environmental sciences and engineering division, king abdullah university of science and technology (kaust), saudi arabia; 2 wageningen university, the netherlands abstract date palm (phoenix dactylifera l.) is an iconic crop plant of desert environments capable to keep ecological dominance under arid conditions and high temperature, but yet very limited knowledge is available on its root microbiome and its contribution to the water and nutritional acquisition by the plant. under the ongoing climate change, the microbial communities associated with the root system is increasingly recognized as an essential resource for development of a sustainable and environmental friendly modern agriculture. recent studies demonstrated that the beneficial-microbiome naturally associated to all the plants accomplishes essential functions and ecological services complementary to the adaptation features encoded by the host plant, but the undergoing mechanisms remain yet elusive. our purpose is to reveal the dynamics and functions of endophytic bacteria associated with the date palm root system in relation to promoting root growth and conferring abiotic stress tolerance. to unravel the mechanisms used by endophytic bacteria, we took advantage of the model system arabidospis thaliana. we found that the selected endophytic strains dramatically influence the root architecture, by increasing the number of lateral roots and by promoting root hair length allowing a larger surface area for resource acquisition. as result, the effects of bacteria on the root system conferred an ‘adaptive advantage’ to the plants exposed to salt stresses. using a series of reporter and mutants lines of a. thaliana, we show that the observed effect in root architecture is strongly linked to the central signaling pathway of growth regulator auxin. our data suggest that beneficial root-associated bacteria can increase date palm tolerance to stresses by directly reprogramming the root growth developmental program through the activation of the auxin signaling pathways. we believe that the beneficial effect observed in both in model and crops plants will be valuable for improving agriculture production under the continuous changing global environmental conditions. 3. optimizing growth and tolerance of date palm (phoenix dactylifera l.) to drought and fusarium oxysporum f. sp. albedinis by application of arbuscular mycorrhizal fungi abdelilah meddich1*, toshiaki mitsui2, and marouane baslam2 1 département de biologie, laboratoire biotechnologie et physiologie végétale, faculté des sciences semlalia, université cadi ayyad marrakech maroc. 2 department of applied biological chemistry, faculty of agriculture, niigata university, niigata 950-2181, japan. *presenting and corresponding author: a.meddich@uca.ma. †corresponding author: mbaslam@ gs.niigata-u.ac.jp. abstract date palm (phoenix dactylifera l.) is an important agricultural and commercial crop in the north of africa and middle eastern countries of asia. date palm trees could be used for generations to come due to its remarkable nutritional, health and economic value in addition to its esthetic and environmental benefits. every part of the date palm is useful and the importance of the date in human nutrition comes from the acceptable taste and its rich composition of carbohydrates, salts and minerals. during the last decade, date palm plantations were subjected to degradation due to an extensive exploitation and to drastic environmental conditions. furthermore, fusarium wilts (`bayoud´) are economically important soil-borne diseases that result in significant crop losses and damage to natural ecosystems. plant-microbe interactions can be either beneficial or detrimental and a fast and accurate assessment of the surrounding organisms is essential for the plant’s survival. arbuscular mycorrhizal fungi (amf) are a major component of soil biofertility and its use can improve crop resistance to biotic and abiotic stresses. our results revealed that mycorrhizal infection rates were higher and slightly affected by water stress. the inoculation by the consortium aoufous, g. monosporus or glomus clarum increased biomass production of date palm instead of the attacks by the fungal pathogen f. oxysporum, whatever the water regime. amf allowed leaf water parameters to be maintained in f. oxysporum-inoculated plants or not under water-limiting conditions. the mortality rate among the date palm trees infected by f. oxysporum was lower in mycorrhizal plants than nonmycorrhizal plants. results showed that amf decrease the deleterious effect of f. oxysporum on date palm, nevertheless the bioprotection effect against the plant pathogen was dependant on the type of amf species. it therefore seems that the indigenous am fungal community 314 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) “aoufous” take advantage to improve crop resistance to those harsh biotic and abiotic conditions. keywords: oasis ecosystem, drought, fusarium oxysporum fsp. albedinis, mycorrhizal symbioses, date palm, tolerance, bioprotector agents. ii. session ii. abiotic stress and water management co-chair: alan walters, southern illinois university, usa 1. vegetation fire as related to leaf shrinkage and water stress salah eddine essaghi* and mohamed yessef iav hassan ii, madinat al irfane, bp 6202-instituts, 10101 rabat, morocco. abstract leaf shrinkage provides insights into the potential variation of foliar surface area-to-volume ratio (svr), within the same species, when leaf moisture content is changing in response to water deficit. since svr is among the most significant plant flammability features, leaf shrinkage would be a relevant component of fuel hazard assessment through its influence on svr, enhancing—if it is taken into account—thereby the wildfire prediction accuracy. the purpose of this work is, first, to consider the leaf shrinkage by characterizing the plant species towards the shrinkability of their leaves, taking account the possible site effect, to characterize the behavior of shrinkage as a function of moisture content and finally to perform a classification for some dominant mediterranean species based on the shrinkage levels. the assessment of the hierarchical relationships between the dimensional shrinkages is also aimed. leaves and needles of thirteen tree and shrub species were harvested from six different sites in western rif mountains. leaves dimensions and moisture content were measured regularly during a gradual drying at the laboratory. dimensional shrinkages were calculated at each moisture content level. dimensional shrinkages behaved similarly whether in leaf or timber and kept the same reporting relationships between each other. among the species sampled in different sites, site effect is significant only in pinus canariensis and pistacia lentiscus. a classification of the plant species was carried out in three separate classes. generally, shrinkage class of the plant species studied gave an idea on its flammability ranking reported in the literature, implying thus a cause-and-effect relationship between both parameters. keywords: dimensional shrinkage, leaves and needles, foliar svr 2. new invasive pest: integrated pest management strategies of the prickly pear cochineal dactylopius opuntiae rachid bouharroud1*, m. el bouhssini2, m. sbaghi3, s. lhaloui2, m. boujghagh1, k. el fakhouri2, and a. sabraoui2 1 centre régional de la recherche agronomique d’agadir, inra, morocco ; 2 international center for agricultural research in the dry areas (icarda), rabat, morocco; 3 division scientifique inra, rabat, morocco. *presenting author: bouharroud@yahoo. fr. abstract in morocco, the prickly pear cactus opuntiae ficus-indica grows in arid and semi-arid areas where it plays an essential role in the ecological system, preventing desertification and preserving biodiversity. the fruits are consumed as a food and cladodes as cattle feed. however, o. f. indica is subjected to several attacks by pests and diseases. the prickly pear cochineal dactylopius opuntiae (hemiptera: dactylopiidae) (cockerell) has been recently reported in morocco. our aim is to reduce the rapid spread of this devastating pest through the development of an integrated management strategy based on the study of biology of this species in the environmental conditions of morocco the use of natural enemies, biopesticides and resistant/tolerant cultivars. the evaluation of 249 cactus ecotypes (inra-agadir cactetum) is being explored for possible tolerance/ resistance to this cochineal. the goal of our efforts would be the implementation of a national integrated pest management strategy to control and limit the rapid spread of this cochineal to uninfested productive areas of cactus pear in morocco. 3. using subsurface drip irrigation for date palm water use effeciency in oasis areas rqia bourziza*, ali hammani, and ahmed bouaziz iav hassan ii, madinat al irfane. bp 6202-instituts, 10101 rabat, morocco. *presenting author: rqia.bourziza@gmail.com. abstract the subsurface drip irrigation (sdi) can be considered as a recent improvement of irrigation water application. the reason given is that it prevents or in most cases considerably reduces losses by direct evaporation, runoff and deep percolation. due to its high efficiency potential, sdi system was recently introduced in morocco. this paper deals with its application to the young date palm in the tafilalet oases (southeastern of morocco) where an appropriate design and irrigation management of this system has to be proposed to farmers. a better understanding in local conditions of the infiltration process around a buried source, and its impact on plant growth is necessarily required. this study aims to improve the water use efficiency by testing the performances of sdi system, especially in areas where water is a limited source. to reach this objective, an experimental test has been installed on a farm plot in the region of erfoud (errachidia province, southeast morocco) to characterize the respective performances of surface and subsurface drip irrigation on young date palm. the results show an increase in root development and in the number of leaves, as well as a substantial water savings due to lower evaporation losses compared to the classic drip irrigation. the results of this study showed that subsurface 315 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 316 drip irrigation is an efficient technique, which allows sustainable irrigation in oasis areas. keywords: subsurface drip irrigation, evaporation, young date palm, performances, oasis aeas. 4. utility of local vegetable crop populations to mitigate yield responses to climate change alan walters1*, mimouni abdelaziz2, rachid bouharroud2, and ahmed wifaya2 1 dept. plant, soil, and agricultural systems, southern illinois university, carbondale, il usa; 2 institut national de la recherche agronomique (inra), agadir center, morocco. *presenting author: awalters@siu.edu abstract future food security challenges must be met in part by developing agricultural technologies to mitigate plant responses to climate change, while at the same time, essential natural resources need to be conserved so that effective food production activities can be sustained for generations. water is an increasingly limited resource influenced by a changing climate and has a definite influence on the long-term productivity of world agriculture. the utilization of more locally adapted crop germplasm (e.g., landraces) to mitigate the effects of drought due to fluctuating water supplies is a strategy that can be used to cope with these ongoing and future food security challenges. however, new crop variety development is generally non-existent in many developing countries, such as morocco, with seeds typically sourced from developed countries. this dependence is troubling as it creates a myriad of problems, especially for smaller subsistence farmers. the selection of locally adapted vegetable crop populations that could be readily adapted by smallholder farmers is an important step to increase food security in a changing climate. although there are limited ongoing efforts to improve crop growth and productivity in developing countries having harsh, dry climates through new variety development, the absence of sustained vegetable breeding programs in these countries will continue to hinder food production, nutritional health and the resulting food security for generations. 5. identification and characterization of desert plant bacterial endophytes inducing abiotic stress tolerance in arabidopsis thaliana axel de zelicourt1,2*, lukas synek, hanin alzubaidy, rewaa jalal, yakun xie, eleonora rolli, santosh satbhai, wolfgang busch, rene geurts, ton bisseling, maged saad, and heribert hirt2 1 institute of plant sciences paris-saclay (ips2), cnrs, inra, université paris-sud, université d’evry, université paris-diderot, université paris-saclay, bâtiment 630, 91405 0rsay, france; 2 division of biological and environmental sciences and engineering, king abdullah university of science and technology kaust, thuwal, saudi arabia. *presenting author: axel.de-julien-dezelicourt@ips2.universite-paris-saclay.fr. abstract food security is of major importance globally and harvest losses due to abiotic stresses amount to more than 60% of total productivity, making abiotic stress tolerance the main goal of crop improvement worldwide. the ability of a variety of plants to cope with stress conditions depends on their association with rhizosphere microbes and can potentially help increase food production in a sustainable way. however, so far, neither the microbial diversity nor the mechanisms of their beneficial interaction with plants are sufficiently understood. our project darwin21 (http://www.darwin21.net) is to isolate and characterize endophyte microbial strains that can help plants to survive and develop in harsh conditions. from an endophyte bacterial library isolated from desert plant roots of the jizan region in saudi arabia, we have established a screening protocol to select strains that can enhance plant tolerance to salt stress in arabidopsis thaliana. using a number of anatomical and physiological parameters, we identified 37 strains, classified as stprs (stress tolerance promoting rhizobacteria). for example, sa187 confers salt, drought and heat stress tolerance in arabidopsis and enhances yield and biomass of crop plants under desert agriculture conditions. a detailed microscopic analysis revealed that sa187 colonizes both surface and inner tissues of arabidopsis roots and shoots. using biochemical, genetic and transcriptomic approaches, the ethylene pathway was found to be crucial for mediating the abiotic stress tolerance by sa187. these results prove that endophytic bacteria can enhance desert agriculture but may also reveal new strategies for breeding crops for enhanced stress tolerance. iii. session iii. date palm ii: ecosystem’s present and future, major diseases, and production systems co-chair: mohamed baaziz, professor, cadi ayyad university, marrakech, morocco co-chair: ikram blilou, professor, wageningen university & research, the netherlands 4. “bayoud” disease in date palm: current status, management, and perspectives adil essarioui* inra, errachidia, morocco abstract date palm (phoenix dactylifera) is an important food source and commercial perennial crop in the sahara and north africa. “bayoud”, a vascular wilt disease, incited by the soilborne fungus fusarium oxyxporum f. sp. albedinis, has become a serious threat to date production in date palm-growing regions in morocco and algeria. since it first appeared in morocco in the late 19th century, the disease has spread throughout morocco and a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 317 into the western half of algerian sahara. attempts to control the disease using systemic fungicides have been unsuccessful. in morocco, date palm groves possess tremendous genetic variability that is the basis for mass selection and identification of many cultivars that combine resistance and good fruit quality. however, some of the cultivars most highly valued by growers for their quality and productivity are highly susceptible to bayoud and require intensive management to control the disease. the discovery of soils that are suppressive to the disease and the isolation of many antagonists presents opportunities for the use of these antagonists as biocontrol agents, particularly in combination with soil solarization and fumigation, and soil amendments. preliminary results indicate that soil solarization and fumigation in combination have the potential to disinfest soils and create an empty niche suitable for rapid colonization by biocontrol agents introduced with soil amendments to protect date palm from the pathogen. research in this area is still in its infancy and bayoud disease continues to prevent growers in morocco from growing the best-but-susceptible cultivars of date palm. 5. use of arbuscular mycorrhizal fungi and bacteria as new strategy to control bayoud disease in date palm fatima jaiti1*, abdelhi dihazi2, reda meziani3, and khalid naamani4 1 equipe protection, amélioration et écophysiologie végétales, faculté des sciences et techniques errachidia, université my ismail, meknes, morocco; 2 laboratoire de biotechnologie, de la valorisation et de la protection des agroressources, université cadi ayyad, faculté des sciences et techniques, marrakech, morocco; 3 laboratoire national de culture des tissus de palmier dattier, institut national de la recherche agronomique, errachidia, morocco; 4 equipe de biotechnologie, biochimie et protection des plantes, faculté des sciences semlalia, université cadi ayyad marrakech, morocco. *presenting author: fatimajaiti@ yahoo.fr. abstract fusarium oxysporum f. sp. albedinis (foa) is a vascular pathogen that causes drastic reduction in cultivation and expansion of date palm in morocco, leading to a considerable socio-economical impact. the control of foa is difficult because chemical treatments are not effective and the prophylactic methods are not of interest due to the contamination of several palm plantations and to their non-durable impact. therefore, planting resistant cultivars constitutes the only efficient and economic method to control this fusarium wilt ‘‘bayoud’’. a promising approach for minimizing the severity of diseases is based on the induction of systemic resistance using localized pre-treatment with elicitors in order to enhance resistance to pathogen infection. arbuscular mycorrhizal fungi (amf) have been shown to protect date palm seedlings against bayoud disease. treatment with amf reduced disease severity by 8–77% depending on the amf isolate used. in addition, the amf induce change in activities of two defencerelated enzymes (peroxidases and polyphenoloxidases) and amplifies the accumulation of non-constitutive hydroxycinnamic acid derivatives, such as the sinapic derivative i2, known to play a crucial role in resistance of date palm to foa. in the second part of the study, two bacteria, bacillus amyloliquefaciens strain ag1 (ag) and burkholderia cepacia strain cs5 (cs), were examined for their potential to control this disease. both bacterial strains inhibited both growth and sporulation of foa. they released compounds into the culture medium, which resulted into cytological changes in foa’s mycelial structure. one month after inoculation, date palm defense reactions against foa were different depending on the bacterium used, and b. cepacia led to higher accumulation of constitutive caffeoylshikimic acid isomers while b. amyloliquefaciens triggered the induction of new phenolic compounds identified as hydroxycinnamic acid derivatives. peroxidase activity has also been stimulated significantly and varied with the bacterial strain used and with foa inoculation. these results highlight the importance of mycorrhizal fungi and selected bacteria as biocontrol agents to combat bayoud disease and improve date palm culture in infected palm groves. keywords: arbuscular mycorrhizal fungi, bacteria, bayoud, biological control, date palm, fusarium oxysporum f. sp. albedinis, bayoud. 6. transpiration and groundwater uptake of date palm (phoenix dactylifera) in tafilalet oasis, morocco wafae elkhoumsi*, ali hammani, and ahmed bouaziz iav hassan ii, madinat al irfane. bp 6202-instituts, 10101 rabat, morocco. abstract improved water management is one of the main challenges for the sustainability of the oasis ecosystem. indeed, there is an urgent need to improve our knowledge concerning the water use by plants. the quantity of water transpired by plant is an important factor in investigating irrigation control and plant-water relations. in addition, the contribution of groundwater table to plant transpiration can be very important in these areas. the objectives of this research is to study the transpiration and groundwater uptake of date palm under oasis conditions. an experiment was conducted in farmland located in the tafilalet oasis. we have been able to calculate transpiration of date palm by using the granierr’s method for irrigated and non-irrigated date palm. the daily average values of transpiration are aboute 0,5 mm/day to 5 mm/day dépending on the climatic demand. we have also established the relationship between transpiration and groundwater uptake. indeed, during the dry season and without irrigation supply, 50% of date palm transpiration comes from groundwater. this relationship is important for control irrigation and water supply, especially since we found that farmers are using more than twice as much water as is needed to irrigate the date palm. keywords: date palm, water use, transpiration, groundwater uptake, tafilalet oasis. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 318 7. effect of regulated deficit irrigation on date palm productivity and water use efficiency cv majhoul a. sabri1*, a. bouaziz2, a. hammani2, m. kuper2,3, a. douaik1, and m. badraoui1,2 1 institut national de la recherche agronomique, rabat, morocco; 2 institut agronomique et vétérinaire hassan ii, rabat, morocco; cirad, montpellier, france. *presenting author: sabri_ inra2004@yahoo.fr. abstract regulated deficit irrigation is one of the ways to save water in regions with limited water resources, while limiting the impact on crop yields. to test the effectiveness of this approach on the productivity of date palm cv majhoul, an experiment was conducted in tafilalet (morocco) for two consecutive years (march 2012-february 2014). the annual cycle of growth and development of this variety was divided into three distinct periods: the first from november to february, the second from march to june and the last from july to october. during these times, seven water regimes were applied under drip irrigation: farmer regime (t0), 100% (t1), 80% (t2), 60% (t3), 80-100-60% (t4), 150% (t5) and 60-100-80% (t6) etm. the measures focused on the monitoring of meteorological parameters, the water irrigation and the evolution of date yields of majhoul variety. the results at the end of the second year of this trial show that: i. water regime has significantly affected the average yields and water use efficiency, ii. water irrigation needs are, on average, 51 m3/tree/year, varying between 30 and 76 m3/tree/ year, and iii. average date yield and water use efficiency by water regime are 31, 61, 46, 39, 43, 45, and 59 kg of dates/ tree/year and 0.44, 1.20, 1.13, 1.30, 1.07, 0.59, and 1.36 kg of dates/m3, respectively for treatments t0, t1, t2, t3, t4, t5, and t6. thus, the water regime t6 has improved the water use efficiency and the conservation has allowed 14% of water irrigation compared to the t1. the regulated water stress strategy contributes to the rationalization of water and sustainability of oasis. keywords: regulated deficit irrigation, date palm, majhoul, water use efficiency. iv. session iv. students oral presentations co-chair: latifa lefrere, ibn zohr university, morocco co-chair: karen miden, southern illinois university 1. evaluation of olive tree resistance to verticillium wilt imane ben salah1*, abderrahman ouzzine1, mouna lamaoui1, abdelghani chakhchar1, ali mhais1, allal douira2 , abdelmajid moukhli3, abdelkarim filali-maltouf4, ahmed tadlaoui ouafi1, and cherkaoui el modafar1 1 laboratoire de biotechnologie, valorisation et protection des agro-ressources, faculté des sciences et techniques guéliz, université cadi ayyad, marrakech, morocco; 2 laboratoire de botanique et protection des plantes, faculté des sciences, université ibn tofail, kénitra, morocco; 3 ur, amélioration des plantes, inra marrakech, morocco; 4 laboratoire de microbiologie et biologie moléculaire, faculté des sciences, université mohammed v agdal, rabat, morocco. *presenting author: bensalah. imane7@gmail.com abstract verticillium wilt of olive (vwo) caused by verticillium dahliae kleb is currently one of the most devastating diseases affecting olive tree in the world. so far, the use of resistant cultivars is the only effective method to control this vascular disease. the aim of this work was the assessment of symptoms of vwo in 20 olive cultivars from the world collection of olive tree located in tassaoute (marrakech, morocco) and the evaluation of their resistance to verticillium dahliae (vd) using a simple, fast and nondestructive method (twigs inoculated with a conidial solution). resistance was evaluated by assessing symptom severity using a 0–4 rating scale. cultivars were classified by combining the final mean severity (fms) of the disease and the percentage of the area under disease progress curves (audpcp). our results showed a high variability of cultivar’s resistance. four groups were obtained; a group of highly susceptible cultivars (arbequina, aggezi shan, doukar, leccino and picual), group of susceptible cultivars (galegavulga, souidi, amphisis, lagoydhera, moroccan picholine, ifiri, chemlal and zaity), group of moderately resistant cultivars (picholine of languedoc, jemribouch, doebli, hojiblanca and koroneiki) and a group of resistant cultivars (chetoui and wateken). this result was compared with that obtained by the principal component analysis (pca) taking into account all variables: fms, audpcp, defoliation, leaf rolling, dieback, mortality and disease incidence. the pca clustered the 20 cultivars into three classes; susceptible, intermediate and resistant class. this analysis confirmed the previous result however, it provided an objective wider grouping of individuals. therefore, to control the vwo, the four resistant cultivars; wateken, chetoui, picholine of languedoc and jemribouch are recommended. keywords: olive tree, verticillium dahliae, resistant cultivars, twigs, world collection of tassaoute. this work is carried out under the project arimnet “pestolive” supported by the ministry of higher education, scientific research a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 319 and executive training and the project “rhizolive” supported by the hassan ii academy of science and technologies. 2. ethnobotanical study of medicinal flora in the southeast morocco: case of ouarzazat “great taznakht” younesse el ouazzani1*, aziz hasib1, abdelali boulli1, and mustapha bouzaid2 1 environment laboratory and valorization of the agro-resources of the faculty of sciences and techniques of beni mellal, morocco; 2 laboratory of bioprocesses and bio-interface of the faculty of sciences and techniques of beni mellal, morocco. *presenting author: y.elouazzani@yahoo.com. abstract medicinal plants represent a treasure for those who have been used to treat human ailments over many centuries and cure their illnesses differently, turning thir backs to the chemical arsenal of current medicine. this study was carried out in the region of ouarzazat, which is renowned for its botanical biodiversity. we distributed 665 surveys during two months on 46 ethnobotanical fields, these survey sheets were completed by traditional practitioners, herbalists and users of medicinal plants of the community who are living in the town of the “great taznakht” as well as in the different villages and surrounding douars. the statistical treatment of the results obtained from the questionnaires sheets, has allowed us to target the plant species suspected having a caregiver power against various diseases, methods of preparation and more. keywords: ethnobotanical survey, medicinal and aromatic plant, traditional practice, biodiversity, pharmacopoeia, ouarzazat, south east of morocco. 3. postharvest control of pathogenic fungi of tomato using two brown seaweed extracts; cystoseira tamariscifolia and bifurcaria bifurcata nadia bahammou1*, o. cherifi1†, h. bouamama2, and k. cherifi3 1 laboratory of food, environment and health, biology department, faculty of sciences and techniques, cadi ayyad university, marrakech, morocco; 2 laboratory of organic and macromolecular chemistry, biology department, faculty of sciences and techniques, cadi ayyad university, marrakech, morocco; 3 laboratory of biotechnology and valorization of natural resources, biology department, faculty of sciences, ibn zohr university, agadir, morocco. abstract fungicides are chemical compounds having toxicological properties, used by the farmers to fight against the phytopathogenic fungi. these fungicides neutralize and reduce the activity of fungi but they remain a worrying and frightening source of pollution and toxicity. to search for biological molecules with antifungal potential, this study was done to investigate the antifungal potentiality of the marine algae collected from the moroccan coast sid bouzid el-jadida. hexane and aqueous extracts of two algae species belonging to phaeophyceae were screened for their antifungal activity against some tomato pathogenic fungi: alternaria sp. and botrytis cinerea. the overall results show that the fractions from hexanic (91,000 ppm) and aqueux extract (49,600 ppm) of cystoseira tamariscifolia and bifurcaria bifurcata exhibits remarkable biological activity with a very small amount of 100 µl. an interesting antifungal effect was observed for the hexanic crude extract of cystoseira tamariscifolia where the inhibition zones were up to 2 cm ± 0.18 and 1.32 ± 0.21 cm against b. cinerea and alternaria sp., respectively. the atlantic coast is a source of bioactive compounds with potential applications in controlling undesired microorganisms of crops. this may encourage the use of natural products to promote agroecology in substitution of chemical compounds in food systems, regarding risks of fungicides on human health and the environment. keywords: macroalgae extracts, phaeophyceae, phytopathogenic fungi, tomato, postharvest. 4. rock phosphate and vesicular-arbuscular mycorrhiza effects on growth and mineral nutrition of acacia gummifera wild fatima zahra lahdachi*, l. nassiri, j. ibijbijen, and f. mokhtari university my ismail, meknes, morocco. *presenting author: fatimazahra.lahdachi@gmail.com. abstract the influence of arbuscular mycorrhizae (am) and rock phosphate (rp) was studied on acacia gummifera, an endemic moroccan species which is experiencing significant losses. they are also a source of firewood and charcoal and precious air fodder in the dry season. acacias were inoculated or not by a mixture of two species of arbuscular mycorrhizal fungi (glomus intrardices and glomus mossae) to which were added two levels of rp: 0, 25 and 0,5g p/kg of soil. the percentage of mycorrhizal infection varied from 10 % to 25 % when rp applications increased. acacia seedlings grew poorly without mycorrhizal colonization and without rp applications. however, am plants with rp applications achieved better results in terms of content of p and n in shoot and root. in contrast, there were not additive effects of inoculation and fertilization on total biomass at any rp applications. however, mycorrhizal acacias took up more p and n at 0.25 g p kg-1 of soil and above. these results suggest that am are able to absorb p from soil and rock phosphate for a better mineral nutrition of acacia gummifera. keywords: acacia gummifera, rock phosphate, arbuscular mycorrhizae. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 320 v. session v. plant genetics, genomics, biotechnology, and bioinformatics i co-chair: my abdelmajid kassem, fayetteville state university, usa co-chair: lamiae ghaouti, iav hassan ii, morocco 1. genetic mapping of qtl for seed isoflavone contents in several soybean recombinant inbred line populations across multiple environments in the usa masum akond1, shiming liu2, stella k. kantartzi2, khalid meksem2, nacer belalloui3, david a. lightfoot2 and my abdelmajid kassem1* 1 plant genomics and biotechnology laboratory, department of biological sciences, fayetteville state university, fayetteville, nc28301-4298, usa; 2 department of plant, soil and agricultural systems, southern illinois university, carbondale, il 629014415, usa; 3 crop genetics research unit, u.s. department of agriculture, agricultural research service, 141 experiment station road, p.o. box 345, stoneville, ms 38776, usa;4 department of plant, soil and microbial sciences, michigan state university, east lansing, mi 48824-1325, usa. *presenting author: mkassem@uncfsu.edu. abstract soybean is one of the most important crops worldwide for its protein and oil as well as the health beneficial phytoestrogens or isoflavone. here, we sumarize three years of genetic and qtl mapping of seed isoflavone contents in three recombinant inbred line (ril) populations across different environments in the usa. three snp-based genetic linkage maps based on the ‘pi 438489b’ by ‘hamilton, ‘md 96-5722’ by ‘spencer’, and ‘hamilton’ by ‘spencer’ ril populations have been constructed and used for qtl mapping of seed isoflavone contents and other agronomic trairs. in the ‘pi 438489b’ by ‘hamilton’ ril population, we identified 6 qtl for seed isoflavone contents on chromosomes 1, 2, 3, 6, 7, 8, 9, and 12. in the ‘md 96-5722’ by ‘spencer’ ril population, we identified 3 qtl for seed isoflavone contents on chromosomes 5, 9, and 14. in the ‘hamilton’ by ‘spencer’ ril population, we also identified 3 qtl for seed isoflavone contents but on chromosomes 6 and 18. these newly assembled soybean linkage maps are useful tools to identify and map qtl for important agronomic traits and enhance the identification of the genes involved in these traits. keywords: soybean, single nucleotide polymorphism, quantitative trait loci, isoflavone, daidzein, genistein, glycitein. 2. the gmsnap18 is the peking-type rhg1-a gene for resistance to soybean cyst nematode shiming liu1, pramod k. kandoth2, naoufal lakhssassi1*, jingwen kang2, vincent colantonio1, robert heinz2, greg yeckel2, zhou zhou1, sadia bekal1, johannes dapprich3, bjorn rotter4, silvia cianzio5, melissa g. mitchum2, and khalid meksem1,† 1 department of plant, soil and agricultural systems, southern illinois university, carbondale, il 62901, usa; 2 division of plant sciences and bond life sciences center, university of missouri, columbia, mo 65211, usa; 3 generation biotech, lawrenceville, nj 08648, usa; 4 genxpro-gmbh, altenhöferallee 3, 60438 frankfurt am main, germany; 5 department of agronomy, iowa state university, ames, ia 50011, usa. *presenting author: naoufal.lakhssassi@gmail.com. †corresponding author: meksem@siu.edu. abstract two types of resistant soybeans [glycine max (l.) merr.] sources are widely used against soybean cyst nematode (scn, heterodera glycine ichinohe): the peking-type soybean resistance requires both the rhg1-a and rhg4 alleles, and the pi 88788type soybean resistance requires only the rhg1-b allele for resistance. we identified, by map based cloning, targeted genome sequencing, haplotyping, and genetic complementation, that the peking-type gmsnap18 alone is the rhg1-a gene conferring resistance to scn at the rhg1-a locus. in gmsnap18, there are nine amino acid differences between resistant (pekingtype soybean cv. forrest and pi 88788) and susceptible (essex) soybeans and five amino acid differences between two resistant soybeans: forrest and pi 88788. our findings reveal that peking-type gmsnap18 is most likely performing a different role in scn resistance than pi 88788-type gmsnap18. to our best knowledge, this is the first report of a gene that evolved to possibly use two mechanisms to ensure the same function within the same species, in this case, resistance to a pathogen. the knowledge gained from this study can be readily used to improve nematode resistance of soybeans. 3. improvement of saffron (crocus sativus) by plant biotechnology mohamed amine serghini1*, k. lagram1, m. ben el caid1, m. lachheb1, l. h. atyane1, l. salaka1, and y. karra2 1 faculté des sciences, university ibn zohr, bp 8106, agadir, morocco; 2 inra, bp 124, inezgane, morocco; *presenting author: maserghini@live.fr. abstract saffron describes both the crocus sativus plant and the spice resulting from its dried stigmas. it is the most expensive spice in the world that is valued in refined culinary art, used in traditional medicine/cosmetic and as dye in textile and woodwork. the commercial part of saffron is the flower style composed of 3 stigmas. 150 to 200 flowers are needed to produce 1 gram of dried spice. its exclusive vegetative propagation considerably reduces its biodiversity and excludes its improvement by classical hybridization. a lot of scientific work has focused on the morphlogical and physiological traits of saffron in terms of mass and shape of corms, the number of shoots and flowers per corm, the form and the mass of the stigma and the effect of tema tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 321 perature and irrigation on flowering. in all these characteristics, variability between cultivars is significant. the in vitro cultivation of saffron can allow the rapid multiplication of healthy selected material. at the molecular level, markers such as rflp, rapd and microsatellites and sequencing were a tool of choice for finding variability in saffron cultivars and defining their molecular identity. the chemistry of saffron is mainly based on crocin, picrocrocin and safranal metabolites. its study by chemical techniques such as uv-vis, hplc and gc attempt to explain the content of saffron secondary metabolites and to establish a chemical identity able to support the hallmarks of quality and to control the falsification of this spice. packaging and storage of saffron have also sparked some experiments focused on the content of crocin which defines, most importantly, the product’s commercial category (iso 3632 international standard). the conference will highlight the most significant scientific results in saffron research and will present a synthesis of research conducted on different axes of saffron in our laboratory. keywords: crocus sativus, saffron, corm, in vitro, molecular markers. 4. evaluation of safflower (carthamus tinctorius l.) accessions using agro-morphological and technological parameters karim houmanat1,2, mohamed el fechtali1, hamid mazouz2, and abdelghani nabloussi1* 1 inra, research unit of plant breeding and plant genetic resources conservation, crra of meknes, po. box 578, meknes, morocco; 2 university moulay ismail, laboratory of biotechnology and molecular biology, faculty of sciences, meknès, morocco. *presenting author: abdelghani.nabloussi@gmail.com. abstract safflower (carthamus tinctorius l.) is a minor hardy crop well adapted to semi-arid conditions. in morocco, it can be cultivated as an alternative for barley based monocropping system in the southern region of the country. recently, a breeding program has been launched in order to develop varieties adapted to local environmental conditions and with high agronomic and technological performances. in 2013-2014, a total of 60 accessions from different origins of the world were evaluated in the inra-experimental station located at douyet (10 km from fez city) for morphological, agronomic and technological traits. they were conducted using a complete-randomized-blocks design (crbd) with two replicates. results indicated large variability among these accessions for spinelessness level (sl), plant height (ph), earliness, branching, leaf rust resistance (rr), thousand seed weight (tsw), seed yield per plant (syp) and seed oil content (soc). generally, genotypes with high seed yield per plant were also characterized by high tsw, early flowering and were dwarf and spiny. average tsw was 15.50 g, with a high variation from 22 to 63 g. seed oil content ranged from 20 to 42.1%, with an average of 28.20%. average seed yield per hectare is about 2.1 t, and the highest yield observed was 7.9 t. the accessions studied were grouped into five pools on the basis of desired traits: seed yield per plant, seed oil content, spineless, earliness and resistance to leaf rust. however, the nature and composition of these groups must be confirmed in other environments. these groups will be considered as genetic pools and selected genotypes from each pool will be used as elite parents in our safflower hybridization program. keywords: morocco, semi-arid, genetic pools, hybridization. vi. session vi. animal sciences chair: mohamed bouslikhane, professor, iav, morocco co-chair: ahmed elamrani, university mohamed i, morocco 1. lipid and fatty acid composition of longissumus dorsi muscle of béni-guil sheep fed on different dryland forages in eastern morocco kamal belhaj1, farid mansouri1, abdessamad ben-moumen1, marianne sindic2, marie-laure fauconnier3, mohamed boukharta4, hana serghini-caid1, and ahmed elamrani1* 1 laboratoire de biologie des plantes et des micro-organismes, faculté des sciences, université mohamed ier, oujda; morocco; 2 laboratoire qualité et sécurité des produits alimentaires, gembloux agro-bio tech, université de liège; belgium; 3 laboratoire de chimie générale et organique, gembloux agro biotech, université de liége; belgium; 4 institut supérieur industriel agronomique, huy, belgium. *presenting author: abstract sheep meat of the beni guil breed is a famous meat in eastern morocco. this sheep breed was integrated into the national program of agricultural development due to the nutritional and sensory properties of its meat widely consumed in eastern morocco however, this good reputation is still only limited to the assessments of tasting panels. thus the objective of this study is to assess from a scientific point of view the nutritional quality of this meat via biochemical analyses, examining major and minor muscle composition of the meat of this breed and specifically fatty acid (fa) composition of the longissumus dorsi muscle in this first study, 10 longissumus dorsi muscle samples were selected and cut with the help of the agents of the national association of sheep and goats (anoc*). the results showed that 100g of fresh meat material contains 25.72% of dry matter, including 5.13% of fat, 19.42% of protein and 0.93% of mineral matter. qualitative and quantitative analyses of fatty acids of 10 samples allowed the identification of 27 components. palmitic, oleic and stearic esters were the main fatty acid methyl esters (fames) identified. saturated fatty acids represented about 41.13% of fas, with the majority being palmitic acid (20.69%) and stearic acid (16%) the unsaturated fatty acids were principally represented by oleic acid (36.06%) and linolenic acid (9.64%). the average content of total fatty acids in meat samples analyzed is 24.98 grams per 100 grams of fat. many reports in the literature have shown that the n-6 and n-3 polyunsaturated fa are important contributors to the savor and flavor of ruminant meat and that this composition is mainly a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 322 correlated with feeding systems used in eastern morocco. keywords: beni guil, sheep, longissumus dorsi muscle, fatty acid. *anoc: association national des ovins et caprins, translated as national association of sheep and goats. 2. can livestock contribute to the ecological intensification of the agriculture in the oasis: a case study from the drâa valley? m. t. sraïri1*, s. mansour1, v. alary2, and m. benidir3 1 hassan ii agronomy and veterinary medicine institute, p.o. box 6202, rabat, morocco; 2 international center of agricultural research for development (cirad), montpellier, france; 3 regional office of agricultural development, ouarzazate, morocco. *presenting author: mt.srairi@iav.ac.ma. abstract recent developments in the global agriculture have been characterized by the emergence of the concept of ecological intensification, as a means to decrease the use of inputs and their impacts on the environment, while increasing and/or stabilizing crops and livestock yields. in the meantime, research studies have emphasized the ability of crops integration with livestock farming systems to adapt themselves to numerous stresses (economic, climate, etc.) in comparison to specialized systems with only crops or livestock. in the oasis, such integration has been traditionally achieved, as it has allowed for centuries the resilience of the cropping activities, in an environment characterized by numerous constraints, particularly frequent water shortages. however, this resilience has nowadays become challenged by increased stresses, above all climate change and its consequences and also social changes, with growing demands of the local population for better livelihoods. therefore, in this study, the focus was on the ability of livestock to contribute to the ecological intensification of the farming systems adopted in the oasis. a sample of twelve (12) farms located in the oasis of the drâa valley was selected and it was representing four types of livestock systems (i.e. 3 farms per system). a follow-up of farms was adopted to characterize inputs uses, water volumes and their origins (rainfall, surface irrigation and groundwater) and incomes from crops. the same methods were used for livestock production. in addition, work needs for crops and livestock were also measured, by determining the contribution of off-farm work uses. the results showed the importance of crop/ livestock association in all the systems, in order to implement a sustainable farming activity. the intensification of livestock systems (retrieved in farms with prominent off-farm feed uses, i.e. d’man breed flocks and dairy cattle) showed the best incomes from crops, and that was mostly remarkable for date palms, which benefitted from the surplus irrigation of the underlying alfalfa, as well as manure from the herds. in the contexts where agriculture intensification was not feasible (due to water, land or capital scarcity), livestock remained the main source of incomes, adding value to vast pastoral areas (as in the area of m’hamid el ghizlane) and to crop by-products (mainly date wastes). altogether, the results imply the need of further research devoted to the performances of the farming systems in the oasis, and the use efficiency of scarce inputs, such as water, land and work. keywords: climate change, crop-livestock integration, ecological intensification, incomes, morocco, oasis. 3. reproduction performances of the female d’man sheep lahsen derqaoui* department of medicine, surgery and reproduction, l.a.v. hassan il, p.o. box 6202, rabat-institutes, 10101, rabat, morocco. *presenting author: l.derqaoui@iav.ac.ma. abstract the d’man is the local sheep breed of the pre-saharan regions of southern morocco. it is located mainly in the oases of the draa (ouarzazat, zagora, tata, guelmime), ziz (errachidia arfoud) and dades valleys. its total population was estimated to some 200 000 head. it is raised in very small size herds (one ram and 3 to 4 ewes, in average) and kept indoors throughout the year. the diet is based on alfalfa and date wastes. the d’man breed is known for its exceptional reproductive traits, namely precocious puberty, high prolificacy and fertility rates, non seasonality and short postpartum anestrous. puberty is commonly defined as the phase of life from which an individual acquires the ability to reproduce. in the female, it corresponds to the age at which it becomes able to ovulate, to manifest signs of heat, to mate and to carry out a gestation. in their native areas, d’man lambs reach puberty at an average age of 5 months (150 days). however, lambing was observed at eight months of age, corresponding to fertile mating at 3 months of age. in ewes born and raised outside the native area, the first estrus occurs at 212 to 229 days of age. the effect of birth season on age at puberty is controversial. moreover, this parameter is genetically transmitted to the crosses between the d’man and seasonal breeds sardi and timahdite. at puberty, the average live weight of the d’man ewe lamb ranges from 18 to 24.5 kg (about 50% of mature weight). the precocity of this breed and its crosses allows the lengthening of their reproductive career. lambing occurs all year around in the d’man sheep, which is in favor of the absence of seasonal anestrous in this breed. experimental studies showed that ewes exhibit continuous sexual activity throughout the year. however, ewes show a period of decreased sexual activity from mid-march to mid-may during their first breeding season. practically, this trait offers the possibility to use the d’man ewe according to a desired breeding program. in the d’man ewe, the duration of the estrous cycle is 1 7.5 days on average (14 to 21 days), with cycles of 1 7 and 1 8 days representing 60% of the cycles. the duration of estrous behavior (heat) varies from 12 to 72 hours with an average of 39 hours in aged females, with 50% of females in heat for 48 hours at least. in the young ewe lambs, estrus is relatively shorter and lasts 33 hours only. the lengthening of the estrus behavior increases the possibilities of mating and improves the fertility of a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 323 the breed. postpartum anestrous is the stage of sexual inactivity after lambing. in the d’man ewe, resumption of estrous and ovarian activities during the postpartum period is precocious. indeed, ovarian and estrous activities resume lactating ewes within 45 days after lambing. the shortening of the post-partum anestrous period makes allowed accelerated lambing program. the d’man sheep is known for its multiovulatory ability. thus, average ovulation rates range from 2.50 to 3.63 (1 to 8) with a higher incidence of double ovulations (32.9%). the average ovulation rate increases with age and then decreases slightly towards the end of the breeding career of the female, i.e. minimal (2.16) in ewes less than two years old maximal (3.63) at 4 to 5 years. the average litter size ranged from 1.90 to 2.27, according to the breeding system. in addition, the incidence of twining was highest (47.1%). likewise, the litter size followed similar pattern as the ovulation rate. given the reproduction performances of the d’man sheep, namely age at puberty, ovulation rate, fertility and prolificacy are genetically transmitted to the breed’s offspring and its crosses with satisfactory heritability rates, the use of this breed is desirable in intensive lamb production systems. 4. epidemiological and clinico-pathological features of edema syndrome in camel dromedaries in southern areas of morocco boutkhoum khadija1*, mohammed bouslikhane1, jaouad berrada1, and faouzi kichou1 1 département de pathologie et santé publique vétérinaires, institut agronomique et vétérinaire hassan ii, bp 10101, rabat, maroc. *presenting author: ?. †corresponding author: f.kichou@ iav.ac.ma abstract this work is a contribution to studying the edema syndrome in camel dromedaries in southern areas of morocco. the aim of the work was to determine the epidemiological and clinico-pathological characteristics of this syndrome and make plausible suspicion relative to the causes implicated. to do so, clinical and epidemiological investigations were carried out in 46 herds of dromedaries in the areas of laayoune-boujdour, guelmim, essmara, oued ed-dahab-lagouira. clinical examinations were performed exclusively on animals suffering from edematous syndrome. additional investigations including hematological (hematocrit) and biochemical (alt and ast, urea, creatinin, total protein and albumin) examinations were performed on blood samples harvested from sick and healthy animals. the results of this work showed a prevalence of 26%of affected flocks by the edema syndrome, an estimated overall individual prevalence of 0.5%and an average individual prevalence of 2.6%. the affected animals showed a chronic generalized passive edema localized mainly in the neck, flank and hindquarters areas. biochemical tests revealed hypoproteinemia in sick animals as shown by a significant decrease in total plasma proteins and albumins. post-mortem examinations carried out on a single case showed generalized edema (severe subcutaneous edema and serous fluid in body cavities), generalized serous atrophy of fat, congested liver of nutmeg appearance, flabby heart and bilateral atrophy and dilation of heart ventricles. the most prominent microscopic lesions were centro-lobular passive congestion and important hemosiderin pigmentation in the liver consistent with the diagnosis of right heart failure. a lympho-plasmocytic enteritis and peri-bronchiolar and interstitial fibrosis of bandlike areas in pulmonary parenchyma were also noted. based on the above findings, the causes of edema syndrome could be trypanosomiasis, haemonchosis, mycotoxicosis, malabsorption syndrome, proteino-energitic malnutrition and/or liver failure. keywords: edema, dromedary, epidemiology, biochemistry, hematology, histopathology, south of morocco. 5. risk factors, clinical, and lesion characteristics of caseous lymphadenitis or abscess disease in sheep in eastern morocco faouzi kichou1*, ahmed mechaal1, mohammed bouslikhane1, ahlam kadiri1, khalil zro2, and jaouad berrada1 1 département de pathologie et santé publique vétérinaires, institut agronomique et vétérinaire hassan ii, bp 10101, rabat, morocco; 2 biopharma, agdal rabat morocco. *presenting author: f.kichou@iav.ac.ma. abstract the aims of this study were to determine the prevalence and risk factors of caseous lymphadenitis in sheep in the orien¬tal region in morocco, and to determine its clinical and lesion characteristics. surveys were conducted in different provinces of the region. they focused on 6376 animals in 107 farms. in addition, search for lesions, and lymph node and abscess collection for histopathological examination were performed in five slaughterhouses of the region. the results showed that almost 100% of the farms (106 out of 107) were affected by caseous lymphadenitis. the overall individual prevalence of the disease was 24%, and the average prevalence in farms and slaughterhouses were 28 and 16%, respectively. besides the poor hygiene and high density of sheep in houses, use of traumatic equipment (metal feeders, drinkers and fences) seemed to be the main factor that lead to the spread of abscesses between animals of a same herd. superficial abscesses, open or not, surrounded by enlarged lymph nodes were present in all affected animals. the location of the lesions varied but about 79% of them were located in the head or the neck. histopathological examination of lymph nodes revealed two types: active abscesses (87%), and lesions of diffuse and severe necrosis and suppuration of the lymphoid tissues (13%). in the lungs, abscesses were well defined and circumscribed. keywords: sheep, caseous lymphadenitis, abscess, risk factor, histopathology, epidemiology, morocco. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 324 vii. session vii plant genetics, genomics, biotechnology, and bioinformatics ii co-chair: lamiae ghaouti, professor, iav morocco co-chair: ahmed bouaziz, professor, iav morocco 5. how large is the genetic diversity in argan (argania spinosa (l.) skeels)? what opportunities present the use of the molecular tools? mohamed louay metougui1*, mimoun mokhtari2, and ouafae benlahbib1 1 department of plant production, protection and biotechnology, hassan ii institute of agronomy and veterinary medicine, rabat, morocco; 2 department of horticulture, hassan ii institute of agronomy and veterinary medicine, horticultural complex of agadir, morocco. abstract argan tree (argania spinosa (l.) skeels) is a multipurpose species of great social and economic interest in morocco. the natural stands are more and more threatened by anthropomorphic pressures and environmental changes which are slowing the natural regeneration of the species. thus, characterization and diversity studies are essential to determine where genetic resources conservation efforts should be focused. in this study, the agro-morphological diversity of argan trees was investigated over three years in the in situ preserved population of admine, a protected area in southwestern morocco. a total number of 122 trees out of 1200 were characterized using 30 quantitative traits. significant differences between genotypes and between years were seen for all traits. correlation analysis between traits showed that vigor traits (leaves and shoots sizes) were positively correlated with fruit traits. cluster analysis using euclidian distances was used to establish the relationships among the argan germplasm and grouped the genotypes into five relatively homogenous clusters. the yield’s genotype × year interaction analysis indicated that the genotype factor contributed more to the yield’s variance than the year. but the genotype × year interaction explained more than 65% of the total variation, revealing specific response of each genotype to each environment and the complexity of this trait. notwithstanding the importance of argan as an emerging alternative oilseed species, and the difficulty of generating reliable data for the selection of stable, plus-trees, and understanding the genetic background of this species from phenotypic data. only a few molecular investigations have been reported in argania and even fewer molecular tools, which are needed for advanced genomic studies and modern plant breeding, have been developed. genomic analysis tools, such as sequence-based molecular markers (e.g., ssrs and snps), genetic linkage maps, and expressed sequence tagged (est) collections have yet to be developed for argania spinosa. development of these molecular resources for argan will have a direct application to argan conservation and potential improvement (i.e. breeding and selection) of the species. 6. genetic diversity of vicia faba l. landraces in morocco: a reservoir for the enhancement of the competitive ability to weed stress lamiae ghaouti1*, n. aqtbouz1, l. belqadi1, and w. link2 1 département de production, protection et biotechnologies végétales, institut agronomique et vétérinaire hassan ii, rabat, morroco; 2 department of crop sciences, georg-august universität, göttingen, germany. abstract faba bean (vicia faba l.), the major legume crop in morocco with an acreage over 190.000 ha, is cultivated mainly as a break crop in cereal based cropping systems. it is an ancient crop in morocco with a large extent of genetic diversity, mainly grown in rain-fed and marginal areas with high level of agrobiodiversity. over 90% of the farmers are cropping local landraces based on traditional farming practices with very low inputs. weeds compete with crop plants for light, nutrients, soil moisture and space. above critical population thresholds, weeds can significantly reduce crop yield and quality. the yield loss attributed to weed stress in faba bean can amount 60 to 70%. mechanical weeding is very rarely used especially for large cropped areas and chemical weeding is mainly used only in case of broomrape infestations. the high level of genetic diversity of the moroccan faba bean landraces constitutes one of the few options available to the farmers to cope with weed stress. indeed, a very valuable option for weed control is the use of genotypes with high competitive ability toward weeds. high grain yield, despite significant weed pressure, is an attribute of competitive crop genotypes. however no study has been carried out so far on the competitiveness of the moroccan landraces toward weed stress. the identification of local populations with high competitive ability is a relevant approach responding to farmer’s need and providing useful germplasm sources for breeders. the objective of the study is (1) to assess the genetic diversity of a collection of moroccan faba bean populations and (2) to evaluate their competitive ability to weed stress. a set of 60 moroccan local populations collected from taounate province, and 2 checks (aguadulce et defez) which are commonly used cultivars, were (1) analyzed molecularly through amplified fragment length polymorphism (aflp) markers based on four selective primer combinations to assess their genetic diversity and (2) tested under field conditions in four environments to assess their competitive ability toward weeds. the trials were laid out as a split-plot design with two treatments (with and without weeds). the white mustard (sinapis alba), a species related to wild mustard (sinapis arvensis) which is a common weed of faba bean, was used as a model weed. aflp analysis revealed a large diversity within the local populations (82%) compared to the diversity between the local populations (18%). both levels of variation were significant. the analysis of variance showed that the differences between genotypes and treatments as well as their interactions were significant to highly significant for maturity, grain yield and grain yield components. two indexes relative a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 325 to yield loss (yl) and mean productivity (mp), were calculated based on grain yield to identify populations that combine both high competitive ability and high productivity. the populations were significantly different for both indexes. yl ranged from 8% to 81% with an average of 67%, whereas mp ranged from 11dt/ha to 31dt/ha with an average of 19dt/ha. aguadulce exhibited a mp of 21dt/ha and a yl of 59%, whereas defez was less performing with a mp of 12dt/ha and a yl of 69%. a range of populations have proven to perform better regarding the indexes than the check cultivars. two local populations exhibited a compromise with a low yl and a high mp. these local populations showing a high competitive ability are of interest for the farming systems where weeding practices are rarely or never applied. keywords: faba bean, genetic diversity, weed stress, competitive ability. 7. sustainable use of halophytes for the rehabilitation of marginal dry areas in morocco salma daoud1* and m. c. harrouni2 1 faculty of sciences, ibn zohr university, agadir, morocco; 2 hassan ii institute of agronomy and veterinary medicine, agadir, morocco abstract salinity in the soil and water is a common feature in arid lands of morocco. moreover, population increase in these areas increases the pressure on fresh water resources which results in less supply for home use. these areas are still inhabited by people who are attached to their land and to their traditions. we present a survey of agriculture in some salt affected areas (coastal and inland) in morocco focusing on adaptation to the constraints and opportunities of saline environments, as well as the potential for survival in the future. some considerations for the identification and the evaluation of the capacity of these marginalised areas to cope with drought and salinity problems based on the capitalization of the local know knowledge and young human resources via appropriate valuing and training. this presentation also demonstrates the potential for the utilization of local and acclimated halophytic species by using a biosaline agriculture approach with the proposition of solutions for the restoration of biodiversity and productivity in salt affected areas in morocco. keywords: morocco, arid lands, drought, salinity, local know-how, halophytes, traditional uses, young human resources, management, biodiversity, productivity. 8. biological control of bacteria onion diseases using a bacterium, pantoea agglomerans 2066-7 el hassan achbani1*, s. sadik1,2, a. bebouazza1, and h. mazouz2 1 la boratoire de bactériologie végétale et de lutte biologique, urpp, crra de méknès (inra), morocco; 2 faculté des sciences de méknès, morocco. *presenting author: achbani105@gmail. com. abstract epiphytic microorganisms isolated from olive knots, apple fruits and trees, quince, compost and water from different areas were screened for antagonistic activity against pseudomonas marginalis, pseudomonas viridiflava, xanthomonas retroflexus and pantoea ananatis, bacteria recently reported on onion in morocco. from 82 microorganisms tested for antagonistic properties against bacteria onion diseases, the strain pantoea agglomerans 2066-7 was selected. this bacterium was very effective against pseudomonas marginalis, pseudomonas viridiflava, xanthomonas retroflexus and pantoea ananatis. complete control at 106 cfu. ml-1 was obtained on wounded onions bulbs inoculated with 105 cfu.ml-1 of pseudomonas marginalis and pseudomonas viridiflava. at 107 cfu.ml-1 pantoea aggolomerans reduced xanthomonas retroflexus and pantoea ananatis decay by more than 90%. keywords: onion, pseudomonas marginalis, pseudomonas viridiflava, pantoae ananatis, xanthomonas retroflexus, biocontrol and morocco. viii. session viii. food, health, and nutrition co-chair: jack turman, professor, indiana state university, usa co-chair: karen midden, prof., southern illinois university, usa 1. the important role of nutrition in early brain and behavior development jack turman jr.* dept. of applied medicine and rehabilitation, college of health and human services indiana state university, usa. *presenting author: jackturman2@hotmail.com. abstract the first 1000 days of life (from conception to the end of the second year of life) is a period of highly orchestrated brain development. the neural circuits that emerge during this period serve as the foundation for life long motor, cognitive, social, and emotional behaviors. brain development during this period is dependent upon adequate nutrition, gene expression patterns, environmental conditions and early life experiences. the first objective of this presentation is to provide a brief overview of basic research studies that elucidate the role that macro and micronutrients play in the cellular processes that underlie brain development. the second objective to is to differentiate severe acute malnutrition, chronic undernutrition, iron deficiency and iodine deficiency, as all of these conditions serve as serious risk factors for altered brain development that results in poor motor, cognitive and emotional development. the third and final objective is to provide examples of strategies used to address these nutritional deficiencies. interestingly, it is now observed that intervention strategies are often not replicated in other regions/ cultures different from their origin. developing an innovative interdisciplinary framework to address these deficiencies in difa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 326 ferent world regions is a global health priority. this presentation aims to stimulate creative discussions between neuroscientists, nutritionists, maternal and child health clinicians and agricultural scientists to design strategies that will successfully address nutritional deficiencies that continue to influence development of some moroccan children. 2. closing the nutritional gap in moroccan maternal and child health through home gardening karen midden*, professor, associate dean, landscape architect college of agricultural sciences, southern illinois university, carbondale, illinois, usa. abstract gardens play a powerful role in the wellbeing of an individual and communities, if the opportunities are understood and utilized. adults and children gain mental and physical benefits being outdoors, connecting with nature, gardening as well as including nutrient rich produce in their diets from their gardens (louv, kuo, taylor, mandel, cobb, simson and straus). the powerful aspect of home gardening for urban or rural people is that the fresh healthy produce they grow can help close serious nutritional gaps leading to malnourishment or undernourishment. it is possible to address health issues generated from lack of specific nutrients by producing and consuming nutrient rich fruits and vegetables in home gardens. the world bank data of 2008, emphasized that attention and changes are needed in morocco to move towards the status of a developed nation and to provide greater health opportunities for children. the data found that 15% of infants in morocco are born with a low birth weight, about 10% are wasted, 43% of those aged 15 and above are overweight or obese and 16% of children under the age of 5 are overweight (world bank). unicef data for morocco dating 2009 – 2013 reported 15% of infants are born with low birth rates, 15% are stunted, 2% are wasted, 3% are underweight, 11% are overweight and there is an infant mortality rate of 18%. the world bank states that there is food insecurity that accompanies the malnourishment resulting in a majority of these problems. vitamin a, folic acid and iron are ‘fore-runner’s in the deficiencies (world bank). all three of these micronutrients are available in common fruits and vegetables that can be easily grown in an economical fashion in a home garden. however, it may be a challenge for the average small farmer or homeowner to have access to seeds that offer the highest value. this problem demands collaborate action to move a nation forward for improved health efforts. the demands include scientists who understand the physical and mental nutrient requirements for a healthy body. it requires scientists in agriculture, through inquiry and research, to provide plant seeds that will fulfill these nutrient needs. to disseminate this information and product, educators at various levels are necessary to be the link from the scientist to the public. the possibility of utilizing home gardening to empower the general public in morocco, especially women and children, to close the nutritional gap is feasible and realistic with the efforts of scientists and educators. 3. seasonal variations of metallothioneins and trace metals in edible cockle cerastoderma edule (l., 1758) in two lagoon ecosystems (khnifiss and oualidia) lefrere latifa1*, z. idardare1, h bergayou1, c. mouneyrac-balocco2, and a. moukrim1 1 laboratoire des « systèmes aquatiques : milieu marin et continental », faculté des sciences, université ibn zohr, agadir, morocco; 2 equipe : « mer molécules santé », ea 2160université de nantes ; faculté des sciences / mms-uco, angers, france. abstract the aim of this study is to evaluate the seasonal variation of metallothioneins (mtlp) and some trace metals in the edible cockle cerastoderma edule (mollusca, bivalvia, linnaeus, 1758) in two moroccan lagoons, oualidia (north) and khnifiss (south). our work also takes into account the correlations between mtlp and metals as well as the influence of some biotic and abiotic parameters on the metal contamination. the samples are taken on a monthly basis, at low tide, from april to january. thirty individuals are taken for quantifying the amount mtlp, while fifty individuals are used to calculate the condition index and quantify trace metals in the whole individual. significant high values of metallothioneins levels are observed in bivalves of oualidia versus those of khnifiss. for metals, the contents of cu and zn are also higher in bivalves of oualidia.; the highest levels of cd were recorded in molluscs of khnifiss. significant relationship exist between mtlp and metals (positive slope). for cd, the mtlp has a high correlation with the metal insoluble fraction; while in the case of zn and cu, the correlation with the mtlp is mainly linked to the metal in soluble fraction. the relationship between the biological (condition index, mtlp) and physico-chemical parameters (temperature, salinity, ph, cd, zn, cu) were performed by pca. two metals, cu and zn, are significantly and negatively correlated with salinity, while they are significantly and positively correlated to mtlp levels. these two metals are also strongly positively correlated with each other. furthermore, the cd levels are significantly and positively correlated with the weight (condition index). mtlp, cu and zn are negatively correlated with weight in c. edule. keywords: bivalves, cerastoderma edule, metallothioneins, moroccan coasts, metals, lagoons. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 327 ix. session ix. olive tree workshop co-chair: ikram blilou, professor, wageningen university & research, the netherlands co-chair: ahmed elamrani, university mohamed i, morocco 1. de novo sequencing brings insight into the biology and evolution of the olive tree genome turgay unver1, mine turktas2, gabriel dorado3, pilar hernandez4, david a lightfoot6, pavan kasarla5, oussama badad*6,7, and hassan ghazal. international olive genome consortium. 1 cankiri karatekin university, cankiri, turkey; 2 cankiri karatekin university, cankiri, turkey; 3 campus de excelencia internacional agroalimentario (ceia3), universidad de córdoba, cordoba, spain; 4 instituto de agricultura sostenible, cordoba, spain; 5 department of plant, soil and agricultural systems, southern illinois university, carbondale, il 62901, usa; 6 department of plant, soil and agricultural systems, southern illinois university, carbondale, il 62901, usa; 7 laboratory of plant physiology, university mohamed the fifth , rabat, morocco; 8 laboratory of biotechnology and genetics, faculty of sciences, mohamed first university, oujda, morocco. *presenting author: oussama.badad@gmail.com. abstract the olive tree (olea europaea l.) is an economically-important fruit and oiltree. the olive tree is a diploid (2n=2x=46), predominantly allogamous, and largely vegetatively propagated species. the genome was sequenced and assembled with 246x coverage. soapdenovo suite was used to assemble reads, which resulted in a draft genome of 1.48 gbp with scaffold n50 of 228 kbp,. a total of 42,843 scaffolds (>1 kbp) were assembled (1.14 gbp). using a newly-constructed genetic map, 50% of the sequences were anchored onto 23 linkage groups, (572 mbp). about 50% of the genome was composed of repetitive dna. transposable elements and interspersed repeats occupied 50% of the genome. protein-encoding gene models were constructed. a total of 50,684 protein-encoding gene models were predicted, of which 31,245 were anchored. heterozygosity was estimated to be about 1.3%. a phylogenetic tree was constructed where the olive-tree clustered with oil-crop sesame (s. indicum l.). genes involved in oil biosynthesis, fruit ripening, secondary metabolite synthesis and alternate fruit bearing were annotated. small rnas and transcription factor genes were annotated. the annotated genes were further analyzed to build a protein-protein interaction network map. the olive tree reference genome is available at the olive genome browser (http:// h3abionet.fso.ump.ma/cgi-bin/gb2/gbrowse/olea_europea/), and will serve as a crucial source for the study of the olive genome, and fruit tree genomics. more effective olive tree breeding programs will be enabled. 2. integrated and sustainable management of the olive tree in the gharb region of morocco nour eddine essafi* regional directorate of agriculture, kenitra, ministry of agriculture and fisheries, morocco *presenting author: essafi_n@yahoo.fr. abstract in the gharb region, the olive grove occupies about 30,000 ha, of which almost all (88%) is located in the north area of sidi kacem province. the olive grove of gharb is dominated by the variety “picholine marocaine” (92%) mainly characterized by its adaptability to various soil and climatic conditions and olive qualities with dual purpose. the new strategy of the green morocco plan (gmp) has given considerable importance to small farmers (pillar ii), which aims to upgrade agriculture solidarity, the fight against poverty and improve the standard of livelihoods. approximately 250 beneficiaries working upon 350 ha are concerned by the integration project of climate change in the implementation of the green morocco plan (piccpmv) funded by the world bank and the global environment. this project aims mainly the conversion of the perimeter with the lowyielding cereal, located in remote and marginal areas, which will be replaced by an orchard of olive tree. new agricultural technologies for adaptation to climate change enhance the resilience of the olive grove in respect of climate change. furthermore, integrated management, variety, supplemental irrigation and inputs are considered priority and sustainable technologies that are considered as part of the pillar ii. these technologies have a positive environmental impact, resulting in a reduction of the effects of erosion, mitigating the effects of climate change and improving water efficiency by supplemental irrigation. keywords: climate change, supplemental irrigation, olive tree, piccpmv, pillar ii. 3. some results of morphological characterization an agronomic evaluation of the main olive tree varieties investigated in various regions of morocco oumkaltoum krimi bencheqroun* and lahcen hssaini inra meknes, morocco. *presenting author: oumkaltoumkrimi@ yahoo.fr. abstract in morocco, the olive cultivation is vested with great socio-economic importance. today, the area devoted to olive cultivation has gone beyond 1 000 000 ha, thus representing more than half of the national arboreal area. furthermore, the contract program for the development of the olive cultivation section which was established by the ministry of agriculture and the green morocco plan, in its 2015 edition, has fixed as one of its major objectives the extension of olive cultivation to an area a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 328 of 1.220.000 ha by the year 2020. on the national scale, the olive cultivation is mostly carried out in a traditional mode. in such conditions, the average harvest per hectare, between 1t/ ha and 1.5t/ha are neatly inferior to the real potential production. the realization of higher levels of production and exportation fixed by the contract program mentioned earlier calls for and necessitates a modernization of olive cultivation. in spain, since 1986, the intensification of the production factors based on the use of performing and adapted varieties, the application of high densities, the drop by drop irrigation and the mechanization of the harvest which allows a substantial improvement, has made it possible to triple the quantity of the production between 1986 and 2003. in 2012, spain has contributed, on its own, to 62% of the overall quantity of olive oil produced by the european union. in morocco, in terms of the research work which is being conducted by inra on olive tree in various fields of experimentation, some national and international varieties of the latter have yielded higher levels of production which swing between 10t/ha and 17t/ha. similarly, other specifications of agronomic interest have been spotted in some samples under investigation. the present communication aims to review some morphological characterization and evaluation results of agronomic performances of some national and international varieties of olive tree which have been studied in various experimentation fields and areas, the ultimate goal being to evaluate the degree of adaptation of these genetic resources to local pedo-climatic conditions. keywords: olive tree, varieties, pedo-climatic, performances, adaptation. 4. quality and chemical profiles of monovarietal olive oils in eastern morocco ahmed elamrani1*, farid mansouri1, marianne sindic2, marielaure fauconnier3, and hana serghini-caid1 1 laboratoire de biologie des plantes et des micro-organismes, faculté des sciences, université mohamed ier oujda; morocco; 2 laboratoire qualité et sécurité des produits alimentaires, gembloux agro-bio tech, université de liège; belgium; 3 laboratoire de chimie générale et organique, gembloux agro bio-tech, université de liége; belgium. *presenting author: ahmed.elamrani@gmail.com. abstract olive oil becomes the object of a considerable amount of research. there are numerous data on chemical-physical characteristics and olive oils quality, of various origins from different world’s production areas, particularly mediterranean countries. paradoxically, very few data, on olive oil produced in the eastern morocco. in this region, olive oil production is considered as an ancient activity, where old olive groves can still be observed, but recently monovarietal groves seem to be increasing and the olive cultivation is being improved by renewing old trees, reducing the association with other crops, selecting the olive varieties suited to local agro-climates and planting new single variety orchards. the extension of olive grove surface areas and improvement of olive oil quality (according to the great *morocco’s green plan) and this is leading to an increase in the prevalence of monovarietal olive oils. thus, the monovarietal oils produced from the ‘arbequina’, ‘arbosana’ and ‘koroneiki’ varieties, which have recently been introduced under intensive cultivation in the eastern morocco, underwent physico–chemical characterization to determine quality criteria, natural antioxidant content, fatty acid composition and triacylglycerol profile. the rancimat test was performed to assess the oxidative stability of these monovarietal oils and their blends to determine the storage stability and the best shelf life of the blends. keywords: monovarietalolive oil, arbequina, arbosana, koroneiki, fatty acid, triacylglycerol, quality. * the olive oil agro-industry is one of the most concerned sector by *morocco’s green plan (2010 2020) designed to promote the development of the entire agricultural and territorial potential and aims to meet new challenges facing morocco’s competitiveness and opening of markets. thanks to “maroc-belgique cooperation” for supporting financially this applied research. x. session x. aromatic, medicinal, and economic plants chair: alan walters, prof., southern illinois university, usa 1. determination of fat and sugar contents in carob pulp from wild and domesticated moroccan trees rababe essoufi1, josé manuel igartuburu2, zineb nejjar el ansari1*, brahim el bouzdoudi1, francisco antonio macias dominguez2, josé maría gonzalez molinillo2, and ahmed lamarti1 1 laboratory of plant biotechnology, department of biology, faculty of sciences, abdelmalek essaadi university, m’hannech ii, b.p.2121, tetouan, morocco; 2 cádiz allelopathy group (inbio, ceia3), departamento de química orgánica, facultad de ciencias, universidad de cádiz, c/ república saharaui, s/n, 11510-puerto real, cádiz, spain. *corresponding and presenting author: zinebnejjarelansari@hotmail.com. abstract ceratonia siliqua l. (carob tree) is a fruit and fodder tree which has potential to enhance soil conservation and rural development. this typical essence of mediterranean flora has many socio-economic, ecological and industrial interests. in order to valorize the moroccan carob, we have carried out a fat and sugar composition study on carob pods (pulp) of 12 wild trees and 8 domesticated ones from 15 different localities in morocco. fat content, identification and quantification of fatty acids on dry pods powder were determined after methylation by gas chromatography (gc), and sugar content was studied through ion chromatography. results show that fat content of carob pods from wild trees varies from 0.053 % to 0.727 %, while it varies from 0.079 % to 0.347 % for domesticated ones. analysis of fatty acids methyl esters by gas chromatography allowed identifying and quantifying 10 fatty acids on dry pod powder. oleic a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 329 acid (c18: 1n-9c) is the most abundant in all powder samples from wild and domesticated trees, except one wild tree whose most abundant fatty acid is palmitic acid (c16: 0). the three most abundant fatty acids are oleic acid (c18: 1n9c), linoleic acid (c18: 2n6c) and palmitic acid (c16: 0). the most abundant saturated fatty acids are palmitic acid (from 8.57 mg/ 100 g of dry matter to 154.05 mg/ 100 g of dry matter) and stearic acid (c18: 0) (from 1.83 mg/ 100 g of dry matter to 38.52 mg/ 100 g of dry matter. oleic acid is the main unsaturated fatty acid in all the samples (from 8.6745 mg/ 100 g of dry matter to 291.56 mg/ 100 g of dry matter), followed by linoleic acid (from 1.36 mg/ 100 g of dry matter to 95.07 mg/ 100 g of dry matter). results obtained for sugar composition show that carob pulp contains up to 40 % of sugars, which are sucrose, glucose and fructose, identified and quantified by ion chromatography. sucrose (non-reducing sugar) is the dominant sugar in the carob pulp, with content varying from 192.10 to 351.60 g/ kg of dry matter for wild trees and from 159.16 to 312.67 g/ kg of dry matter for domesticated ones, followed by reducing sugars: glucose and fructose. glucose content varies from 3.92 to 81.34 g/kg of dry matter for wild trees and from 3.57 to 36.03 g/kg of dry matter for domesticated ones. fructose content varies from 12.48 to 92.55 g/kg of dry matter for wild trees and from 22.95 to 60.13 g/kg of dry matter for domesticated ones. keywords: ceratonia siliqua l., pod powder, gas chromatography, ion chromatography, fat, fatty acid, sugar, sucrose, glucose, fructose. 2. in vivo wound healing potential and identification of bioactive compounds from pistacia lentiscus leaves by hplcpda-esi-ms detection halima saiah1,4*, rachida allem1, meriem mokhtar2, wassila saiah3, fatima zohra el kebir4 1 faculty of snv, department of biology, university of hassiba ben bouali, chlef, algeria; 2 laboratory of beneficial microorganisms, functional food and health, faculty of natural sciences and life, university of abdelhamid ibn badis, mostaganem, algeria; 3 department of biological sciences and physiology, laboratory of bioenergetics and intermediary metabolism, university of sciences and technology houari boumediene, algiers, algeria; 4 department of biology, faculty of life and natural sciences, university of oran i ahmed benbella, oran, algeria. *presenting author: halimasaiahbio@yahoo.com. abstract the present study was carried out to explore the in vivo wound healing potential of algerian pistacia lentiscus and to identify the active compounds that may be responsible for its action. polyphenols were extracted with 80% methanol. a 10% ointment was made from methanol extract of p. lentiscus. studies were performed on excision wound model using the test ointment. the healing potential was assessed by measuring wound contraction rate and hydroxyproline content. in addition, histological evaluations of full thickness wounds on the 16th post wounding day were performed. hplc-pad-esi-ms analyses were used for identification and confirmation of bioactive compounds. the test ointment-treated groups healed significantly faster, which was indicated by improved contraction rate (93.45±3.08) % in comparison to control group (73.12±3.51 %). moreover, biochemical analyses revealed a significant increase in hydroxyproline contents of the ointment-treated wounds in comparison to control group. the results obtained from the histological evaluation indicated that ointment treatment of wounds promotes the process of wound healing by influencing fibroblasts proliferation, collagen synthesis and deposition and enhancing the rate of reepithelialisation. the hplc-pda-esi-ms studies revealed the presence of catechin as a major compound. this study had demonstrated that the methanol extract promoted the acceleration of the healing process when compared to the control group. this might be due to the combined effect of the constituents present in the extract. keywords: pistacia lentiscus, polyphenols, wound healing, hplc-pda-esi-ms. 3. biological activities of thymus leptobotrys and thymus satureioides, two moroccan aromatic and medicinal plants abdellah el hamdaoui1,2*, asmaa bouglad1,2, fouad msanda1, abdellah ait ben aoumar2, hassan boubaker2, yassine el maati1, abdelkhaleq el moslih1,2,3, fouad achemchem3, el hassan boudyach2, and abdelhamid el mousadik1 1 laboratoire de biotechnologies et valorisation des ressources naturelles, faculté des sciences, agadir, morocco; 2 laboratoire de biotechnologies microbiennes et protection des végétaux, faculté des sciences, agadir, morocco; 3 laboratoire des sciences de l’ingénieur et le management de l’energie, est, agadir, morocco. *presenting author: hmd_abdl@hotmail.com. abstract in the last decades, the essential oils and various extracts of plants have been of great interest as they have been the sources of natural products. the antimicrobial activities of essential oils and plant extracts constitute the basis of many applications, including food preservation, pharmaceuticals, alternative medicine and natural therapies. the moroccan flora has more than 7000 species and subspecies among which approximately 800 are aromatic and medicinal plants. these plants are a potential source of natural bioactive molecules and can contribute to the economic development of rural populations. essential oils of thymus species are classified among the most powerful substances due to their strong antimicrobial activity, particularly against pathogenic microorganisms resistant to antibiotics. thus, the aim of this study was to evaluate the antioxidant, insecticidal and antibacterial activities of essential oils of two moroccan aromatic herbs, thymus leptobotrys and thymus satureioides. the dpph radical scavenging ability and power reducing assay were used to evaluate the antioxidant activity of essential oils. while, the agar disc diffusion assay and broth macro-dilution method were employed for the determination of antibacterial activity against six bacteria, namely listeria innocua (cect 4030), lisa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 330 teria monocytogenes (cect 4032), staphylococcus aureus (cect 976), bacillus subtilis (dsm 6633), proteus vulgaris (cect 484) and pseudomonas aeruginosa (cect 118). the results showed that thymus leptobotrys oil was found the most active one as antioxidant product with an ic50 value of 0.148 mg/ml for dpph assay and 0.023 mg/ml for power reducing assay. concerning antibacterial activities, results showed that both oils inhibited growth of all the tested bacteria with minimal inhibition concentration (mic) and minimal bactericide concentration (mbc) lower than 2mg/ml. both oils showed a moderate insecticidal activity against tribolium castaneum. keywords: essential oil, aromatic and medicinal plants, biological activity, antioxidant activity, insecticidal activity, antibacterial activity. xi. session xi. us-morocco research and higher education collaboration co-chair: khalid meksem, southern illinois university, usa co-chair: james miller, macece, morocco 1. undergraduate and graduate studies in the us my abdelmajid kassem* plant genetics, genomics, and biotechnology lab, dept. of biological sciences, fayetteville state university, nc, usa. *presenting author: mkassem@uncfsu.edu. abstract in this presentation, i will discuss undergraduate and graduate studies in the us and opportunities for moroccan students to attend us institutions of higher education including community colleges, four-year colleges, and universities. emphasis will be on the graduate school and the cost of attending as an international student including tuition, room and board, and additional expenses will be discussed with detailed examples from several us institutions. opportunities for scholarships, work study, and other financial aid opportunities will also be discussed. 2. the graduate school opportunities at southern illinois university, united states karen midden*, professor, associate dean, landscape architect college of agricultural sciences, southern illinois university, carbondale, illinois, usa abstract southern illinois university (siu) graduate school has a commitment to enhance advanced education through high quality instruction, experience and research for students. siu is ranked by the carnegie foundation as being in the top 5% of all higher education research institutions in the united states, a ranking that must be earned and maintained through quality research and grantsmanship. the graduate school offers master’s degrees in over sixty programs and the doctoral degree in over thirty programs. graduate students are fortunate to study and research under the leadership of graduate faculty as they pursue advanced study leading to outstanding careers. there are ten colleges with programs and numerous departments within the colleges, one of which is the college of agricultural sciences. this college houses four departments with many disciplines within each department and they work collaboratively with outside departments. this presentation will provide general information about siu, the graduate school, requirements and opportunities to apply, study and research at siu. an overview of the colleges will be provided with more specific focus on the faculty, areas of study and highlights of past graduates of the college of agricultural sciences graduate program. 3. my life experiences in the usa oussama badad* laboratory of biotechnology and genetics, faculty of sciences, mohamed first university, oujda, morocco. *presenting author: oussama.badad@gmail.com. abstract far beyond the scientific exchange, the fulbright experience is a cultural crossroad between the united states of america and the kingdom of morocco. the moroccan students are not only research fellows they are also young ambassadors of the moroccan history and culture. the fulbright social experience was about discovering and melting into a new culture with all its components: the people, the food the music, and all the traditions. travelling north, south east and west to more the 20 states in 12 months while still doing great research, visiting the most significant places in the history of the us: capitols, white house(s), museums, battle fields, cemeteries and music studios. this experience revealed some sides of my personality that i have never seen before brought joy and tears but made me the person i am today. 4. the moroccan american commission for educational & cultural exchange (macece) mission james miller macece, rabat, morocco abstract not provided. 5. fifty years of iav in morocco mohamed bouklikhane iav hassan ii, rabat, morocco a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 331 abstract not provided. 6. networking and opportunuities khalid meksem southern illinois university, usa abstract not provided. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 332 poster presentations abstracts wednesday & thursday december 14 & 15, 2016 poster # 001. isolation and identification of entomopathogenic fungi of the mediterranean fruit fly (ceratitis capitata) ayoub hallouti1*, hicham karim1, abdelaziz zahidi2, abdelhamid el mousadik2, abdellah ait ben aoumar1, and hassan boubaker1 1 laboratory of microbial biotechnologies and plant protection, department of biologie, ibn zohr university, faculty of sciences, po box 8106, agadir, morocco ; 2 laboratory of biotechnology and valorisation of natural resources, department of biologie, ibn zohr university, faculty of science, po box 8106, agadir, morocco. *corresponding author: department of biologie, ibn zohr university, faculty of sciences, agadir, morocco. *presenting author: hallouti.ayoub@gmail.com. abstract this work forms part of biological control against crop pests and has the objectives of search, isolation and identification of entomopathogenic fungi and the assessment of their pathogenic power against the mediterranean fruit fly (ceratitis capitata). search, isolation and estimation of the abundance of entomopathogenic fungi were carried out on soil samples of argania spinosa forests and citrus orchards. to trap the insect pathogenic soil fungi, we adopted the technique of using larvae of the fruit fly (ceratitis capitata) as baits. the resulting fungal isolates are identified, based on macroscopic and microscopic criteria. after trapping, isolation and identification of fungal strains, a pathogenicity tests were used to select several fungal strains that have significant pathogenic powers against the mediterranean fruit fly. the degree of virulence was estimated based on the ability of the fungus to induce the disease and / or death in the insect and also by calculating the lethal time 50 (lt50). the results obtained demonstrated clearly the sensitivity of medfly of the different treatments tested and particularly to strains of aspergillus niger and epicoccum sp that have shown high mortality rates (more than 84%) and strains of fusarium sp, trichoderma harzianum of scedosporium sp. and ulocladium sp with more than 70% of mortality. furthermore these strains showed short lt50 (less than 83 hours). all these results confirm firstly the presence of insect pathogenic fungi of the fruit fly ceratitis capitata in the argan and citrus orchards soil, and prove at secondly the potential of insect pathogenic fungi in the fight against this pest. keywords: biological control, ceratitis capitata, entomopathogenic fungi, citrus, argania spinosa. poster #002. lipid peroxidation of argania spinosa selected lines tolerant to salt and water stress mouna lamaoui1, imane ben salah1*, raja benlaouane1, abdelghani chakhchar1, abderrahim ferradous3, said wahbi2, abdelhamid el mousadik4, saâd ibnsouda koraichi5, abdelkarim filali-maltouf6, and cherkaoui el modafar1 1 laboratoire de biotechnologie valorisation et protection des agroressources, faculté des sciences et techniques guéliz, université cadi ayyad, 40 000 marrakech, morocco; 2 laboratoire de physiologie végétale, faculté des sciences semlalia, université cadi ayyad, 40 000 marrakech, morocco; 3 centre régional de la recherche forestière marrakech. bp 12360 ain itti ennakhil, marrakech, morocco; 4 laboratoire de biotechnologie et valorisation des ressources naturelles. faculté des sciences, université ibn zohr. agadir, morocco; 5 laboratoire de biotechnologie microbienne. faculté des sciences et techniques. université sidi mohamed ben abdellah. fès, morocco; 6 laboratoire de microbiologie et biologie moléculaire. faculté des sciences. université mohammed v agdal. rabat, morocco. *presenting author: mounalamaoui@gmail.com. abstract lipids play an important role as the structural constituent of most of the cellular membranes. it is well known that free radicalinduced peroxidation of lipid membrane is a sign of stress induced damage at cellular level. therefore, the level of malondialdehyde (mda), produced during peroxidation of membrane lipids, is often used as an indicator of oxidative damage. argania spinosa (l.) callus tolerant to water and salt stress were selected. the effect of the procedure of selection followed on the levels of hydrogen peroxide (h2o2) and the degrees of the oxidative damage to the membrane lipids were examined. analysis indicated that selected calli for both salt and water stresses maintained high levels of h2o2 compared to the nonselected calli while the differences in the content of malondialdehyde (mda) was insignificant. which indicates the lower level of oxidative damage caused after the selection procedure and the mobility of antioxidant system activity in order to limit cellular damages. keywords: argania spinosa, callogenesis, salt stress, water stress, hydrogen peroxide, malondialdehyde, lipid peroxidation. this work is supported by the hassan ii academy of science and technology and the ministry of higher education and scientific research (morocco). poster #003. anti-stress molecules in plants saadia karbou1,2,3*, y. el bahloul2 , b. benaji3, m. bouksaim2, and m. taoudi benchekroun1 1 fsac, université hassan ii, casablanca, morocco; 2 inra, crra, rabat, morocco; 3 enset, rabat, morocco. *presenting author: skarbou@gmail.com. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) abstract for the adaptation to climate change, it is now essential to find new sources for food security mostly with the rapid growth of the world population. in the extreme conditions for life on earth, certain vegetal species presents a great capacity for survival in the arid climate and the sahara. this property attracts the curiosities of the scientific world to study the biological mechanisms involved in this survival power. our work will try the identify and analyze the biological molecules involved in one of the metabolic pathways of one of these species, argan which plays an essential role both in the adaptation and the product yield of this plant species. this research will help in defining the molecules involved in stress resistance in arid climates. keywords: antistress molecule, biological mechanisms, resistance, adaptation. poster #004. pheophytinization of chlorophyll in argan tree under drought stress abdelghani chakhchar1, mouna lamaoui1, imane ben salah1*, salama aissam1, abderrahim ferradous3, said wahbi2, abdelhamid el mousadik4, saâd ibnsouda koraichi5, abdelkarim filali-maltouf6, and cherkaoui el modafar1 1 laboratoire de biotechnologie valorisation et protection des agroressources, faculté des sciences et techniques guéliz, université cadi ayad, 40 000 marrakech, morocco; 2 laboratoire de physiologie végétale, faculté des sciences semlalia, université cadi ayyad, 40 000 marrakech, morocco; 3 centre régional de la recherche forestière marrakech. bp 12360 ain ittiennakhil, marrakech, morocco; 4 laboratoire de biotechnologie et valorisation des ressources naturelles. faculté des sciences, université ibn zohr. agadir, morocco; 5 laboratoire de biotechnologie microbienne. faculté des sciences et techniques. université sidi mohamed ben abdellah. fès, morocco; 6 laboratoire de microbiologie et biologie moléculaire. faculté des sciences. université mohammed v agdal. rabat, morocco. *presenting author: bensalah.imane7@gmail.com. †corresponding author: chakhchar.ckr@gmail.com. abstract the objective of this study was undertaken to characterize the pigment composition and to quantify the degradation of chlorophyll in the argan tree under drought conditions. two-year old plants taken from contrasting ecotypes were exposed to three different irrigation regimes during two months. the concentration of anthocyanin and pheophytin increased significantly in all ecotypes under drought stress (p< 0.001). however, the chl a/b ratio has decreased as the drought stress increased. in fact, our results showed high increase in percentage of pheophytinised chlorophyll suggesting that drought stress destabilize the chloroplastic pigment-protein complexes, rendering the chlorophyll susceptible to pheophytinization. the drought stress probably caused membrane disorganization in argan tree and enhanced membrane permeability, which led to a kind of cascade reaction resulting in a lower ph and promoting pheophytinization. intra-specific differences were observed in pigment composition among argan tree ecotypes studied. according to three-way anova analysis, a significant ecotype x watering regime x time interaction was recorded for the anthocyanin and pheophytin contents (p< 0.05). keywords: argan tree, drought stress, pheophytin, anthocyanin. *** this work is supported by the hassan ii academy of science and technology and the ministry of higher education and scientific research (morocco). poster #005. evaluating the eating behavior of type 1 diabetic patients receiving functional insulin therapy in the endocrinology department of errazi hospital uh med vi marrakesh asma oouzaka1*, nawal el ansari1, nabil layachi2, and ghizlane el mghari tabib3 1 head of the endocrinology department, uh med vi, marrakech, morocco; 2 uh ibn sina, rabat, morocco; 3 endocrinology department uh med vi, marrakech, morocco. abstract functional insulin therapy (fit) is a therapeutic educational method that helps ameliorate the glycemic control; it frees t1d patients from treatment constraints and improves their life quality. this study aims at evaluating the eating behavior of t1d patients receiving fit, changes in metabolic parameters, and the frequency of hypoglycemia. this is an observational study conducted in the endocrinology department of the university hospital med vi in marrakech. it covered 20 t1d patients receiving fit (60% women/ 40% men; 75% between 19 and 37 years old; 60% of all the cases have had t1d for or less than 5 years / 40% for more than 5 years; hba1c initial average : 9 (±2.05% ). concerning metabolic data: the frequency of hypoglycemia has decreased; 67% among the cases who had one session per month. a considerable modification is also noticed in the hba1c; 3 months after fit, hba1c average: 8.24 (±1.47%) and 6 months after fit, hba1c average: 7.47 (±0.8%). concerning nutritional evaluation, ½ of the participants of the current study have bmi > 25kg/m² vs bmi average: 24kg/m². current weight average: 69 (±9kg) vs ideal weight average 61.4 (±6.61kg). total energy intake average 2830 (±80.35 kcal/ day) vs energetic needs average: 2550 (±394.03 kcal/day). concerning life quality, 100% of t1d patients receiving fit are pleased to have participated in the fit as they have adapted their diet to fit their lifestyle. it is noticed that life quality of t1d patients receiving fit has improved. thanks to this method, t1d patients could consume food said to be “forbidden” for them earlier without any deterioration at the level of the glycemic control. in the end, t1d patients have truly become actors in their own health. 333 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 334 poster #006. chemical tests for available soil phosphorus nora chaouqi1,2*, m. el gharous2, z. naceri3, and m. bouzziri1 1 laboratory of applied chemistry and environment, faculty of sciences and techniques, university hassan 1st, settat, morocco; 2 inra, regional center for agricultural research, settat, morocco; 3 phosphorus-morocco, safi-direction. quality control department, safi, morocco. *presenting author: nora.chaouqi@ gmail.com. abstract the research goal was to develop a simplified method for estimating the available phosphorus for routine analysis. this study compared the measured soil-p using the icp-nahco3 with the simplified extraction method (sm-p). the correlation (r=0.99) and the regression(using xlstat-pro) were employed for comparing the data of available phosphorus content in soil samples for a variety of moroccan soil types, with contrasted physicochemical characteristics: ali moumen, oued qibane, ouled said, settat, dower lhfaya, and had ghoualem (are located using arcgis 10.1 and fertimap). sm-p is most suited for soils with ph >= 7 and caco3 content above 5%. in this experiment, several parameters are modified, the fineness, the type and degree of mechanical agitation, the color development solution (2.5% (nh4)6mo7o24.4h2o, in 5 mol.l-1 h2so4), 1 %(w/v) ascorbic acid solution), and the adaptation of the reading at 860 nm. the accuracy of p analysis was insured, and the high correlation of this method with icp-nahco3 content indicated as such. the results of this experiment showed that sm-p can be the best method for predicting the available phosphorus, simple, quick, and easy to execute. keywords: fertilizer phosphate, extraction methods, available phosphorus, moroccan soils poster #007. micropropagation of thymus broussonetii boiss., threatened medicianl and aromatic plant in morocco zineb nejjar el ansari, brahim el bouzdoudi*, rajae benkaddour, ibtissam boussaoudi, and ahmed lamarti laboratory of plant biotechnology, department of biology, faculty of sciences, abdelmalek essaadi university, m’hannech ii, b.p.2121, tetouan, morocco. *corresponding and presenting author: bchiri2008@gmail.com. abstract within the labiate family, there are about 220 genera, and the genus thymus is one of the eight most important genera with regard to the number of species included, although this number varies depending on the taxonomical point of view. in morocco, 15 species of this genus have been identified. these are rare, endemic and endangered species. indeed, people used intensively and exploited haphazardly thyme populations. moreover, the interests of the pharmaceutical and food industries focus on specific chemotypes especially thymol. in such a situation, cultivated thyme represent an indispensable alternative, via the selection of endangered species and the implementation of culture techniques under modified environmental conditions (ex vitro culture) or controlled conditions in laboratories (in vitro culture). in vitro vegetative multiplication is a good method for reproduction of genotypes and selected chemotypes of certain species. plants cultivated in vitro can be used for many purposes and then collection from their natural shelters could be avoided, also, model systems to study the production and accumulation of metabolites with a great interest could be developed. the present study reports an efficient protocol for in vitro clonal propagation of thymus broussonetii boiss., threatened medicinal and aromatic plant in morocco. initially, seeds collected from wild plants were used for in vitro culture establishment on gautheret basal salts medium without plant growth regulators. then, aseptic and living explants were transferred to shah and dalal medium for culture multiplication. afterwards, we have selected one stable clone (the longest one) and we have evaluated the effect of six macronutrients. after that, seven cytokinins in different concentrations have been evaluated. moreover, the effect of three polyamines has been investigated. thereby, seeds germination started from the fourth day with a survival rate of 24-28%. furthermore, margara medium has been proved the most favorable for plantlets growing. higher number of buds (24.083±1.401) was obtained on maragara medium supplemented with 0.93 µm of dpu, it also ensures good shoot multiplication with an average number of 1.833±0.177 and good root development with an average number of 8.208±0.942 and a length of 1.254±0.085 cm. also, 5 µm of spermine gives higher number of buds (26.833±1.789), a good shoot multiplication (1.958±0.153) and a good root development (an average number of 8.917±0.782 and a length of 1.821±0.120 cm). plantlets with well developed roots were successfully acclimatized to ex vitro conditions and an in vitro propagation was again established from the acclimatized and well grown plants. the in vitro culture system successfully established for thymus broussonetii boiss. offers a viable tool for mass micropropagation and conservation of this very threatened species. keywords: medicinal and aromatic plants, thymus broussonetii, micropropagation, cytokinins, polyamines. poster #008. artificial regeneration of cork oak: difference of root system btissame bouther1,2*, y. abbas3, m. el ouajdi2, and m. arahou1 1 faculty of science of rabat, university mohamed v, rabat, morocco; 2 forest research centre rabat, morocco; 3 faculty of science of beni-mellal, morocco. *presenting author: bouther. btissame@gmail.com. abstract nature is a source of life and survival condition but the man who lived in communion with nature has become a danger for it. he recognizes its value since the rift which was created between him and his environment by the industrialization and the a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 335 multiple aggressions. since the beginning of the 20th century, the regeneration of cork oak has always been a problematic in the forest of maâmora. despite of all efforts to the preservation and conservation of the forest, ecosystems were constantly degraded over time. several tests of regeneration have been initiated in recent decades by acorn or by plant. although less studied than the aerial parts, the root system of the cork oak has been the work of the forest research centre (frc rabat). if successful plantation is based first and foremost on the choice of species and genetic origins adapted to the plot to restore the quality of introduced plants also influences the recovery of plantations, in this context, our study is based on the production of plants of cork oak from different origins and to compare between them which is the most adapted and resistant for the climate change. also, we wanted to study the root system and see the differences between the origins of cork oak. keywords: cork oak, root system, climate change, ecosystem, maâmora, artificial regeneration. poster #009. synthesis and characterization of acrylonitrile and polyacrylic acid membranes for the water treatment equipment ely cheikh mahmoud* laboratory of water and environment, dept. of chemistry, faculty of sciences, chouaib doukkali university, el-jadida, morocco. *presenting author: cheikhatti@gmail.com. abstract today, water sources are overused and poorly protected in many parts of the world. demography and human activity provide increase in water demand while misuse of water resources can also alter availability of clean water. membrane processes are increasingly used in environmental protection including the production of drinking water and medical applications. here we present a simple and effective method for the manufacture of ion exchange ultrafiltration membranes for removal of water pollutants. for synthesis of membranes, poly acrylonitrile (an69) and poly acid acrylic (paa) were dissociated separately in the solvent n, n-diméthylformamid (dmf), and mixed in a known portion. after the two polymers are blended, poured films are then placed on a glass plate and brought to defined temperature and duration. the ftir spectrum of the membrane an69 / paa shows a band at 1683 cm-1 that can be assigned to the vibration of the co group indicating the presence of paa and a peak very end to 2239 cm-1. this shows the presence function cn in structure of an69, the spectrum also rose moderately broadband in 1174 cm-1 which correspond to the groups asymmetrical sulfonate so3na. later we will study the morphology of the membranes using sem and om. poster #010. production of saffron corms (crocus sativus l., iridaceae) by in vitro culture rajae benkaddour1*, amal rahmouni1, mounira lage2, and ahmed lamart1 1 laboratory of plant biotechnology, department of biology, faculty of sciences, abdelmalek essaadi university, m’hannech ii, b.p.2121, tetouan, morocco; 2 department of plant breeding and laboratory of physiology (inra), bp. 415, avenue la victoire, rabat, morocco. *presenting author: benkaddourrajae27@gmail.com. abstract our purpose is to produce on a large scale of viable saffron microcorms to meet the needs farmers. we tested a cytokinin (kinetin) in three concentrations (6, 9 and 12 mg/l) and an auxin (2 4dichlorophenoxyacetic acid, 2 4-d) at 0.1 mg/l on the direct production of microcorms within slices after 6 months of culture. in the darkness, explants react to 100%, but to the light, this percentage decreases in 52% (for 6 mg/l of kinetin) and in 92% (for 12 mg/l of kinetin). the number of microcorms formed by explant is not influenced by the dose of the kinetin; it is one for explant cultivated in the light and of two for those placed in the darkness. the diameter of microcorm is maximal in the darkness (14.6 ± 1.21 mm) in the presence of 12 mg/l of kinetin. however, it is minimal in the light (6.9 ± 0.4 mm). the combination of two other plant regulators, 2,4-d in 0.1 mg/l and ba with various concentrations (6, 9, and 12 mg/l) does not lead formation of microcorm in the light (0%). but, the explants placed in the darkness are 100% reactive and form a single microcorm of diameter between 2.8 ± 0.71 mm (ba in 6 mg/l) and 3.8 ± 1.03 mm (ba in 12 mg/l). lastly, microcorms were grown in a hydroponic room (photoperiod 16 hours light / 8 hours darkness; 25°c), and after 5 weeks, 100% of microcorms germinated. keywords: saffron, crocus sativus l., corm production, tissu culture. poster #011. in vitro inhibitory effect of fungicides against colletotrichum acutatum, the causing agent of strawberry anthracnose rababe es-soufi*, mohamed l’bachir el kbiach, and ahmed lamarti laboratory of plant biotechnology, department of biology, faculty of sciences, abdelmalek essaadi university, m’hannech ii, b.p.2121, tetouan, morocco. abstract anthracnose is one of the most important strawberry (fragaria x ananassa duch.) diseases and can be a limiting factor for cultivation, because of its devastating nature, the sensitivity of cultivars and little available control. since 2012, three fungicides basultra, thiramchim and thiramic (based on thiram) were a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 336 approved for agricultural use by onssa (database, approval of chemical inputs). to prevent this disease, farmers in loukous area use a product based on copper (bordeaux caffaro) as a plant protector. the aim of this study is to determine the impact of these fungicides on the different stages of the development of colletotrichum acutatum. c. acutatum has been collected from strawberries attacked with anthracnose and purified in laboratory of plant biotechnology in the faculty of sciences of tetouan. the study of the inhibitory effect of the fungicides cited above against colletotrichum acutatum has shown that the thiram has a big effectiveness on germination and moderately effective on mycelial growth and sporulation. copper has shown effectiveness on germination as variable activity in other life stages. keywords: strawberry antracnose, fungicides, thiram, colletotrichum acutatum, inhibitory effect. poster #012. determination of total polyphenols and gallic acid content in carob pulp ibtissam boussaoudi*, brahim el bouzdoudi, and ahmed lamarti laboratory of plant biotechnology, department of biology, faculty of sciences, abdelmalek essaadi university, m’hannech ii, b.p. 2121, tetouan, morocco. *presenting author: ibtissam.boussaoudi@gmail.com. abstract extraction effeciency of total polyphenols (tp) of carob pods (ceratonia siliqua l., leguminosae family) was examinated by performing extractions with various solvent systems, in order to evaluate and optimize the conditions for the recovery of polyphenols. maximum quantities of polyphenolic components were found in 80% acetone extracts. by contrast, water was inefficient in extracting polyphanols. total polyphenol compounds were identified with a yield of 11.19 g/kg (dry weight) as evaluated by measuring total polyphenol content using the folin-ciocalteu method. the profile was dominated by gallic acid (45.01 % of polyphenols by dry weight). carob leaves and the diffrent parts of fruits were also analyzed for their total polyphenols and gallic acid content using hplc. it was presumed that the leaves contain high values of phenolic compounds and gallic acid (45.26 g/kg and 17.012 g/kg, respectively. tegument contained appreciable amounts of polyphenols and gallic acid (26.30 g/kg and 2.512 g/kg of gallic acid), while only traces were detected in germ (germ meal) and endosperm (1.33 g/kg and 0.80 g/ kg tp ; 0.997 g/kg and 0.715 g/kg of gallic acid respectively). it should be noted that there are some additional phenolic compounds present whose structures still need to be determined. keywords: carob tree, ceratnoia siliqua l., leguminosae, extraction, polyphenols, gallic acid, hplc. poster #013. use of feces micro-histological analysis to study atlas deer diet (cervus elaphus barbarous) in tazekka national park (province of taza, morocco) brahim ismaili* and mohammed diouri moulay ismail university, meknes, morocco abstract atlas deer is a flagship species in morocco. it was reintroduced in tazekka national park in 1994 to rehabilitate the natural state in the regions of middle atlas and rif. very little research has been made on this species and its diet in morocco. therefore, a diet study was recommended by the national strategy for ungulates in order to get useful data for the feasibility of a subsequent release. in this context, we aimed at studying the diet of atlas deer and its seasonal variation. feces were collected, during the years 2013 and 2014, in bab klati 520-ha reserve located in the west of the aforementioned park. the collection was performed in the existing four types of environments: that of quercus faginea, that of quercus suber, scrublands and clearings. fecal samples were micro-histologically analyzed based on a reference epidermis catalog of all existing plants in the reserve. poaceae species, the main representatives of the herbaceous category, were consumed at 28%, 37% and 43% of the diet in autumn, winter and spring, respectively. in summer, their consumption did not exceed 2%; because of their limited availability and the vegetation drying out. pteridium aqualinium was consumed especially in summer (6%). shrubs were represented by three main species: ulex boivinii and cytisus triflorus (paplionaceae) and lavandula steochas (lamiaceae). consumption of u. boivinii was high in autumn (41%) and spring (31%) and low in winter (16%) and summer (6%). as for c. triflorus, the consumption was maximal in winter (30%), average in autumn (19%) and spring (18%) and lowest in summer (2%). l. steochas was consumed mainly in autumn (8%). during the summer, trees were the main component of the diet and were represented by the oak species q. faginea (61%), q. rotandifolia (13%) and q. suber (5%). keywords: tazekka national park, diet, atlas deer, micro-histology, feces, morocco. poster #014. induction of secondary somatic embryogenesis of moroccan cork oak (quercus suber l.) safaâ rahmouni*, brahim el bouzdoudi, mohamed l’bachir el kbiach, and ahmed lamarti laboratory of plant biotechnology, department of biology, faculty of sciences, abdelmalek essaadi university, m’hannech ii, b.p.2121, tetouan, morocco. *presenting author: safaarahmouni@hotmail.fr. abstract cork oak (quercus suber l., fagaceae) is one of the most important species of the mediterranean basin due to its ecologia tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 337 cal and socio-economical interests. nevertheless, the increasing demand for cork and the low natural regeneration provides for a rapid loss of cork oak forests. vegetative propagation of trees by somatic embryogenesis contributes to the regeneration of forests and their development. this technique allows the formation of a high number of somatic embryos (large scale multiplication). our study consists in testing the somatic embryogenesis technique on moroccan cork oak (quercus suber l.). we have induced primary somatic embryogenesis from leaves obtained from epicormic shoots forced to sprout from segments of branches of quercus suber l. branches from quercus suber l. trees were sterilized and cultured in specific conditions. after 7 days of culture, branches began to emit epicormic shoots. secondary, leaves obtained from epicormic shoots were used for induction of somatic embryogenesis. after series of transfers in culture media on various nutritive mediums with or without plant growth regulators (ana and ba), primary somatic embryos were obtained. these embryos were multiplied by secondary somatic embryogenesis by a number of factors. keywords: cork oak, quercus suber l., fagaceae, secondary somatic embryogenesis. poster #015. the impact of organic and biological amendements on date palm seedlings salt stress tolerance mohamed ait el mokhtar*, said wahbi, raja benlaouane, and abdelilah meddich† laboratory of biotechnology and plant physiology, faculty of sciences, university cadi ayyad, marrakech, morocco. *presenting author: mohamed.aitelmokhtar@gmail.com. †corresponding author: a.meddich@uca.ma. abstract the date palm has suffered from the devastating effects of multiple constraints including salt stress. in this study we will evaluate the impact of two amendments, namely compost and mycorrhizal fungi (amf) on tolerance of the date palm seedlings under salt stress. six treatments were applied: compost, amf and compost + amf with (14 g/l of nacl) and without (0 g/l of nacl) salt stress. after 8 months of mycorrhization (5 months from the application of salt stress), amf and compost seems to have a positive effect on tolerance of the date palm subjected to salt stress compared to control. as a matter of fact, the two amendments, separate or combined, enhance growth and water parameters of the date palm with a high efficiency of the combination of the two amendments. in the absence of salt stress, the growth parameters (aerial elongation, leaf area, shoot and root dry matter) were improved by compost 54%, by amf 77% and by compost + amf 100% compared to control. the water parameters (stomatal conductance, content water and water potential) were improved by compost 95%, by amf 168% and by compost + amf 224%. under salt stress, the growth parameters were enhanced by compost 57%, by amf 100% and by compost + amf 117% compared to control. the water parameters were enhanced by compost 140%, by amf 202% and by compost + amf 230%. keywords: date palm, compost, mycorrhizal fungi, salt stress. poster #016. effect of salt and water stress on morphological parameters, physiological and agronomic of four medicinal plants mohammed chetouani*, i. mzabri, a. aamar, a. boukroute, n. kouddane, and a. berrichi laboratoire de biologie des plantes et des microorganismes, faculté des sciences, université mohamed premier, b.p. 717, oujda 60.000, morocco. *presenting author : chetouanimohammed@gmail.com abstract salinity is a common factor in the environmental stress seriously affecting crop production in different regions of the world. morocco, which is known by the scarcity of rainfall in semi-arid and arid regions accentuate the salinization of irrigated areas and make them unsuitable for crops. under these conditions, changes in mophophysiological and biochemical characteristics (leaf water potential, chlorophyll fluorescence, relative water content and sugar proline content) are effective parameters sensing plant response vis-à-vis the stress, and also realize the water status of the plant and its ability to incorporate water, especially under stress conditions. as part of this approach and to explore the effect of salt stress on the behavior of the plant, we are interested in four medicinal and aromatic plants, namely salvia officinalis, cytrus cymbopogon, lavandula dentata and rosmarinus officinalis have various agronomic and socio-economic interests in the eastern region. nacl concentrations were used in order to assess salt stress and essential oil and its chemical composition. the preliminary results show that these plants are resistant to salt stress for lower doses of 100 mm (0.086mm) nacl beyond this value the plants wither. poster #017. effect of environmental conditions on morphological characteristics of saffron (crocus sativus l.) moroccan cultivars salaka latifa*, m ben el caid, k lagram, m lachheb, lh atyane, a el mousadik, m fallah, and ma serghini laboratory of biotechnology and valorization of natural resources, ibn zohr university, sciences faculty, agadir, 80000, morocco. *presenting author: latifasalaka@gmail.com. abstract saffron is the most expensive spice in the world. in morocco, saffron cultivation is localized in the taliouine-tazanakht region. the depth knowledge of moroccan saffron cultivars and the selection of those with good performances constitute a solid base for the development of this sector and the conservation of this plant. this study aims to evaluate the morphological diversity of taliouine’s saffron cultivars in two sites and to select those with a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 338 the best quality. to reach this objective, corms of saffron from four provenances (sidi hssaine, agadir melloul, zagmouzen and askaouen) were planted in two different sites (taliouine and faculty of science, agadir (fsa) and were followed for four years. the measured morphological parameters are the number, weight and length of different plant organs, namely, corms, leaves, flowers and stigmas. the multiple analyses highlighted significant differences between saffron provenances for different parameters. the number of flowers produced depends significantly on the corms provenance; it can be five times more important. the length, fresh and dry weight of their stigmas can show a difference 6 times higher. this agro-morphological characterization has revealed the existence of significant morphological variability within the moroccan saffron. this variability exists despite of the mode of vegetative propagation of the species. during the 4 years of study, the rate of corm proliferation and stigma production now better understood. keywords: saffron, crocus sativus l., morphological characterization, cultivars. poster #018. improved micropropagation method for in vitro cormlet production through indirect organogenesis of moroccan saffron (crocus sativus l.) lagram khalid*, m. ben el caid, l.h. atyane, l. slaka, m. lachhab, r. el boulani, a. el mousadik, and m.a. seghrini laboratory of biotechnology and valorization of natural resources, sciences faculty, ibn zohr university, agadir, morocco. *corresponding and presenting author: lagram.khalid@gmail. com. abstract saffron is the most expensive spice in the world. this spice, also called ‘red gold’, consists of the dried stigmas of crocus sativus l. it is a male-sterile and triploid plant (2n = 3x = 24) which belongs to irridaceae family. it spreads vegetatively thought the formation of daughters’ corms. saffron is popular for its color, taste and aroma property due to its essential metabolites that are respectively crocin, picrocrocin and safranal. the auto-triploid nature of saffron creates problems for sexual reproduction and renders improvement by breeding practically impossible. application of biotechnology, especially tissue culture, can improve the quality and quantity of the saffron product, by the large production of healthy selected saffron corms. in this work, an in vitro regeneration protocol was optimized through indirect organogenesis for the efficient multiplication of selected moroccan saffron cultivars. the calluses had been initiated after one month on ms medium supplemented with 3% sucrose, 100 mg/l ascorbic acid and different combinations of pgrs. as result, we noted that the combination of 1 mg/l bap and 1 mg/l naa was efficient for callus initiation from the rectangular sections of upper part of corms tissue. additionally, for shoots formation from calluses, maximum rates were founded in the combination of 2 mg/l tdz with 0.5 mg/l naa. shoots obtained previously from callus, were then transferred to the corms and roots induction medium. as a result, we observed the formation of adventitious corms and roots in ½ ms medium under the obscurity. keywords: corms, in vitro, organogenesis, crocus sativus, callus, propagation, tissue culture. poster #019. molecular identification of moroccan saffron (crocus sativus l.) with microsatellite markers mohamed lachheb*, m. ben el caid, l. salaka, k. lagram, a. el mousadik, and m.a. serghini laboratory of biotechnology and valorization of natural resources (lbvrn), faculty of sciences, agadir, morocco. *presenting author: lachheb.mohamed91@gmail.com abstract crocus sativus l. is a male sterile plant. its flower produces stigmas that when dried, form the most expensive spice in the world commonly known as ‘red gold’. the saffron name is usually used to refer both to the spice and the plant itself. in morocco, saffron is primarily cultivated in the area of taliouine/taznakht. the improvement of the moroccan saffron imposes it’s morphological, biochemical and molecular characterization. the present study consists of an investigation and an evaluation of the genetic variability of moroccan saffron by molecular markers. it aims to develop its molecular identity and to get a database allowing better management, selection, exploitation and protection of natural heritage. samples were collected from four different selected areas (provenances) in the region of taliouine/taznakht: sidi hssaine, agadir melloul, taznakht and askaouen. genomic dna, was extracted from 40mg of leaves, using ctab method and it has used as the matrix for pcr amplification. the microsatellite markers were selected for the identification and analysis of the genetic variability of cultivars of the moroccan saffron, taking in account their large polymorphism, their specificity, their codominance and their reproducibility and stability. three hundred and fifty nine (359) est sequences of genomic dna of the saffron were collected from the national center for biotechnology information (cnib). the ssrit software has allowed the identification of 12 est-ssr (ssre) and 10 ssr from the genomic dna (ssrg). the specific primers for each ssr region were determined by the software primer 3 version 4.0.0. in addition, 12 pairs of primers developed by nemati et al. (2012) among the iranian saffron and three pairs of specific primers of iris ensata, were also selected for the molecular characterization of the moroccan saffron. preliminary results show that moroccan saffron is genetically different compared to the iranian saffron and the provenance of sidi hssaine is not only agromorphologically but also genetically different compared to the other provenances. this data is invaluable for the improvement of the productivity of moroccan saffron via ssr markers assisted selection. keywords: microsatellite, saffron (crocus sativus), molecular markers, genetic diversity. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 339 poster #020. evaluation and comparison of standards of quality of the plants of the thuja tetraclinis articulata vahl masters products in three moroccan forest nurseries amal el khaddari¹,²*, mohamed ouajdi¹, saloua el antry¹, jamila dahmani², zine el abidine³, and abbas younes¹ ¹ centre nationale de la recherche forestières rue omar ibn al khattab bp 763,10050 rabat-agdal, morocco; ² laboratoire de botanique, biotechnologie et protection des plantes université ibn tofail faculté des sciences bp. 133 kenitra, morocco; ³ ecole nationale forestière des ingénieures. bp. 511, tabriquet, salé, morocco; 4 université sultan moulay slimane, faculté poly disciplinaire de béni mellal, département de biologie-géologie, mghila b.p. 592, béni mellal, morocco. *presenting author: elkhaddaribiologiste@gmail.com. abstract the thuja (tetraclinis articulata vahl masters) is an endemic species of morocco; it occupies the fourth most important place after the aleppo pine, the green oak, and the cork oak. it presents a great ecological interest which contributes to the socio-economic development of the country. however, it is threatened as a result of multiple factors related to climate change and human activities. in this regard to preserve this species, the office of the high commissioner to the waters and forests has undertaken a few strategic measures by installing a large number of forest nurseries in morocco in order to rectify the faults recorded in relation to the natural regeneration of this species. however, the results on the grounds of reforestation remain unsatisfactory and often, it assigns this failure to the poor quality of the plants produced in nurseries. there is interest in producing quality plants in order to optimize their rate of regeneration once put in the soil. in this context, three different moroccan nurseries (sidi yahia, bni souhane and droua) were the subject of this study by comparing the quality attributes of plants products. for each nursery, 21 plants were sampled. the morphological characters were assessed through measures of height, root collar diameter and biomass. the results obtained from the different settings have found that the production of seedlings of red cedar differs from one nursery to another. also, the nursery of sidi yahia is the one that produces the highest quality compared to other nurseries. but this result does not justify that this nursery is the most effective in terms of plant production. it was found that the plants products are not balanced, which reflects failures at the level of the technical measures adopted during their cultuvation (watering, fertilization…). it would be essential to review production methods of red cedar seedlings in the nursery to propose other more effective standards that can be used to better assess and enhance this production. keywords: thuja (tetraclinis araticulata), comparison,, growth, height, diameter, nursery poster #021. biofilm formation of lactic acid bacteria on stainless steel surfaces mariem zanzan1*, f. achemchem1, f. hamadi2, h. latrache3, l. amzil2, a. elmoslih2, and r. mimouni2 1 laboratoire de sciences de l’ingenieur et managment de l’energie, ecole national de sciences appliquées, agadir, morocco; 2 laboratoire de biotechnologie et valorisation des ressources naturelles, faculté des sciences, université ibn zohr, agadir, morocco; 3 laboratoire de bioprocédés et biointerfaces, faculté des sciences et techniques, université sultan moulay slimane, béni-mellal. *presenting author : mariem.zanzan@ gmail.com. abstract the biofilm formation of pathogenic bacteria creates major problems in food industry such as food contamination and materials deterioration. in the literature we found some successful examples of using nonpathogenic bacteria generally recognized as safe (gras) to ensure food safety and to protect against biofilms formed by undesirable spoilage and/or pathogenic bacteria. the aims of this work were, to evaluate the ability of thirteen lactic acid bacteria to form biofilm on abiotic surfaces (stainless steel 316 and stainless steel 304), and to determine the physicochemical properties of substratum and bacterial surfaces using contact angle measurements. the results obtained show that all lactic acid bacteria have higher ability to form biofilm on two substratum. keywords: lactic acid bacteria, biofilm, stainless steel, physicochemical properties. poster #022. potential antagonism of some trichoderma strains isolated from moroccan soil against three phytopathogenic fungi of great economic importance (fusarium oxysporum, verticillium dahlia, and rhizoctonia solani) wafaa mokhtari1*, n. chtaina1, e. halmschlager2, h. volgmayr3, c. stauffer2, and w. jaklitsch3 1 institute of agronomy and veterinary sciences, environment and pesticides laboratory, production, protection and biotechnology department, rabat, morocco; 2 university of natural resources and life science, institute of forest entomology, forest pathology and forest protection (ifff), hasenauerstrasse 38, 1190 vienna, austria; 3 university of vienna, department of systematic and evolutionary botany, faculty centre ofbiodiversity, rennweg 14, 1030 vienna, austria. abstract in this study, 17 trichoderma strains were isolated from different soils (crop fields and argan forests) in morocco. purified singlespore cultures were identified to species-level using molecular methods and tested for their potential antagonism against three phytopathogenic fungi of great importance in morocco (fusarium oxyxporum, verticillium dahlia and rhizoctonia solani). after a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 340 dna extraction translation elongation factor (tef1) was amplified in extracts of 17 strains, sequenced and compared with their ex-types. as a result, three species were identified among the strains, which clustered in two different subclades of trichoderma. the species, t. afroharzianum and t. guizhouense, belong to the harzianum clade, while t. longibrachiatum belongs to the longibrachiatum clade. investigation of potential antagonistic effects of these strains against the soil-borne phytopathogens fusarium oxysporum, rhizoctonia solani and verticilium dahliae was conducted in a dual culture plate assay. all trichoderma isolates showed effective antagonistic performance by decreasing ra: radial of pathogens’ mycelium confronting trichoderma isolates. trichoderma afroharzianum showed significant differences when comparing control radii rc with the radii confronting antagonist ra (p< 0.05). the highest percentage inhibition of radial growth (pirg, 80.64 %) was obtained for isolate t2.1 against fusarium. in comparison, t9i12 which is a reesei species shows a high radial inhibition of pathogens’ mycelium. keywords: trichoderma spp., fusarium oxysporum, verticillium dahliae, rhizoctoniasolani, antagonistic fungi. poster #023. nitrogen, phosphorus and potassium exports assessment in wheat and validation model ‘nutrient expert’ for the recommendation of fertilizers in rain fed and irrigated condition in morocco hanaa bellasri1*, m. elgharouss2, a. ibnlfassi1, and h. boulal2 1 laboratory of environmental sciences and development, faculty of science and techniques, university hassan 1 , casablanca road , km3.5 , bp577, 26000, settat, morocco; 2 international plant nutrition institute (ipni). *presenting author: hanaa.bellasri@gmail.com. *presenting author: abstract wheat is the second most important cereal in morocco after barley. it is grown primarily under rainfed conditions. in morocco, grain production varies from less than 10 to more than 70 quintals / ha and this wide range occurs due to prevailing climatic conditions and cultivation techniques. fertilization contributes to over 50% improvement to wheat production. also, surveys of farmers in four regions of morocco namely abda, chaouia, tadla and sais showed that the quantities of fertilizers applied to wheat is generally weak and unbalanced which affects the quantity and quality yields. fertilization is an important step in the production of wheat and must be best used to improve land productivity and water use efficiency, and to lower the damage to soil and groundwater. thus, in response to the increased cost of fertilizer and the awareness of the risks of environmental degradation, the fertilizer must be adapted and adjusted better to the soil characteristics and culture. farmers must adopt the concept ‘4b’ which is based on providing the right fertilizer at the right dose at the right time and place. the main objectives of our research are using the best fertilization practices for wheat in rainfed and irrigated areas of morocco under the concept ‘4b’. this study assesses nitrogen, phosphorus and potassium, and validation of the recommendation model fertilizers ‘nutrients expert’ at the farmer field locations in four regions of morocco: abda, chaouia, saïs and tadla. field trials were conducted using seven treatments, contribution of npk, contribution of pk (-n ), contribution of nk (-p ), contribution of np(k ), recommendations based on the soil analysis, recommendations based on the model ‘ nutrient expert ‘ and the practice of the farmer. keywords: fertilization, wheat, nutrient expert. poster #024. identification of mineral content of 8 khalts and varieties of date palm in south of morocco abderrahim alahyane1*, jamal ayour1, ahmed ait oubahou2, and mohamed benichou1 1 food sciences laboratory; faculty of science semlalia; cadi ayyad university marrakech, morocco; 2 horticultural laboratory; agricultural and veterinary institute hassan ii complex horticulture agadir, morocco. *presenting authro: ab.alahyane@ gmail.com. abstract the date palm (phoenix dactylifera l) is one of the oldest fruit species in morocco with high ecological significance and socioeconomic impact. dates, are sources of minerals that could constitute a significant raw material for food. many studies have focused on the nutrition and agro-industrial characterization of moroccan dates from the known varieties of date palm (phoenix dactylifera) while those dedicated to the characterization of unknown clones called khalts remain far fewer. the objective of our work was to determine the mineral composition by icp of eight cultivars “khalts and varieties” of southern morocco. our study included a comparison of these fruits in minerals content. the pulp of the eight fruits studied was more rich in minerals (calcium, magnesium, potassium, iron, manganese) than some widely consumed fruits. the content of some trace elements (zinc, copper) has not exceeded the toxic threshold, indicating that the consumption of these fruits would not be detrimental to public health. the results of the analyzes are very promising and deserve further investigation in order to select clones with great nutritional and health interest and may also be valued at the agro-industrial level. keywords: date palm; fruit quality; minerals; valuation. poster #025. performance of a solar pumping system harkani assia1*, a. el aissaoui2, and h. fihri fassi1 1 laboratory of engineering, industrial management and innovation fst settat, morocco; 2 laboratory of agricultural machinery, inra settat, morocco. *presenting author: a.harkani@uhp. ac.ma. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 341 abstract in the last few years, photovoltaic energy sector has consistently grown in several countries, due to its economic and environmental benefits. nowadays, economic and social development in morocco is based on improving water resources management and promoting efficient irrigation technologies as a part of a global and integrated policy development. this study aims to evaluate the performance of a solar pumping system using maximum power tracking technology (mppt) according to variability of the daily solar radiation and its impact on the pump response. the evaluation of mppt behavior showed that its efficiency kept low (80%) according to deficiency of its algorithm to improve the outputs of the photovoltaic panel (pu-max = 300w) and the dc diaphragm pump shurflo (p= 60w). keywords: photovoltaic energy, mppt , solar irradiation, dc pump, effeciency. poster #026. monitoring and microbiological bioremediation of pesticides in soils of fez-meknes region of morocco mohamed maldani*, f. aliyat, o. zennouhi, l. nassiri, and j. ibijbijen environment & soil microbiology unit, faculty of sciences, moulay ismail university b.p. 11201 zitoune, meknes, morocco. *presenting author: maldani.mohamed@hotmail.com. abstract for decades, the majority of modern crop protection practices were based on the use of chemicals for the plant protection. the excessive use of chemicals was considered indispensable for the success of agricultural development which has the aim to increase food production. despite their importance, the misuse of chemicals can cause direct and indirect threats for humans, animals and for the entire environmental system. these risks are related to the chemicals that are toxic and can have a negative impact on the environment. the soil is oftentimes the portion of the environmental ecosystem that is the most threatened by chemical contamination. soils are very low renewable resource, and although contamination can be fast; however, it recives time to regenerate. this contamination or pollution directly influences (runoff or leaching by seepage) water (groundwater or surface: rivers, lakes, ground water, etc). once these two essential components of the environmental ecosystem are contaminated by chemical inputs, the rest of the components will also be affected. man is affected either directly by consuming contaminated products or indirectly through intermediate channels. according to who the increase of the diseases rate (such as colorectal cancer, leukemia, allergies, parkinson, etc.) can be caused by environmental pollution mainly by chemical inputs. the meknes region is one of the largest agricultural areas in morocco which explain the excessive use of chemicals. in this context, and with the aim to participate to the sustainable development of agricultural practices, we have to find a new alternative based on the study of bacterial strains able to degrade the active molecules of the chemical products. these bacterial strains will present an important biotechnological application for the bioremediation of the contaminated soils. keywords: soil; chemicals; pollution; bacteria; bioremediation. poster #027. evaluation of seed production of interspecific wild and cultivated crosses of beets siham oumous1,3*, ilham rahmouni1, ghizlane tobi1, hikmat tahiri3, yasmina el bahloul1† 1 national institute of agronomic research (inra), genetic resources and plant breeding unit, crra-rabat, bp 6570, 10101 rabat instituts, morocco; 2 laboratory eco valuation, crra-rabat, bp 6570, 10101 rabat instituts, morocco; 3 mohamed v university, faculty of sciences rabat, morocco. *presenting author: . †corresponding author: yasmina.elbahloul@inra.org.ma. abstract the genetic structure of species is the primary way to improve sugar beet genetic potential. these plant genetic resources are an important genetic reservoir used to supply sugar beet germplasm via inter-specific crosses through the transfer of genes of interest. selected offspring genotypes have a remarkable interest from a food perspective, for biotic and abiotic stress tolerance. thirteen crosses have been realized in the experimental field of merchouch a favorable site for vernalization required for the sugar beet seed production. results showed successful crosses between wild and cultivated beets with a yield per plant ranging from 50 to 80g. evaluation based on morphological and genetic traits showed a difference in inherited characters between the crosses according to the parent genotypes. hybrids issued from the selected crosses are being evaluated for ploidy and biotic stress résistance. keywords: interspecific crosses, genetic structuration, beet populations, sugar beet, wild beet. poster #028. the use of citrus aurantium peel powder to fight against callosobruchus maculatus (coleoptera, bruchinae) lalla fatima douiri1*, nada lafkih1, yasmine ghouati2, hayat bouhareb1, chakir meryem3, mohieddine moumni1 1 department of biology sciences faculty, moulay ismail university, p.b 11201, meknès, morocco; 2 food technology and biochemistry unit, national school of agriculture, meknès, morocco; 3 department of biology sciences faculty, ibn tofail university, kenitra, morocco. *presenting author: fatimadouiri@yahoo.fr. abstract aromatic and medicinal plants are used not only as a source of medical care but also to fight against many pests including callosobruchus maculatus, pest of legume seeds. during the storage process, most vegetables’ seeds are affected by several pests such as callosobruchus maculatus. this study focuses on the inseca tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 342 ticidal activity of citrus aurantium powders against c. macualtus feeding on cicer arietinum seeds. a laboratory experiment was carried out under controlled conditions (27°c, 70 ±5% relative humidity and 12 hours of light). the results obtained for the studied parameters indicate that powders extrated from the orange peel have an insecticidal activity against weevils. after (1 to 5) days exposure the lowest lc50 and lc99 range respectively from 74.59 to 2.19 and 257.24 to 16,52 mg/50 graines for females and 42.06 to 0,99 mg/50 graines and from 89,79 to 15.74 mg/50graines for males. in fact, en effet, longevity obtained with the control is 9,1 to 7,73 days, for males 5 to 3,7days and for females de 6 à 4,7days. the citrus powders exert negative effects on all the parameters of the weevil, and can be used against c. maculatus. in addition, they are derived from botanical products, biodegradable, renewable source, economic and have low environmental impact which is often undetectable. keywords: citrus aurantium, cicer arietinum, powders, callosobruchus maculatus. poster #029. lipid and fatty acid composition of longissumus dorsi muscle of béni-guil sheep fed on different dryland forages in eastern morocco kamal belhaj1*, farid mansouri1, abdessamad ben moumen1, marianne sindic2, marie-laure fauconnier3, mohamed boukharta1, hana serghini-caid1, ahmed elamrani1 1 laboratoire de biologie des plantes et des micro-organismes, faculté des sciences, université mohamed ier, oujda; morocco; 2 laboratoire qualité et sécurité des produits alimentaires, gembloux agro-bio tech, université de liège; belgium; 3 laboratoire de chimie générale et organique, gembloux agro biotech, université de liége; belgium. *presenting author: belhaj. kamal90@gmail.com. abstract sheep meat of the beni guil breed is one of the main moroccan local meats, integrated into the program of agricultural development due to the savory, nutritional and sensory properties of sheep meat of moroccan highlands (beni guil). however, this good reputation is still only limited to the assessments of tasting panels. thus, the objective of this study is to assess from a scientific point of view the nutritional quality of this meat via biochemical analyses, examining major and minor muscle composition of the meat of this breed and specifically fatty acid (fa) composition of the longissumus dorsi muscle. in this first study, 10 longissumus dorsi muscle samples were selected and cut with the help of the agents of the national sheep and goat association. the results showed that 100g of fresh meat material contains 25.72% of dry matter, including 5.13% of fat, 19.42% of protein and 0.93% of mineral matter. qualitative and quantitative analyses of fatty acids of 10 samples allowed the identification of 27 components. palmitic, oleic and stearic esters were the main fatty acid methyl esters (fames) identified. saturated fatty acids represented about 41.13% of fas, with the majority being palmitic acid (20.69%) and stearic acid (16%). the unsaturated fatty acids were principally represented by oleic acid (36.06%) and linolenic acid (9.64%). the average content of total fatty acids in meat samples analyzed is 24.98 grams per 100 grams of fat. many reports in the literature have shown that the n-6 and n-3 polyunsaturated fa are important contributors to the odor and flavor of ruminant meats and that this composition is mainly correlated with feeding systems used. keywords: beni guil, sheep, longissumus dorsi muscle, fatty acid. poster #030. general chemical composition of almonds (prunus amygdalus miller) grown in eastern morocco nadia houmy1,2*, f. mansouri1, a. ben-moumen1, m. sindic2, m-l. fauconnier3, h. serghini-caid1, and a. elamrani1 1 laboratoire de biologie des plantes et des micro-organismes, faculté des sciences, ump, oujda ; morocco; 2 laboratoire qualité et sécurité des produits alimentaires, gembloux agrobio tech, ulg, belgium; 3 unité de chimie générale et organique, gembloux agro-bio tech, ulg ; belgium. *presenting author: houmy.nadia@gmail.com. abstract the production of almond has increased more and more in eastern morocco, from 136 000 t in 2012 to 183000 t in 2015. the chemical composition of five varieties (marcona, fournat, ferragnes, ferraduel and bedi) were determined. total sugar ranged between 6,2086 for marcona and 8,5462% for fournat. total protein was changed from 19,4167 for ferragnes ferradul to 22,261% for beldi. total fiber was varied from 15,4197 for marcona to 18,150 % for beldi. ashes ranged between 2,9079 for marcona and 3,5606 % for beldi. oil content varieed between 51,4741 for beldi and 56,5688% for ferragnes ferraduel. this oil content requires us to determine the characteristics of this almond oil. fatty acid profile was identified. oleic acid, linoleic acid, palmitic acid and stearic acid ranged from 60,76 for marcona to 69,306% for ferragnes ferraduel, between 20,545 for ferragnes ferraduel and 27,45% for marcona, between 7,0692 for ferragnes ferraduel and 8,010 for marcona and from 2,009 for beldi to 2,743 for marcona, respectively. oxidative stability was highest in ferragnes ferraduel. keywords: almond oil, fatty acid, sugar, protein, fiber oil content, ashes. poster #031. root growth of date palm (phoenix dactylefera l.) under drought stress hanane m’hamdi*, fouzia alla, kawtar jdaini, abdelmajid belabed, and mohammed aziz el houmaizi laboratory of plant biology and microorganisms, department of biology, faculty of sciences, bp 717 oujda, morocco. *presenting author: hanane57hana@gmail.com. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 343 abstract the palm date tree is a symbolic species of mediterranean countries and has been cultivated for its fruits for centuries. this species establishes the base of human and animal food and creates creates a microclimate allowing the development of diverse animal and plant life in desert. numerous studies have been made on the palm tree and the production of dates. however, knowledge concerning the root system of the palm date tree are very limited. indeed, not much information is available concerning root structure. it is probably due to the difficulty of the observation and the collection of the precise and coherent information concerning the subterranean part. the architectural analysis of the system root is essential to describe its main functions. it consists of classifying the various axes making up the plant according to their morphology, position or according to their dynamics of growth, is a way to try to understand certain effects of ecological and agronomic problems. the main objective of this project is to follow the spatiotemporal development of the root system of the palm date tree by the observation of the root parameters of the palm date tree of three cultivars, and to establish a 3 d root model using xplo software. poster #032. the variability of the main components of dates depending on soil factors and cultural practices kawtar jdaini1*, h. m’hamdi1, f. alla1, h. rey2, c. jourdan2, r. lecoustre2, a. belabed1, and m. a. elhoumaizi1 1 laboratory of biology of plants and microorganisms, faculty of sciences, university mohamed first, oujda, morocco; 2 agricultural research centre for international development, montpelier, france. *presenting author: jdaini91@gmail.com. abstract the date palm is one of the oldest cultivated plants, and is the backbone of the oasis ecosystem in view of its economic, social, ecological and cultural. its fruit contains high nutrients and is a good source of energy, and has high levels of carbohydrates, proteins, minerals, vitamins and fiber. this composition varies depending on the cultivar, soil parameters, cultural practices and the maturation phases. the south east moroccan economy heavily depends on the date palm groves. the average annual production of dates in morocco exceeds 100,000 tones, 25% high quality dates, 35% average quality and 40% low quality. the composition of moroccan dates has been widely studied but none have been conducted on the variability based on cultural and edaphic factors. the objective of our study is to evaluate all these parameters and their influence on the main components of dates and subsequently their quality. to achieve, a biometric characterization, a physicochemical and a microbiological analysis will be conducted on 5 main cultivars from palm groves situated in figuig, tinjdad, erfoud, rissani, zagora tata namely ‘aziza bouzid’, ‘assiane’, ‘boufeggous’, ‘jihel’ and ‘majhool’, respectively. we will then proceed to an assessment of physicochemical soil parameters such as texture, limestone and salinity, and an assessment of cultural practices such as irrigation modes, fertilizer types and pollination modes. this project will contribute to the development of good agricultural practices, help improve the quality and increase the quantity of moroccan date production. poster #033. estimating sap flux densities in date palm trees using the heat dissipation method faouzi alla*, k. jdaini, h. m’hamdi, a. belabed, and m. a. elhoumaizi laboratory of plant biology and microorganisms, department of biology, faculty of sciences, bp 717 oujda, morocco. *presenting author: fouzia.alla@gmail.com. abstract water is essential for life of living beings. in plants, the water requirement measurement is a crucial part of understanding the physiology of tree, and dynamics of water transfers in forest stands. improved water management in oasis systems are one of the main challenges for the preservation of its agricultural ecosystem. the date palm is the backbone of the oasis ecosystem saharan and pre-saharan regions of morocco. this tree is one of the oldest fruit species with high ecological and socioeconomic importance. water consumption of the palm is an essential indicator of water management constraints and the overall water balance. the present study is interested in continuous measuring sap flow densities in date palm using the heat dissipation method, to ensure optimum water consumption for quality production and good profitability. keywords: water balance, sap flow, date palm, thermal method. poster #034. saffron (crocus sativus l.) yield parameter assessment of stressed corms stored in low temperature ibtissame mzabri*, m. legsayer, m. chetouani, a. aamar, n. kouddane, a. boukroute, i. bekkouch, and a. berrichi laboratory of biology of plants and microorganisms, faculty of sciences, oujda, morocco. *presenting author: btissammzabri@ gmail.com. abstract saffron (crocus sativus l.) is the most expensive spice in the world. it is cultivated in morocco for centuries and used for culinary, medicinal and cosmetic purposes. this study aims to optimize saffron yield parameters (saffron morphology, flowering and corm yield) focusing on its performance for semi-arid region of eastern morocco using low temperature storage, and salt and drought stresses. corms obtained from salt and drought stresses and stored under cold storage at 4 °c for 7 and 14 days (with a control kept at room temperature) and were cultivated in open fields at the experimental station of the faculty of sciences of oujda. the number of flowers that formed, the yield of saffron a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 344 spice per corm and daughter corm yield depended on storage temperature and cold-storage duration. flowers from the corm cold-stored for 14 days formed earlier than other treatments. flower number, fresh stigma yield decreased gradually with increasing duration of coldstorage. similarly, leaf number and plant height showed the same decrease with increasing of cold storage period. diameter of produced daughter corms was largest in control. however, increasing cold storage time increases the number of small daughter corms per plant. cold storage induces precocious dormancy. overall, no benefit resulted from coldstorage of corms; also, corms from stressed plants had no effect on the studied parameters. keywords: saffron, cold storage, stressed corms, flower yield, corm yield. poster #035. the effect of different concentrations of indole3-butyric acid (iba) and the season on the resumption release date palm (phoenix dactylifera variety: aziza bouzid) ayoub aamar1*, m. chetouani, i. mzabri, a. berrichi, n. kouddane, and a. boukroute laboratoire de biologie des plantes et des microorganismes, faculté des sciences, université mohamed premier, oujda, morocco. *presenting author: ayoubaamar@gmail.com. abstract the farmers of figuig have great difficulties in the multiplication of date palm sucker, especially the variety of aziza bouzid whose regeneration rate is very low and does not exceed 10%. in this work, two factors influencing this regeneration were studied: the season of transplantation and hormonal dose of indole-3-butyric acid (iba). the results showed that the warmer spring (april) and summer (july) are the most favorable for a rooting release season of the variety studied. iba treatments improved, in a very remarkable way, the rate of regeneration with a rate of 100% for the concentration of iba in the spring season. keywords: date palm, regeneration rate, iba, season, variety aziza bouzid. poster #036. antioxidant activity of the essentiel oil of an endemic species of morocco: thymus marrocanus mohamed ouknin1,2*, abderrahmane romane2, and lhou majidi1 1 laboratoire des substances naturelles & synthèse et dynamique moléculaire, faculté des sciences et techniques, université moulay ismail, errachidia, morocco; 2 laboratoire de chimie appliquée, faculté des sciences-semlalia, bp. 2390, université cadi ayyad, marrakech, morocco. *presenting author: medoknine@gmail.com. abstract the lamiaceae family which includes 7200 species distributed in 240 genera, is divided into 7 subfamilies. the thymus marrocanus genus belongs to the subfamily nepetoides. this study was designed to examine the phytochemical screening and antioxidant activity of the essential oils of thymus marrocanus stem, flower and leaf. thymus marrocanus aerial parts were the valuable organs (flower and leaf), for the essential oil production representing a yield of 2.1% (w/w). the phytochemical screening of thymus marrocanus stem, leaf, and flower was characterized by high proportions of reducers compounds, flavonoids and catechin tannin compounds. antioxidant activities of the essential oil from aerial part of thymus marrocanus were evaluated by using three methods: free radical scavenging activity dpph (2,2-diphenyl-1-picrylhydrazyl), the ferric reducing power (frap), and β-carotene. the first method showed that the essential oil of the aerial part has an ability important to reduce the iron compared to bht. the antioxidant activity obtained by the second method showed that the essentials oil from the aerial part of thymus marrocanus (ic50= 0.542±0.0012 µg/ml) is more important than those obtained from antioxidants used in food and pharmaceutical industries namely butylated hydroxytoluene (bht) (ic50= 0.854± 0.0041 µg/ml). keywords: essentiel oil; lamiaceae; thymus marrocanus; dpph; frap; β-carotene. poster #037. influence of arbuscular mycorrhiza and rhizobia strain on the growth and physiological parameters of alfalfa (medicago sativa) under salt stress raja ben laouane1,2,3*, a. meddich1, m. faghire3, k. oufdou2, f. anaya1, r. fghire1, n. bechtaoui2, f. el amerany, m. ait el mokhtar1, and s. wahbi1 1 laboratoire de biotechnologie et physiologie végétale fssm, université cadi ayyad, marrakech, morocco; 2 laboratoire de biologie et biotechnologie des microorganismes fssm-université cadi ayyad, morocco; 3 laboratoire de biotechnologie végétale faculté des sciences université ibn zohr agadir, morocco. *presenting author: benlaouaneraja@yahoo.fr. abstract the interactions benefits of the dual inoculation of legumes with nodule bacteria and arbuscularmycorrhizae (am) are established. however, plant responses to this dual inoculation vary depending on the plant. the current study aimed to compare the effectiveness of autochtonous mycorhizal isolated from palm grove of tafilalet in morocco and/or rhizobial strain rhol1 on two medicago sativa varieties (demnate and a variety australiene). under 0mm nacl the dual symbiosis formed by am fungi and rhizobia were able to improve significantly dry matter production of variety australiene in comparison with the single inoculation and the control, although, no significant difference was detected between the single and dual symbiosis for demnate. for both varieties, salt stress significantly reduced plant biomass and physiological parameters. however, plants water parameters didn’t show any significant variation. but, these effects can be reduced by the single or dual inoculation with arbuscular-mycorrhizal autochthonous (am) fungi and rhizobia strain. our results suggested that medicago sativa inoculation with rhizobia strain (rhol1) and/ or arbuscular-mycorrhizal a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 345 autochthonous (am) fungi could help to alleviate salt stress effects. keywords: salinity, dual inoculation, growth parameters, physiological parameters, alfalfa. poster #038. effect of fluorescent pseudomonas on the two spotted spider mite tetranychus urticae (acari: tetranychidae) redouan qessaoui1,2*, r. bouharoud2, b. chebli1, s. hakou2, a. amarraque2, a. ajerrar1,2, and h. mayad3 1 equipe génie de l’environnement et de biotechnologie, ecole nationale des sciences appliquée, agadir, morocco; 2 research unit of integrated crop production, centre régional de la recherche agronomique d’agadir (inra), morocco; 3 laboratoire des biotechnologies végétales, faculté des sciences d’agadir, université ibn zohr, morocco. *presenting author: qessaoui_bio@ hotmail.fr. abstract the two spotted spider mite tetranychus urticae (acari: tetranychidae) is a phytophagous pest that can cause significant yield losses in many agricultural crops, including fruits, cotton, vegetables, and ornamentals. this mite has been recorded to feed on more than 180 plants species in both greenhouse and outdoor environments. to control t. urticae, farmers spray usually chemicals (miticides) despite their harmful effects on the environment and human health. this study investigated the effects of pseudomonas isolates (q036b, q110b and q172b) as a biological control agent of t. urticae. the bacteria were isolated from tomato rhizospheric soil. leaf dip bioassay was adopted to test the miticide effect of pseudomonas isolates. five concentrations of bacteria were used, 102, 104, 106, 108 and 1010 cfu/ ml. the mortality rates were assessed 24, 48 and 72h after treatment. control leaves were dipped in sterilized distillate water. ten to 15 adults of t. urticae were introduced in petri dish containing treated leaves and 3 replicates were used for each concentration. the statistic analysis shows that all bacteria and their concentrations significantly reduce the total numbers of t. urticae adults compared to control at 72 h. the mortality rates were 49.9 and 97.8 % for q172b and q036b, respectively. the results of this study indicate that the fluorescent pseudomonas isolates have an efficient activity to control t. urticae and can be promising tools to bild up a biological control strategy. keywords: tetranychus urticae, biological control, miticide, pseudomonas. poster #039. biodiversity and abundance of ants (formicidae) in the argane (argania spinosa) forest in the south-west of morocco abdelhadi ajerrar1,2*, r. bouharroud2, m. zaafrani1, b. chebli1, a. amarraque2, r. qessaoui1,2, and el h. mayad3 1 ecole nationale des sciences appliquée, agadir, morocco; 2 centre régional de la recherche agronomique d’agadir, inra, morocco ; 3 faculté des sciences ibn zohr, agadir, morocco. abstract ants (formicidae) have an important role in the functioning of the terrestrial ecosystem. ants clean and enrich the soil, contribute to spread of seeds and to prey on other organisms. this study focused on comparing the biodiversity and abundance of ants in two argane forest areas in the southwest morocco. the first is located in aoulouz (taroudant) and the second in sidi-ifni. sampling was carried out using mouth aspirator during late spring (may). litter of two frames of 1 m² each for every tree were collected and then kept in ethanol 75%. in each site, seven locations were sampled and two samples were collected for each location (total= fourteen samples per site). preparation and slide mounting of specimens were conducted in the plant protection laboratory at national institute for agricultural research (inra), agadir. identification was performed using a binocular microscope and based on specific key for each family, subfamily, genus and species. a total of 10 species of ants were identified in sidi-ifni and only 3 in aoulouz. the shannon indexes (h’) estimated were respectively 1.37 and 0.15. the most important genus identified was monomorium which represented 97% in aoulouz and 60.7% in sidi ifni. this work will contribute to understand different ecological relationships in the argane forest ecosystem. keywords: biodiversity, ants, monomorium, argane forest, shannon index. poster #040. biennial seed production tendency evaluating sugar beet genotypes performances through selection cycle ghizlane tobi1,2*, siham oumouss1, ilham rahmouni1, ouafae benlahbib2, and yasmina el bahloul1,2,† 1 unité de recherche en amélioration des plantes conservation et valorisation des ressources phytogénétiques, centre régional de la recherche agronomique (crra), rabat, morocco; 2 unité de recherche en biotechnologies appliquées en agriculture, agrobiodiversité et produits de terroir, institut agronomique et vétérinaire hassan ii, rabat, morocco. *presenting author: tobighizlane@gmail.com. †corresponding author: yasmina.elbahloul@inra.org.ma. abstract seed production analysis in plant breeding has a basic role in the selection of best performing germplasm. so, conducting astudy related to this on the sugar beet (beta vulgaris l.) in moroccan climate conditions is an important step to determine adapted cultivars, for breeding program. before a maternal pedigree selection method is adopted, seed production require particular conditions to initiate bolting for the reproductive stage. low temperatures (vernalization) followed by long photoperiod are needed for flowering induction. the present study aims to evaluate the biennial seed production tendency of 40 selected sugar beet genotypes, derived from the second cycle of selection. these individuals were transplanted into the experimental field at merchouch as a vernalizing site and were carried out by polycross design block during the 2013-2014 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 346 and 2014-2015. collected data showed a total response to the vernalization and resistance to early bolting, during 20132014; seed yield per plant showed a high variability ranging between 68,80g and 410g. 1000-seed-weight was between 18g and 36g. during the second year, seed yield varied between 56 g/plant and 300g/plant, with 1000-seeds-wieght between 16g and 28,50g. germination tests showed between 80% and 100% for both years, after 3 days incubation. results prove the genotypes capacity for biennial production of quality seeds with some variation in yield components. keywords: sugar beet, genotypes performances, seed production, breeding program, selection cycle. poster #041. valorization of solid sludge from phosphates in arboriculture and forestry using microorganisms fatima zahra aliyat*, m. maldani, o. zennouhi, l. nassiri, and j. ibijbijen environment & soil microbiology unit faculty of sciences moulay ismail university, meknes, morocco. *presenting author: aliyatfatimazahra@gmail.com. abstract morocco is the world’s leading exporter and the third largest producer of crude phosphates. it contains 75% of the known phosphate reserves on the planet. the ocp group (the cherifien phosphates office) specializes in the extraction, recovery and marketing of phosphates and their by-products. each year, more than 23 million tones of minerals are mined. the treatment of phosphates, by wet process, generates a lot of sludge. this pulp containing 10% solids represents 90% of the rejects from the factory. the volume of this sludge is all the more important, as the production of phosphates increases. the presence of phosphorus in this sludge means loss of phosphates during treatment, from the industrial point of view, and this loss obviously leads to lower yields. however, it is mainly from the environmental point of view that this loss has serious consequences because of the large mass of sludge released into nature. they constitute a major source of environmental pollution, which can have a negative impact on the natural environment. the ocp group is committed to promoting the use of phosphates as part of a vision of sustainable agricultural development that respects the environment. consequently, the search for appropriate solutions to the pollution risk posed by this sludge and the recovery of some of this lost phosphorus is critical. the objectives of this work are: to use the sludge generated as a growing substrate, which can be used in nurseries in the production of fruit plants and forests; and the evaluation of the effects of substrates with microorganisms (bacteria) on the growth, development and vigor of plants. poster #042. the convective solar drying of cherries rachida ouaabou1,2*, hanine.hafida2, ali idlimam3, mohammed kouhila3, abdelkader lamharrar3, and mostafa mahrouz1 1 eriddecv (research team of innovation and sustainable development & expertise in green chemistry), department of chemistry, cadi ayyad university, b.p. 2390, marrakesh 40000, morocco; 2 laboratory of valorization and safety of food products, faculty of sciences and technics, university sultan moulay slimane, beni mellal, morocco; 3 laboratory of solar energy and medicinal plants, teacher’s training college, cadi ayyad university bp 2400 marrakesh, morocco. *presenting author: rachaouaabou@gmail.com. abstract fruits are important to human health as a food source. they are an important source of nutrients and an essential part of the human diet. cherry is one of the most popular fruit by consumers due to its precocity and excellent quality as a food. sweet cherry fruit has relatively low caloric content, low glycemic response, high antioxidant activity and major amounts of important phytochemicals: anthocyanins, quercetin, hydro-xycinnamates, vitamin c, carotenoids and melatonin, as well as nutritive compounds such as sugars and organic acids. cherry fruit have a limited life and does not exceed 21 days in storage. this requires postharvest methods to ensure their presence throughout the year. convective drying is one of the oldest methods of food preservation, and as often realized in natural conditions such as solar drying. the hot air drying method has several advantages that support its wide use in industry and in food industry, especially as it is relatively cheap and an easy operated drying technology. the process is based on partially solar convective drying. therefore, this study was conducted to understand mechanisms influencing the cherry drying process. for this, we studied the behavior of the product dried in a partial solar convective dryer operating continuously. the experiments were performed under real weather conditions. we studied the effect of drying temperature using different temperatures ranging from 60 to 75° c with air flow drying of 300m3/h. the main aim of the studies was to find the best drying conditions, by which the drying time was shortened and the product quality degradation was minimized. keywords: drying, solar energy, cherry, conservation process. poster #043. biological activity of some aromatic and medicinal plants parts extracts against eutetranychus orientalis (acari : tetranychidae) under laboratory conditions hassan alahyane1,2*, h. abousaid1,2, k. oufdou1,3, and s. el messoussi1,2 1 faculty of science semlalia, cadi ayyad university, bp 2390, 40000 marrakech; 2 laboratory of molecular modeling and ecophysiology; 3 laboratory of biology and biotechnology of microorganisms. *presenting author: alahyaneingeco@gmail. com. abstract one of the main pests of citrus in morocco is the phytophagous mite eutetranychus orientalis (acari : tetranychidae) [1;2]. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 347 to manage this pest, synthetic chemicals have traditionally been used, some of which are well known to be potentially toxic to the environment and humans [3; 4]. therefore, alternative strategies for pest management in citrus orchards have been developed in recent years, including biological control with natural enemies such as parasitoids, predators and entomopathogenic microorganisms as well as chemical control using plant extracts. such extracts have shown toxicity to insects which has positioned them as a common alternative in programs of integrated pest management [5]. the objective of this study was to evaluate the effect of aqueous extracts of four aromatic and medicinal plants on female of e. orientalis under laboratory conditions. all the extracts exhibited significant female adult mortality in comparison with the control. zingerber officinalis l. (root), jatropha curcas l. (seeds) extracts had significantly higher mortality rates, greater to 90%, than pelargonium graveolens l. (leaves) and rosmariums officinalis l. (leaves), that did not exceed 67%. our results showed that several plant extracts have good potential for acaricidal activity and are worth further investigation. keywords: eutetranychus orientalis, aromatic and medicinal plants, aqueous extract, acaricidal activity. references [1] a. mazih,. 2011. citrus ipm in morocco: current status. in proceedings of meeting of working group “integrated control in citrus fruit crops” (2010, agadir, morocco). iobc/wprs bulletin, (62), 266 (abstr.) [2] m. benziane, m. abbassi, and a. jahaj. 2001. comparaison de deux méthodes de lutte intégrée contre les principaux ravageurs associés aux agrumes dans la région du gharb. in: ampp (ed.), proceeding du symposium sur «la protection intégrée des cultures dans la région méditerranéenne», rabat, 29-31 mai 2001: 257-269. [3] r. kumar, 1991, la lutte contre les insectes ravageurs. la situation de l’agriculture africaine. editions karthala et cta, paysbas, paris, 310 p. [4] n. stumpf and r. nauen. 2002. biochemical markers linked to abamectin resistance in tetranychus urticae (acari-tetranychidae). pestic. biochem. physiol. 72: 111-121 [5] isman m.b. 2006: the role of botanical insecticides, deterrents, and repellents in modern agriculture and an increasingly regulated world. annual review of entomology, 51: 45–66. poster #044. importance of bituminaria bituminosa to restore defraded sylvopastoral systems btissam ben messaoud*, imane aboumerieme, laila nassiri, and jamal ibijbijen faculty of sciences moulay ismail university, bp 11201 zitoune, meknes, morocco. abstract seven million moroccans abuse forests and place them under heavy pressure. the population increase and land requirement for food crops and wood for domestic and industrial use also provide pressure for deforestation. moreover, 93% of the national territory is subject to desertification and soil erosion. these natural changes cause environmental, social and economic problems. deforestation must be slowed in order to preserve the sylvopastoral systems, and we must look for alternative strategies based on the use of biological tools such as the use of plant adapted to drought and low soil fertility mainly when it’s combined with plant growth promoting rhizobacteria. the aim of present study is to select the best complex bituminaria bituminosa-plant growth promoting rhizobacteria combination that shows the best growth (symbiotic relationship). the choice of this leguminous shrub due to its importance and use as good fodder. the experiment was conducted in the greenhouse in the faculty of sciences of meknes. the experiment was setup in a completely randomized block. each pot contains sandy soil which is nutrient-poor. for that we prepared the inoculation treatment, in the presence and absence of phosphorus fertilizer. five repetitions are done for each treatment. the inoculum was prepared with two plant growth promoting rhizobacteria in erlenmyer. inoculation alone showed a low beneficial effect on the height, the biomass production and the mineral nutrition. while the effect of the inoculation combined to the presence of phosphorus fertilization was most important. the use of bituminaria bituminosa-plant growth promoting rhizobacteria will constitute good solution in semi-arid and arid regions and thus it will contribute to the preservation of the environment. keywords: phosphorus fertilization, inoculation, bituminaria bituminosa, plant growth promoting rhizobacteria. poster #045. the fertilizing components of seaweeds soufana safih*, k. makroum, m. belfaiza, a. bakkali, a. ezzaher, a. alwan, l. a. rifai, m. l. nedjar, s. esserti, a. smaili, m. faize, e. m. kabil, and t. koussa laboratory of plant biotechnology, ecology and ecosystem valorization, department of biology, faculty of sciences, university chouaib doukkali, el jadida, morocco. *presenting author: soufana.safih@gmail.com. abstract morocco has a high abundance and diversity of seaweeds (e.g. more than 143 algal species in el jadida’s coastline). for centuries seaweeds were applied to increase the fertility of soil, and foliar applications of algal extracts have shown many beneficial effects. seaweed extracts have potential as fertilizers and they were not well exploited. seaweed extracts improve rest break, bud burst and yields, upgrading the resistances to biotic and abiotic stresses and the stimulation of the vegetal defense. now several products with seaweeds are marketed, but these uses are much limited comparatively to the richness of their composition. the biochemical characterization of seaweeds revealed answers about their beneficial effects on plants, tests conducted determined the quantity of total sugars and protein in four species of seaweeds that used preserved in various conditions (fresh a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 348 samples, frozen samples, dries samples). the study showed differences in the contents of parameters studied according to seaweeds species and according to the state of matter. the analysis and the quantification of the components of seaweeds in particular in protein and in total sugars will help to adapt their use in agriculture. poster #046. mycorrhizal colonization response to water and salt stresses on two plant species to be used in degraded sand dunes’ rehabilitation programs abdessamad fakhech*, lahcen ouahmane, and mohamed hafidi ecology and environment laboratory, faculty of sciences semlalia, cadi ayyad university. *presenting author: abdessamad. fakhech@edu.uca.ma. abstract in this study, mycorrhizal colonization rates of essaouira sand dunes arbuscular mycorrhizal fungi were observed in response to water and salt stresses on two plant species: retama monosperma and acacia gummifera. these species were selected due to their high plasticity and tolerance for abiotic stresses, and to be used in essaouira sand dunes ecosystem restoration. mycorrhizal inoculum was prepared from a mix, sampled from the rhizospheric soil of retama monosperma and juniperus phoenicea, which are well established species on the dunes of essaouira. the experiment lasted 6 months. the results were then compared and tested with an anova for a p-value of 50%. the stresses had a little but significant impact on the colonization rates of plant roots. overall, the mycorrhization rates showed some distinction between extreme treatments and almost no significant difference between close range treatments. keywords: water stress, salt stress, mycorrhization, nitrogen, phosphorus, colonisation. poster #047. correlation between climatic conditions and severity of potato soft rot in morocco nisrine sbai idrissi*, aicha ouarzan, mohamed amine senhaji, meryem meftah elkhir, said élantri; and amine abdessamad laboratoire de biochimie, environnement et agro-alimentaire, faculté des sciences et techniques de mohammedia, university hassan ii, morocco. *presenting author: svtnisrine@gmail.com. abstract potato (solanum tuberosum), the third biggest crop in morocco, is prone to a wide range of diseases which drastically reduce yield and quality. the most common pathogens which affect potato are pectolytic enterobacteria (pectobacterium atrosepticum, pectobacterium carotovorum) which cause stem and tuber rot in potato (czjakowski et al., 2011). the symptoms developed on the plant are not strain specific but are dependent on the climatic conditions prevailing at the infection stage. the main objective of this study is monitoring the prevalence of this disease in the different regions of morocco known by a difference of climatic conditions. during 2015 to 2016, different potato fields and the most important potato storages in morocco (moyen atlas, loukkos, doukkala, haute moulouya, haouz, chaouia, sais, elgharb, sous, oriental) were surveyed and samples were collected from tubers and plants suffering from soft rot disease. seventy five fields were visited. different symptoms of soft rot, black leg, and wilting were found. prevalence varies from 0 to 14%. in the same region, prevalence could vary from 1 to 8% (haute moulouya) and from 0 to 9 % (haouz). this study shows that in morocco, there is not always a correlation between the climatic conditions and the degree of severity of the disease (prevalence percentage). keywords: potato, climatic conditions, prevalence, soft rot. poster #048. drought tolerance gene integration in immature embryos of moroccan durum wheat by agrobacterium tumefaciens mediated transformation ahansal khadija1,2*, aadel hanane1,2, abbdelwahd rabha2, gaboun fatima2, udupa m. sripada3, ibriz mohammed1, and iraqi driss2 1 ibn tofail university, faculty of sciences, laboratory of agrophysiology and in vitro culture, kenitra, morocco; 2 biotechnology unit, national institute of agronomic research (inra). avenue de la victoire, b.p. 415, rabat, morocco; 3 icarda-inra cooperation research project, inetrnational center for agriculture research in the dray areas (icarda), b.p. 6299, rabat, morocco. *presenting author: kh.ahansal@gmail.com. abstract wheat improvement by genetic engineering requires the delivery, integration and expression of defined foreign genes into suitable regenerable explants. in our experiment, we focused first on the effect of auxins 2,4-d and picloram for the plants regeneration. further, we used agrobacterium as mediated transformation technique with phva1-fl plasmid containing drought tolerance gene hva1, and selectable marker bar gene. in the first experiment, we used immature embryos (ie) from three durum wheat varieties (amria, chaoui and marouane) for callus induction. picloram regenerate a higher number of plants compared to 2,4-d. therefore, picloram was selected for the genetic transformation experiment and added in induction medium. the transformation step was rbegan by using intact immature embryos, half immature embryos and small pieces of immature embryo of the same three varieties of durum wheat. the percentage of regeneration of ‘chaoui’ was high when we used half embryos, a similar regeneration rate was observed for variety ‘amria’ in all explants; further, variety ‘marouane’ was regenerated only in half embryos. a successful integration of hva1 gene was confirmed by analyzing the plantlets using pcr amplification. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 349 poster #049. effect of two biostimulants on the productivity of mejhoul date palm in tafilalet oases hakim outghouliast1,2*, messaoudi zerhoune1, touhami ouzzani amina2, and lhoussain ait haddaou1 1 laboratory of pomology, arboriculture-viticulture, national school of agriculture, meknes, morocco; 2 laboratory of botany, biotechnology and plant protection, faculty of sciences, university ibn toufail, kenitra, morocco. *presenting author: hakim. agro.ena@gmail.com. abstract nowadays, in the dry climate zones, such as oases, the improvement of date production as well as its physical and chemical characteristics, consider one of the fundamental research goals. an experiment was conducted out during 2016 season, in a conventional production of palm dates mejhoul, under the climatic conditions of tinejdad, errachidiya region, morocco. the aim of this study was to evaluate the effect of two biostimulants including protifert lmw 6.3 (4 ml.l-1) and vitazyme® (1.3 ml.l-1) on yield and quality of dates. three treatments were compared (control, protifert, and vitazyme). the applications of two biostimulants were repeated three times after pollination in particular late april, mid-july and mid-august. the experimental design was completely random block with 3 replicates and 3 trees per treatment (a total of 27 palm trees). observations were made on fruit dimensions (width and length), fruit weight, flesh weight, seed weight, seed/fruit ratio, pulp/ fruit report and yield. data shows that the treatment has affected significantly all of the parameters studied. indeed, protifert lmw 6.3 has enhanced yield per tree by 18 and 27 %, respectively compared to vitazyme and control. as for fruit dimensions, protifert had longer fruits than vitazyme and control, whereas, both biostimulants induced fruit of the same width but larger than those from the control trees. in addition, fruit weight ranged from a maximum of 14.51g by protifert lmw 6.3 and a minimum of 10.32g observed in the control. however, fruits of treated trees vitazyme were richer in flesh (92.62%) compared to those obtained by untreated palm (90.78%), besides their environmentally safe effect as non-chemical tool. keywords: biostimulants, date palm, mejhoul, production, quantity, quality. poster #050. optimization of growth and development of date palm seedlings by using organic and biological fertilizers mohamed anli1*, mimoun el kaoua2, abdelillah meddich1, mohamed ait el mokhtar1, mounir mansouri2, amine faarouk ibrahim2, raja benlaouane1, yousef ait rahou1, fatima el amerany1, and abdessamad fakhech1 1 laboratory of biotechnology and plant physiology, faculty of sciences semlalia, cadi ayyad university, marrakech, morocco; 2 laboratory of biotechnology, valorization and protection of agro-resources, faculty of sciences and technology, cadi ayyad university, marrakech, morocco. *presenting author: mohamedbacoanli@gmail.com. abstract the aims of our study is to evaluate the effect of compost produced locally and seaweed liquid extract on four parameters: growth, physiology, histology and mineral nutrition of seedlings of the date palm (phoenix dactylifera). after 4 months of greenhouse cultivation, we evaluated the parameters on palm trees subjected to different treatments. the results showed the importance of the two amendments on the date palm grown in greenhouse. indeed, the production of fresh and dry matter of the aerial part is significantly improved by different treatments applied compared to the control and 20% compost-extract combination. in addition, chlorophyll fluorescence (fv/fm) was enhanced in the all treatments compared to the control. however, stomatal conductance was highly affected positively for seedlings amended to 5% compost compared to the control. in addition, the number of conductive tissues (xylem and phloem) was increased by the various treatments compared to the control. in the same way, the number of sclerenchyma fibers also improved in plant treated (except to those treated with the algal extract) compared to control plants. furthermore, the phosphorus content of the aerial part is improved in the palm trees treated with algal extract, 5% compost-extract combination and 5% compost compared to the control. the potassium content of the aerial part was also improved by 5% compost. finally, the compost with a low dose (5%) seems to be the best amendment for improving the growth and vigor of young palm trees. keywords: date palm, compost and algal extract. poster #051. genetic identification of adaptation and impact of genotype by environment interactions on argan tree ecotypes ilham rahmouni1,2,3*, mohammed el massoudy1,2, siham oumouss1,2, ghizlane tobi1, najib bendaou3, mohammed bouksaim2, and yasmina el bahloul1 1 unité de recherche en amélioration des plantes conservation et valorisation des ressources phytogénétiques, centre régional de la recherche agronomique crra, rabat, morocco; 2 laboratoire eco valorisation, centre régional de la recherche agronomique crra, rabat, morocco; 3 université mohammed v, faculté des sciences, rabat, morocco. *presenting author : ilham. rahmouni@gmail.com. abstract argania spinosa, skeels. is one of endemic species to morocco, is currently extended in arid and semi-arid areas in southwest morocco region. it plays a very important role for local populations. in addition, it’s an essential component of forest ecosystems biodiversity. for the purpose of preserving and understanding the distribution of this tree species, a study of the variation and the influence of environmental conditions on the morphology was a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 350 carried out on different ecotypes of moroccan argan forest. this study is based on surveys and collections of argan genotypes as well as on the analysis of climatic and soil factors (temperature, rainfall, altitude, soil components, etc.). individuals were selected previously to be tested on different environments. trials are conducted in four regions of morocco, where the soil and environmental conditions are different, to evaluate genotype x environment interactions. evaluation is based on 20 morphological traits and 12 molecular ssr species specific markers. results of this study will contribute to identify genetic ecotype of argan forest and to determine the adaptability of different genotypes, for argan breeding programs. keywords: argania spinosa, adaptability, soil factor, genotype x environment interactions. poster #052. evaluation of some fruit quality traits of eight tomato varieties (solanum lycopersicum) grown in four different moroccan regions amraoui rajae*, abid malika, ahmed elamrani, hana caid serghini, addi mohamed, and mihamou aatika laboratory of biology of plants and microorganisms, faculté des sciences oujda – université mohammed premier, bp-717 oujda, morocco. abstract the organoleptic and nutritional quality characterization of eight tomato varieties cultivated in four different regions in morocco (berkane, hoceima, figuig and rissani) were evaluated. several physicochemical parameters such as ph, titratable acidity (ta) and total phenolic compounds were analyzed on mature tomato fruits. the ph did not change between varieties. however, titratable acidity ranged between 2.1 g/l for white rissani variety to 5.6 g/l for black rissani. for total polyphenols content, figuig 2 and hoceima showed the greatest value ranged between 125 and 122 mg/100g, respectively. results of this analysis showed that physicochemical parameters could be used for varietal characterization, and morphological and molecular tests indicated that figuig and hoceima varieties were the most interesting of those tested. keywords: tomato, physicochemical parameters, ph, titratable acidity (ta), total phenolic compounds. poster #053. evaluation of some date palm males used in pollination in figuig province imane zahiri* and mohammed aziz el houmaizi laboratory of biology of plants and microorganisms, faculty of sciences, university mohamed i, oujda, morocco. *presenting author: zahiri-imane@hotmail.fr. abstract date palm (phoenix dactylifera l.) is a dieocious plant with separate male and female trees. the high nutritive value of the date fruits and the substantial quantities produced justify the need for ameliorating the production by using new methods for breeding and selection of new male palm cultivars which have a high viability and compatibility with female cultivars, and positive influence on quantity or quality of fruits produced. the principal characteristics in a male palm are: fertility, blooming early in season, abundance of flowers and pollen, and production of good characters in the fruits. in the present study, several visits were carried out to the date palm farms in figuig region, and 40 male date palm trees were selected from different local farms. pollen viability was determined by in vitro germination, and results indicated that maximum and minimum germination varied from 18% to 90%. several morphological characteristics of the selected date palm males inflorescences were studied. these characteristics included spathe length, spathe width, pedencule length, pedencule width, flower spik length, strands length and number of strands by spathe. keywords: pollen, viability, morphological characteristics, invitro germination poster #054. essential oil: adsorption properties of thymus willdenowii boiss. as a green inhibitor for the corrosion of mild steel in 1m hcl. mohamed ouknin1,2*, abderrahmane romane2, and lhou majidi1 1 laboratoire des substances naturelles & synthèse et dynamique moléculaire, faculté des sciences et techniques, université moulay ismail, errachidia, morocco; 2 laboratoire de chimie appliquée, faculté des sciences-semlalia-bp. 2390, université cadi ayyad, marrakech, morocco. *presenting author: medoknine@gmail.com. abstract there are numerous medicinal and aromatic plants and in the process of understanding their individual benefits requires specific knowledge and expertise of each group of plants and products. according to the definition of the world health organization (who), medicinal plants include plant species of which one part or more parts contain biologically active substances that can be used for therapeutic purposes or for chemical and pharmaceutical synthesis. therefore, medicinal and aromatic plants include plant species containing biologically active substances with practical applications in pharmaceutical, food and cosmetics industry, and industry additives such as aromatic preparations, etc. the analysis of thymus willdenowii (tw) essential oil by gas chromatography (gc) and gas chromatography-mass spectrometry (gc/ms) allowed the identification of 32 components which accounted to 97.6% of the total amount. among them, the main components were thymol, p-cymene, ϒ-terpinene, borneol and carvacrol. the inhibitive effect of this essential oil on the corrosion of mild steel in 1m hcl solution has been investigated by weight loss measurement, as well as potentiodynamic polarization and electrochemical impedance spectroscopy (eis) techniques. the gravimetric results indicate that tw oil exhibits good inhibition efficiency in 1m hcl solution. electrochemical impedance spectroscopy measurements revealed that the charge a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 351 transfer resistance increases with increase in the concentration of essential oil. linearity of langmuir isotherm adsorptions indicated the monolayer formation as inhibitor on mild steel surface. keywords: essential oil; thymus willdenowii; mild steel; corrosion inhibitor; hcl. poster #055. action of physiological factors on the in vitro and in vivo development of the argan tree (argania spinosa l. skeels.) hassania farhoune1,2*, souad cherkaoui1, and alaoui-mdarhri meriem2 1 mohammed v university, faculty of sciences rabat, morocco; 2 national institute of agronomic research (inra-maroc), biotechnology research unit, crra-rabat, bp 6570, 10101 rabat instituts, morocco. *presenting author: fhassania@gmail.com. abstract the argan tree (argania spinosa l. skeels) is an endemic tree of southwestern morocco. it is very appreciated for the quality of its oil and wood. it plays important roles in the socio-ecological, environmental, economic, medicinal and cosmetic domains. currently, the argan forest tree suffers from alarming degradation. since natural regeneration cannot ensure the reconstitution of this species, several methods of classical multiplication have been tested. however, the results obtained were insufficient. to respond to the strategy of the “green morocco plan”, aimed at the improvement and mass production of this species, the use of biotechnology and more particularly the in vitro micropropagation technics and in vivo multiplication were studied. the work done and the results obtained seem encouraging and promising. keywords: argan tree, argania spinosa, micropropagation, in vivo, in vitro. poster #056. cystic echinococcosis: epidemiology, socio-economic impact and consequent control actions undertaken in morocco ikhlass el berbri*; ouaffa fassi fihri; anne françoise petavy; wissal maher; franck boué; allal dakkak; and mohammed bouslikhane iav hassan ii, rabat, morocco. *presenting author: abstract echinoccocus granulusus, is the causal agent of cystic echinococcosis in humans, and in domestic and wild herbivores. disease transmission occurs by ingestion of parasite’s eggs living in the small intestine of canids, especially dogs. it is a matter of disease and causes major problems to human health, and economic losses. according to the latest who estimates, this disease is responsible of annual losses of 1-3 million dalys (disability adjusted life expectancy years) and of $ 3 billion (us $) for the treatment of patients and for livestock losses. in morocco, an annual average surgical incidence of approximately 4.2 cases per 100 000 inhabitants was recorded (2009-2013), involving an estimate of over 20 million mad (≈ € 2 million) for annual medical expenses. in livestock, losses associated with infected organs seizure in slaughterhouses, were estimated at 10 million mad / year (≈ € 1 million / year). to address this situation, the main undertaken actions in the field, were conducted mainly by the iav hassan ii team. the targeted regions were the province of khénifra (2000-2007) and, recently, the province of sidi kacem (2009-2014), as part of major research american and european projects, bafi and iconz respectively. field studies evaluate the disease control approaches and alternatives. the results of these actions were very promising. however, as they were located and project related, sustainability was not ensured. thus, cystic echinococcosis is still prevailing in the country with socioeconomic and public health consequences. it was concluded that any selected control action should include health education and community based approaches. poster #057. an ethnopharmacological survey of plants used in traditional diabetes treatment in the region of beni mellal – khenifra, morocco younesse el ouzzani1,2*, aziz hasib1, abdelali boulli1, and mustapha bouzaid2 1 environment laboratory and valorization of the agro-resources of the faculty of sciences and techniques of beni mellal, morocco; 2 laboratory of bioprocesses and bio-interface of the faculty of sciences and techniques of beni mellal, morocco. *presenting author: y.elouazzani@yahoo.com. abstract this study was carried out in the region of beni mellal – khenifra which has a sizeable botanical biodiversity as determined by herbalists and traditional healers. for about forty years ago, diabetes has been a real public health problem in the world. it is the most common metabolic pathology characterized by chronic hyperglycemia resulting from a partial or complete deficit of the pancreas cells to synthesize insulin and/or tissue resistance to insulin. many plants are traditionally considered as antidiabetic. some of them have led to the development of several medicines, and in front of the considerable increase in the number of diabetics and the elevation of the financial value of certain synthetic drugs, research has begun to evaluate the pharmacological actions of these medicinal plants anchored in the traditions of the population. the statistical treatment of the results obtained from the question sheets, allowed us to target the plant species suspected to have a curing power against diabetes. keywords: phytotherapy, medicinal flora, biodiversity, ethnobotany, antidiabetic. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 352 poster #058. value addition of some moroccan regional products by liquid fermentation: production of date and apple vinegars majid mounir1,2* 1 iav hassan ii, rabat, morocco; 2 université de liège, belgium. *presenting author: mounirmajid@gmail.com. abstract six strains of acetic acid bacteria were isolated from moroccan local products and evaluated for their industrial vinegar production potential. two of them, namely af01 and cv01, isolated from apple (malus domestica) and cactus (opuntia ficus indica) fruits, respectively, were selected and investigated for their thermoresistant and thermophilic properties. a biochemical and molecular study based on a 16srdna gene sequence analysis confirmed that the two isolated strains belong to acetobacter pasteurianus with 99 % of homology. their growth and acetic acid production capacity were compared with (1) the thermoresistant reference strain a. senegalensis isolated from mango (senegal) and (2) other, wild acetic strains belonging to a. pasteurianus (lmg 1632, lmg 1607, lmg 1701, dsm 2324 and dsm 4491). the two strains showed very good growth and noticeable acetic acid production ability at high temperatures (38 to 41°c). a thermophilic character was noted for af01 strain. indeed, this bacterium grew better at 38°c than at 30°c. subsequently, a semi-continuous fermentation was performed in a 20l bioreactor for both selected strains. a total amount of 7.64% and 10.08% acetic acid was achieved at the end of fermentation, respectively for af01 and cv01 strains. process fermentation of cv01 strain was marked by the ability to produce gluconic acid during acetic fermentation. finally, an enzymatic study was carried out on the selected strains af01 and cv01 to confirm the tolerance of their enzymes responsible for acetic fermentation process to high temperature. results clearly showed that the two strains exhibited high adh and aldh enzyme activity at either 30 or 38 °c as compared to the mesophilic reference strain lmg 1632 which exhibited marked thermal inactivation rate. it is concluded that the two selected strains are well-suited to industrial use for vinegar production in morocco. keywords: vinegar; acetobacter; thermoresistance; bioreactor; enzyme activity; morocco. poster #059. soybean mutations mapping: applications in functional gene analysis and soybean improvement naoufal lakhssassi*, shiming liu, zhou zhou, and khalid meksem department of plant soil and agricultural systems, southern illinois university, carbondale, il, usa. *presenting author: naoufal.lakhssassi@siu.edu. abstract the alteration of fatty acid profiles to improve soybean oil quality has been a long time goal of many researches through the world. biodiesel is an up and coming trend in energy production. breeding effort can be undertaken in order to produce higher energy profile soybean oil. using the ethyl-methanesulfonate (ems) mutagenesis effects on dna, significant changes to the genes and gene network underlying oil profile can be achieved, these changes are hard to accomplish under standard breeding techniques. high content linolenic acid is very important for fuel and biodiesel production, but disadvantageous for food production due to the fact that the oil is oxidized easily and the foods go rancid quickly. however, soybean oil with elevated oleic acids is desirable for food. oleic acid is a monounsaturated fatty acid that improves the nutrition and oxidative stability of soybean oil compared to other oils. the main purposes of this work were to mutation breed new soybean [glycine max (l.) merr.] sources containing high level of seed oleic acid good for the soy industrial and food production, on a background conferring the resistance to soybean cyst nematode (scn), the most devastating pest in soybean production. a subset of the ems mutagenized developed population containing about 1000 families was screened using forward genetics approach to identify mutants within the fad2 genes. mutations within the fad2 genes encoding the fatty acid desaturase were identified. five soybean mutants with high levels of oleic acid were identified presenting 2.5 times more content than the forrest wt. these mutants were further identified to carry one mutation each in both fad2-1a and fad2-1b isoforms. poster #060. influence of drought stress on several root traits and their correlation with seed protein and oil contents in soybean ambrocio zenis1*, stella kantartzi2, khalid meksem2, and my abdelmajid kassem1† 1 plant genetics, genomics, and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc 28301, usa; 2 department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901, usa. *presenting author: zambrocio@uncfsu.edu. †corresponding author: mkassem@uncfsu.edu. abstract the important crop soybean [glycine max (l.) merr.] is cultivated worldwide and the us is its number one exporter. however, farmers face many challenges in cultivating soybeans, including drought and diseases that reduce yields drastically. the root system is very important for plants, including crops, because it receives water and minerals from the soil so that the plant/crop can photosynthesize, grow, and increase its yield. the objective of this study was to grow the ‘md 96-5722’ by ‘spencer’ recombinant inbred line (ril) population (n=86) in the greenhouse under normal (group i) and drought stress (group ii) conditions, and compare the root length (rl), root surface area (rsa), average root diameter (ard), and average root volume (arv) in the two groups of plants. whinrhizo software was used to measure the root traits and spsstm was used to evaluate population a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 353 performance under normal and drought conditions. jmptm was used to compare the root traits under normal and drought conditions, and to analyze the correlation between root traits, protein and oil contents. the results showed that there is a huge variation in these traits among the parents ‘md 96-5722’ and ‘spencer’, and among their rils. for group i plants, the rl of parents and rils ranged from 20.67 cm to 2,327.88 cm; the rsa ranged from 4.57 cm 2 to 1,176.79 cm2; the ard ranged from 0.38 mm to 4.04 mm; and the arv ranged from 0.08 cm3 to 47.34 cm3. for group ii plants, the rl of parents and rils ranged from 15.70 cm to 3,562.42 cm; the rsa ranged from 4.15 cm2 to 829.72 cm2; the ard ranged from 0.24 mm to 5.74 mm; and the arv ranged from 0.03 cm3 to 23.67 cm3. it is clear from the results that group i plants have higher means of rl [572.58 cm vs. 537.33 cm], rsa [201.20 cm2 vs. 165.50 cm2], ard [3.96 mm vs. 1.45 mm], and arv [6.31 cm3 vs. 5.61 cm3] compared to group ii plants which demonstrates that droughtstressed plants have reduced overall plant growth and development. however, statistically, these differences were not significant; therefore, further studies with several replicates should be conducted both in the greenhouse and the field in order to determine the effects of drought stress on the ‘md 96-5722’ by ‘spencer’ rils. moreover, studies of quantitative trait loci (qtl) mapping of the root traits studied here are underway to genetically map qtl for these root traits in this soybean ril population. keywords: root length (rl), root surface area (rsa), average root diameter (ard), average root volume (arv), md 96-5722, spencer. poster #061. genetic analysis of yield components in the pi 438489b by ‘hamilton’ recombinant inbred line (ril) population of soybean [glycine max (l.) merr.] ragin bobby1*, richard bazzelle1, willsheana clark1, stella k. kantartzi2, khalid meksem2, masum akond1, and my abdelmajid kassem1† 1 plant genomics and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc, usa; 2 department of plant, soil, and agricultural systems, southern illinois university, carbondale, il, usa. *presenting author: bragin@uncfsu.edu. †corresponding author: mkassem@uncfsu.edu. abstract yield is a multi-factorial trait determined by several genetic traits and highly correlated with important agronomic traits in many crops including soybean. [glycine max (l.)]. plant height, seed and pod numbers, and seed weight are all components of yield and polygenic in nature. the objective of this study was to identify quantitative trait loci (qtl) for days to germination, days to flowering, plant height, pod number, seed number, 100-seed weight, and total seed weight in soybean using the using the pi 438489b by ‘hamilton’ recombinant inbred line (ril) population (pixh, n=50). a total of 18 qtl were found on 10 different chromosomes. three qtl for days to germination (qdg001-qdg003) have been identified on chromosomes 5b, 6, and 13b. two qtl (qdf001 and qdf002) have been identified on chromosomes 9 and 13b, respectively. on qtl for plant height (qph001) have been identified on chromosome 6. four qtl for pod number (qpn001-qpn004) had been identified on chromosomes 2, 6, and 8 (2 qtl), respectively. two qtl for seed number (qsn001 and qsn002) have been identified on chromosomes 5b and 11b, respectively. five qtl for 100-seed weight (qsw001 to qsw005) have been identified on chromosomes 5a, 6, 8, 9, and 11c, respectively. two qtl for total seed weight (qtsw001 and qtsw002) have been identified on chromosomes 5b and 17c, respectively. the qtl identified here may be introduced in breeding programs to develop soybean cultivars with high yield potential. keywords: soybean, qtl, days to germination, days to flowering, plant height, pod number, seed number, seed weight. poster #062. forward and reverse genetic screening of genes underlying stearic acid content in soybean naoufal lakhssassi*, shiming liu, zhou zhou, and khalid meksem department of plant soil and agricultural systems, southern illinois university, carbondale, il, usa. *presenting author: naoufal.lakhssassi@siu.edu. abstract stearoyl-acyl carrier protein desaturase (sacpd), which has four isoforms in soybean: sacpd-a, -b, -c and –d, converts stearic acid into oleic acid, influencing and controlling the accumulation of seed stearic acid. stearic acid is a desirable constituent of oils for food use because it is less likely to be incorporated into cholesterol esters and has a neutral effect on the concentration of the blood serum ldl cholesterol. stearic acid may be useful to eliminate the need for chemical hydrogenation of soybean oil in the production of trans-free margarines and shortenings. the available soybean lines with high level of seed stearic acid are mostly susceptible to soybean cyst nematode (scn), one of the most devastating pests in soybean production. in order to improve the quality of soybean oil and processed foods, chemically mutagenized soybean using ems has been developed in this work. a subset of the mutagenized population containing about 1000 families was screened using reverse (tilling) and forward genetic screening approaches to identify mutants within the sacpd genes. using a forward screening approach, we were able to identify four mutants: f605, f620, f714, and f813, all of which contain high level of seed stearic acid with an increased level of up to 2.4 times. each of these four mutants was identified to carry one missense or nonsense mutation of sacpd-c: q83* (f605), l79f (f620), d77n (f714), and p102l (f813), among them, q83*, l79f, and p102l are the new sacpd-c alleles identified in soybean. however, none of the four mutants exhibited mutations in sacpd-a, sacpd-b, and sacpd-d. furthermore, reverse genetics was used to identify mutants within the other sacpd isoforms. thus, we identified one missense sacpd-a mutant: f1396 (d277g), three missense sacpd-d mutants: f2360 (g263e), f2374 (e37k), and f2487 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 354 (t297m), and all the four lines were measured to contain the same level of seed stearic acid as the wild type forrest. thus, only the mutations of sacpd-c isoform perfectly correlate with the alteration of soybean seed stearic acid phenotype, suggesting sacpd-c is most likely responsible for the accumulation of stearic acid in soybean seed. the international american moroccan agricultural sciences conference (amas conference; www.amas-conference.org) is an international conference organized by the high council of moroccan american scholars and academics (hc-masa; www.hc-masa.org). atlas journal of biology 2019, pp. 628–654 doi: 10.5147/ajb.v0i0.201 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) mirabilis expansa grown in southern illinois: amino acid percentages relative to soil amendments miriam kritzer van zant1*, william j. banz2, gary apgar2, and david a. lightfoot1 1 department of plant, soil and general agriculture, southern illinois university, carbondale, carbondale, il 62901-4415, usa; 2 department of animal science, food, and nutrition, southern illinois university, carbondale, il 62901-4317, usa received: january 16, 2019 / accepted: october 5, 2019 __________________________________________________ * corresponding author: mkvzant@gmail.com 628 abstract effects of plant structure and soil amendments, on percentages of amino acids, were examined with three hypotheses for two crop varieties, of rare, ancient, drought resistant, indigenous, andean, mirabilis expansa (ruiz and pav.) standl. (nyctaginaceae), grown in southern illinois. anova and ls-means analyses were run as pair-wise comparisons. percentages of amino acids, crude protein, and total protein were dependent variables. soil amendments, structure, and variety, were independent variables. independent variable levels were percentages of soil amendments peat and steer manure, used to amend sand plots when constructed, growth above vs below ground, and each variety. significance for p-values was at .05 or less. anovas indicated several amino acids were present in significantly different amounts between roots and leafy parts of plants. there were statistically inconsistent effects from peat, and slight significance from steer manure. exploratory analyses also indicated significantly different amounts of some amino acids between varieties, and for both above and below ground growth. keywords: amino acid profiles; mirabilis expansa; soil amendments; variety; structure; nyctaginaceae. abbreviations: indispensable amino acids: arg, arginine; his, histidine; iso, isoleucine; leu, leucine; lys, lysine; met, methionine; phe, phenylalanine; thr, threonine; try, tryptophan; val, valine. dispensable amino acids: ala, alanine; asp, aspartic acid; cys, cysteine; glu, glutamic acid; gly, glycine; hpr, hydroxyproline; hly, hydroxylysine; lan, lanthionine; orn, ornithine; pro, this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction changing growth conditions have been shown to affect percentages of amino acids in plants (thanapornpoonpong et al., 2008; li et al., 2013). two mirabilis expansa (ruiz and pav.) standl. (nyctaginaceae) crop varieties have been shown to be protein machines, that need little water or fertilizer to produce large amounts of high quality, complete protein (kritzer van zant 2016a, 2016b, 2017; kritzer van zant et al. 2018, 2019). there is a need for modern processing methods for m. expansa to become more important to modern agriculture (kritzer van zant 2016a, 2016b; kritzer van zant et al. 2019). percentages of indispensable amino acids produced in two crop varieties of m. expansa during a growth study in southern illinois (kritzer van zant, 2016a; kritzer van zant et al., 2018, 2019), were examined for effects of independent variables in three hypotheses. independent variables were soil amendments peat and steer manure, added to sand plots used to grow the m. expansa material at the time of construction, as well as plant structures including herbage and roots, and the two varieties. results are reported from anovas and tukey-adjusted lsmeans analyses. tukey-adjusted ls-means results showed which variable level combinations supported significance for each pair of independent variables. two additional independent proline; ser, serine; tau, taurine; tyr, tyrosine. total amino acids: crdp, crude protein; totp, total protein. ext, extended, applies to tables in appendix c-7 of kritzer van zant’s dissertation (2016a); si, supporting information. table abbreviations are given in the notes for each table. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) variables, variety, for which both varieties were submitted to statistical analyses simultaneously, and coa (combined organic amendments), which incorporated all of the plots containing peat, steer manure, or both, simultaneously, were also analyzed in anovas and tukey-adjusted ls-means. dependent variables were percentages of each amino acid tested, crdp (crude protein) and totp (total protein). significant p-values were less than or equal to 0.05. due to small sample sizes, differences in years of production between varieties, and variation in amounts of amendments in plots used for growing each variety, all analyses were run as pair-wise comparisons of the independent variables. these discrepancies were covered previously (kritzer van zant, 2016a; kritzer van zant et al., 2018, 2019) and summarized in the supporting information (si) for this paper. si also included consideration of the exploratory results which were not applied to the three hypotheses, and included discussion of results for interaction terms produced in some of the anovas. in addition, though positive results are given in the main text of this paper, arranged for each indispensable and dispensable amino acid, crdp and totp, they are reconsidered, arranged for each independent variable, in the si. detail for some positive results summarized in the main text are therefore only given in si. results were extensive. therefore, though positive results are presented in the text, tables for all statistical results for all amino acids, including positive and negative results, are currently only available in appendix c-7 of kritzer van zant’s (2016a) dissertation (extended (ext) tables 1-20). from herein in this paper and in its si, all extended tables from appendix c-7 (kritzer van zant 2016a) are only identified by the abbreviated ‘ext table(s)’ designation, without repeating their source. lys was singled out for summary data tables included in this paper (tables 1 and 2), because lys is the consensus indispensable amino acid used as an indicator of protein quality, and of special importance for vegetarian diets (reeds, 2000; who, 2007; woolf et al., 2011). crdp (tables 3 and 4) and totp (tables 5 and 6) were also presented in summary tables in the body of the paper, because percentages by weight of each were outstanding in m. expansa, particularly for leaves, relative to other protein sources examined (kritzer van zant et al., 2019). percentages of most individual amino acids found in m. expansa were competitive with those in other protein sources to which they were compared, including all ten indispensable amino acids (kritzer van zant, 2016a; kritzer van zant, 2019). hypotheses and levels of variables are detailed in materials and methods below. information on the growth of m. expansa in southern illinois, used for this research, was detailed previously (kritzer van zant et al., 2018), as well as summarized later (si). m. expansa’s amino acid profiles were also previously presented (kritzer van zant et al., 2019). introductory information used previously (kritzer van zant, 2016a, b, 2017; kritzer van zant et al., 2018, 2019) also applies to the analyses in this paper. earlier publications summarized information on m. expansa from the latin american literature (kritzer van zant, 2016a, 2017), and the history of use and biochemical research on the 629 family nyctaginaceae (kritzer van zant, 2016a, b). recent papers on mirabilis included research on peptides derived from m. jalapa l. for their potential as biopesticides (maulina et al., 2018), an update on ongoing agronomic and other research on m. expansa in the andes (seminario et al., 2019), and information from a recent study of the current status of m. expansa amongst indigenous farmers, and attempts to revive interest in consuming and growing m. expansa, primarily in peru (gendall et al. 2019). materials and methods plants used for the amino acid profiles were grown in southern illinois, lyophilized, and submitted for amino acid profiling (horwitz and latimer, 2006, 2011; kritzer van zant, 2016a; kritzer van zant et al., 2018, 2019). plant structure above vs below ground, and soil amendments in which those plants were grown (kritzer van zant, 2016a; kritzer van zant et al., 2018), were examined for their potential effect on the percentages of amino acids produced in m. expansa (kritzer van zant, 2016a; kritzer van zant et al., 2019). plots used to grow this material were at least 95% sand. percentages of soil amendments in these plots were utilized as levels of independent variables for statistical purposes. in addition, structures grown above vs. below ground were levels for the independent variable structure. percentages of lys, crdp and totp (tables 1-6) and other profiled m. expansa amino acids (ext tables 1-20) are the results from statistical analyses in anovas and tukey adjusted ls-means. these analyses were performed in sas 9.3 (sas institute inc., cary, nc), to examine relationships between quantities of amino acids produced and the percentages of peat and steer manure added to the sand plots used to grow the plants, at the time of their construction. results of the analyses were interpreted relative to each applicable hypothesis in the main text. all results were presented for all indispensable and dispensable amino acids assayed, and for both crdp and totp, in ext tables 1-20 in appendix c-7 (kritzer van zant 2016a). positive results that were not used to resolve the hypotheses are in the si for this paper. plants grew as masses of vines, and stored starch and protein in enlarged roots, stems, and underground rhizomes. once engorged with starch, vascular arrangements of these structures became obscured and difficult to distinguish even in slides of cross-sections under magnification. in addition, the primary storage area in m. expansa for starch, is the crown of the plant where these structures meet just below the soil line. it was difficult to see what portions of the crown are truly roots, rhizomes, or the base of emerging stems. for these reasons, plant structure was separated based on growth above versus below ground for statistical purposes. ext anova tables 1-3 and ext ls-means tables 11-13, all contained data for the pair-wise comparison of the variables structure and variety. in ext tables 1 and 11, data for var. ‘t’ grown in the greenhouse, separately for leaf and stem, were included in the analyses of data from field-grown material. in ext tables 2 and 12, the var. ‘t’ greenhouse-grown leaf data a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 630 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) was included while the var. ‘t’ greenhouse-grown stem data was excluded. in ext tables 3 and 13, neither leaf nor stem data for var. ‘t’ grown in the greenhouse was included. the only differences among the results described within these two sets each of three tables, were those to be expected as a result of the inclusion or exclusion of more categories of data. therefore, of the six versions of the analyses of structure x variety, only data from ext tables 1 and 11 were actually considered in the main paper, as they are the most complete of each set. all six of these ext tables were referred to by their separate table numbers so the differences among the data they contain could be addressed in the discussion. additionally, data tables from analyses of pair-wise comparisons that resulted in no significant data retained consecutive table numbers in the text of kritzer van zant’s dissertation (2016a) though they were not presented in table form and only briefly mentioned in that text (kritzer van zant 2016a). this kept the relative numbering system intact for the same pairs of independent variables, which remains intact in this paper and its si. for example, anova ext table 2 contains data for the same pair of independent variables as ls-means ext table 12, anova ext table 6 is for the same pair-wise comparison as ls-means ext table 16, etc. this was done to avoid confusion over missing tables and analyses due to the lack of results. some tables had significant results from anovas which had no correlating positive or computable results in the matching ls-means analyses, and visa-versa (kritzer van zant 2016a). using table numbers in the text (kritzer van zant 2016a), that matched with extended table numbers in the description also kept table numbers consistent with graphs of the anovas for the same pair-wise analyses, given as figures 1-1000 in appendix c-8 of kritzer van zant’s dissertation (2016a). therefore, numbering for those graphs can also be matched to the data from the ext tables referenced in this paper and si. raw percentage data from the amino acid profiles were also given previously (kritzer van zant et al., 2019) in the form of tables, and the data in those tables later used for each pair-wise comparison in the ext tables (kritzer van zant 2016a). in the main text of this paper, anova (tables 1, 3, 5) and ls-means (tables 2, 4, 6) combination tables have positive results from all statistical analyses summarized for lys (tables 1, 2), crdp (tables 3, 4) and totp (tables 5, 6). positive ls-means results were also extensive, so only text descriptions of positive tukey-adjusted ls-means results are given in this paper and si. levels of ls-means results are arranged from least to most significant for each amino acid. only anova results that were significant or approaching significance for at least one of the hypotheses, at the .05 level of probability or less, are presented in the main text. each independent variable applicable to any of the hypotheses, was first addressed through each pair-wise comparison in anovas that included it, for analyses both with and without the interaction term request. next, the same independent variable was examined in each ls-means analyses which included it, described in greater detail in the si for this paper. positive results of analyses were presented separately and consecutively from the ext tables for each amino acid, crdp or totp. this process was continued until every independent variable had been considered for every pair-wise comparison in which it was included. results were discussed in the main text, arranged by amino acid, crdp or totp. discussion of the same results, arranged by independent variable, are in the si, though the data itself was not repeated. variety and coa were analyzed in the same way as other independent variables (si), though neither addressed any hypothesis. statistical principles discourage setting up hypothesis after applicable analyses have been run. variety and coa had already been included in earlier analyses with each variety run separately to address the hypotheses. additionally, coa plots had been considered separately for their peat and steer manure percentages to address the hypotheses. later a decision was made to run exploratory analyses that treated both varieties at the same time in spite of their discrepancies, and separately, to run analyses that kept coa plots distinct and also included data from all plots containing peat or steer manure by themselves. interaction results from anovas that included an interaction term request were also discussed separately (si). 1. hypotheses for amino acid profiling three hypotheses were established prior to running the anovas and ls-means analyses on the amino acid profiles data. below ground structures were labeled roots, and above ground material labeled shoots, for purposes of the hypotheses, for reasons explained in the above introduction. shoots included leaves for var. ‘l’ and leaf and stem separately for var. ‘t’ (si). first is the structure hypothesis: h0 = there will not be a significant difference in quantity for each amino acid, between above and below ground plant parts; and h1 = there will be a significant difference in quantity for each amino acid, between above and below ground plant parts. second is the peat hypothesis: h0 = amount of peat added at the time of plot construction, has no effect on the amount of amino acid produced, considered individually, in roots and separately in shoots; and h1 = amount of peat added at the time of plot construction has an effect on the amount of amino acid produced, considered individually, in roots and separately in shoots. third is the steer manure hypothesis: h0 = amount of steer manure added at the time of plot construction has no effect on the amount of amino acid produced, considered individually, in roots and separately in shoots; and h1 = amount of steer manure added at the time of plot construction has an effect on the amount of amino acid produced, considered individually, in roots and separately in shoots. percentages of individual amino acids, crdp and totp, in the anovas and ls-means analyses, supported h0 if not significant and h1 if significant, in applicable hypotheses. interpretation of these analyses was limited by discrepancies within the raw data (kritzer van zant, 2016a, b, 2017; kritzer van zant et al. 2018, 2019), summarized in the si. conflicting results among pair-wise analyses, for the same independent variables, were also problematic for resolving the hypotheses, despite considerable amounts of significance. some of this may have been due to conflicts in the raw data. all discussion of analyses for structure, peat or steer manure paired with variety or coa (combined organic amendments), or plots containing both peat and steer manure, are also in the si. results from the analyses held contradictions for each independent variable. therefore, it became necessary to consider each independent variable separately across all analyses instead of considering each set of pair-wise analyses separately. the summary tables of results from each of the anova (tables 1, 3, 5) and ls-means analyses (tables 2, 4, 6) for lys (tables 1, 2), crdp (tables 3, 4) and totp (tables 5, 6) showed the method of breakdown of data for each independent variable, and included only positive results, if any, from each set of pairwise analyses. however, percentages of peat and steer manure in coa plots, were each separately included in the peat only and steer manure only analyses paired with other independent variables. ignoring that the 1% peat or 3% steer manure data came from plots that contained both, in the peat only or steer manure only analyses, for some raw data may have skewed results. however, this effect appeared minimal when the results of the individual analyses for peat and steer manure were considered against each other and against the results of the exploratory coa analyses (si). examination of the figures in appendix c-8 (kritzer van zant 2016a) that accompany the extended tables of the anova results, can assist with understanding the role that 1% peat and 3% steer manure data, which came from plots that contained both, had on the overall results. 2. amino acids twenty-three amino acids, crdp and totp, were the dependent variables in the anovas (ext tables 1-10) and tukey-adjusted ls-means (ext tables 11-20) analyses. all amino acid profiling results were given as percent dry weight per 100 g of sample in all tables referenced in this paper and the ext tables from which they were summarized. all ten indispensable amino acids were profiled and statistically analyzed arginine (arg), histidine (his), isoleucine (iso), leucine (leu), lysine (lys), methionine (met), phenylalanine (phe), threonine (thr), tryptophan (trp) and valine (val). thirteen dispensable amino acids were also profiled and statistically analyzed alanine (ala), aspartic acid (asp), cysteine (cys), glutamic acid (glu), glycine (gly), hydroxyproline (hpr), hydroxylysine (hly), lanthionine (lan), ornithine (orn), proline (pro), serine (ser), taurine (tau) and tyrosine (tyr). these abbreviations were used in the remaining text, tables (1-6), ext tables 1-20, and in si for this paper. crdp is a measure of the percentage of all nitrogen containing compounds in each sample. totp measures the percentage of nitrogen in samples of only amino acids. some totp was lost as is typical during the hydrolysis step of the percent amino acid assays. percentages of amino acids per crdp have been presented by some authors (zarkadas, 1997; house et al., 2010). a table was given previously with percent amino acids/ crdp for m. expansa (kritzer van zant et al., 2019). crdp and totp were each only considered separately here. 3. statistical data analysis each data point represented more than one plant from more than one plot which received the same percentage of a soil amendment, or more than one cloned individual grown exclusively in the greenhouse. var. ‘t’ data was only statistically analyzed in pair-wise comparisons that included variety as an independent variable (si). anova and tukey-adjusted ls-means p-values were considered significant at 5% or less. independent variables were analyzed in pairs in anovas, without and with requests for interaction terms, separately for the same data, which usually altered the degrees of freedom and therefore modified some of the significance in the anova results. anova results for each dependent variable were first compared without and with the interaction term request, then compared to anovas for the same dependent variable in analyses in which they were paired with other independent variables, and lastly comparison was made with tukey-adjusted ls-means results for the same pairs of independent variables. this was done to reduce the effect of the imbalances in the data. interactions could only be considered for pairs of variables separately, due to the small size of data sets, and were also discussed separately to reduce repetition (si). tukey adjusted ls-means analyses examined relationships among pairs of levels for each pair-wise comparison of independent variables. ls-means results (tables 2,4,6; ext tables 1120) were reported with standard errors, degree of significance, and accompanying tukey-kramer letters, if generated. tukeykramer letter assignments indicate proximity of the means to each other for each pair-wise comparison of levels of independent variables, and were computed separately, if at all, for each amino acid, crdp and totp. ls means with the same letter assignment in the ext tables (kritzer van zant 2016a), were not significantly different from each other for different variable and level combinations. tukey-kramer letters were computed separately for each amino acid, crdp and totp. in tables 1-6, anova and ls-means ext tables 1-20, and in the ls-means results detailed in si, asterisks indicated the degree of significance (kritzer van zant 2016a). if an asterisk was lacking, there was no significant result. one asterisk indicated a probability of .05 % or less, and was described in the text as significant. two asterisks indicated a probability equal to or less than .0001 % which was described in the text as highly significant. results were presented to two digits after the decimal. though numbers six through nine were rounded up, the number 5 was rounded down. this made a slight reduction in the anova and ls-means results values as well as in the error term, compared with rounding five up. some noise dropped out of the results of the analyses because of this rounding change. despite limitations, statistics gave insight into which of the independent variables appear to have influenced quantities of certain individual amino acids, and which clearly did not. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 631 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 632 results and discussion imperfect sample matches created imbalances in the data. despite these imbalances, some rough comparisons in the anovas were still possible that otherwise would have been more difficult to consider. in addition, it was possible to make sense of some relative values in the ls-means analyses that were not resolved in the anovas. positive results are presented from the anovas of the pairwise comparisons of the independent variables for structure x variety with greenhouse leaf and stem data for var. ‘t’ (ext table 1; si 2.1-2.11), variety by steer manure (ext table 6), structure x coa (ext table 7), structure x peat (ext table 8), and structure x steer manure (ext table 9). tukey adjusted ls-means pair-wise analyses of independent variables resulted in significant results from structure x variety with greenhouse leaf and stem data for var. ‘t’ (ext table 11), variety x coa (ext table 14; see si 3.6), variety x peat (ext table 15; see si 3.6 and tables 2 and 4), variety x steer manure (ext table 16; see si 3.6 and tables 2 and 4), structure x peat (ext table 18; see results in si 3.3 and tables 2, 4 and 6), structure x steer manure (ext table 19; see si 3.4 and tables 2, 4 and 6), and peat x steer manure (ext table 20). details of positive ls-means results for various level combinations were given previously in tables 2, 4, and 6 in the main text, as well as in si 3.2. also in si are discussions of the differences between anova ext table 1, vs anova ext tables 2 and 3, and ls-means ext table 11 vs ext tables 12 and 13 (si 2.1-2.11, 3.1 and 3.8, and in the main text tables 2, 4 and 6), all six ext tables contained data from structure x variety pair-wise comparisons. ext tables 1 and 11 had all of the field data for structure for both varieties, and all of the greenhouse data for var. ‘t’. ext tables 2, 3, 12, and 13, all contained results from different versions of the structure x variety analyses (si 2.1-2.11). no greenhouse leaf data for var. ‘t’ was included though stem data for var. ‘t’ was included for ext tables 2 and 12. ext tables 3 and 13 results had no greenhouse data included for var. ‘t’ leaf or stem. anovas variety x coa (ext table 4), variety x peat (ext table 5), and peat x steer manure (ext table 10), had no significant results for any amino acid, crdp or totp. only tukey adjusted ls-means analyses structure x coa (ext table 17; see si 3.6) lacked any significant results for any amino acid, crdp, totp, among the combinations of their levels. analyzing each pair of variables with and without an interaction term request for the anovas, changed the resulting degrees of freedom and error terms for models, and in some cases for individual independent variables as components of those models. tables that included structure as an independent variable (ext tables 13, 79, 1113, 17-19) included leaf (above ground) and root (below ground) as levels for both varieties, and stem (above ground) as a level only for var. ‘t’. tables also included results for percentages of steer manure (ext tables 6, 9, 16, 19), and peat (ext tables 5, 8, 10, 15, 18, 20). peat x steer manure were also separately examined (ext tables 10 and 20) and those results also considered below. as there were many positive results, often in conflict for interpreting the hypotheses, table 1. lysine, combined results from anovas for m. expansa. i or none indpv depv f value df1, df2 p-value = pr>f for anova p-value significance at .05 level ext table 1: structure x variety with greenhouse leaf and stem data for var. ‘t’: strc lys 243.28 2, 8 <.0001 ** i strc lys 552.51 2, 7 <.0001 ** i v lys 5.74 1, 7 0.0477 * ext table 2: structure x variety with greenhouse leaf data not including stem for var. ‘t’: strc lys 475.07 1, 8 <.0001 ** i strc lys 1087.67 1, 7 <.0001 ** i v lys 5.74 1, 7 0.0477 * i strc*v lys 20.93 1, 7 0.0026 * ext table 3: structure x variety with no greenhouse data for var. ‘t’ strc lys 1410.33 1, 7 <.0001 ** i strc lys 1410.33 1, 7 <.0001 ** ext table 4: variety x coa – no positive results ext table 5: variety x peat – no positive results ext table 6: variety x steer manure – only dispensable hly had positive results ext table 7: structure x coa: strc lys 2980.12 1, 3 <.0001 ** ext table 8: structure x peat: strc lys 2602.27 1, 5 <.0001 ** i strc lys 2221.78 1, 3 <.0001 ** ext table 9: structure x steer manure: strc lys 758.89 1, 5 <.0001 ** i strc lys 540.08 1, 3 0.0002 * ext table 10: peat x steer manure – no positive results abbreviations for independent variables and levels: coa = combined organic amendments = peat plots and steer manure plots and plots containing both; strc = structure = plant part; v = variety; strc*v = interaction. amino acid abbreviation: lys= indispensable lysine. other abbreviations: depv = dependent variable; df2 = error; i = interaction requested vs none = blank space = no interaction requested; indpv= independent variable. key to symbols: one asterisk (*) indicates significance at the .05 to 0.001 level of probability; two asterisks (**) indicates high significanceat the .0001 or less level ofprobability. 633 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) results and discussion of all pair-wise comparison analyses having variety and/or coa as independent variables, are also only discussed in si. other pairs of independent variables discussed in the main text below were structure x peat (ext tables 8, 18), structure x steer manure (ext tables 9, 19), and peat x steer manure (ext tables 10, 20). variable levels for these analyses were for structure, leaf with some fine stem material for both varieties, root for both varieties, and from the greenhouse material stem without leaves only for var. ‘t’. for peat considered alone variable levels were 0% peat for each variety, 1% peat from the same plots as 3% steer manure only for var. ‘l’, 2% peat only for var. ‘t’, and 3% peat only for var. ‘l’. for steer manure considered alone variable levels were 0% steer manure only for var. ‘l’, 3% steer manure from the same plots as 1% peat only for var. ‘l’, 4% steer manure only for var. ‘t’, and 5% steer manure only for var. ‘l’. therefore, a single variable level, along each of the continuums for percentages of peat and steer manure, additionally had some of the other soil amendment, which were also separately analyzed as the coa plots for var. ‘l’. despite these short comings, in varieties ‘l’ and ‘t’ grown outdoors for a single temperate summer in southern illinois, lack of variation between the variable levels still indicates that the amounts of particular amino acids were intrinsic to the varieties and structures of the plants. in addition, it was show that under the given conditions, relative quantities of some amino acids were either affected or not by variation in quantities of peat and steer manure. though discussion below includes some of the information from the ls-means extended tables (kritzer van zant 2016a), the details of often lengthy positive results from these ls-means ext tables are in si unless given in the summary tables in the main text. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg )table 2. lysine, combined results from tukey adjusted ls-means for m. expansa. lys structure var. ‘l’ ls means var. ‘t’ ls means ext table 11: structure x variety with greenhouse leaf and stem data for var. ‘t’: lys leaf 1.45 ± 0.02a** 1.25 ± 0.04b** lys root 0.37 ± 0.02c** 0.43 ± 0.03c** lys stem 0.41 ± 0.04c** ext table 12: structure x variety with greenhouse leaf data not including stem for var. ‘t’: lys leaf 1.45 ± 0.02a** 1.25 ± 0.04b** lys root 0.37 ± 0.02c** 0.43 ± 0.03c** ext table 13: structure x variety with no greenhouse data for var. ‘t’: lys leaf 1.45 ± 0.02a** lys root 0.37 ± 0.02b** 0.43 ± 0.03b** ext table 14: variety x coa –no positive results for lysine ext table 15: variety x peat: lys control 0.91 ± 0.34a* 0.38 ± 0.68a lys p 0.92 ± 0.48a lys pp 0.49 ± 0.68a lys ppp 0.90 ± 0.48a ext table 16: variety x steer manure: lys control 0.89 ± 0.34a* 0.49 ± 0.68a lys sss 0.92 ± 0.48a lys ssss 0.38 ± 0.68a lys sssss 0.93 ± 0.48a ext table 17: structure x coa – no positive results ext table 18: structure x peat: lys control 1.46 ± 0.02a** 0.37 ± 0.02b* lys p 1.47 ± 0.03a** 0.38 ± 0.03b* lys pp 0.49 ± 0.03b* lys ppp 1.41 ± 0.03a** 0.39 ± 0.03b* ext table 19: structure x steer manure: lys control 1.42 ± 0.04a** 0.41 ± 0.04b* lys sss 1.47 ± 0.06a* 0.38 ± 0.06b* lys ssss 0.38 ± 0.06b* lys sssss 1.49 ± 0.06a* 0.38 ± 0.06b* ext table 20: peat x steer manure – no positive results for any indispensable amino acid notes: tukey adjusted ls means include sds among levels of independent variables for lys, letters showing similar tukey adjusted ls means, and asterisks indicating degrees of significance; leaf and root were levels of structure forboth varieties. abbreviations for independent variables and their levels: coa = combined organic amendments = peat plots and steer manure plots and plots containing both; control = all sand; steerm = steer manure; p = 1% peat (same as psss plots) pp = 2% peat; ppp = 3% peat; psss = 1% peat with 3% steerm in the same plot; sss= 3% steerm (same as psss plots); ssss = 4% steerm; sssss = 5% steerm; varieties 'l' and 't' were levels of variety. amino acid abbreviation: lys= lysine. o ther abbreviations: ext = extended table(s) from appendix c-7 of kritzervan zant’s (2016a)dissertation. key to symbols: one asterisk (*) indicates significance at the .05 to 0.001 level of probability; two asterisks (**) indicates high significanceat the .0001 or less level ofprobability.no asterisk means no significance. 634 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) table 3. crude protein, combined results from anovas for m. expansa. 1. positive results for structure in the structure x peat anovas with no greenhouse data for var. ‘t’ positive results for structure for the indispensable amino acids and totals in the anovas for the independent variables of structure x peat with no greenhouse data for var. ‘t’ (ext table 8) without the interaction term request were all highly significant for: arg (f = 2441.551,5, p = <.0001), his (f = 2425.501,5, p = <.0001), iso (f = 1798.831,5, p = <.0001), leu (f = 2492.581,5, p = <.0001), lys (f = 2602.271,5, p = <.0001), met (f = 1834.101,5, p = <.0001), phe (f = 2617.631,5, p = <.0001), thr (f = 1395.091,5, p = <.0001), trp (f = 938.941,5, p = <.0001), val (f = 1940.431,5, p = <.0001), crdp (f = 1224.831,5, p = <.0001), and totp (f = 2943.521,5, p = <.0001). positive results for structure for the indispensable amino acids and totals in the anovas for the independent variables of structure x peat with no greenhouse data for var. ‘t’ (ext table 8) with the interaction term request were all highly significant for: arg (f = 2575.741,3, p = <.0001), his (f = 2463.521,3, p = <.0001), iso (f = 2652.121,3, p = <.0001), leu (f = 3168.401,3, p = <.0001), lys (f = 2221.781,3, p = <.0001), met (f = 16274.801,3, p = <.0001), phe (f = 7220.701,3, p = <.0001), thr (f = 2468.521,3, p = <.0001), trp (f = 1368.101,3, p = <.0001), val (f = 1978.721,3, p = <.0001), crdp (f = 817.391,3, p = <.0001), and totp (f = 4841.271,3, p = <.0001). therefore, for structure in the anovas for structure x peat (ext table 8) without and with the interaction term request, all ten indispensable amino acids and both crdp and totp, were significant or highly significant in full agreement with h1 in the structure hypothesis. 2. positive results for peat in the structure x peat anovas with no greenhouse data for var. ‘t’ positive results for peat for the indispensable amino acids and totals in the anovas for the independent variables of structure x peat with no greenhouse data for var. ‘t’ (ext table 8) without the interaction term request was significant for his (f = 9.343,5, p = 0.0172), crdp (f = 9.343,5, p = 0.0171), and totp (f = 05.493,5, p = 0.0487). positive results for peat for indispensable amino acids and totals in the anovas for independent variables structure x peat i or none indpv depv f value df1, df2 p-value = pr>f for anova p-value significance at .05 level ext table 1: structure x variety with greenhouse leaf and stem data for var. ‘t’: strc crdp 60.12 2, 8 <.0001 ** i strc crdp 110.12 2, 7 <.0001 ** i strc*v crdp 18.97 1, 7 0.0033 * ext table 2: structure x variety with greenhouse leaf data not including stem for var. ‘t’: strc crdp 116.24 1, 8 <.0001 ** i strc crdp 214.48 1, 7 <.0001 ** i strc*v crdp 18.97 1, 7 0.0033 * ext table 3: structure x variety with no greenhouse data for var. ‘t’: strc crdp 361.02 1, 7 <.0001 ** i strc crdp 361.02 1, 7 <.0001 ** ext table 4: variety x coa – no positive results ext table 5: variety x peat – no positive results ext table 6: variety x steer manure – only dispensable hly had positive results ext table 7: structure x coa: strc crdp 2049.44 1, 3 <.0001 ** coa crdp 11.54 5, 3 0.0357 * ext table 8: structure x peat: strc crdp 1224.83 1, 5 <.0001 ** peat crdp 9.34 3, 5 0.0171 * i strc crdp 817.39 1, 3 <.0001 ** ext table 9: structure x steer manure: strc crdp 175.15 1, 5 <.0001 ** i strc crdp 111.92 1, 3 0.0018 * ext table 10: peat x steer manure – no positive results abbreviations for independent variables: coa = combined organic amendments = peat plots and steer manure plots and plots containing both; strc = structure = plant part; v = variety; strc*v = interaction. amino acid abbreviation: crdp = crude protein. o ther abbreviations: depv= dependent variable; df2 = error; ext = extended table(s) from appendix c-7 of kritzer van zant’s (2016a) dissertation; i = interaction requested vs none = blank space = no interaction requested; indpv= independent variable. key to symbols: one asterisk (*) indicates significance at the .05 to 0.001 level of probability; two asterisks (**) indicates high significance at the .0001 or less level of probability. no asterisk means no significance. 635 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) with no greenhouse data for var. ‘t’ (ext table 8) with the interaction term request was significant for his (f = 10.943,3, p = 0.0401), met (f = 30.533,3, p = 0.0095), and totp (f = 09.833,3, p = 0.0463). for peat, in the structure x peat (ext table 8) anovas: without and with the interaction term request, there was significance for indispensable his, dispensable glu, and totp; without the interaction term request there was significance for crdp; and with the interaction term request there was significance for indispensable met and cys. these results (ext table 8) supported h1 for the peat hypothesis. for peat in the structure x peat anovas (ext table 8): without and with the interaction term request there was no significance for indispensable arg, iso, leu, lys, phe, thr, trp and val; without the interaction term request there was no significance for indispensable met and dispensable cys; and with the interaction term request there was no significance for crdp. these anova results (ext table 8) were in support of h0 for the peat hypothesis. thus, results from the structure x peat anova (ext table 8) were split in their support and negation of the peat hypothesis. 3. positive results in the structure x peat ls-means with no greenhouse data for var. ‘t’ in the structure x peat ls-means analyses (ext table 18): most indispensable and dispensable amino acids and crdp were highly significant for all levels of peat for leaf while significant for all levels of peat for root; only indispensable met was highly significant for all levels of peat for both leaf and root; and totp was highly significant for all levels of peat for leaf and for the control for root while significant for other levels of peat. these results supported h1 in both the structure and peat hypotheses and are described in more detail in si. table 4. crude protein, combined positive results from tukey adjusted ls-means for m. expansa. crdp structure var. ‘l’ ls means var. ‘t’ ls means ext table 11: structure x variety with greenhouse leaf and stem data for var. ‘t’: var. ‘l’ var. ‘t crdp leaf 29.37 ± 0.76a** 22.52 ± 1.52b** crdp root 8.89 ± 0.76c** 11.42 ± 1.08c** crdp stem 8.78 ± 1.52c* ext table 12: structure x variety with greenhouse leaf data not including stem for var. ‘t’: var. ‘l’ var. ‘t crdp leaf 29.37 ± 0.76a** 22.52 ± 1.52b** crdp root 8.89 ± 0.76c** 11.42 ± 1.08c** ext table 13: structure x variety with no greenhouse data for var. ‘t’: var. ‘l’ var. ‘t crdp leaf 29.37 ± 0.76a** crdp root 8.89 ± 0.76b** 11.42 ± 1.08b** ext table 14: variety x coa –no positive results for crude protein ext table 15: variety x peat: var. ‘l’ var. ‘t’ crdp control 19.09 ± 6.49a* 8.94 ± 12.99a crdp p 19.37 ± 9.18a crdp pp 13.91 ± 12.99a crdp ppp 18.97 ± 9.18a ext table 16: variety x steer manure: var. ‘l’ var. ‘t’ crdp control 18.63 ± 6.48a* 13.91 ± 12.97a crdp sss 19.37 ± 9.17a crdp ssss 8.94 ± 12.97a crdp sssss 19.88 ± 9.17a ext table 17: structure x coa – no positive results ext table 18: structure x peat: leaf root crdp control 29.68 ± 0.68a** 8.64 ± 0.56b* crdp p 29.43 ± 0.96a** 9.31 ± 0.96b* crdp pp 13.91 ± 0.96b* crdp ppp 28.70 ± 0.96a** 9.25 ± 0.96b* ext table 19: structure x steer manure: leaf root crdp control 28.70 ± 1.82a* 10.35 ± 1.49a* crdp sss 29.43 ± 2.58a* 9.31 ± 2.58a* crdp ssss 8.94 ± 2.58a* crdp sssss 30.66 ± 2.58a* 9.11 ± 2.58a* ext table 20: peat x steer manure – no positive results for crude protein notes: tukey adjusted ls means include sds among levels of independent variables for crdp, letters showing similar tukey adjusted ls means, and asterisks indicating degrees of significance; leaf and root were levels of structure forboth varieties; percentages were levels ofpeat and steermanure, respectively. abbreviations for independent variables and their levels: coa = combined organic amendments = peat plots and steer manure plots and plots containing both; control = all sand; steerm = steer manure; p = 1% peat (same as psss plots) pp = 2% peat; ppp = 3% peat; psss = 1% peat with 3% steerm in the same plot; sss= 3% steerm (same as psss plots); ssss = 4% steerm; sssss = 5% steerm. amino acid abbreviation: crdp = crude protein. o ther abbreviations: ext = extended table(s) from appendix c-7 of kritzer van zant’s (2016a)dissertation. 636 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 4. positive results for structure in the structure x steer manure anovas with no greenhouse data for var. ‘t’ positive results for structure for the indispensable amino acids and totals in the anovas for the independent variables of structure x steer manure with no greenhouse data for var. ‘t’ (ext table 9), without the interaction term request, were all highly significant for: arg (f = 2025.801,5, p = <.0001); his (f = 349.071,5, p = <.0001); iso (f = 1142.191,5, p = <.0001); leu (f = 1351.601,5, p = <.0001); lys (f = 758.891,5, p = <.0001); met (f = 691.671,5, p = <.0001); phe (f = 1960.961,5, p = <.0001); thr (f = 1176.571,5, p = <.0001); trp (f = 517.861,5, p = <.0001); val (f = 1138.651,5, p = <.0001); crdp (f = 175.151,5, p = <.0001); and totp (f = 680.191,5, p = <.0001). positive results for structure in the structure x steer manure anovas (ext table 9) with the interaction term request are: highly significant for indispensable amino acids and totals arg (2384.871,3, p = <.0001), iso (f = 1272.031,3, p = <.0001), leu (f = 1383.861,3, p = <.0001), met (f = 632.511,3, p = 0.0001), phe (f = 2014.621,3, p = <.0001), thr (f = 1348.531,3, p = <.0001), val (f = 1216.601,3, p = <.0001), and crdp (f = 111.921,3, p = 0.0018); and significant for his (f = 253.921,3, p = 0.0005), lys (f = 540.081,3, p = 0.0002), trp (f = 515.861,3, p = 0.0002), and totp (f = 512.931,3, p = 0.0002). for structure in the anovas for structure x steer manure (ext table 9) without and with the interaction term request, all ten indispensable amino acids and both crdp and totp, were again significant or highly significant in full agreement with h1 in the structure hypothesis. this was in full agreement for structure with the results from structure x peat (ext table 8). in addition, for structure in the anovas for structure x steer manure (ext table 9), without the interaction term request indispensable his, lys and trp were also highly significant in full agreement with h1 in the structure hypothesis, and with the interaction term request were significant in agreement with h1 in the structure hypothesis. 5. results for steer manure in the structure x steer manure anovas with no greenhouse data for var. ‘t’ results for steer manure in the structure x steer manure anovas (ext table 9) had no significance, consistently supporting h0 for the steer manure hypotheses. table 5. total protein, combined results from anovas for m. expansa. i or none indpv depv f value df1, df2 p-value = pr>f for anova p-value significance at .05 level ext table 1: structure x variety with greenhouse leaf and stem data for var. ‘t’: strc totp 76.21 2, 8 <.0001 ** i strc totp 407.95 2, 7 <.0001 ** i v totp 34.99 1, 7 0.0006 * i strc*v totp 68.60 1, 7 <.0001 ** ext table 2: structure x variety with greenhouse leaf data not including stem for var. ‘t’: strc totp 149.56 1, 8 <.0001 ** i strc totp 807.52 1, 7 <.0001 ** i v totp 34.99 1, 7 0.0006 * i strc*v totp 68.60 1, 7 <.0001 ** ext table 3: structure x variety with no greenhouse data for var. ‘t’: strc totp 1346.86 1, 7 <.0001 ** i strc totp 1346.86 1, 7 <.0001 ** ext table 4: variety x coa – no positive results ext table 5: variety x peat – no positive results ext table 6: variety x steer manure – only dispensable hly had positive results ext table 7: structure x coa: strc totp 1980.51 1, 3 <.0001 ** ext table 8: structure x peat: strc totp 2943.52 1, 5 <.0001 ** peat totp 5.49 3, 5 0.0487 * i strc totp 4841.27 1, 3 <.0001 ** i peat totp 9.83 3, 3 0.0463 * ext table 9: structure x steer manure: strc totp 680.19 1, 5 <.0001 ** i strc totp 512.93 1, 3 0.0002 * ext table 10: peat x steer manure – no positive results abbreviations for independent variables and their levels: coa = combined organic amendments = peat plots and steer manure plots and plots containing both; strc = structure; v = variety; strc*v = interaction. amino acid abbreviation: totp = total protein. o ther abbreviations: depv= dependent variable; df2 = error; ext = extended table(s) from appendix c-7 of kritzer van zant’s (2016a) dissertation; i= interaction requested vs none = blank space = no interaction requested; indpv= independent variable. key to symbols: one asterisk (*) indicates significance at the .05 to 0.001 level of probability; two asterisks (**) indicates high significance at the .0001 or less level of probability. 637 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 6. positive results in the structure x steer manure ls-means with no greenhouse data for var. ‘t’ in the structure x steer manure ls-means analyses (ext table 19; see results in si 3.4 and tables 2, 4 and 6): most indispensable and dispensable amino acids were highly significant for leaf while significant for structure for all levels of steer manure; indispensable met and trp were only highly significant for leaf for the control for steer manure, while significant for all other level combinations for leaf and all level combinations for root; totp was highly significant for the control for leaf, significant for all other levels of steer manure for leaf, and for all levels of steer manure for root; and crdp was significant for all levels of steer manure for leaf and root. these results (ext table 19; see results in si 3.4 and tables 2, 4 and 6) supported h1 for both the structure and steer manure hypothesis, and were in contradiction to the anovas (ext table 9) for the same pair-wise comparison of independent variables. specifics for these results are given in si. 7. results for peat and steer manure, in the peat x steer manure anovas with no greenhouse data for var. ‘t’ there were no positive results in the anovas for the independent variables peat x steer manure (ext table 10). detailed results were given from the peat x steer manure tukeyadjusted ls-means (si). for peat in the peat x steer manure (ext table 10) analyses, without and with the interaction term request, there was no significance for any amino acid, crdp or totp, which was in full support of h0 for the peat hypothesis. however, in the structure x peat (ext table 18) ls-means analyses, most amino acids and crdp (see si 3.3, and tables 2, 4 and 6) supported h1 in the peat hypothesis. this may have indicated that there was an effect of peat on amino acid production. peat in the peat x steer manure (ext table 20) ls-means analyses, was not significant for any level combination for all ten indispensable amino acids, most dispensable amino acids, and was not significant for either crdp or totp. therefore, in the peat x steer manure table 6. total protein, combined results from tukey adjusted ls-means for m. expansa. totp structure var. ‘l’ ls means var. ‘t’ ls means ext table 11: structure x variety with greenhouse leaf and stem data for var. ‘t’: totp leaf 23.80 ± 0.35a** 16.80 ± 0.70b** totp root 5.70 ± 0.35c** 6.87 ± 0.49c** totp stem 5.99 ± 0.70c** ext table 12: structure x variety with greenhouse leaf data not including stem for var. ‘t’: totp leaf 23.80 ± 0.35a** 16.80 ± 0.70b** totp root 5.70 ± 0.35c** 6.87 ± 0.49c** ext table 13: structure x variety with no greenhouse data for var. ‘t’: totp leaf 23.80 ± 0.35a** totp root 5.70 ± 0.35b** 6.87 ± 0.49b** ext table 14: variety x coa –no positive results for total protein ext table 15: variety x peat – no positive results for total protein ext table 16: variety x steer manure – no positive results for total protein ext table 17: structure x coa – no positive results ext table 18: structure x peat: totp control 23.97 ± 0.25a** 5.62 ± 0.20b** totp p 24.43 ± 0.35a** 5.98 ± 0.35b* totp pp 7.76 ± 0.35b* totp ppp 22.84 ± 0.35a** 5.94 ± 0.35b* ext table 19: structure x steer manure: totp control 23.26 ± 0.77a** 6.35 ± 0.63b* totp sss 24.43 ± 1.09a* 5.98 ± 1.09b* totp ssss 5.98 ± 1.09b* totp sssss 24.25 ± 1.09a* 5.55 ± 1.09b* ext table 20: peat x steer manure – no positive results for total protein notes: tukey adjusted ls means include sds among levels of independent variables for totp, letters showing similar tukey adjusted ls means, and asterisks indicating degrees of significance; leaf and root were levels of structure for both varieties; percentages were levels of peat and steer manure, respectively. abbreviations for independent variables and their levels: coa = combined organic amendments = peat plots and steer manure plots and plots containing both; control = all sand; steerm = steer manure; p = 1% peat (same as psss plots) pp = 2% peat; ppp = 3% peat; psss = 1% peat with 3% steerm in the same plot; sss= 3% steerm (same as psss plots); ssss = 4% steerm; sssss = 5% steerm. abbreviation for amino acid: totp = total protein. o ther abbreviations: ext = extended table(s) from appendix c-7 of kritzer van zant’s (2016a)dissertation. key to symbols: one asterisk (*) indicates significance at the .05 to 0.001 level of probability; two asterisks (**) indicates high significanceat the .0001 or less level ofprobability. 638 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) ls-means analyses most dependent variables supported h0 in both the peat and steer manure hypotheses. indispensable amino acids for the most part, and crdp and totp, were present in higher amounts in leaf over root. results for structure were most frequently in support of h1 for the structure hypothesis. this was seen with and without the interaction term request in the structure x peat (ext table 8) and structure x steer manure (ext table 9) anovas. considerable significance for level combinations and thus for h1 in the peat hypothesis was also seen in the structure x peat ls-means analyses (ext table 18; see si 3.3 and tables 2, 4 and 6). additionally, there was some significance amongst level combinations in the structure x steer manure ls-means analyses (ext table 19; see results in si 3.4 and tables 2, 4 and 6), which further gave support to h1 in the structure hypothesis. results for steer manure consistently supported h0 for the steer manure hypotheses from the structure x steer manure anovas (ext table 9). however, results from the structure x steer manure ls-means analyses (ext table 19) supported h1 in the steer manure hypothesis. this contradicted the anovas (ext table 9) for steer manure for the same pair-wise comparison of independent variables. detailed results for steer manure from other analyses are in si. it can be said that in the anovas, most of the results concerned with peat vs. steer manure, showed a mild influence from peat on the amino acid profiles for m. expansa. it will require larger experiments and proper mathematical consideration to learn if the effect of steer manure is truly non-existent. yet, especially from the ls-means results, it appears that either soil amendment may have given a slight benefit to production of at least some amino acids. this discrepancy may have been because soil amendments increased water retention in the root zone, which m. expansa reacts poorly to, vs. amendments as nutrient sources. as amendments had been added to plots years before the work on m. expansa commenced, effects may have been magnified successfully by the uniform additions of a small amount of mushroom composted horse manure, under each plant at planting time. there was contradiction in results for peat and steer manure from other analyses (si). however, those analyses included variety as an independent variable, so were not used to resolve the hypotheses. there were also additional considerations covered in the si. southern illinois research on m. expansa was unfunded, and native southern illinois soils retain water too readily for the crop to survive. these issues limited the scale of production and slowed the speed of many steps along the way. however, this research has increased understanding of the growth and nutritional value of this ancient, endangered crop. funding is recommended for research on m. expansa to: find more horticultural and wild varieties, save and protect all known remaining horticultural and wild varieties; complete taxonomic work on wild andean species by kritzer van zant and publication of that work; expand taxonomic work on the existing horticultural varieties to the basic taxonomic work on wild species; begin molecular research in several areas relevant to wild m. expansa, the crop, and its potential uses; identify and test more locations with seemingly suitable conditions to grow the crop; examine the potential of m. expansa and other mirabilis spp. for floriculture and landscaping in arid regions, and for roof top gardens as these plants are tolerant of high uv conditions and high and low temperature extremes, support multiple species of native pollinators, and require little water; investigate m. expansa and other mirabilis spp. for their potential to stabilize loose soils on cliffs and hillsides, with their unusual ability to send rhizomes deeply into or toward water tables while remaining anchored by their roots within dry root zones in loose soils; test the effectiveness of fermentation and methods of separation of protein and starch in m. expansa for dealing with anti-nutrients; test mature m. expansa varieties grown under different conditions and for longer periods of time than material previously tested, for the presence of useful micro-molecules including volatile micro-molecules; test for medicinal and further agricultural benefits from the known enzyme inhibitors in m. expansa and other mirabilis spp., and their component peptides, some already shown to have species specific effects on agriculturally significant bacteria and fungi; examine the epigenetic variation in mirabilis, including m. expansa wild and crop varieties; support indigenous and andean agency efforts to continue growing traditional varieties of the crop where they still remain; begin modern breeding programs for the crop in locales with sufficient drainage and other appropriate conditions around the world; and further investigate the value of m. expansa for forage and feed. more than one of these recommendations may be relevant to finding solutions to address the effects of climate change on food production. m. expansa can serve as an important test case for why old crops matter, even when to date, there is no variety of the same taxa commonly in production. 8. supporting information description si includes information from ext tables 1-20. ext tables 1-10 contained positive results from anova analyses. ext tables 11-20 contained positive results from tukey adjusted ls-means analyses. ext table 1 correlates with ext table 11, ext table 10 with ext table 20, etc., so that tables of the same pair-wise comparisons of independent variables were numbered ending in the same digit (kritzer van zant 2016a). extended table numbers were also used in short descriptions of pair-wise comparisons that did not produce positive results. this kept ending digits consistent for sets of pair-wise comparisons and matched earlier number designations for extended tables with negative and non-computed data in appendix c-7 (kritzer van zant 2016a) and for graphs depicting the anova results in appendix c-8 (kritzer van zant 2016a). si for this paper also includes discussion of interaction terms from some of the anovas, and all analyses with coa or variety as independent variables, as there were no hypotheses for those variables. conflicting results for both occurred frequently, as coa and variety had more caveats for the raw data. additional information is given in si, for materials and methods summarized from previous papers for the reader’s convenience. additional considerations are also given for statistics. all text description of specifics for positive results from the tukey adjusted ls-means analyses not in the summary tables in the main paper, are also 639 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) in the si due to their length. ext tables 1-3 and 11-13 were for different data configurations of structure x variety. those differences were also addressed in the si along with reasons for only considering the versions of the exploratory structure x variety tables with the most complete data, in the main text above. acknowledgements thank you to jim persinger, bill barthalomew, john haddock, ken diesburg, john miller, and the horticultural research center, all at siu-c. advice and assistance on statistics for this chapter, were given by john d. reeve of southern illinois university. thank you in memoriam to abraham kritzer. author contributions ground preparation, planting and weeding of plots, lyophilization, statistical analyses, preparation and sending of samples for profiling, and writing, were done by miriam kritzer van zant. david a. lightfoot assisted with lab space for extractions, access to sand plots and the greenhouse, advice and encouragement. william j. banz made arrangements for the profiling and helped with editing. gary apgar also assisted with the statistical analyses. references gendall, h, j seminario, m sørensen, and i theilade (2019) unearthing the “lost” andean root crop “mauka” (mirabilis expansa [ruíz & pav.] standl.). econ. bot. xx(x): 1-18. downloaded 20 aug 2019 from: https://link.springer.com/article/10.1007/s12231-01909467-y. horwitz, w, gw latimer (2006, 2011) chapter 45.3.05 official method 982.30 e (a, b, c). in w horowitz, gw latimer (eds.) official methods of analysis of aoac international 18th edition, revision 1. aoac international: gaithersburg, md. house, jd, j neufeld, and g leson (2010) evaluating the quality of protein from hemp seed (cannabis sativa l.) products through the use of the protein digestibility-corrected amino acid score method. j agric food chem 58: 11801-11807. downloaded 11 apr 2017 from: http://pubs.acs.org/doi/abs/10.1021/jf102636b. kritzer van zant, m (2016a) analysis and development of mirabilis expansa (ruiz and pav.) standl.; for potential as a new root crop outside the andes (doctoral dissertation). proquest dissertations and theses accession order no. 12694, pp. i-xxvii, 1-352, and 9 appendices on cd. kritzer van zant, m (2016b) review of the economic and ethnobotany of the family nyctaginaceae. atlas j of bio doi: 10.5147/ ajb.2016.0139, 249-266. downloaded 8 jun 2017 from: http:// www.atlas-publishing.org/wp-content/uploads/2013/07/35-ajbvan_zantl_2016b-139.pdf. kritzer van zant, m (2017) history of mirabilis expansa (ruiz and pav.) standl.; growth and use in the andes. atlas j of bio doi: 10.5147/ajb.2016.0138, 236-248. downloaded 8 jun 2017 from: http://www.atlas-publishing.org/wp-content/uploads/2013/07/34ajb-van_zantl_2016-0138.pdf. kritzer van zant, m, k diesburg, and da lightfoot (2018) mirabilis expansa growth, yield, and quality traits: first us field trials for an ancient, endangered, andean crop. atlas j of bio doi: https:// doi.org/10.5147/ajb.v0i0.174, 551-574. kritzer van zant, m, wj banz, hb chai, g apgar, da lightfoot (2019) amino acid profiles and cytotoxicity of mirabilis expansa (ruiz and pav.) standl.; baseline data for a rare indigenous andean crop. adv nutri and food sci: anafs-126: 1-18. downloaded 18 apr 2019 from: https://www.kosmospublishers.com/wp-content/ uploads/2019/04/amino-acid-profiles-and-cytotoxicity-of-mirabilis-expansa-ruiz-and-pav.-standl.-baseline-data-for-a-rareindigenous-andean-crop.pdf. li, x, yb kim, m romij uddin, s lee, s-j kim, and su park (2013) influence of light on the free amino acid content and γ-aminobutyric acid synthesis in brassica juncea seedlings. j agric food chem 61: 8624-8631. downloaded 9 feb 2017 from: https://www.researchgate.net/publication/255174673_influence_of_light_on_ free_amino_acids_content_and_g-aminobutyric_acid_synthesis_in_brassica_juncea_seedlings. maulina, d, sb sumitro, m amin, sr lestari (2018) identification of peptides compounds from mirabilis jalapa l. (caryophyllales: nyctaginaceae) potentially as a biopesticide iop conf. series: journal of physics: conf. series 1093 (2018) 012009 doi :10.1088/1742-6596/1093/1/012009. reeds, pj (2000) criteria and significance of dietary protein sources in humans. j nutr 130: 1835s-1840s. downloaded 9 feb 2017 from: http://jn.nutrition.org/content/130/7/1835s.full?related-urls=yes&l egid=nutrition;130/7/1835s. schmidt, ja, s rinaldi, a scalbert, p ferrari, d achaintre, mj gunter, pn appleby, tj key, and rc travis (2016) plasma concentrations and intakes of amino acids in male meat-eaters, fish-eaters, vegetarians and vegans: a cross-sectional analysis in the epic-oxford cohort. eur j clin nutr 70(3): 306-312. doi:10.1038/ejcn.2015.144 downloaded 9 feb 2017 from: http://www.nature.com/ejcn/journal/ v70/n3/full/ejcn2015144a.html. seminario, j, d chalampuente-flores, h gendall, m. sørensen (2019) the agronomy of mauka (mirabilis expansa (ruíz & pav.) standl.) – a review. j of plant gen and crop research 1(2) downloaded 1 jul 2019 from: https://openaccesspub.org/jgrc/article/997. thanapornpoonpong s-n, s vearasilp, e pawelzik, and s gorinstein (2008) influence of various nitrogen applications on protein and amino acid profiles of amaranth and quinoa. j agric food chem 56: 1164-11470. downloaded 9 feb 2017 from: http://pubs.acs.org/ doi/abs/10.1021/jf802673x. who (2007) protein and amino acid requirements in human nutrition; who technical report series 935. who, singapore, pp. xi, 265. downloaded 9 feb 2017 from: http://apps.who.int/iris/bitstream/10665/43411/1/who_trs_935_eng.pdf?ua=1. woolf, pj, ll fu, a basu (2011) vprotein: identifying optimal amino acid complements from plant-based foods. plos one 6(4): e18836 doi: doi.org/10.1371/journal.pone.0018836. downloaded 9 feb 2017 from: http://journals.plos.org/plosone/article?id=10.1371/ journal.pone.0018836. zarkadas, cg (1997) assessment of the protein quality of native white floury maize, designated iapo-13, by amino acid analysis. j agric food chem 45: 1062-1069. downloaded 11 apr 2017 from: http:// pubs.acs.org/doi/pdf/10.1021/jf960573o. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 640 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) van zant et al. (2019) supporting information supporting information (si) si materials and methods despite imperfect matches for plot types, some differences in results for percentages of amino acids between the two andean horticultural varieties of m. expansa were clear. this clarity was particularly noticeable in the structure x variety lsmeans exploratory analyses (ext (extended) tables 11, 12 and 13). table numbers that were used previously (kritzer van zant 2016a), are described as ext tables with the same table numbers, in the main text above and si, for the same pairs of variables. var. ‘t’ material was not grown in the exact same kinds of plots and for only one of the same years as var. ‘l’ (kritzer van zant et al., 2018), therefore there was no hypothesis for the independent variable variety, though data for both varieties were subjected to anovas and tukey-adjusted ls-means analyses to explore differences in amino acid percentages between them. varieties ‘l’ and ‘t’ were the levels of the independent variable variety. only analyses including variety as an independent variable incorporate data for var. ‘t’ grown in 2009 (kritzer van zant et al. 2018). this was the single good year of its production in illinois (kritzer van zant et al., 2018). var. ‘l’ data was the product of growth in 2008 and 2009 (kritzer van zant et al., 2018). though variety was analyzed as an independent variable, the anovas and ls-means analyses for variety were not statistically meaningful for any of the hypotheses presented, and were only an exploration of differences between the two varieties. var. ‘t’ was grown outdoors in plots with slightly different percentages of peat and steer manure than plots used to grow var. ‘l’. despite this dissimilarity, percentages of peat and steer manure were each consecutively dispersed. this made comparison of levels for each variety easier. discrepancies within the amino acid profile data also included imperfect matching from each variety having been grown in different years. in addition, some var. ‘t’ material came from greenhouse-grown plants that were never in the field. independent variable variety was examined in the structure x variety (ext tables 1-3, 11-13), variety x coa (ext tables 4, 14), variety x peat (ext tables 5, 15) and variety x steer manure (ext tables 6, 16) anovas (ext tables 1-6) and ls-means analyses (ext tables 11-16). results from these tables are given in the text in full below, and included data for dispensable amino acids included in the assays. coa analyses included all individual plots planted in the 2009 repeat experiment with var. ‘l’, containing different percentages of peat and steer manure separately, as well as the plots containing a mixture of both. effects from the plots containing both soil amendments could have been due to peat, steer manure or synergistically from both. peat vs. steer manure was considered in a separate set of anovas (ext table 10) and ls-means (ext table 20) analyses, which combined data for both varieties. again, these analyses were not statistically meaningful, and were only explored for obvious patterns. si statistics one set of anova analyses (ext table 1) and one set of ls-means analyses for structure x variety (ext table 11), were run including both exclusively greenhouse-grown var. ‘t’ above ground samples. structure x variety was reanalyzed in anovas and ls-means twice more, first with the lone var. ‘t’ stem sample from the green house excluded from the analyses (ext tables 2, 12), and then excluding both greenhouse-grown data points for var. ‘t’ (ext tables 3, 13). only the most inclusive of the three structure x variety anovas (ext table 1) and ls-means analyses (ext table 11) were used for text description and comparison to other pairs of independent variables, for both structure and variety. results of all analyses using variety or coa as even one of the independent variables, are only discussed in any detail below. all other pairings of independent variables, other than structure x variety, excluded both stem and leaf data for var. ‘t’ exclusively grown in the greenhouse, as these other analyses addressed independent variables only applicable to field grown material. the generally preferred analysis in sas for percentage data, at the time these analyses were run, was compositional analysis (john d. reeve, personal communication, 2011). however, this would have required a larger number of data points than numbers of amino acids, so a full scale compositional analysis was not possible. compositional analysis would have been possible for one or two amino acids at a time, if a larger number of individual data points had been available, even if only for at least one control organism. this was not the situation for the southern illinois data, and not found for comparable data from other research on m. expansa or other crops compared to it (kritzer van zant, 2017; kritzer van zant et al., 2018, 2019). utilizing a type of anova called canova (canonical analysis of variance), which shares some aspects with regression, was also considered. initially, canova was run in sas to compare means for differences among amino acids between varieties, between above and below ground portions of the plants, and for levels of organic amendments. the data was too unbalanced for the number of samples, to allow the canova pro641 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) grams to do more than analyze pairs of independent variables as ordinary anovas, which is how it is reported. had the same data been grouped for similar levels of amendments for both varieties, the subtle differences due to quantities of soil amendments within varieties would have been lost. in most cases, there would have been a control group vs a single all-inclusive treatment group. this is less useful than running each level separately. however, should larger scale growth trials become possible with m. expansa, compositional analysis and canova may be possible, and are worthwhile statistical options to plan for. si results and discussion si 1. additional details there were few data points for the m. expansa amino acid profile. each data point represented a combination of plants from the same kinds of plots from different terraces or from the greenhouse (kritzer van zant 2018). therefore, two plot samples were combined for leaf, and two for root, for var. ‘l’ from the field, and two for var. ‘t’ root from the field, and one stem, and one leaf sample, for var. ‘t’ from the greenhouse, which were then submitted for amino acid profiling. the single enlarged var. ‘t’ stem sample produced above ground, though expected to be most similar to var. ‘t’ leaf, were most similar to roots of either variety for percentages of individual amino acids, crdp (crude protein), and totp (total protein). this shows consistency for amino acid percentages within each variety, for all locations engorged with starch. coa analyses only accounted for var. ‘l’ plots, including those having a combination of steer manure and peat, and all peat and all steer manure plots. however, additionally running the percentages of steer manure and peat inside coa plots as separate variables within the separate analyses for peat and steer manure, allowed us to examine all of the levels for each of these variables side-by-side for both varieties. examination of the figures of graphs of the analyses in appendix c-8 of kritzer van zant (2016a), made it possible to visually compare how including or ignoring peat or steer manure from combined plots affected the outcomes, which gave some confidence in our interpretation of those results. as the largest differences for var. ‘l’ amino acids were usually for treated plots vs control plots, the inclusion of peat or steer manure separately, from the combined plots, was of less importance. had the opposite been true these results would be less useful. hopefully, these baseline results will be further studied in future research with fewer caveats for the data. si 2. tables of positive anova results for all amino acids and interactions anova ext tables 1-10 of m. expansa f data gave the effects of independent field variables on percentages of amino acids. interactions in these tables were for two independent variables with percentage of amino acids within subjects. pvalues reported in the tables from these anovas were corrected with the huynh-feldt-lecoutre epsilon test of interaction. ext tables 11-20 were for results of the tukey adjusted ls-means analyses. the two sets of tables correlate with each other in their order of independent variables used for pair-wise comparisons. for example, ext table 1 was paired with ext table 11, and ext table 4 was paired with ext table 14, etc. tables that included variety as an independent variable (ext tables 16, 11-16) had two levels, ‘l’ and ‘t’. tables also included percentage of coa (ext tables 4, 7, 14, and 17). as there were many positive results, often conflicting for interpretation of the hypotheses, results and discussion of all pair-wise comparison analyses having variety and coa as independent variables were only discussed in the si. variety was included in the structure x variety comparisons for both anovas (ext tables 1-3) and ls-means (ext tables 11-13). as results were so similar, only results from ext tables 1 and 11, which were the most inclusive for data for ext tables 1-3, and for ext tables 11-13, were used for comparison to other data, though all six tables were mentioned in discussion. discussion for coa or variety did not directly apply to any hypothesis. in addition to the structure x variety analyses (ext tables 1-3, 11-13), remaining pairs of independent variables were variety x coa (ext tables 4, 14), variety x peat (ext tables 5, 15), variety x steer manure (ext tables 6, 16), and structure x coa (ext tables 7, 17), which includes peat and steer manure addressed individually, usually added alone to a plot though sometimes added to the same plot. variable levels for these are: two for variety –var. ‘t’ and var. ‘l’; four for coa for field grown material of var. ‘l’ 0% coa, 3% peat, 1% peat with 3% steer manure, and 5% steer manure, and two for coa for field grown var. ‘t’ 2% peat and 4% steer manure. for coa, peat considered alone, and steer manure considered alone, there was no exact match for any of the variable levels between varieties so they must be understood along respective continuums of percentage. each of the structure x variety analyses included or excluded part or all of the var. ‘t’ data from the greenhouse-grown plants. when compared amongst themselves (ext tables 1-3), most results were the same. however, there were fewer results in the tables based on less raw data. structure x variety tukeyadjusted ls-means t-values were given in the si text below (ext tables 11-13). for all three versions of the anovas (ext tables 1-3) for structure x variety, including those with any greenhouse data for var. ‘t’, there were shifts in degrees of freedom and many of the f and p values, though there were very few changes in the degrees of significance. the few differences in the amount of significance were briefly described. ext table 2 had additional details on shifts in f’s, p-values, and degrees of freedom for the structure x variety anovas, also seen in graphs in figs. 101-200 in appendix c-8 of kritzer van zant (2016a). differences between ext tables 1 and 2 were primarily between f-values and error terms for individual independent variables, though the degrees of significance were mostly the same, and p-values between ext tables 1 and 2 for variety and the interaction were nearly identical. differences between the three structure x variety anova tables (ext tables 1-3) were a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 642 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) further distinguished by the interaction. si 2.1. comparison of ext tables 1 and 2 for differences in structure trp without the interaction term request for structure was significant in ext table 1 (f = 31.532,8, p = 0.0002) and highly significant in ext table 2 (f = 61.921,8, p = <.0001), and dispensable glu had the same results as trp for structure. pro with the interaction term request had significance for structure in ext table 1 (f = 5.862,7, p = 0.0319) and no significance in ext table 2. si 2.2. comparison of ext tables 1 and 2 for differences in variety in the structure x variety anova analyses, with greenhouse leaf and stem data for var. ‘t’ (ext table 1) and with greenhouse leaf data and no stem for var. ‘t’ (ext table 2), without and with the interaction term request, there were no differences between the p-values for any dependent variable for variety. si 2.3. comparison of ext tables 1 and 2 for differences in the interaction in the structure x variety anova analyses, with greenhouse leaf and stem data for var. ‘t’ (ext table 1) and with greenhouse leaf data and no stem for var. ‘t’ (ext table 2), without and with the interaction term request, there were no differences between the p-values for any dependent variable for the interaction. si 2.4. comparison of ext tables 1 and 3 for differences in structure trp without the interaction term request was significant for structure (f = 31.532,8, p = 0.0002) in ext table 1, though highly significant for structure (f = 625.001,7, p = <.0001) in ext table 3, and dispensable glu without the interaction term request was also significant for structure (f = 30.702,8, p = 0.00002) in ext table 1 though highly significant for structure in ext table 3 (f = 27.801,7, p = 0.0012). si 2.5. comparison of ext tables 1 and 3 for differences in variety without the interaction term request, dispensable tau (f = 10.281,8, p = 0.0125) and tyr (f = 8.451,8, p = 0.0197) were significant for variety in ext table 1, and not significant for the same in ext table 3. his, without the interaction term request, was significant for variety in ext table 3 (f = 5.861,7, p = 0.0460) though not significant for the same in ext table 1, as was dispensable glu. arg (f = 51.841,7, p = 0.0002), iso (f = 37.081,7, p = 0.0005), leu (f = 39.951,7, p = 0.0004) , lys (f = 5.741,7, p = 0.0477), met (f = 27.731,7, p = 0.0012), thr (f = 23.531,7, p = 0.0019), trp (f = 49.001,7, p = 0.0002) and val (f = 39.571,7, p = 0.0004), as well as dispensable ala (f = 8.941,7, p = 0.0202), asp (f = 51.761,7, p = 0.0002), gly (f = 48.871,7, p = 0.0002), ser (f = 23.851,7, p = 0.0018), tau (f = 10.121,7, p = 0.0154), and totp (f = 34.991,7, p = 0.0006), with the interaction term request, were significant for variety in ext table 1 though not significant for the same in ext table 3. phe (f = 100.501,7, p = <.0001) and tyr (f = 138.921,7, p = <.0001), with the interaction term request, were highly significant for variety in ext table 1 and not significant for the same in ext table 3. si 2.6. comparison of ext tables 1 and 3 for differences in the interaction indispensable leu (f = 70.191,7, p = <.0001), phe (f = 122.901,7, p = <.0001), trp (f = 68.061,7, p = <.0001), val (f = 63.101,7, p = <.0001) as well as dispensable asp (f = 70.731,7, p = <.0001), glu (f = 92.111,7, p = <.0001), gly (f = 71.791,7, p = <.0001) and tyr (f = 84.041,7, p = <.0001), and totp (f = 68.601,7, p = <.0001), with the interaction term request, were highly significant for the interaction in ext table 1 though not significant for the same in ext table 3. indispensable his (f = 36.221,7, p = 0.0005), iso (f = 52.341,7, p = 0.0002), lys (f = 20.931,7, p = 0.0026), met (f = 41.431,7, p = 0.0004), thr (f = 31.121,7, p = 0.0008) and dispensable ala (f = 17.121,7, p = 0.0044), asp (f = 70.731,7, p = <.0001), pro (f = 12.131,7, p = 0.0102), and ser (f = 21.491,7, p = 0.0024), and crdp (f = 18.971,7, p = 0.0033), with the interaction term request were significant for the interaction in ext table 1 though not significant in ext table 3. si 2.7. comparison of structure x variety analyses to other pair-wise analyses of the three structure x variety anova analyses (ext tables 1-3), the version which includes leaf and stem var. ‘t’ data (ext table 1) was more informative and therefore of these three tables only ext table 1 positive results were presented in full in the si above, and it was the only of the three (ext tables 1-3) used below for comparison with other pairings of independent variables which had included either structure or variety. si 2.8. positive results for structure in the structure x variety anovas with greenhouse leaf and stem data for var. ‘t’ all positive results for structure for the indispensable amino acids and totals in the anovas for the independent variables of structure x variety with no greenhouse data for var. ‘t’ (ext table 1) were without the interaction term request: highly significant for structure in arg (f = 146.202,8, p = <.0001), his (f = 88.282,8, p = <.0001), iso (f = 106.812,8, p = <.0001), leu (f = 132.282,8, p = <.0001), lys (f = 243.282,8, p = <.0001), met (f = 80.522,8, p = <.0001), phe (f = 81.062,8, p = <.0001), thr (f = 171.482,8, p = <.0001), val (f = 61.921,8, p = <.0001), crdp (f = 60.122,8, p = <.0001) and totp (f = 76.212,8, p = <.0001); and significant for trp (f = 31.532,8, p = 0.0002). all positive results for structure for the indispensable amino 643 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) acids and totals in the anovas for the independent variables of structure x variety with no greenhouse data for var. ‘t’ (ext table 1) were with the interaction term request highly significant for: arg (f = 964.712,7, p = <.0001); his (f = 278.652,7, p = <.0001); iso (f = 471.102,7, p = <.0001); leu (f = 775.382,7, p = <.0001); lys (f = 552.512,7, p = <.0001); met (f = 280.732,7, p = <.0001); phe (f = 749.602,7, p = <.0001); thr (f = 514.772,7, p = <.0001); trp (f = 141.502,7, p = <.0001); val (f = 280.561,7, p = <.0001); crdp (f = 110.122,7, p = <.0001); and totp (f = 407.952,7, p = <.0001). si 2.9. positive results for variety in the structure x variety anovas with greenhouse leaf and stem data for var. ‘t’ all positive results for variety for the indispensable amino acids and totals in the anovas for the independent variables of structure x variety with no greenhouse data for var. ‘t’ (ext table 1) were with the interaction term request: highly significant only for phe (f = 100.501,7, p = <.0001); and significant for arg (f = 51.841,7, p = 0.0002), his (f = 9.321,7, p = 0.0185), iso (f = 37.081,7, p = 0.0005), leu (f = 39.951,7, p = 0.0004), lys (f = 5.741,7, p = 0.0477), met (f = 27.731,7, p = 0.0012), thr (f =23.531,7, p = 0.0019), trp (f = 49.001,7, p = 0.0002), val (f = 49.001,7, p = 0.0002), and totp (f = 34.991,7, p = 0.0006); though crdp without and with the interaction term request was not significant for variety. si 2.10. positive results for the interaction in the structure x variety anovas with greenhouse leaf and stem data for var. ‘t’ all positive results for the interaction for the indispensable amino acids and totals in the anovas for the independent variables of structure x variety with no greenhouse data for var. ‘t’ (ext table 1) were: highly significant for arg (f = 79.111,7, p = <.0001), leu (f = 70.191,7, p = <.0001), phe (f = 122.901,7, p = <.0001), trp (f = 68.061,7, p = <.0001), val (f = 68.061,7, p = <.0001), and totp (f = 68.601,7, p = <.0001); and significant for his (f = 36.221,7, p = 0.0005), iso (f = 52.341,7, p = 0.0002), lys (f = 20.931,7, p = 0.0026), met (f = 41.431,7, p = 0.0004), thr (f = 31.121,7, p = 0.0008), and crdp (f = 18.971,7, p = 0.0033). most amino acids in the structure x variety anovas without a request for an interaction term, would have agreed with h0, had there been a similar hypothesis for variety to the hypotheses for structure, peat, and steer manure. this was true even though with the interaction term request the structure x variety anovas had significance in agreement with h1 for variety. despite this contradiction, these results are still worth discussing. this was because in the same analyses, both dispensable tau and indispensable tyr were significant or highly significant without and with the interaction term request for variety. therefore, a lack of significant results for variety without the interaction term request was not a uniform issue for every amino acid. this appears to indicate that these anovas produced meaningful results for variety, without as well as with the interaction term request. si 2.11. positive results for variety and for the interaction, in the variety x steer manure anovas with greenhouse leaf and stem data for var. ‘t’ positive results for variety for the indispensable amino acids and totals in the anova for the independent variables of variety x steer manure with no greenhouse data for var. ‘t’ (ext table 6) without the interaction term request, as well as for the interaction, were identical and significant only for dispensable hly (f = 08.001,5, p = 0.0367). there was no significance for the independent variable steer manure with or without the interaction term request, nor for variety with the interaction term request. si 2.12. positive results for structure in the structure x coa anovas with no greenhouse data for var. ‘t’ positive results for structure for the indispensable amino acids and totals in the anovas for the independent variables of structure x coa with no greenhouse data for var. ‘t’ (ext table 7) only without the interaction term request were: highly significant for arg (f = 2126.121,3, p = <.0001), his (f = 2448.091,3, p = <.0001), iso (f = 1437.481,3, p = <.0001), leu (f = 1749.601,3, p = <.0001), lys (f = 2980.121,3, p = <.0001), met (f = 1046.291,3, p = <.0001), phe (f = 1705.121,3, p = <.0001), thr (f = 961.001,3, p = <.0001), val (f = 1773.711,3, p = <.0001), crdp (f = 2049.441,3, p = <.0001) and totp (f = 1980.511,3, p = <.0001); and significant only for trp (f = 535.711,3, p = 0.0002). si 2.13. positive results for coa in the structure x coa anovas with no greenhouse data for var. ‘t’ positive results for coa for the indispensable amino acids and totals in the anovas for the independent variables of structure x coa with no greenhouse data for var. ‘t’ (ext table 7) was significant only for crdp and only without the interaction term request (f = 11.545,3, p = 0.0357), though there were no positive results in the ls-means analyses for the same pair of independent variables (ext table 17; see si 3.6), and in spite of several positive results in the same anova analyses for structure (ext table 7). si 2.14. results for the interaction in the structure x coa anovas with no greenhouse data for var. ‘t’ results for the interaction for the indispensable amino acids and totals in the anovas for the independent variables of structure x coa with no greenhouse data for var. ‘t’ (ext table 7) with the interaction term request were not significant. si 2.15. positive results for the interaction in the structure x peat anovas with no greenhouse data for var. ‘t’ positive results for the interaction for peat for the indispensable amino acids and totals in the anovas for the independent variables of structure x peat with no greenhouse data for var. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 644 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) ‘t’ (ext table 8) was only significant for met (f = 25.302,3, p = 0.0132). si 2.16. results for the interaction, in the structure x steer manure and peat x steer manure anovas, each with no greenhouse data for var. ‘t’ there were no positive results in the anovas for the interaction, from the structure by steer manure (ext table 9) or peat x steer manure (ext table 10) anovas. these analyses both excluded greenhouse data for var. ‘t’. ext table 9 had positive results for both individual independent variables though ext table 10 did not. tukey-adjusted ls-means analyses of both pair-wise comparisons, also had positive results (ext tables 19, 20). si 2.17. anova tables named in discussion not containing significant data there were no positive results with or without the request for the interaction term, from the variety x coa (ext table 4), variety x peat (ext table 5), or peat x steer manure (ext table 10) anovas. distribution of negative and uncomputed results in ext tables 4, 5 and 10 are available in appendix c-7 of kritzer van zant (2016a). si anova tables 4, 5 and 10 correlate consecutively with si ls-means tables 14, 15 and 20 for the consecutively same pair-wise comparisons, which all have positive results presented below. all other positive anova data was discussed in the text of the main paper above, so was not detailed again in discussion in the si. si 3. ext tables 11–20 tukey adjusted ls-means of m. expansa data; effects of independent field variable levels on percentages of amino acids ext tables 11-20 showed positive data for the tukey adjusted ls-means t-table data with levels for the same variables seen in anova ext tables 1-10, and matched to them in the order in which dependent variables were presented for each pair-wise comparison. supporting ls-means tables were presented in this form to save white space, fit into a standard word document, and for easier comparison with text description. excel formatted tables of the same ls-means data were also previously given (kritzer van zant 2016a). si 3.1. comparison of structure x variety ls-means analyses structure x variety were run in the tukey-adjusted lsmeans analyses incorporating data from the field grown plants, and also incorporating both leaf and stem data for var. ‘t’ from greenhouse-grown plants (ext table 11), and then only also additionally incorporating the var. ‘t’ leaf data (ext table 12), and finally with only field data while excluding the greenhouse data (ext table 13). differences among the results from the three versions of the structure x variety ls-means analyses, mostly consisted of shifts in the assigned tukey-kramer letters, as consecutively fewer variable level combinations were included as more data was excluded. relative assignments of tukey-kramer letters did not lose relative meaning among level combinations in the structure x variety ls-means analyses as data was excluded. there were also some slight differences in assigned tukey-kramer letters among the structure x variety ls-means t-values, after changes were made to the rounding of all final digits after the decimal 5, from rounding up to rounding down (ext tables 11-13). structure x variety anova f-values were given above (ext table 1), also representing data for ext tables 2 and 3, which were for the same pair-wise comparison as ext table 1. however, ext table 2 was based on the same data as ext table 1, including green-house leaf data for var. ‘t’ though lacking greenhouse stem data for var. ‘t’. ext table 3 lacked all of the green-house data for var. ‘t’. the same was true for the consecutively matching tukey-adjusted ls-means analyses (ext tables 11, 12 and 13), so of these three versions of the structure x variety ls-means analyses, only fully or partially positive results for level combinations for the independent variables from ext table 11, are detailed in full below. si 3.2. positive results in the structure x variety ls-means with greenhouse leaf and stem data included for var. ‘t’ positive results for the indispensable amino acids and totals in the tukey-adjusted ls-means analyses for the independent variables of structure x variety with greenhouse data for var. ‘t’ (ext table 11) were: arg highly significant for all variable level combinations which included var. ‘l’ root 0.23 ± 0.01c**, var. ‘t’ root 0.27 ± 0.02c**, var. ‘t’ stem 00.27 ± 0.03c**, var. ‘t’ leaf 0.99 ± 0.03b**, and var. ‘l’ leaf 1.32 ± 0.01a**; his highly significant for all variable level combinations which included var. ‘l’ root 0.13 ± 0.01c**, var. ‘t’ stem 00.15 ± 0.02c**, var. ‘t’ root 0.17 ± 0.01c**, var. ‘t’ leaf 0.42 ± 0.02b** and var. ‘l’ leaf 0.55 ± 0.01a**; iso highly significant for all level combinations which included var. ‘l’ root 0.25 ± 0.02c**, var. ‘t’ stem 00.26 ± 0.03c**, var. ‘t’ root 0.28 ± 0.02c**, var. ‘t’ leaf 0.84 ± 0.03b**, and var. ‘l’ leaf 1.16 ± 0.02a**; leu highly significant for all variable level combinations which included var. ‘l’ root 0.37 ± 0.02c**; var. ‘t’ root 0.44 ± 0.03c**; var. ‘t’ stem 0.45 ± 0.05c**; var. ‘t’ leaf 1.49 ± 0.05b**; var. ‘l’ leaf 1.99 ± 0.02a**); lys highly significant for all variable level combinations which included var. ‘l’ root 0.37 ± 0.02c**, var. ‘t’ stem 0.41 ± 0.04c**, var. ‘t’ root 0.43 ± 0.03c**, var. ‘t’ leaf 1.25 ± 0.04b** and var. ‘l’ leaf 1.45 ± 0.02a**; met highly significant or significant for var. ‘l’ root 0.10 ± 0.01c**, var. ‘t’ stem 0.11 ± 0.02c*, var. ‘t’ root 0.12 ± 0.01c**, var. ‘t’ leaf 0.34 ± 0.02b** and var. ‘l’ leaf 0.49 ± 0.01a**; phe highly significant for all variable level combinations which included var. ‘l’ root 0.24 ± 0.02c**, var. ‘t’ root 0.27 ± 0.02c**, var. ‘t’ stem 0.29 ± 0.03c**, var. ‘t’ leaf 0.92 ± 0.03b** and var. ‘l’ leaf 1.42 ± 0.02a**; thr highly significant for all variable level combinations which included var. ‘l’ root 0.27 ± 0.01c**, var. ‘t’ root 0.28 ± 0.02c**, var. ‘t’ stem 0.32 ± 0.03c**, var. ‘t’ leaf 0.83 ± 0.03b** and var. ‘l’ leaf 1.04 ± 0.01a**; trp highly significant or significant for all variable level combinations which included var. ‘t’ stem 0.04 ± 0.01c*, 645 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) var. ‘l’ root 0.05 ± 0.01c**, var. ‘t’ root 0.06 ± 0.01c*, var. ‘t’ leaf 0.15 ± 0.01b** and var. ‘l’ leaf 0.30 ± 0.01a**; val highly significant for all variable level combinations which included var. ‘l’ root 0.30 ± 0.02c**, var. ‘t’ stem 0.33 ± 0.04c**, var. ‘t’ root 0.35 ± 0.03c**, var. ‘t’ leaf 1.01 ± 0.04b** and var. ‘l’ leaf 1.40 ± 0.02a**; crdp highly significant or significant for all variable level combinations which included stem of var. ‘t’ 8.78 ± 1.52c*, root of var. ‘l’ 8.89 ± 0.76c**, root of var. ‘t’ 11.42 ± 1.08c**, leaf of var. ‘t’ 22.52 ± 1.52b** and leaf of var. ‘l’ 29.37 ± 0.76a**; and totp highly significant for all variable level combinations which included root for var. ‘l’ 5.70 ± 0.35c**, stem for var. ‘t’ 5.99 ± 0.70c**, root for var. ‘t’ 6.87 ± 0.49c**, leaf for var. ‘t’ 16.80 ± 0.70b** and leaf for var. ‘l’ 23.80 ± 0.35a**. si 3.3. positive results in the structure x peat ls-means with no greenhouse data for var. ‘t’ positive results for the indispensable amino acids and totals in the tukey-adjusted ls means analyses for structure x peat with no greenhouse data for var. ‘t’ (ext table 18) were: arg was highly significant for leaf and significant for root for all variable level combinations which included: 0% peat for root 0.23 ± 0.02b*, 1% peat for root 0.24 ± 0.03b*, 3% peat for root 0.26 ± 0.03b*, 2% peat for root 0.28 ± 0.03b*, 3% peat for leaf 1.28 ± 0.03a**, 0% peat for leaf 1.32 ± 0.02a** and 1% peat for leaf 1.36 ± 0.03a**; his with no greenhouse data for var. ‘t’ was highly significant for leaf and significant for root for all variable level combinations which included 0% peat for root 0.13 ± 0.01b*, 1% peat for root 0.13 ± 0.01b*, 3% peat for root 0.13 ± 0.01b*, 2% peat for root 0.20 ± 0.01b*, 3% peat for leaf 0.53 ± 0.01a**, 0% peat for leaf 0.55 ± 0.01a** and 1% peat for leaf 0.57 ± 0.01a**; iso was highly significant for leaf and significant for root for all variable level combinations which included 0% peat for root 0.25 ± 0.01b*, 1% peat for root 0.25 ± 0.02b*, 3% peat for root 0.27 ± 0.02b*, 2% peat for root 0.31 ± 0.02b*, 3% peat for leaf 1.11 ± 0.02a**, 0% peat for leaf 1.16 ± 0.02a** and 1% peat for leaf 1.20 ± 0.02a**; leu was highly significant for leaf and significant for root for all variable level combinations which included 0% peat for root 0.37 ± 0.02b*, 1% peat for root 0.38 ± 0.04b*, 3% peat for root 0.39 ± 0.04b*, 2% peat for root 0.47 ± 0.04b*, 3% peat for leaf 1.90 ± 0.04a**, 0% peat for leaf 2.01 ± 0.03a** and 1% peat for leaf 2.03 ± 0.04a**; lys was highly significant for leaf and significant for root for all variable level combinations which included 0% peat for root 0.37 ± 0.02b*, 1% peat for root 0.38 ± 0.03b*, 3% peat for root 0.39 ± 0.03b*, 2% peat for root 0.49 ± 0.03b*, 3% peat for leaf 1.41 ± 0.03a**, 0% peat for leaf 1.46 ± 0.02a** and 1% peat for leaf 1.47 ± 0.03a**; met was highly significant for all variable level combinations for both leaf and root, which included 0% peat for root 0.10 ± 0.00d**, 1% peat for root 0.11 ± 0.00cd**, 3% peat for root 0.11 ± 0.00cd**, 2% peat for root 0.14 ± 0.00c**, 3% peat for leaf 0.46 ± 0.00b**, 0% peat for leaf 0.49 ± 0.00a** and 1% peat for leaf 0.51 ± 0.00a**; phe in the ls-means analyses for structure x peat with no greenhouse data for var. ‘t’ (ext table 18), was highly significant for leaf and significant for root for all variable level combinations which included 0% peat for root 0.24 ± 0.01b*, 1% peat for root 0.24 ± 0.02b*, 3% peat for root 0.27 ± 0.02b*, 2% peat for root 0.29 ± 0.02b*, 3% peat for leaf 1.36 ± 0.02a**, 0% peat for leaf 1.42 ± 0.01a** and 1% peat for leaf 1.46 ± 0.02a**; thr was highly significant for leaf and significant for root for all variable level combinations which included 0% peat for root 0.26 ± 0.01b*, 1% peat for root 0.27 ± 0.02b*, 3% peat for root 0.30 ± 0.02b*, 2% peat for root 0.31 ± 0.02b*, 3% peat for leaf 1.01 ± 0.02a**, 1% peat for leaf 1.04 ± 0.02a** and 0% peat for leaf 1.06 ± 0.01a**; trp was highly significant or significant for all variable level combinations which included 0% peat for root 0.05 ± 0.01b*, 1% peat for root 0.05 ± 0.01b*, 3% peat for root 0.05 ± 0.01b*, 2% peat for root 0.08 ± 0.01b*, 0% peat for leaf 0.29 ± 0.01a**, 3% peat for leaf 0.31 ± 0.01a** and 1% peat for leaf 0.32 ± 0.01a**; val was highly significant for leaf and significant for root for all variable level combinations which included 0% peat for root 0.30 ± 0.02b*, 1% peat for root 0.31 ± 0.03b*, 2% peat for root 0.38 ± 0.03b*, 3% peat for root 0.32 ± 0.03b*, 0% peat for leaf 1.40 ± 0.02a**, 1% peat for leaf 1.44 ± 0.03a** and 3% peat for leaf 1.34 ± 0.03a**; crdp was highly significant for leaf and significant for root for all variable level combinations which included 0% peat for root 8.64 ± 0.56b*, 3% peat for root 9.25 ± 0.96b*, 1% peat for root 9.31 ± 0.96b*, 2% peat for root 13.91 ± 0.96b*, 3% peat for leaf 28.70 ± 0.96a**, 1% peat for leaf 29.43 ± 0.96a** and 0% peat for leaf 29.68 ± 0.68a**; and totp for variable level combinations was highly significant for leaf and one of four root results, and significant for three of four root, which included 0% peat for root 5.62 ± 0.20b**, 3% peat for root 5.94 ± 0.35b*, 1% peat for root 5.98 ± 0.35b*, 2% peat for root 7.76 ± 0.35b*, 3% peat for leaf 22.84 ± 0.35a**, 0% peat for leaf 23.97 ± 0.25a** and 1% peat for leaf 24.43 ± 0.35a**. si 3.4. positive results in the structure x steer manure lsmeans with no greenhouse data for var. ‘t’ positive results for the indispensable amino acids and totals in the tukey-adjusted ls means analyses for structure x steer manure with no greenhouse data for var. ‘t’ (ext table 19) were: arg was highly significant for leaf or significant for root for all variable level combinations which included 5% steer manure for root 0.23 ± 0.03b*, 3% steer manure for root 0.24 ± 0.03b*, 0% steer manure for root 0.25 ± 0.02b*, 4% steer manure for root 0.26 ± 0.03b*, 0% steer manure for leaf 1.29 ± 0.02a**, 5% steer manure for leaf 1.35 ± 0.03a** and 3% steer manure for leaf 1.36 ± 0.03a**; his was significant for all variable level combinations which included 3% steer manure for root 0.13 ± 0.03b*, 5% steer manure for root 0.13 ± 0.03b*, 4% steer manure for root 0.14 ± 0.03b*, 0% steer manure for root 0.15 ± 0.02b*, 0% steer manure for leaf 0.53 ± 0.02a*, 5% steer manure for leaf 0.56 ± 0.03a* and 3% steer manure for leaf 0.57 ± 0.03a*; iso was highly significant or significant for all variable level combinations which included 3% steer manure for root 0.25 ± 0.03b*, 4% steer manure for root 0.25 ± 0.03b*, 5% steer manure for root 0.26 ± 0.03b*, 0% steer manure for root 0.27 ± 0.02b*, 0% steer manure for leaf 1.12 ± 0.02a**, 5% steer manure for leaf 1.19 ± 0.03a** and 3% steer a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) manure for leaf 1.20 ± 0.03a**; leu was highly significant for leaf or significant for root for all variable level combinations which included 5% steer manure for root 0.36 ± 0.06b*, 3% steer manure for root 0.38 ± 0.06b*, 0% steer manure for root 0.40 ± 0.03b*, 4% steer manure for root 0.41 ± 0.06b*, 0% steer manure for leaf 1.94 ± 0.04a**, 3% steer manure for leaf 2.03 ± 0.06a** and 5% steer manure for leaf 2.05 ± 0.06a**; lys for variable level combinations was highly significant for one of three and significant for two of three leaf, and significant for all of three root, which included 3% steer manure for root 0.38 ± 0.06b*, 4% steer manure for root 0.38 ± 0.06b*, 5% steer manure for root 0.38 ± 0.06b*, 0% steer manure for root 0.41 ± 0.04b*, 0% steer manure for leaf 1.42 ± 0.04a**, 3% steer manure for leaf 1.47 ± 0.06a* and 5% steer manure for leaf 1.49 ± 0.06a*; met for variable level combinations was highly significant for one and significant for two of three levels of leaf, and was significant for four of four levels of root, which included 4% steer manure for root 0.10 ± 0.02b*, 5% steer manure for root 0.10 ± 0.02b*, 3% steer manure for root 0.11 ± 0.02b*, 0% steer manure for root 0.12 ± 0.01b*, 0% steer manure for leaf 0.47 ± 0.01a**, 5% steer manure for leaf 0.50 ± 0.02a* and 3% steer manure for leaf 0.51 ± 0.02a*; phe was highly significant for leaf and significant for root for all variable level combinations which included 3% steer manure for root 0.24 ± 0.03b*, 4% steer manure for root 0.25 ± 0.03b*, 5% steer manure for root 0.25 ± 0.03b*, 0% steer manure for root 0.26 ± 0.02b*, 0% steer manure for leaf 1.38 ± 0.02a**, 5% steer manure for leaf 1.44 ± 0.03a** and 3% steer manure for leaf 1.46 ± 0.03a**; thr was highly significant for leaf and significant for root for all variable level combinations which included 4% steer manure for root 0.26 ± 0.03b*, 5% steer manure for root 0.26 ± 0.03b*, 3% steer manure for root 0.27 ± 0.03b*, 0% steer manure for root 0.29 ± 0.02b*, 0% steer manure for leaf 1.02 ± 0.02a**, 3% steer manure for leaf 1.04 ± 0.03a** and 5% steer manure for leaf 1.09 ± 0.03a**; trp for variable level combinations was highly significant for one and significant for two of three leaf and significant for four of four root results, which included 3% steer manure for root 0.05 ± 0.01b*, 4% steer manure for root 0.05 ± 0.01b*, 5% steer manure for root 0.06 ± 0.01b*, 0% steer manure for root 0.06 ± 0.01b*, 5% steer manure for leaf 0.28 ± 0.01a*, 0% steer manure for leaf 0.30 ± 0.01a** and 3% steer manure for leaf 0.32 ± 0.01a*; val was highly significant for leaf and significant for root for all variable level combinations which included 3% steer manure for root 0.31 ± 0.04b*, 5% steer manure for root 0.31 ± 0.04b*, 4% steer manure for root 0.32 ± 0.04b*, 0% steer manure for root 0.33 ± 0.02b*, 0% steer manure for leaf 1.35 ± 0.03a**, 3% steer manure for leaf 1.44 ± 0.04a** and 5% steer manure for leaf 1.44 ± 0.04a**; crdp was significant for all variable level combinations, which included 4% steer manure for root 8.94 ± 2.58a*, 5% steer manure for root 9.11 ± 2.58a*, 3% steer manure for root 9.31 ± 2.58a*, 0% steer manure for root 10.35 ± 1.49a*, 0% steer manure for leaf 28.70 ± 1.82a*, 3% steer manure for leaf 29.43 ± 2.58a* and 5% steer manure for leaf 30.66 ± 2.58a*; and totp for variable level combinations was highly significant for one and significant for two of three leaf, and significant for four of four root, which included 5% steer manure for root 5.55 ± 1.09b*, 3% steer manure for root 5.98 ± 1.09b*, 4% steer manure for root 5.98 ± 1.09b*, 0% steer manure for root 6.35 ± 0.63b*, 0% steer manure for leaf 23.26 ± 0.77a**, 5% steer manure for leaf 24.25 ± 1.09a* and 3% steer manure for leaf 24.43 ± 1.09a*. si 3.5. positive results in the peat x steer manure ls-means with no greenhouse data for var. ‘t’ there were no positive results for any of the indispensable amino acids and totals in the tukey-adjusted ls means for peat x steer manure with no greenhouse data for var. ‘t’ (ext table 20). for peat x steer manure significant results were only found for dispensable amino acids hly, hpr, pro and tau. of these, only hly was significant for all variable level combinations. positive results for the dispensable amino acids in the tukeyadjusted ls means for peat x steer manure with no greenhouse data for var. ‘t’ (ext table 20) were: hly was significant for independent variable level combinations 0% peat and 0% steer manure 0.15 ± 0.02a*, 2% peat and 0% steer manure 0.24 ± 0.03a*, 3% peat and 0% steer manure 0.14 ± 0.02a*, 1% peat and 3% steer manure 0.13 ± 0.02a*, 0% peat and 4% steer manure 0.13 ± 0.03a*, 0% peat and 5% steer manure 0.12 ± 0.02a*; hpr was significant for independent variable level combinations 0% peat and 0% steer manure 0.06 ± 0.01a*, 2% peat and 0% steer manure 0.06 ± 0.02a*, 3% peat and 0% steer manure 0.07 ± 0.01a*, 1% peat and 3% steer manure 0.09 ± 0.01a*, 0% peat and 5% steer manure 0.09 ± 0.01a*, and not significant for 0% peat and 4% steer manure; pro was significant for: 0% peat and 0% steer manure 1.11 ± 0.35a*, 3% peat and 0% steer manure 1.06 ± 0.35a*, 1% peat and 3% steer manure 1.33 ± 0.35a*, 0% peat and 5% steer manure 1.16 ± 0.35a*, and not significant for both 2% peat and 0% steer manure, and 0% peat and 4% steer manure; tau was significant for independent variable level combinations 0% peat and 0% steer manure 0.05 ± 0.01a*, 2% peat and 0% steer manure 0.08 ± 0.02a*, 1% peat and 3% steer manure 0.05 ± 0.01a*, 0% peat and 4% steer manure 0.08 ± 0.02a*, and not significant for both 3% peat for 0% steer manure, and 0% peat for 5% steer manure. si 3.6. results in the variety x coa, variety x peat, variety x steer manure, variety x coa and structure x coa anovas and ls-means with no greenhouse data for var. ‘t’ there were no hypotheses for variety or coa. there were no significant results for the variety x coa (ext table 4) or variety x peat (ext table 5) anovas. anovas for variety x steer manure (ext table 6) both without and with the interaction term request were significant only for dispensable hly and only for variety, with the exact same results both without and with the interaction term request, given previously in si 2.11. for these reasons, results were summarized in this one si section for the variety x coa (ext table 14), variety x peat (ext table 15) and variety x steer manure (ext table 16) tukey-adjusted ls-means. there were several significant results for structure in the structure x coa anovas (ext table 646 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 7; see si 2.12 and si 3.2), as well as a single positive result for coa for crdp in the same analyses, as coa was an independent variable. no significant results were computed in the tukey-adjusted ls-means for structure x coa (ext table 17, see si 2.13). positive results for the indispensable amino acids and totals in the tukey-adjusted ls means analyses for variety x coa with no greenhouse data for var. ‘t’ (ext table 14) were: hly significant for all variable level combinations which included var. ‘l’ control 0.15 ± 0.02a*, var. ‘l’ 5% steer manure 0.12 ± 0.02a*, var. ‘t’ 4% steer manure 0.13 ± 0.03a*, var. ‘l’ 1% peat and 3% steer manure 0.13 ± 0.02a*, var. ‘t’ 2% peat 0.24 ± 0.03a*, and var. ‘l’ 3% peat 0.14 ± 0.02a*; hpr significant for five of six variable level combinations which included var. ‘l’ control 0.06 ± 0.01a*, var. ‘l’ 5% steer manure 0.09 ± 0.01a*, var. ‘l’ 1% peat and 3% steer manure 0.09 ± 0.01a*, var. ‘t’ 2% peat 0.06 ± 0.02a*, and var. ‘l’ 3% peat 0.07 ± 0.01a*, and not significant for var. ‘t’ 4% steer manure; and pro significant for four of six variable level combinations which included var. ‘l’ control 1.11 ± 0.35a*, var. ‘l’ 5% steer manure 1.16 ± 0.35a*, var. ‘l’ 1% peat and 3% steer manure 1.33 ± 0.35a*, var. ‘l’ 3% peat 1.06 ± 0.35a*, and not significant for var. ‘t’ 4% steer manure and var. ‘t’ 2% peat; tau significant for four of six variable level combinations which included var. ‘l’ control 0.05 ± 0.01a*, var. ‘t’ 4% steer manure 0.08 ± 0.02a*, var. ‘l’ 1% peat and 3% steer manure 0.05 ± 0.01a*, and var. ‘t’ 2% peat 0.08 ± 0.02a*, and not significant for var. ‘l’ 5% steer manure and var. ‘l’ 3% peat. positive results for the indispensable amino acids and totals in the tukey-adjusted ls means analyses for variety x peat with no greenhouse data for var. ‘t’ (ext table 15) were: indispensable thr significant only for var. ‘l’ 0% peat 0.66 ± 0.24a*, and not significant for var. ‘t’ 0% peat, var. ‘l’ 1% peat, var. ‘t’ 2% peat, and var. ‘l’ 3% peat; dispensable ala significant only for var. ‘l’ 0% peat 0.83 ± 0.29a*, and not significant for var. ‘t’ 0% peat, var. ‘l’ 1% peat, var. ‘t’ 2% peat, and var. ‘l’ 3% peat; hly significant for var. ‘l’ 0% peat 0.14 ± 0.01a*, var. ‘t’ 0% peat 0.13 ± 0.03a*, var. ‘l’ 1% peat 0.13 ± 0.02a*, var. ‘l’ 1% peat 0.14 ± 0.02a*; var. ‘t’ 2% peat 0.24 ± 0.03a*, var. ‘l’ 3% peat 0.14 ± 0.02a*; hpr significant for var. ‘l’ 0% peat 0.07 ± 0.01a*, var. ‘l’ 1% peat 0.09 ± 0.01a*, var. ‘t’ 2% peat 0.06 ± 0.02a*; var. ‘l’ 3% peat 0.07 ± 0.01a*, and not significant for var. ‘t’ 0% peat 0.04 ± 0.02a*; pro significant for var. ‘l’ 0% peat 1.14 ± 0.22a*, var. ‘l’ 1% peat 1.33 ± 0.32a*, var. ‘t’ 2% peat 1.33 ± 0.45a*, var. ‘l’ 3% peat 1.06 ± 0.32a*, and not significant for var. ‘t’ 0% peat 0.91 ± 0.45a; ser significant only for var. ‘l’ 0% peat 0.60 ± 0.22a*, and not significant for var. ‘t’ 0% peat, var. ‘l’ 1% peat, var. ‘t’ 2% peat, and var. ‘l’ 3% peat; tau significant for all variable level combinations which included var. ‘l’ 0% peat 0.04 ± 0.01a*, var. ‘t’ 0% peat 0.08 ± 0.02a*, var. ‘l’ 1% peat 0.05 ± 0.01a*, var. ‘t’ 2% peat 0.08 ± 0.02a*, and not significant var. ‘l’ 3% peat; and crdp significant only for var. ‘l’ 0% peat 19.09 ± 6.49a*, and not significant for var. ‘t’ 0% peat, var. ‘l’ 1% peat, var. ‘t’ 2% peat, and var. ‘l’ 3% peat. positive results for the indispensable amino acids and totals in the tukey-adjusted ls means analyses for variety x steer manure with no greenhouse data for var. ‘t’ (ext table 16) were: indispensable lys significant for all variable level combinations which included var. ‘l’ 0% steer manure (see table 4), and was not significant for var. ‘t’ 0% steer manure, var. ‘l’ 3% steer manure, var. ‘t’ 4% steer manure, and var. ‘l’ 5% steer manure; thr significant only for var. ‘l’ 0% steer manure 0.65 ± 0.24a*, and was not significant for var. ‘t’ 0% steer manure, var. ‘l’ 3% steer manure, var. ‘t’ 4% steer manure, and var. ‘l’ 5% steer manure; dispensable ala significant only for var. ‘l’ 0% steer manure 0.84 ± 0.29a*, and was not significant for var. ‘t’ 0% steer manure, var. ‘l’ 3% steer manure, var. ‘t’ 4% steer manure, and var. ‘l’ 5% steer manure; hly highly significant for var. ‘l’ 0% steer manure 0.15 ± 0.01a**, and significant for var. ‘t’ 0% steer manure 0.24 ± 0.03a*, var. ‘l’ 3% steer manure 0.13 ± 0.02a*, var. ‘t’ 4% steer manure 0.13 ± 0.03a*, var. ‘l’ 5% steer manure 0.12 ± 0.02a*; hpr significant for var. ‘l’ 0% steer manure 0.06 ± 0.01a*, var. ‘t’ 0% steer manure 0.06 ± 0.02a*, var. ‘l’ 3% steer manure 0.09 ± 0.01a*, var. ‘l’ 5% steer manure 0.09 ± 0.01a*; and not significant for var. ‘t’ 4% steer manure; pro significant for var. ‘l’ 0% steer manure 1.08 ± 0.22a*, var. ‘t’ 0% steer manure 1.33 ± 0.45a*, var. ‘l’ 3% steer manure 1.33 ± 0.32a*, var. ‘t’ 4% steer manure 0.91 ± 0.45a*, and var. ‘l’ 5% steer manure 1.16 ± 0.32a*; ser significant only for var. ‘l’ 0% steer manure 0.59 ± 0.22a*, and not significant for var. ‘t’ 0% steer manure, var. ‘l’ 3% steer manure, var. ‘t’ 4% steer manure, and var. ‘l’ 5% steer manure; tau significant for var. ‘l’ 0% steer manure 0.04 ± 0.01a*, var. ‘t’ 0% steer manure 0.08 ± 0.02a*, var. ‘l’ 3% steer manure 0.05 ± 0.02a*, and not significant for var. ‘t’ 4% steer manure and var. ‘l’ 5% steer manure; and crdp significant only for var. ‘l’ 0% steer manure 18.63 ± 6.48a*, and not significant for var. ‘t’ 0% steer manure, var. ‘l’ 3% steer manure, var. ‘t’ 4% steer manure, and var. ‘l’ 5% steer manure. si 3.7. comparison of the tukey-adjusted ls-means and anova results for the peat x steer manure analyses with no greenhouse data for var. ‘t’ there were no positive results in the anovas for the independent variables of peat x steer manure with no greenhouse data for var. ‘t’ (ext table 10). therefore, results for the peat x steer manure tukey-adjusted ls-means (ext table 20) were considered alone. si 3.8. tukey-adjusted ls-means tables named though not presented in the supporting information positive results were not presented in full from the structure x variety tukey-adjusted ls-means analyses that included leaf, however, no greenhouse stem data for var. ‘t’ (ext table 12) and also for the ls-means analyses that did not include any greenhouse data for var. ‘t’ (ext table 13). no positive results were generated in the tukey-adjusted ls-means analyses of structure x combined organic amendments (ext table 17; see si 3.6). this was despite significance for structure in the structure x coa anovas (ext table 7), for all ten indispensable amino acids, crdp, totp and for nine of the 13 dispensable amino acids assayed. distribution of negative results and un647 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) computed results for ext tables 12, 13 and 17 have been presented previously in appendix c-7 of kritzer van zant (2016a). ls-means ext tables 12, 13 and 17 correlated consecutively with ext anova tables 2, 3 and 7. as only results from anova ext table 1 of anova ext tables 1-3 were presented in the si, the lack of consideration of results from ext tables 2 and 3 for comparison to ext tables 12 and 13 had no meaning here, though the negative results from all extended tables are available for consideration in kritzer van zant (2016a). ext tables 1 and 11 were also for the pair-wise comparison of structure x variety, and both had positive results that were presented below and considered relative to each other. the anova for structure x combined organic amendments (ext table 7) had positive results that were considered alone, as they could not be compared with positive tukey-adjusted ls-means results for the same pair of variables, since they were lacking. si 4. summaries of independent variables si 4.1. independent variable structure the structure hypothesis for var. ‘l’, was addressed for the independent variable structure, using the structure x peat (ext tables 6, 16), structure x steer manure (ext tables 9, 19) and structure x coa (ext tables 7, 17) analyses, as appropriate in the body of the paper. in addition, the structure x variety (ext tables 1, 11) anova and ls-means analyses were examined below. discussion for structure is organized around the high return of significant results in the structure x variety anovas, even though all pairs of independent variables including variety, were considered separately from the hypotheses. structure had the most significance of any independent variable. the hypothesis for structure is repeated here for convenience. h0 = there will not be a significant difference in quantity for each amino acid, between above and below ground plant parts, and h1 = there will be a significant difference in quantity for each amino acid, between above and below ground plant parts. si 4.1.1. anova analyses for the independent variable structure for the independent variable structure, in the anovas for structure x variety (ext table 1) without and with the interaction term request, all ten indispensable amino acids, arg, his, iso, leu, lys, met, phe, thr, and val, and both crdp and totp were consistently highly significant in full agreement in the structure hypothesis with h1. for the independent variable structure, in the anovas for structure x coa (ext table 7), without the interaction term request, indispensable arg, his, iso, leu, lys, met, phe, thr, val, and both crdp and totp, were highly significant while indispensable trp was significant, in agreement in the structure hypothesis with h1. for the independent variable structure, in the anovas for structure x coa (ext table 7), with the interaction term request, no results were generated for any dependent variable. for the independent variable structure, in the anovas for structure x peat (ext table 8), without and with the interaction term request, all ten indispensable amino acids and both crdp and totp, were highly significant in full agreement for the structure hypothesis with h1. for the independent variable structure, in the anovas for structure x steer manure (ext table 9), supporting h1 in the structure hypothesis were: indispensable arg, iso, leu, met, phe, thr and val, which were highly significant without and with the interaction term request; and indispensable his, lys and trp, and both crdp and totp, which were highly significant without the interaction term request and significant with the interaction term request. in summary, for the structure hypothesis, most indispensable amino acids fully supported h1 meaning that there was a distinction in the amounts of most amino acids and crdp and totp between above and below ground plant parts. the only exceptions were dispensable hly, hpr and tau which supported h0, indicating there was no distinction in the amounts of these amino acids between above and below ground plant parts. si 4.1.2. ls-means analyses for the independent variable structure in the structure x variety ls-means analyses with all greenhouse data included (ext table 11), nearly every indispensable amino acid and both categories were highly significant for all level combinations. the structure x variety ls-means analyses supported h1 for the structure hypothesis and indicated that variety may have also had meaning for differences in the amino acid profiles for m. expansa. in the structure x variety ls-means analyses with all greenhouse data included (ext table 11), most dispensable amino acids were highly significant for all level combinations though ala and glu dropped to significant for var. ‘t’ stem, which was still in support of h1 for the structure hypothesis and indicated that variety may also have had meaning for differences in the amino acid profiles for m. expansa. in the structure x variety ls-means analyses with all greenhouse data included (ext table 11), the highest amounts of all ten indispensable amino acids were found in leaf of var. ‘l’, then leaf of var. ‘t’, which was in support of h1 in the structure hypothesis. in the structure x coa ls-means analyses with no greenhouse data included (ext table 17; see si 3.6), none of the level combinations had significant differences so they were not detailed in discussion. this lack of significance in the structure x coa ls-means may have supported h0 in the structure hypothesis, or may just have been due to too many imbalances in the data for these analyses to resolve that combination of independent variables. for the independent variables structure and peat, in the structure x peat ls-means analyses with no greenhouse data included (ext table 18; see results in si 3.3 and tables 2, 4 and 6), h1 was supported in both the structure and peat hypotheses by: indispensable met which was the only amino acid highly significant for all levels of peat for both leaf and root; most indispensable and dispensable amino acids, and crdp, as they 648 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) were highly significant for all levels of peat for leaf, and significant for all levels of peat for root; and totp which was highly significant for all levels of peat for leaf and for the control for root, and significant for the other levels of peat. in the structure x peat ls-means analyses with no greenhouse data included (ext table 18; see results in si 3.3 and tables 2, 4 and 6), indispensable amino acids for the most part, and crdp and totp, were present in higher amounts in leaf over root for all levels of peat, which gave support to h1 for the structure hypothesis. in the structure x steer manure ls-means analyses with no greenhouse data included (ext table 19; see results in si 3.4 and tables 2, 4 and 6), for both structure and steer manure in support, of h1, respectively for the structure and steer manure hypothesis: most indispensable and most dispensable amino acids were highly significant for leaf and significant for structure for all levels of steer manure; indispensable met and trp were only highly significant for leaf for the control for steer manure, and were significant for all other level combinations for leaf and all level combinations for root; totp was highly significant for the control for leaf and significant for all other levels of steer manure for leaf and for all levels of steer manure for root; and crdp was significant for all levels of steer manure for leaf and root. in the structure x steer manure lsmeans analyses with no greenhouse data included (ext table 19), all indispensable and most dispensable amino acids were present in consistently higher amounts in leaf over root, which gave support to h1 for the structure hypothesis. for the steer manure hypotheses, these same ls-means results were in contradiction to the anova results, which consistently supported h0 for the steer manure hypotheses, though these analyses were not applied to that hypothesis in the body of the paper. si 4.2. independent variable variety for the independent variable variety, examination of anovas could only be made for non-statistical obvious patterns in the data. the two varieties were only grown in the same year once, and with similar yet different amounts of soil amendments. variety was included in pair-wise comparisons in the structure x variety (ext tables 1, 11), variety x coa (ext tables 4, 14), variety x peat (ext tables 5, 15) and variety x steer manure (ext tables 6, 16) anovas (ext tables 1, 4, 5, 6) and ls-means (ext tables 11, 14, 15, 16) analyses. no hypothesis was given for variety, so it was only examined for obvious patterns of significance. si 4.2.1. anova analyses for the independent variable variety for the independent variable variety, in the structure x variety anovas (ext table 1) with the interaction term request, indispensable phe was highly significant and indispensable arg, his, iso, leu, lys, met, thr, trp and val, and totp, were significant. for the independent variable variety, in the structure x variety anovas (ext table 1) without the interaction term request, indispensable arg, his, iso, leu, lys, met, phe, thr, trp and val, and both crdp and totp, and with the interaction term request, crdp, were not significant. variety x coa anovas (ext tables 4, 14) were too imbalanced to produce any significant results for variety, also for the interaction between variety and coa for any amino acid or category. therefore, the structure x coa anovas were not analyzed further. for the independent variable variety, in the variety x peat anovas (ext table 5) without and with the interaction term request, all ten indispensable amino acids, and both crdp and totp, were not significant. though this seemed to have fully supported h0 in both the structure and peat hypotheses, variety had no statistical significance for either. for the independent variable variety, in the variety x steer manure anovas (ext table 6) without and with the interaction term request, all ten indispensable amino acids were not significant. in summary, though there was no hypothesis considered for variety, all indispensable amino acids in the structure x variety anovas were at least significant with the interaction term request, and so were most of the dispensable amino acids. however, the opposite was true without the interaction term request and in the variety x peat and variety x steer manure anovas. it may be possible to distinguish varieties of m. expansa from each other with amino acid profiles, though only after using meaningful calculations to analyze sufficient data for that purpose. si 4.2.2. ls-means analyses for the independent variable variety structure x variety tukey adjusted ls-means (ext table 11) analyses were already examined above under ls-means for structure. variety x coa ls-means (ext table 14; see si 3.6) analyses were not significant for any indispensable amino acid, and not for crdp or totp. variety x peat adjusted ls-means (ext table 15; see si 3.6 and tables 2 and 4) analyses had significance only for var. ‘l’ for the control for peat for indispensable lys and thr, and for crdp. variety x peat adjusted ls-means (ext table 15) analyses had no significance for other indispensable amino acids, nor for remaining level combinations for the previously mentioned amino acids, and crdp, and no significance for totp. variety x steer manure tukey-adjusted ls-means (ext table 16; see si 3.6) analyses for var. ‘l’ for the control for steer manure were highly significant for dispensable hly and significant for indispensable lys and thr, and for crdp. variety x steer manure ls-means (ext table 16) analyses for var. ‘l’ for all other amino acids and level combinations were not significant for any other indispensable amino acids and not for totp. 649 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) si 4.3. independent variable peat independent variable peat was addressed for the peat hypothesis in the variety x peat (ext tables 5, 15), structure x peat (ext tables 8, 18) and peat x steer manure (ext tables 10, 20) analyses. peat x steer manure analyses were added after the other pairwise variables were analyzed, so do not apply directly to any of the original hypotheses. the peat hypothesis is repeated here for convenience. h0 = amount of peat added at the time of plot construction, has no effect on the amount of amino acid produced, considered individually, in roots and separately in shoots, and h1 = amount of peat added at the time of plot construction has an effect on the amount of amino acid produced, considered individually, in roots and separately in shoots. si 4.3.1. anova analyses for the independent variable peat for the independent variable peat, in the variety x peat anovas (ext table 5) without and with the interaction term request, all ten indispensable amino acids, and both crdp and totp, were not significant in full support of h0 for the peat hypothesis. for the independent variable peat, in the variety x peat anovas (ext table 5) without and with the interaction term request, all amino acids with significant results were computed to be fully in support of h0 for the peat hypothesis. for the independent variable peat, in the structure x peat (ext table 8) anovas, which were in support of h1 for the peat hypothesis were: significant without and with the interaction term requestindispensable his and glu, and totp; without the interaction term requestsignificant crdp; and with the interaction term requestsignificant indispensable met and cys. for the independent variable peat, in the structure x peat (ext table 8) anovas, lacking significance in support of h0 for the peat hypothesis were: without and with the interaction term requestindispensable arg, iso, leu, lys, phe, thr, trp and val; without the interaction term requestindispensable met and dispensable cys; and with the interaction term requestcrdp. for the independent variable peat, in the peat x steer manure (ext table 10) anovas, without and with the interaction term request, there was no significance for any of the ten indispensable amino acids, and both crdp and totp, which fully supported h0 for the peat hypothesis. si 4.3.2. ls-means analyses for the independent variable peat independent variable peat was addressed for the peat hypothesis in the variety x peat (ext table 15; see si 3.6 and tables 2 and 4), structure x peat (ext table 18), structure x steer manure (ext table 19) and peat x steer manure (ext table 20) tukey-adjusted ls-means analyses. independent variable peat was addressed for the peat hypothesis in the variety x peat (ext table 15), ls-means analyses, in the ls-means analyses section for variety, above. independent variable peat was addressed for the peat hypothesis in the structure x peat (ext table 18) ls-means analyses, in the ls-means analyses section for structure, above. in structure x peat (ext table 18) ls-means analyses, most indispensable and dispensable amino acids, and crdp, supported h1 in the peat hypothesis, which indicated that there was an effect of peat on amino acid production. independent variable peat in the peat x steer manure (ext table 20) ls-means analyses, was not significant for all level combinations for all ten of the indispensable amino acids, most dispensable amino acids, and was not significant for either crdp or totp. therefore, in the peat x steer manure lsmeans analyses most dependent variables supported h0 in both the peat and steer manure hypotheses. si 4.4. independent variable steer manure independent variable steer manure was addressed for the steer manure hypotheses, in the variety x steer manure (ext tables 6, 16), structure x steer manure (ext tables 9, 19) and peat x steer manure (ext tables 10, 20) analyses. as the peat x steer manure analyses were added after the other pair-wise variables were analyzed, peat x steer manure did not apply directly to any of the original hypotheses. peat x steer manure could only be discussed without statistical meaning. the steer manure hypothesis is repeated here for convenience. h0 = amount of steer manure added at the time of plot construction has no effect on the amount of amino acid produced, considered individually, in roots and separately in shoots, and h1 = amount of steer manure added at the time of plot construction has an effect on the amount of amino acid produced, considered individually, in roots and separately in shoots. this hypothesis was addressed for the independent variable steer manure, in the structure x steer manure anovas (ext table 9) and ls-means (ext table 19; see results in si 3.4 and tables 2, 4 and 6) analyses. si 4.4.1. anova analyses for the independent variable steer manure for the independent variable steer manure, in the variety x steer manure (ext table 6) anovas, without and with the interaction term request, all ten indispensable amino acids, as well as both crdp and totp, were not significant which fully supported h0 in the steer manure hypothesis. for the independent variable steer manure, in the structure x steer manure (ext table 9) anovas, without and with the interaction term request, all ten indispensable amino acids, and crdp and totp, were not significant which fully supported h0 in the steer manure hypothesis. for the independent variable steer manure, in the peat x steer manure (ext table 10) anovas, without and with the interaction term request, all ten indispensable amino acids, and both crdp and totp, were not significant, which fully supported h0 in the steer manure hypothesis. 650 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) si 4.4.2. ls-means analyses for the independent variable steer manure independent variable steer manure was addressed in the variety x steer manure (ext table 16; see si 3.6 and tables 2 and 4) tukey-adjusted ls-means analyses, above, under the heading, si 4.2.2. ls-means for the independent variable variety. independent variable steer manure was also addressed in the structure x steer manure (ext table 19; see results in si 3.4 and tables 2, 4 and 6) tukey-adjusted ls-means analyses above, under the heading, si 4.1.2. ls-means for the independent variable structure. independent variable steer manure was addressed in the peat x steer manure (ext table 20) tukey-adjusted ls-means under the heading si 4.3.2. ls-means for the independent variable peat. si 4.5. independent variable coa both the peat and steer manure hypotheses partially examined the independent variable coa. coa was utilized in the variety x coa (ext tables 4, 14) and structure x coa (ext tables 7, 17), analyses. in addition, both peat and steer manure were considered in the peat x steer manure (ext tables 10, 20) analyses. these analyses were considered above under the anova and ls-means heading for the individual independent variables, so will not be repeated here. the meaning of coa had to be weighed against the totality of the imbalances in the data, as most of them came into play in the coa analyses. it could be said that in the anovas, most results concerned with peat vs. steer manure showed that there was a mild influence on the amino acid profiles for m. expansa from peat, which will require sufficient data and proper mathematical consideration to find if the effect of peat is consistent, if possible. also, the effect of steer manure was mostly non-existent. yet, especially from the ls-means results, it appeared that either soil amendment may have a limited benefit. we speculated that this was due to soil amendments having had the effect of increasing water retention in the root zone, which m. expansa reacts poorly to, and that it was less likely to have been from the presence of the amendments as nutrient sources. as the amendments had been added to the plots years before the work on m. expansa commenced, it may be that this effect was magnified by additions of equal amounts of mushroom composted steer manure, equally aged for each year, into the hole dug for every plant. this amendment was only added at the time of planting. the increased age of the steer manure may have also affected the outcome between years and therefore to some extent between varieties. si 4.6. interactions in the anovas as it was necessary to separate the independent variables for purposes of discussing the data for m. expansa in a meaningful manner, it was also necessary to separate the meaning of interactions in all of the anova analyses. this was done separately for each anova analyses and presented in the order of the anova tables though there was some discussion of interactions above. interactions for structure and variety, in the structure x variety anovas (ext table 1), for indispensable arg, phe, trp and val, and dispensable asp, glu, gly and tyr, and totp were highly significant, while indispensable his, leu, lys, met and thr, and crdp, were significant. interactions were not computed for significance for any dependent variable in the variety x coa (ext table 4), variety x peat (ext table 5), variety x steer manure (ext table 6), structure x coa (ext table 7) and peat x steer manure (ext table 10) anovas, which indicated that imbalances in the data were too great for those analyses to compute the interaction results. interactions for structure and variety, in the structure x variety anovas (ext table 1), for all ten indispensable amino acids, and both crdp and totp were at least significant, which was in keeping with the large amounts of significance for both independent variables. interactions for structure and peat, in the structure x peat anovas (ext table 8), were only significant for indispensable met and dispensable cys and tyr, and were not significant for any other indispensable or dispensable amino acid, and not for either crdp or totp, and were not computed for dispensable lan and orn. as peat had already been shown to probably have had a slight effect on amino acid production, it may be that structure was another contributor to the uncertainty of the effect of peat, and that peat may have influenced the balance of amino acids in the upper and lower parts of m. expansa grown in southern illinois. interactions for structure and steer manure, in the structure x steer manure anovas (ext table 9), were not significant for all dependent variables. this was a consistent outcome. taken as a whole, results of these interactions show that structure and variety affect each other, and to a more limited extent so do structure and peat, while structure and steer manure have no effect on each other. it was not possible to determine whether there were interaction effects for any of the other independent variable combinations. si 5. summary of additional considerations for resolution of hypotheses for the structure hypotheses, in the anova analyses for structure x variety including all positive data reported (ext table 1), structure x coa (ext table 7), structure x peat (ext table 8) and structure x steer manure (ext table 9), without and with the interaction term request, all ten indispensable amino acids, and both crdp and totp, supported h1, further supporting that structure had an effect on amino acid production. most amino acids also had good support for h1 for the structure hypothesis in the tukey-adjusted ls-means results. variety gave mixed results and was without statistical meaning for any of the given hypotheses. the presence or absence of an interaction term request caused results to change from supporting an effect from variety to not supporting that effect. larger sample sizes and improvements in the balance of raw 651 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) data may give clearer results. in the ls-means for variety results were mixed, depending on which other independent variable was paired with variety. in the ls-means for variety x peat (ext table 15; see si 3.6 and tables 2 and 4), indispensable lys and thr, and crdp, were the only significant dependent variables, and the only dependent variables to have had any significance for the control. results for peat were contradictory among pairs of independent variables in the anovas, for the same dependent variables. in the structure x peat ls-means (ext table 18; see si 3.3 and tables 2, 4 and 6), all indispensable amino acids, and both crdp and totp, supported h1 for the peat hypothesis. in the variety x peat anovas (ext table 5), indispensable amino acids were all not significant, which fully supported h0 for the peat hypothesis, and indicated there was no effect of peat on amino acid production. however, analyses with variety as an independent variable were not statistically meaningful relative to the hypotheses. in the variety x peat ls-means (ext table 15; see si 3.6 and tables 2 and 4) only indispensable lys and thr, along with crdp, appeared to have supported h1 for the peat hypothesis, as they were all significant. in the variety x peat ls-means analyses (ext table 15) once again it appeared that peat did not have a significant effect on amino acid production for most amino acids and totp. peat x steer manure anovas (ext table 10) and ls-means (ext table 20) were an afterthought, which did not apply to either the peat or steer manure hypotheses. even so, both the anovas (ext table 10) and ls-means (ext table 20) for peat x steer manure seemed to have given full support to a lack of effect by peat. for steer manure in the variety x steer manure anovas (ext table 6) all independent variables lacked significance, which fully supported h0 for the steer manure hypothesis, and indicated that there was no effect of steer manure. for steer manure in the variety x steer manure ls-means analyses (ext table 16; see si 3.6 and tables 2 and 4), indispensable lys and thr, and crdp, had significance though only for the control for var. ‘l’. for steer manure in the variety x steer manure lsmeans analyses (ext table 16), eight of the ten indispensable amino acids and totp lacked significance for all level combinations. for steer manure in the structure x steer manure anovas (ext table 9), full support by all independent variables was for h0 in the steer manure hypothesis, indicating there was no effect of steer manure on amino acid production. however, for the structure x steer manure ls-means analyses (ext table 19; see results in si 3.4 and tables 2, 4 and 6) overwhelming support was for h1 in the steer manure hypothesis, indicating that there was an effect of steer manure on amino acid production in m. expansa. peat x steer manure anovas (ext table 10) and lsmeans (ext table 20) were not applicable directly to the steer manure hypothesis because they were added as an afterthought. for steer manure in the peat x steer manure anovas (ext table 10), all ten indispensable amino acids, and both crdp and totp, supported h0 in the steer manure hypothesis, which indicated a lack of effect from steer manure on amino acid pro652 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) duction. for steer manure in the peat x steer manure ls-means analyses (ext table 20), all ten indispensable amino acids, as well as both crdp and totp, were not significant for all level combinations, which would have also supported a lack of effect by steer manure on amino acid production. there was no hypothesis for coa prior to running the coa analyses. in addition, these analyses included plots with both steer manure and peat coded separately for each amendment. coding the same plot twice while disregarded the effect of the other amendment each time, overemphasized the importance of each amendment as it was being coded. perhaps these canceled each other out. however, this may also have compounded their individual impact. for coa in the variety x coa anovas (ext table 4), all ten indispensable amino acids, and both crdp and totp, were not significant. for coa in the variety x coa ls-means analyses (ext table 14; see si 3.6), all ten indispensable amino acids, and neither crdp or totp had any amount of significance for any level combination, indicating a lack of effect from peat or steer manure. for coa in the structure x coa anovas (ext table 7), all ten indispensable amino acids and totp, were not significant without the interaction term request, and with the interaction term request no significant results were computed. for coa in the structure x coa anovas (ext table 7), only crdp was significant and only without the interaction term request, and with the interaction term request no results that were computed had significance. coa in the structure x coa ls-means analyses (ext table 17; see si 3.6) had no significance for any independent variable. results of the variety x coa (ext tables 4, 14) and structure x coa (ext tables 7, 17) anovas (ext tables 4, 7) and ls-means analyses (ext tables 14, 17; see si 3.6) seemed to have supported that neither peat nor steer manure had an effect on the production of most individual amino acids. this contrasted with limited support for an effect by peat, and less by steer manure, for most dependent variables, when each of these independent variables was considered alone. there were also conflicting results without the interaction term request, for crdp for coa, which had no result with the interaction term request. it seemed that there was a real effect of peat for the peat hypothesis, and there may also have been a slight effect of steer manure for the steer manure hypothesis. however, the individual effects of peat and steer manure were vague and hard to pin down with the data available for these analyses. it may be that the time span since the plots were constructed and/or imbalances in the data contributed to conflicting results for the effects of peat and steer manure on amino acid production. it was also possible that peat and/or steer manure had an effect on percentages of amino acids and their total production by m. expansa. it may be that any organic soil amendment that contributes nutrients can assist or limit the growth of m. expansa. a little was helpful. however, too much causes problems for the plant. it may be that the high amount of water sensitivity observed for m. expansa was a greater factor for the effect of both peat and steer manure than any contribution either may 653 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) make as a source of nutrients. these results also seem to point to a low need for fertilizer for m. expansa. si 6. other considerations high amounts of significance for structure indicated that all ten indispensable amino acids, and both crdp and totp, were present in higher amounts in leaf over root. though this is typical for most plants, it was less certain for m. expansa as it produces enlarged stem as well as enlarged root. however, it was also shown that the single sample of enlarged stem from variety ‘t’ had an amino acid profile more similar to those of root, than to those of leaf samples, even if leaf samples contained un-enlarged stem. this was true for var. ‘t’ enlarged stem compared with roots and shoots of either variety for most amino acids. in spite of marked differences in quantity, it appears that the crop produced and/or stored a very similar quality of protein both above and below ground. m. expansa produced a good representation of dispensable as well as indispensable amino acids. the presence of the dispensable amino acids allows consumers of this crop to utilize more of the indispensable amino acids as they are, without having to convert them to dispensable amino acids, to make protein. there were considerable and varied amounts of significance in four of the sets of analyses, all addressing the independent variable structure. however, some pairs of independent variables had little in common for patterns of significance without and with the interaction term request, and significance in the ls-means analyses often contradicted one or both anovas for the same pair of independent variables. in addition, the lsmeans analyses mostly showed little meaning for the amounts of significance for increasing or decreasing increments of soil amendments. the greatest differences were between the controls and other treatments for field grown var. ‘l’. for every independent variable in every anova, there were differences in degrees of significance and exceptions to the general pattern of significance for different dependent variables. all ten indispensable amino acids had at least significant results for structure in the anova analyses, as did crdp and totp. only dispensable tau was present in higher amounts in root than in leaf and may be of use in distinguishing the part of the plant from which ground up material originated. there may be a limited use for dispensable hly in distinguishing var. ‘l’ from var. ‘t’, at least when grown under specific conditions, and for monitoring peat sensitivity in some future m. expansa studies. these potential uses for hly require further testing. hly used alone is unlikely to be useful as an indicator for monitoring effects due to variety or peat. dispensable lan did not produce meaningful results in any of the pair-wise combinations for any model, as it was absent from the samples even though it was addressed in the assays. dispensable orn production was extremely low and undetected in the field. orn was only produced in the green house, indicating that at least for this one amino acid, growth conditions may turn production on or off in the crop. perhaps there is an epigenetic component to orn production and this might hold implications for production of other amino acids in m. expansa as well. this would be in keeping with the epigenetic flexibility suspected by kritzer van zant for morphological expression by m. expansa. epigenetic variation is also a possible explanation for repeated variations in other nyctaginaceae such as the tree genus neea, which varies the number of leaves at a node. the family has many similar examples of morphological flexibility. or, it may just be that the amount of orn produced in that individual sample was just sufficient in quantity to cross the threshold of sensitivity in the amino acid profile assay, while production in other samples was present in amounts just below the threshold for recognition. several combinations of independent variables without any of the variety ‘t’ greenhouse data, gave no significant results in any anova for either independent variable. further, these same pairs of variables often produced no results at all for many dependent variables. variety x coa (ext table 4), produced no results for variety both with and without the interaction term request in the anovas, and produced no significant results for the interactions. in the anovas for variety x peat (ext table 5), variety x steer manure (ext table 6), and peat x steer manure (ext table 10), each independent variable and interaction term request either had no results, or were not significant for any interaction. for the independent variable structure within the anovas for structure x variety (ext tables 1, 2 and 3), most of the indispensable amino acids were highly significant with and without the interaction term. only indispensable trp reflected its own pattern for structure within the anova for structure x variety, significant without the interaction term request and highly significant with the interaction term request, and only in table 1 which had all of the greenhouse data included. in ext tables 2 and 3, all indispensable amino acids, crdp and totp, were highly significant. data for whole models was included in kritzer van zant (2016a), so the interested reader can see how the pair-wise component variables affected anovas overall, and differences between models with and without requests for interaction terms. however, based on the other analyses using the same variables, one of the paired variables was typically considerably more responsible than the other variable in the pair, for the presence and degree of or lack of significance. imbalances in the data compelled the one-by-one approach to the independent variables. causes of imbalances included similar though mismatched treatments with organic amendments in plots for the two varieties, breakdown of coa plots to their component amendments for statistical purposes while ignoring the presence of the other amendment present in only some of the plots included in the analyses, lack of a stem sample for var. ‘l’, only one leaf field sample for var. ‘t’, using field material for the two varieties grown in different years and submitting them for amino acid profiling separately in different years; utilizing greenhouse-grown material for one leaf sample and for the stem sample for var. ‘t’ to represent the above ground material for that variety while all var. ‘l’ samples were field grown, compounded by stem having a profile closer to root than to leaf, the very small number of samples profiled, and the lack of randomness in selection of the remaining samples for amino acid profiling. much of the south american information on growing m. expansa (kritzer van zant, 2016a, b, 2017) was not available to us until after the southern illinois outdoor growth trials were coma tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) pleted (kritzer van zant et al., 2018). in addition, the south american material on growing m. expansa was less detailed than the information from the southern illinois growth study (kritzer van zant, 2016a, b, c; kritzer van zant et al., 2018). figuring out how to grow m. expansa outdoors, based on what we had learned while growing it in the greenhouse, was the original intent of the field study, though hypotheses were written prior to running the anova and ls-means analyses. vivanco, flores and colleagues work at colorado state university was done on a mixture of imported m. expansa from south america and colorado greenhouse-grown material, as it did not survive in colorado in the field (j.m. vivanco, personal communication, 2008). we suspect that the colorado researchers were not aware of the unusual watering needs of the crop, and its high sensitivity to too much water, which became obvious during the southern illinois field and greenhouse work. the plants cannot tolerate standing water, which is characteristic of most southern illinois native soils at various times during the growing season. thus, a special area of artificially constructed sand plots was the only place where the plants were grown successfully in the field. the choice and availability of these plots was limited and not fully consistent from year to year. it was necessary to use most of the same plots used for var. ‘l’ for two years, to obtain repeat data for statistical purposes. var. ‘t’ was grown in 2009 on some other plot types, which had also been used to grow var. ‘l’ on a terrace not available for this purpose in 2008. however, those plots did not produce a harvest in 2009, leaving only var. ‘t’ grown on slightly different treatments than var. ‘l’, to analyze. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 654 atlas journal of biology 2018, pp. 575–582 doi: 10.5147/ajb.v0i0.179 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) morphological and pomological variability analysis of walnut (juglans regia l.) genetic resources from the middle and high atlas of morocco ghizlane kabiri, said bouda*, mohamed elhansali, and abdelmajid haddioui laboratory of biotechnologies and valorization of plant gnetic resources, university of sultan moulay slimane, faculty of sciences and techniques, p.b. 523, beni mellal, morocco received: october 12, 2018 / accepted: december 1, 2018 __________________________________________________ * corresponding author: saidbouda@yahoo.fr 575 abstract the walnut (juglans regia l.) is conducted yet as a traditional fruit crop in different environments of moroccan mountains. besides, little data is available on its genetic diversity. thus, twenty-one morphological and pomological characters of 11 moroccan accessions were used to assess phenotypic variation of this species. significant differences were found between accessions for the most examined traits, indicating a high phenotypic diversity. multivariate analyses lead to identify three groups of accessions. the first group composed by the accessions of both middle and high atlas mountain with high weight and percentage of kernel and low thickness of shell. the second group is made of accessions from the high atlas mountain characterised by large leaf, high nut dimension and large nut size. the third group is composed of two accessions; one belongs to the middle atlas mountain and the other originating from high atlas mountain and characterized by small leaf and nut. then, the structuration of accessions in three groups was not correlated to the mountain range type. so, this study showed a significant phenotypic heterogeneity between the eleven studied accessions which opens the way for the strategies for their conservation and the selection of efficient genotypes with the desired traits. keywords: walnut; juglans regia l; morocco’s accessions; leaf variability; fruit variability. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. introduction the walnut species (juglans regia l.) is one of the most widely cultivated and economically important species of edible nuts in the temperate regions of the world (mcgranahan and leslie, 1990). this species is originated from areas of central asia (amiri et al., 2010) and its cultivation and production were centered in usa and china (angmo et al., 2013). throughout history, the nuts have been a staple food providing energy, protein, essential fatty acids, vitamins and minerals. they are also being investigated for their potential health benefits (dreher et al., 1996). the walnut tree has a great nutritional value and the noble wood. moreover, walnuts have significant economic value and medicinal importance for human health, because of their biochemical composition of polyunsaturated fatty acids, especially linoleic acid and alpha-linolenic and protein value (savage et al., 2001). the first introduction of this species, as a traditional fruit crop, to the great maghreb was attributed to the romans (germain, 1992). in morocco, walnut trees cover an area of 7600 ha and are cultivated in different environments (lansari et al., 2001). the trees can be found in humid and warm conditions, in the rif and atlas mountains (high and middle atlas) and in arid regions in southeastern morocco. more than half of the plantings resulted from the prevailing way of seed propagation practiced by farmers, since grafting is less adopted (lansari et al., 2001). genetic research on moroccan juglans regia has received little attention and few studies have concerned its genetic diversity. only one study of phenotypic variability of few south-eastern populations was carried out by kodad et al. (2014). consequently, it would be very interesting to find more a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) discriminating criteria that could provide additional information on genetic diversity of moroccan walnut populations. thus, the present study was conducted to characterize and evaluate the genetic variability of walnut accessions using some morphological and pomological traits and to look for any space structuring of such accessions according to their distribution on the atlas mountains in morocco. materials and methods plant material and phenotyping traits during september 2014, fresh leaves and mature nuts were collected from eleven moroccan accessions representing the main cropping area of walnut. table 1 and figure 1 present the accessions investigated as well the characteristics of their geographical and ecological origins. for each accession, 10 trees were randomly chosen and sampled. from each tree, ten developed leaves and 20 nuts were collected from different sites and elevation of the tree to be subject of observations and measurements considered in the present study. 576 according to the ipgri and upov descriptors (ipgri, 1994; upov, 1999), 31 parameters related to leaves and fruits (table 2) were considered, following instructions given by the ipgri and upov descriptors. all traits on the kernel have been measured one month after harvest when the water content was below 8% (upov, 1999). statistical analyses the obtained data was submitted to analysis of variance (anova) to search for any significant differences among accessions for the parameters analyzed. when significant differences occurred among accessions, a lsd test was used to determine if there were significant differences between individual accessions. association between traits was established using pearson correlation coefficient. all these analyses were performed by using statistical analysis system program (sas, 1999). accessions ordination and classification were performed using the principal component analysis (pca)( xlstat, 2015) and the hierarchical cluster analysis (statistica statsoft, 1997). the pca and the cluster analyses were carried out on the matrix of mean values of measured characters. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 1. morocco map showing sampling sites of the analyzed walnut accessions. table 1. geographic origin and ecological characteristics of the 11 walnut (juglan rejia l.) accessions. accessions code geographic origin altitude (m) latitude n longitude w zone rainfall average (mm) aghbala agh 32 km north east of aghbala 1673 32°32’ 5°39’ middle atlas 450 naour nao central naour 1300 32°29’ 5°58’ middle atlas 600 taghzirte tag 12 km east of tagzirte 650 32 26 6° 12’ middle atlas 700 ait bougamez abz ait bougamez centre 1996 31°38’ 6° 28’ high atlas 580 ait m’hamed amd 20 km south east of azilal 1728 31° 25’ 2° 28’ high atlas 450 demnate dem 3 km south east of demnate 932 31° 43’ 6° 58’ high atlas 350 imlil iml 17 km south of asni 1763 31° 8’ 7° 55’ high atlas 459 anougal ang 40 km south of amzmiz 1569 31° 9’ 8° 15’ high atlas 681 beram ber 5 km south of midelt 1521 32° 40’ 4° 44’ high atlas 210 amouguer amg 40 km west of rich 1569 32° 12’ 5° 8’ high atlas 250 tabrijjate tbr 70 km east of imilchil 1831 32° 16’ 4° 56’ high atlas 319 577 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) -leaf traits width of pad on suture wps leaf length ll prominence of pad on suture pps leaf width lw shape of base perpendicular to suture sbps number of leaflet nlet shape of apex perpendicular to suture saps leaflet shape lets prominence of apical tip pat leaflet width letw structure of surface of shell sssh leaflet length letl shell colour shc leaflet margin letm shell strength shs leaf colour lc adherence of two halves of shell athsh rachis colour rc thickness of shell tsh -nut traits -kernel traits nut shape in longitudinal section through suture nsls ease of removal of kernel erk nut shape in longitudinal section perpendicular to suture nslps kernel weight kw nut width nwth kernel percentage kp* nut length nl kernel fill kf nut weight nw kernel colour kc nut: position of pad on suture npps kernel flavor kfv kernel percentage = kernel weigh/nut weight*100 table 2. morphological and pomological traits analyzed in the 11 walnut accessions. results mean values of morphological and pomological characters measured and their standard deviations are presented in table 3. results of analysis of variance indicated significant differences between accessions for all parameters except number of leaflets and leaflet margin. leaf morphological traits morphological traits related to leaf show large variability between accessions. in fact, leaf length varied among accessions from 37.98 cm for aghbala to 44.25 cm for anougal with an average of 41.52cm, the leaf width, with a general mean of 25.85cm, ranged from 24.46 cm for taghzirte to 27.17 cm for beram and the number of leaflets scaled from 7.38 for naour to 8.75 for taghzirte with an average of 8.24. in addition, leaflet length, having an average of 10.75 cm, varied from 9.37 cm for taghzirte to 11.16 cm for imlil. with a general mean of 5.48 cm, the highest value of leaflet width was recorded in tabrijjate (6.22 cm) and the lowest was observed in taghzirte (4.98 cm). however, the number of leaflets per leaf has varied narrowly among accessions, within a range of 7.3 for naour to 8.75 for taghzirte, and averaged 8.24. furthermore, with an entire leaflet margin for almost all, the accessions did not show any difference for this trait. in general, the color of leaf was green with a rachis color varied between green, yellow to red. fruit pomological traits the nut weight for the accessions tested varies within the range of 8.26 g for aghbala to 10.20 g for tabrijjate, with an average of 9.48 g. the average nut length was 35.46 mm and ranged from 32.08 mm for demnate to 38.11 mm for imlil. for nut width, the lowest value was obtained for demnate (27.39 mm) and the highest for tabrijjate (31.50 mm) with a mean of 30.10 mm. the shell thickness of the studied accessions in this investigation varied between 1.33 mm for amouguer and 1.83 mm for ait m’hamed with a value of 1.60 mm as average. regarding the kernel weight, the highest value was observed in amouguer (4.13 g), while the least was obtained for aghbala (2.86 g), with a mean of 3.71g. the kernel percentage ranged from 33.65% in demnate to 43.76% in amouguer and averaged 38.28%. for nut shape, in general, it was elliptic board with a medium shell color and intermediate shell strength. concerning kernel color, 9% of the walnut accessions were revealed with light kernel, 36% with amber kernel and 55% with light amber kernel. for kernel flavor, it was revealed satisfactory for all walnut accessions. for the remaining analyzed traits, they have varied widely among accessions indicating the existence of high pomological variability in moroccan walnut germplasm. association between parameters studied correlation among all morphological and pomological traits is summarized in table 4. the strongest negative correlations were observed between, on one hand, kernel weight (kw) and, on the other hand, leaf length, (ll), leaf width (lw), leaflet length (letl), nut length (nl), nut weight (nw), shape of base perpendicular to suture (sbps) and kernel colour (kc), with respective coefficients of -0.95, -0.93, -0.93, -0.92, -0.91, -0.95 and -0.96. however, strong positive correlations were found between kernel weight (kw) and number of leaflet (nlet) (r=0.97), nut shape (nslps) (r=0.97), prominence of apical a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 578 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) ta bl e 3. m ea n va lu es a nd st an da rd d ev ia tio n fo r l ea f, nu t a nd k er ne l t ra its m ea su re d in 1 1 m or oc ca n w al nu t a cc es si on s. tr ai ts ta gh zi rt e ta br ijj at e am ou gu er an ou ga l ai t m ha m ed im lil be ra m ag hb al a n ao ur ai t b ou ga m ez d em na te m ea n f ll ( cm ) 39 .1 4± 3. 19 a 42 .1 4± 3. 76 b 39 .3 9± 4. 14 a 44 .2 5± 6. 51 b 43 .3 5± 5. 54 b 44 .1 4± 5. 88 b 42 .8 0± 4. 19 b 37 .9 8± 3. 85 a 39 .2 8± 4. 06 a 40 .1 7± 4. 75 a 44 .0 8± 3. 12 a 41 .5 2 4. 68 ** * lw ( cm ) 24 .4 6± 2. 20 ab 25 .9 1± 4. 29 a 27 .1 1± 3. 06 c 25 .4 7± 3. 95 a 24 .9 0± 2. 99 a 27 .0 3± 3. 67 c 27 .1 7± 3. 09 c 24 .7 ±4 .1 1a 25 .5 0± 6. 42 b 25 .4 6± 3. 20 a 26 .6 5± 4. 09 c 25 .8 5 6. 23 ** * n le t 8. 75 ± 0. 62 b 7. 5 ± 1. 06 a 8. 05 ±1 .0 3a c 8. 34 ±0 .9 0b c 8. 62 ±0 .6 8b 8. 56 ±0 .9 8b 8. 67 ±0 .9 4b 7. 6± 1. 03 a 7. 38 ±1 .6 6a 8. 37 ±0 .8 3b c 8. 60 ± 0 .7 7b 8. 24 1. 77 le ts 2. 46 ±0 .7 6a b 2. 95 ±0 .2 0c 2. 52 ±0 .6 4b 2. 42 ±0 .6 5a b 2. 77 ±0 .4 2b c 2. 93 ±0 .2 4c 2. 52 ± 0 .6 6b 2. 20 ±0 .8 4a 2. 3± 0. 59 ab 2. 67 ±0 .7 0b c 2. 93 ±0 .3 6c 2. 61 3. 33 ** * le tw (c m ) 4. 98 ±0 .7 7a 6. 22 ±0 .9 6c 5. 31 ±0 .6 7a b 5. 75 ±1 .5 1b c 5. 59 ±0 .7 8b 5. 49 ±0 .8 6b 5. 34 ±0 .8 0a b 5. 35 ±0 .9 5a b 5. 56 ±1 .0 2b 5. 27 ±0 .8 1a b 5. 40 ±1 .2 5a b 5. 48 4. 33 ** * le tl (c m ) 9. 37 ±1 .4 2a 11 .0 2± 1. 68 b 10 .7 8± 1. 37 b 10 .7 2 ± 1. 84 b 10 .5 1± 1. 14 b 11 .1 6± 1. 57 b 10 .5 3± 1. 56 b 10 .4 8± 1. 74 b 10 .6 5± 2. 01 b 10 .5 9± 1. 40 b 10 .4 9± 1. 65 b 10 .5 7 3. 09 ** le tm 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1± 00 a 1. 00 1. 22 lc 5. 68 ±1 .0 9b 5. 66 ±0 .9 6b 4. 78 ±1 .0 1a 5. 45 ±1 .2 9b 5± 00 a 4. 81 ±1 .5 5a 5± 00 a 5. 33 ±1 .0 6b 5± 00 a 5. 12 ±0 .4 9a 5± 00 a 5. 17 3. 75 ** * rc 3. 68 ±1 .3 0a 3. 33 ±0 .9 6a 4. 63 ±0 .7 8c 3± 00 a 4. 18 ±1 .0 0b c 4. 43 ±1 .8 4b c 4. 11 ±1 .0 0b 4± 1. 36 b 6. 86 ±0 .5 0d 4. 06 ±1 .0 1b 3. 06 ±0 .3 6a 4. 12 4. 14 ** * n sl s 6. 32 ±1 .7 4c d 4. 97 ± 1. 97 a 6. 43 ±2 .0 2c 5. 27 ±1 .9 6a 5. 78 ±2 .1 3c 5. 34 ±2 .5 8b 5. 86 ±1 .9 2c 6. 57 ±1 .1 3c 5. 57 ±2 .0 5a 6. 23 ±1 .7 0c 6. 97 ±1 .6 5d 5. 95 10 .3 4* ** n sl ps 6. 65 ±0 .7 2d 4. 77 ± 2. 39 a 6. 70 ±1 .3 0c 5. 94 ±1 .5 6c 5. 54 ±2 .1 1c 5. 01 ±2 .4 4b 6. 09 ±1 .7 1c 6. 65 ±1 .1 0c 5. 54 ±2 .2 5a 6. 35 ±1 .8 7d c 6. 26 ±1 .8 1d 5. 97 6. 67 ** * n w th (m m ) 29 .6 7± 2. 47 b 31 .5 0± 3. 17 a 29 .1 8± 2. 69 b 31 .2 4± 2. 74 c 31 .3 6± 4. 60 a 31 .3 9± 2. 55 c 29 .7 7 ± 3. 29 ab 29 .5 0± 1. 95 b 30 .1 3± 2. 87 b 30 .3 0± 1. 94 a 27 .3 9± 8. 81 a 30 .1 0 19 .8 7* ** n l (m m ) 34 .1 3± 2. 81 b 35 .0 7± 3. 62 bc 35 .1 1± 3. 53 c 37 .0 2± 3. 36 d 36 .4 5± 4. 63 a 38 .1 1± 3. 27 e 34 .8 4± 4. 36 ac 35 .2 3± 2. 12 ac 36 .0 8± 3. 77 a 36 .7 4± 3. 13 d 32 .0 8± 10 .8 2a 35 .4 6 17 .2 7* ** n w (g ) 9. 24 ±1 .6 1c 10 .2 0± 2. 11 b 9. 42 ±2 .8 3c 9. 84 ±2 .1 4b 10 .1 4± 4. 00 b 9. 28 ±2 .1 2c 9. 71 ±2 .6 4b 8. 26 ±2 .0 5b 9. 79 ±2 .9 4b 9. 54 ±1 .5 5b 8. 91 ±3 .5 1b 9. 48 7. 61 ** * n pp s 2. 15 ±0 .7 5d 1. 58 ±0 .7 5b 1. 61 ±0 .9 1b 1. 22 ±0 .4 8a 1. 73 ±0 .6 8b 1. 74 ±0 .7 6b 1. 86 ±0 .7 7c 1. 96 ±0 .9 3c 2. 15 ±0 .6 5d 2. 44 ±0 .7 9e 2. 03 ±1 .0 4c 1. 86 13 .1 2* ** w ps 3. 94 ±0 .9 9b 4. 68 ±0 .8 0e f 4. 84 ±0 .7 3f 5. 24 ±0 .8 6g 4. 60 ±1 .5 3e 5. 04 ±0 .9 4g 4. 36 ±0 .9 7c d 4. 34 ±0 .9 4d 4. 12 ±1 .3 1c 3. 59 ±0 .9 3a 3. 70 ±0 .9 5a 4. 40 8. 95 ** * pp s 4. 32 ±1 .1 0a 4. 94 ±0 .5 8c 4. 60 ±1 .1 3b 4. 86 ±0 .8 6c 4. 64 ±0 .7 6b 5. 02 ±0 .9 2c d 5. 26 ±1 .1 2d 4. 98 ±0 .7 7c 4. 19 ±0 .9 9a 4. 48 ±0 .9 6b 4. 51 ±1 .3 7b 4. 71 6. 2* ** sb ps 1. 77 ±0 .5 0b 2. 15 ±0 .6 2d 1. 45 ±0 .5 5a 2. 19 ±0 .5 1d 2. 02 ±0 .4 7c 2. 20 ±0 .6 3d 1. 78 ±0 .7 1b 1. 54 ±0 .7 3a 2. 06 ±0 .9 3c 1. 72 ±0 .6 9b 1. 82 ±1 .2 0a 1. 88 9. 25 ** * sa ps 3. 06 ±0 .6 4d 2. 84 ±0 .5 8b c 2. 68 ±0 .4 6b 2. 69 ±0 .5 4b 2. 41 ± 0 .5 5a 2. 24 ±0 .8 0a 2. 85 ±0 .8 5c 2. 25 ±0 .5 3a 2. 17 ±0 .8 7a 2. 62 ±0 .7 3b 3. 03 ±0 .9 4c 2. 62 11 .3 ** * pa t 3. 05 ±0 .6 4a 3. 87 ±0 .9 9b 3. 54 ±0 .9 5a 3. 94 ±1 .0 9b 3. 72 ±1 .0 3a b 4. 9± 0. 77 d 3. 99 ±1 .2 0b 4. 4± 1. 16 c 3. 58 ±0 .9 2a 4. 02 ±1 .1 0b 3. 89 ±1 .0 9b 3. 90 3. 05 ** * ss sh 2± 00 bc 2. 38 ±0 .4 9d 1. 76 ±0 .6 5a 2. 35 ±0 .6 4d 2. 62 ±0 .8 6e f 2. 51 ±0 .6 4e 2. 70 ±0 .8 1f 2. 65 ±0 .5 5e f 2. 16 ±0 .3 6c 2. 14 ±0 .3 7c 2. 10 ±0 .3 0b c 2. 31 18 .3 2* ** sh c 6. 42 ±1 .4 8f 4. 40 ±2 .4 8c d 3. 96 ±1 .7 1b c 4. 15 ±1 .1 9c 5. 15 ±2 .2 0e 5. 25 ±1 .4 5e 3. 86 ±1 .4 5b 3. 88 ±1 .6 6b 4. 40 ±1 .9 9d 3. 37 ±1 .6 5a 4. 97 ±0 .6 1e 4. 53 40 .7 9* ** sh s 4. 70 ±0 .7 7b 5. 48 ±1 .1 5d 4. 68 ±0 .9 3b 5. 53 ±1 .3 5d 5. 27 ±0 .9 1d 4. 34 ±1 .1 0a 5. 32 ±0 .8 8c d 5. 1± 0. 99 c 5. 18 ±0 .7 0c d 4. 33 ±1 .0 0a 5. 71 ±1 .8 3d 5. 06 7. 19 ** * at h sh 4. 65 ±0 .7 8b 5. 24 ±0 .7 3d 4. 47 ±0 .8 8b 4. 18 ±1 .3 0a 5. 21 ±1 .0 2d 4. 30 ±1 .2 5a b 5. 15 ±1 .1 5c d 5. 12 ±1 .0 1c d 5. 5± 0. 99 e 4. 35 ±1 .0 0a b 4. 96 ±0 .8 4c 4. 83 9. 84 ** * ts h (m m ) 1. 57 ±0 .3 2c 1. 71 ±0 .3 0d 1. 33 ±0 .4 0a 1. 69 ±0 .4 6d 1. 83 ±0 .5 1e 1. 55 ±0 .3 8c 1. 66 ±0 .4 1d 1. 50 ±2 .8 8b c 1. 61 ±0 .3 6c d 1. 44 ±0 .3 8b 1. 72 ±0 .3 4d 1. 60 4. 07 ** * d rk 4. 3± 1. 01 a 5. 7± 1. 22 a 4. 56 ±0 .9 8b 5. 28 ±1 .1 8d 4. 87 ±1 .3 6c 4. 7± 1. 34 bc 4. 66 ±1 .4 1b c 4. 3± 1. 16 a 4. 79 ±1 .1 4c 4. 25 ±0 .9 9a 5. 24 ±1 .3 2d 4. 79 8. 58 ** * k w (g ) 3. 71 ±1 .1 2b c 4. 02 ±1 .1 5c e 4. 13 ±1 .4 8e 3. 62 ±1 .3 8b c 3. 61 ±1 .3 6b c 3. 87 ±1 .3 6c 3. 78 ±1 .2 7b c 2. 86 ±1 .1 1a 3. 77 ±1 .6 9b c 4. 01 ±1 .1 2c e 3. 45 ±1 .2 5b 3. 71 5. 56 ** * k p (% ) 39 .4 5± 8. 94 cd 38 .7 2± 7. 23 c 43 .7 6± 9. 74 e 35 .4 9± 9. 40 ab 36 .7 1± 11 .1 7b 41 .1 6± 10 .2 3d 38 .8 ±8 .1 4c 33 .6 1± 10 .0 3a 37 .7 3± 9. 67 c 42 .0 1± 8. 42 de 33 .6 5± 9. 90 ab 38 .2 8 5. 3* ** k f 6. 39 ±1 .1 7d 6. 45 ±1 .2 4d 5. 82 ±1 .5 0b c 5. 26 ±1 .5 8a 6. 31 ±1 .3 3c d 5. 60 ±1 .6 2b 6± 1. 37 c 5. 56 ± 1 .6 8b 6. 27 ±1 .1 2d 6. 45 ±1 .1 5d 6. 24 ±1 .3 4c d 6. 03 9. 55 ** * k c 3. 6± 0. 58 2. 72 ±0 .9 6 3. 32 ±0 .6 6 3. 21 ±0 .7 6 2. 94 ±0 .7 8 3. 36 ±0 .8 9 3. 22 ±0 .6 9 2. 57 ±0 .9 9 3. 25 ±0 .8 2 2. 73 ±0 .7 8 3. 25 ±0 .5 1 3. 11 26 .0 7* ** k fv 1. 06 ±0 .2 5 1. 04 ±0 .2 0 1. 05 ±0 .2 2 1. 30 ±0 .4 6 1. 06 ±0 .2 5 1. 15 ±0 .3 6 1. 07 ±0 .2 6 1. 31 ±0 .4 6 1. 02 ±0 .1 4 1. 02 ±0 .1 4 1. 08 ±0 .2 8 1. 11 7. 83 ** * si gn ifi ca nc e le ve l: ** * :p < 0. 00 1; * *: p < 0. 01 ; * : p< 0. 05 . d iff er en t l et te rs n ot e si gn ifi ca nt d iff er en ce s ( ls d a t 0 .0 5 le ve l). in b ol d ar e m in im um a nd m ax im um v al ue s a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 579 ll lw n let le ts le tw le tl le tm lc r c n sl s n sl ps n w th n l n w n pp s w ps pp s sb ps sa ps pa t ss sh sh c sh s a th sh ts h d r k k w k p k f k c lw 0, 97 ** * n let -0 ,9 5* ** 0, 93 ** * le ts -0 ,9 5* ** 0, 93 ** * 0, 98 ** * le tw -0 ,5 9* ** 0, 58 ** * 0, 73 2* ** 0 ,7 1* ** le tl 0, 96 ** * 0, 95 ** * -0 ,9 3* ** 0, 93 ** * -0 ,5 6* ** le tm -0 ,8 3* ** 0, 82 ** * 0, 85 ** * 0, 84 ** * 0, 62 ** * -0 ,8 2* ** lc 0, 15 ** * 0, 14 ** * -0 ,1 0* ** 0, 13 ** * -0 ,0 2 0, 17 ** * -0 ,0 7 r c -0 ,3 6* ** 0, 32 ** * 0, 38 ** * 0, 42 ** * 0, 29 ** * -0 ,3 7* ** 0, 24 ** * -0 ,2 4* ** n sl s -0 , 0 4 0, 01 0, 03 0, 05 0, 05 -0 ,0 3 -0 ,0 6 -0 ,0 7 0, 48 ** * n sl ps -0 ,9 5** * -0 ,9 3* ** 0, 97 ** * 0, 97 ** * 0, 69 ** * -0 ,9 3* ** 0, 84 ** * -0 ,1 3* ** 0, 38 ** * 0, 05 n w th -0 ,5 9* ** 0, 57 ** * 0, 68 ** * 0, 7* ** 0, 64 ** * -0 ,5 6* ** 0, 56 ** * 0, 00 1 0, 36 ** * 0, 06 0, 68 ** * n l 0, 95 ** * 0, 93 ** * -0 ,9 1* ** 0, 93 ** * -0 ,5 1* ** 0, 93 ** * -0 ,8 0* ** 0, 19 ** * -0 ,3 8* ** -0 ,0 1 -0 ,9 1* ** 0, 47 ** * n w 0, 92 ** * 0, 90 ** * -0 ,9 0* ** 0, 91 ** * -0 ,5 8* ** 0, 92 ** * 0 ,8 1* ** 0, 17 ** * -0 ,3 7* ** -0 ,0 2 -0 ,9 1* ** 0, 49 ** * 0, 94 ** * n pp s -0 ,7 2* ** 0, 72 ** * 0, 73 ** * 0, 73 ** * 0, 52 ** * -0 ,7 1* ** 0, 83 ** * -0 ,0 6 0, 15 ** * -0 ,0 8* 0, 74 ** * 0, 52 ** * -0 ,6 9* ** 0, 71 ** * w ps -0 ,2 3* ** 0, 21 ** * 0, 25 ** * 0, 24 ** * 0, 20 ** * -0 ,1 7* ** 0, 21 ** * 0, 41 ** * -0 ,0 5 -0 ,1 3* 0, 25 ** * 0, 29 ** * -0 ,1 7* ** 0, 13 ** * 0, 22 ** * pp s -0 ,2 7* ** 0, 23 ** * 0, 30 ** * 0, 34 ** * 0, 28 ** * -0 ,2 9* ** 0, 17 ** * -0 ,2 1* ** 0, 62 ** * 0, 33 ** * 0, 30 ** * 0, 30 ** * -0 ,2 9* ** 0, 28 ** * 0, 09 * -0 ,0 9* sb ps 0, 98 ** * 0, 95 ** * -0 ,9 5* ** 0, 95 ** * -0 ,6 0* ** 0, 95 ** * -0 ,8 3* ** 0, 16 ** * -0 ,3 8* ** -0 ,0 05 -0 ,9 5* ** 0, 59 ** * 0, 95 ** * 0, 92 ** * -0 ,7 2* ** 0, 21 ** * -0 ,2 6* ** sa ps -0 ,4 7* ** 0, 45 ** * 0, 59 ** * 0, 56 ** * 0, 57 ** * -0 ,4 5* ** 0, 50 ** * 0, 01 0, 17 ** * -0 ,0 5 0, 57 ** * 0, 56 ** * -0 ,4 2* ** 0, 45 ** * 0, 43 ** * 0, 25 ** * 0, 11 ** -0 ,4 4* ** pa t -0 ,9 4* ** 0, 93 ** * 0, 96 ** * 0, 98 ** * 0, 67 ** * -0 ,9 2* ** 0, 83 ** * -0 ,1 5* ** 0, 43 0, 06 0, 96 ** * 0, 70 ** * -0 ,9 2* ** 0, 90 ** * 0, 72 ** * 0, 23 ** * 0, 36 ** * -0 ,9 4* ** 0, 59 ** * ss sh -0 ,7 9* ** 0, 77 ** * 0, 84 ** * 0, 84 ** * 0, 74 ** * -0 ,7 8* ** 0, 70 ** * -0 ,1 5* ** 0, 38 ** * 0, 07 0, 84 ** * 0, 67 ** * -0 ,7 2* ** 0, 76 ** * 0, 61 ** * 0, 17 ** * 0, 31 ** * -0 ,7 9* ** 0, 66 ** * 0, 87 ** * sh c 0, 87 ** * 0, 85 ** * -0 ,8 4* ** 0, 84 ** * -0 ,5 3* ** 0, 89 ** * -0 ,7 6 * ** 0, 17 ** * -0 ,3 3* ** -0 ,0 02 -0 ,8 4* ** 0, 47 ** * 0, 85 ** * 0, 86 ** * -0 ,6 8* ** -0 ,1 4* * -0 ,2 4* ** 0, 88 ** * -0 ,3 4* ** 0, 84 ** * -0 ,6 9* ** sh s 0, 89 ** * 0, 87 ** * -0 ,8 5* ** 0, 87 ** * -0 ,5 1* ** 0, 87 ** * -0 ,6 5* ** 0, 19 ** * -0 ,4 2* ** -0 ,0 9 * -0 ,8 6* ** 0, 50 ** * 0, 88 ** * 0, 85 ** * -0 ,5 2* ** -0 ,1 4* * -0 ,3 4 * ** 0, 90 ** * -0 ,2 8* ** 0, 86 ** * -0 ,7 0* ** 0 ,8 2* ** a th sh -0 ,8 7* ** 0, 85 ** * 0, 87 ** * 0, 87 ** * 0, 54 ** * -0 ,8 4* ** 0, 76 ** * 0, 1* 0, 30 ** * -0 ,0 07 0, 87 ** * 0, 59 ** * -0 ,8 5* ** 0, 81 ** * 0, 67 ** * 0, 43 ** * 0, 21 ** * -0 ,8 6* ** 0 ,5 1* ** 0, 88 ** * 0, 72 ** * -0 ,7 4* ** 0, 76 ** * ts h -0 ,1 1* * -0 ,0 7 0, 15 ** * 0, 19 ** * 0, 15 ** * -0 ,1 6* ** 0, 03 -0 ,0 8* 0, 54 ** * 0, 37 ** * 0, 15 ** * 0, 2* ** -0 ,1 5* ** 0, 15 ** * -0 ,0 1 -0 ,1 6* ** 0, 60 ** * -0 ,1 3* * 0, 07 0, 20 ** * 0, 19 ** * -0 ,1 3* * -0 ,2 1* ** 0, 07 5 d r k -0 ,3 8* ** 0, 31 ** * 0, 37 ** * 0, 40 ** * 0, 23 ** * -0 ,3 8* ** 0, 23 ** * -0 ,1 5* ** 0, 52 ** * 0, 31 ** * 0, 37 ** * 0, 27 ** * -0 ,3 9* ** 0, 35 ** * 0, 14 ** * 0, 05 0, 43 ** * -0 ,3 8* ** 0, 13 ** 0, 40 ** * 0, 33 ** * -0 ,3 4* ** 0, 43 ** * 0, 33 ** * 0, 55 ** * k w -0 ,9 5* ** 0, 93 ** * 0, 97 ** * 0, 97 ** * 0, 69 ** * -0 ,9 3* ** 0, 84 ** * -0 ,1 3 * * 0, 38 ** * 0, 05 0, 97 ** * 0, 67 ** * -0 ,9 2* ** 0, 91 ** * 0, 74 ** * 0, 24 ** * 0, 29 ** * -0 ,9 5* ** 0 ,5 7* ** 0, 97 ** * 0, 84 ** * -0 ,8 4* ** 0, 86 ** * 0, 88 ** * 0, 15 ** * 0, 37 ** * k p -0 ,9 5 * ** 0, 93 ** * 0, 96 ** * 0, 98 ** * 0, 68 ** * -0 ,9 3* ** 0, 83 ** * -0 ,1 4* ** 0, 41 ** * 0, 06 0, 97 ** * 0, 70 ** * -0 ,9 2* ** 0, 91 ** * 0, 73 ** * 0, 23 ** * 0, 34 ** * -0 ,9 5* ** 0 ,5 5* ** 0, 98 ** * 0, 84 ** * -0 ,8 4* ** 0, 86 ** * 0, 87 ** * 0, 20 ** * 0, 41 ** * 0, 98 ** * k f 0, 94 ** * 0, 92 ** * -0 ,9 1* ** 0, 92 ** * -0 ,5 0* ** 0, 92 ** * -0 ,7 9* ** 0, 19 ** * -0 ,3 7* ** -0 ,0 3 -0 ,9 1* ** 0, 48 ** * 0, 97 ** * 0, 93 ** * -0 ,6 8* ** 0, 17 ** * -0 ,3 0* ** 0, 94 ** * -0 ,4 2* ** 0, 91 ** * -0 ,7 1* ** 0 ,8 4* ** 0, 88 ** * -0 ,8 4* ** 0, 16 ** * -0 ,3 8* ** 0, 90 ** * -0 .9 1* ** k c 0, 97 ** * 0, 95 ** * -0 ,9 6* ** 0, 97 ** * -0 ,6 2* ** 0, 96 ** * -0 ,8 3* ** 0, 17 ** * -0 ,4 0* ** -0 ,0 3 -0 ,9 6* ** 0, 58 ** * 0, 97 ** * 0, 95 ** * -0 ,7 2* ** 0, 21 ** * -0 ,3 2* ** 0, 97 ** * 0 ,4 9* ** 0, 96 ** * -0 ,8 0* ** 0 ,8 8* ** 0, 89 ** * -0 ,8 7* ** 0, 17 ** * -0 ,4 0* ** 0, 96 ** * -0 ,9 6* ** 0, 97 ** * k fv 0, 81 ** * 0, 79 ** * -0 ,7 9* ** 0, 81 ** * -0 ,4 8* ** 0, 79 ** * -0 ,5 8* ** 0, 15 ** * -0 ,3 7* ** -0 ,0 9* -0 ,8 0 * ** 0, 41 ** * 0, 84 ** * 0, 84 ** * -0 ,4 7* ** -0 ,1 1* -0 ,3 5* ** 0, 81 ** * -0 ,3 5* ** 0, 81 ** * -0 ,6 7 * ** 0 ,7 1* ** 0, 85 ** * -0 ,7 2* ** 0, 22 ** * -0 ,4 0* ** 0, 79 ** * -0 ,8 0* ** 0, 87 ** * 0, 86 ** * ta bl e 4. c or re la tio n co effi ci en ts a m on g le af , n ut a nd k er ne l t ra its in th e st ud ie d ac ce ss io ns . si gn ifi ca nc e le ve l: ** * :p < 0. 00 1; * *: p < 0. 01 ; * : p< 0. 05 . a bb re vi at io ns a s i n ta bl e 1. a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 580 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) tip (pat) (r=0.97) and kernel percentage (kp) (r=0.98). furthermore, nut weight (nw) was revealed to be positively and strongly correlated with leaf length (ll) (r=0.92), leaf width (lw) (r=0.90), leaflet length (letl) (r=0.92), nut length (nl) (r=0.94), shape of base perpendicular to suture (sbps) (r=0.92), kernel fill (kf) (r=0.93) and kernel colour (kc) (r=0.95), while it has a negative correlation with number of leaflet (nlet) (r=0.90), shape of nut (nslps) (r=-0.91), prominence of apical tip (pat) (r=-0.90), kernel weight (kw) (r=-0.91) and kernel percentage (kp) (r=-0.91). multivariate analysis the eigenvalues obtained by principal component analysis (pca) indicate that the first two components provide a good summary of the data. they explained 71.29% of the total variation with each component explaining respectively 45.88% and 25.41%. the first component is defined negatively by weight and length of nut, color and fill of kernel and by length and width of leaf; and positively by weight and percentage of kernel and by shape and number of leaflet. the second component is positively correlated to thickness of shell, ease of removal the kernel and the color of rachis. figure 2 illustrated accessions on the plot of the first two axis spaces, shows that accessions may constitute three groups. the first one is composed of two accessions of middle atlas mountain: naour (nao) and taghzirte (tag) and two other accessions of high atlas: ait bougamaz (abz) and amouguer (amg), characterized by high weight and percentage of kernel and low thickness of shell. the second group is made of five accessions of high atlas mountain namely ait m’hamed (amd), imlil (iml), beram (ber), tabrijjate (tab) and anougal (ang), characterized with large leaf, high nut dimension and large nut size. the third group is composed of two accessions; one belongs to middle atlas (aghbala, (agh)) and the other arising from high atlas (demnate (dem)) which are characterized by small leaf and nut. hierarchical cluster analysis identified three distinct groups (figure 3) confirming the pca results. the first group is composed of taghzirte (tag) and naour (nao) accessions originating from middle atlas mountain and amouguer (amg) and ait bougamaz (abz) coming from high atlas mountain. the second group includes ait m’hamed (amd), imlil (iml), barem (bar), tabrijjate (tbr) and anougal (ang) accessions, all belonging to high atlas mountain. the demnate (dem) and aghbala (agh) accessions represented the third group and seem to diverge significantly from the others. figure 2. plot on the two first principal components of 11 walnut accessions. abbreviations as in table 1. 581 a tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) figure 3. dendrogram of 11 moroccan walnut accessions based on morphological and pomological traits. abbreviations as in table 1. discussion the results of this study show that the accessions studied could be a very interesting source of walnut genetic diversity. the majority of morphological traits of leaves showed a great difference among walnut accessions, especially for the length and width of leaf. furthermore, the pomological traits exhibited the highest variability among accessions, which was established for most of nut and kernel traits. according to consulted literature, nut weight, kernel percentage and color of kernel have been considered as the most important indices of quality and economic yield in walnut trees, which should be further investigated. in the moroccan accessions studied, the nut weight varied from 8.26 to 10.20 g. values that are within the consulted literature are found between 6 and 16.89 g in iranian genotypes (arzani et al., 2008; ghasemi et al., 2012; ahandani et al., 2014), 7.46 and 15.21 g in turkish genotypes (karadag and akça, 2011) and from 6.9 to 16.27 g in romanian walnut (cosmulescu, 2013). in addition, moroccan accessions have an average kernel percentage between 33.65% and 43.76%. these values are lower than those obtained for some walnut genotypes from turkey (46.15 to 63.16%) (karadag and akça, 2011) and iran (48 to 59%) (ahandani et al., 2014). according to color of kernel, our result showed that 55% of the walnut accessions investigated were revealed with light amber kernel. this result is in accordance with that reported in iranian genotypes by arzani et al. (2008), finding 52% of genotypes with light amber kernel. in fact, the light kernel color is a primary breeding objective for walnut. on other hand, the shell thickness of the moroccan accessions analysed in this work varied from 1.33 to 1.83 mm, which is in agreement with that obtained by karadag and akça (2011) (0.95 to 1.60 mm) and akça et al. (2015) (1.11 to 2.33 mm) in turkish walnut, but greater than that reported by sharma et al. (2014) in indian walnut (1.24 mm). these results showed a considerable phenotypic diversity in the moroccan walnut germplasm in comparison to that of other countries. this variability may be due, first, to genotypic variation or environmental conditions (ghasemi et al., 2012). second, walnuts species are monoecious and heterodichogamous, favoring outcrossing over selfing (ebrahimi et al., 2016). third, reproduce by seeds of this species causes a very important genetic variability that appears at the flowering period for pomological characters, vigor of the tree and type of fructification, which allowed each geographic region to maintain a diverse population (lansari et al., 2001). besides, diaz et al. (2005) stated that the geographic location and the growing conditions affect fruit and kernel traits of walnut populations from western spain. conclusion these results show that moroccan walnut presents a high variability in comparison to that of others countries with similar ecological conditions. this investigation showed significant differences among accessions, permitting discrimination between them according to morphological traits related to leaf, nut and kernel. these findings should be exploited to select the best walnut accession with the wanted traits to be multiplied in order to encourage the agriculture and raise the production of walnut. taking into account the relevance of high kernel weight, kernel percentage and low thickness of shell, the wala tla s j ou rn al o f b io lo gy is sn 2 15 891 51 . p ub lis he d b y a tla s p ub lis hi ng , l p (w w w. at la spu bl is hi ng .o rg ) 582 nut accessions from amouguer and imlil should be considered as the first accessions to be useful as seed sources or multiplied vegetatively for walnut propagation in morocco. it is obvious that survey of genetic diversity in moroccan walnut using biochemical and molecular markers are projected and would be a complement for this study. references ahandani ea, h ramandi, j sarmad, m samani, a yavari and ra ahandani (2014) evaluation of morphological diversity among some persian walnut accessions (juglans regia l.) in guilan, northern iran. international journal of biotechnology 5(2): 21-30. akca y, y bilgen and s ercisli (2015) selection of superior persian walnut (juglans regia l.) from seedling origin in turkey. acta scientiarum polonorum hortorum cultus 14(3): 103-114. amiri r, k vahdati, s mohsenipoor, mr mozaffari and ch leslie (2010) correlations between some horticultural traits in walnut. horticultural science 45(11): 1690–1694. angmo s, p acharyya and ma hasan (2013) performance of indigenous walnut selections under climatic conditions of ladakh. international symposium on agri-foods for health and wealth. bangkok, thailand, p.167-176. arzani k, h mansouri-ardakan, a vezvaei and mr roozban (2008) morphological variation among persian walnut (juglans regia l.) genotypes from central iran. new zealand journal of crop and horticultural science 36: 159–168. cosmulescu s (2013) phenotypic diversity of walnut (juglans regia l.) in romania – opportunity for genetic improvement. south western journal of horticulture, biology and environment 4(2): 117-126. diaz r, e alonso and j fernadez-lopez (2005) genetic and geographic variation in seed traits of common walnut (juglans regia l.) among twenty populations from the west of spain. acta horticultura 705: 137-141. dreher m, cv maher and p kearney (1996) the traditional and emerging role of nuts in healthful diets. nutrition reviews 54(8):241–245. ebrahimi a, a zarei, sh lawson, k woeste and mjm smulders (2016) genetic diversity and genetic structure of persian walnut (juglans regia l.) accessions from 14 european, african, and asian countries using ssr markers. tree genetics and genomes 12:114. germain e (1992) le noyer. in: amélioration des espèces végétales cultivées, objectifs et critères de sélection, inra editions. paris, p. 620-632. ghasemi m, k arzani and d hassani (2012) evaluation and identification of walnut (juglans regia l.) genotypes in markazi province of iran. cell biology,; 2(2): 119-124. ipgri, (1994) descriptors for walnut (juglan spp.). international plant genetic resources institute, rome, italy. karadağ h and y akça (2011) phenological and pomological properties of promising walnut (juglans regia l.) genotypes from selected native population in amasya province. the african journal of biotechnology 10(74): 16763-16768. kodad o, r socias i company and m sindic (2014) kernel quality in a local walnut (juglan sregia l.) population grown under different ecological conditions in morocco. fao-ciheam nucisnewsletter, number 16, p 27-31. lansari a, e hassani, d nabil and e germain (2001) preliminary results on walnut germplasm evaluation in morocco. acta horticultura 544: 27–35. mcgranahan gh and c leslie (1990) walnuts (juglans). acta horticultura, 290: 905–951. sharma rm, k kour, b singh, s yadav, n kotwal, jc rana and r anand (2014) selection and characterization of elite walnut (juglans regia l.) clone from seedling origin trees in north western himalayan region of india. australian journal of crop science 8(2): 257-262. upov (1999) guidelines for the conduct of tests for distinctness, uniformity and stability. walnut (juglans regia l.), geneva: international union for the protection of new varieties of plants. atlas journal of biology 2023, pp. 768–829 https://doi.org/10.5147/ajb.vi.239 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) proceedings of the fifth international american moroccan proceedings of the fifth international american moroccan agricultural, health, and life sciences conference (amahls v agricultural, health, and life sciences conference (amahls v conference) and the first cannabis & hemp sciences and conference) and the first cannabis & hemp sciences and entrepreneurship day (chse i), december 13-14, 2022, tangier, entrepreneurship day (chse i), december 13-14, 2022, tangier, moroccomorocco my abdelmajid kassem1*, s. alan walters2, john w. groninger2, karen s. midden2, and khalid meksem3 1 plant genetics, genomics, and biotechnology lab, department of biological and forensic sciences, fayetteville state university, fayetteville, nc 28301, usa; 2 school of forestry and horticulture, southern illinois university, carbondale il 62901-4411, usa; 3 school of agricultural sciences, southern illinois university, carbondale il 62901-4415, usa received: april 10, 2023 / accepted: may 14, 2023 __________________________________________________ * corresponding author: mkassem@uncfsu.edu 768 abstractabstract the international american moroccan agricultural, health, and life sciences conference (amahls conference; https:// amahls.org) is an international conference organized by the high council of moroccan american scholars and academics (hc-masa; www.hc-masa.org) in collaboration with various universities and research institutes in morocco. amahls v was held on december 13-14, 2022 in tangier, morocco and comprises the first cannabis and hemp sciences and entrepreneurship day (chse i). the current proceedings summarizes abstracts from 80 oral presentations and 50 posters that were presented during amahls v conference. keywords:keywords: amahls conference, hc-masa, cannabis day, agricultural, health, life sciences. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. session i: plant sciences isession i: plant sciences i co-chair: alan walters, southern illinois university, usaco-chair: alan walters, southern illinois university, usa co-chair: ahmed elamrani, university mohamed i, moroccoco-chair: ahmed elamrani, university mohamed i, morocco o1. valorization of moroccan saffron (o1. valorization of moroccan saffron (crocus sativuscrocus sativus l.) by l.) by plant biotechnologyplant biotechnology mohammed amine serghini*, soumaya el merzougui, khadija lachguer, imane boudadi, mohamed ben el caid, khalid lagram, rachida el boullani and mohamed lachheb laboratory of biotechnologies and valorization of natural resources, faculty of sciences, ibn zohr university, agadir, morocco. *corresponding and presenting author : m.serghini@ uiz.ac.ma. abstractabstract the word saffron refers both to the crocus sativus l. plant and to the spice consisting of the dried stigmas of its flower. saffron is one of the most important local products in morocco. its production in morocco is around 7 t per year and ranks our country 3rd in terms of producers in the world after iran oral presentations abstractsoral presentations abstracts tuesday & wednesday tuesday & wednesday december 13-14, 2022december 13-14, 2022 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) and greece. almost all moroccan saffron is produced in the taliouine and taznakht regions of the respective provinces of taroudant and ouarzazate. saffron is the most expensive spice, earning it the name ‘red gold’. the characterization and valuation of this resource have used plant biotechnologies and are based on different moroccan accessions and their comparison with accessions from other countries. the conference will focus on (1) the agro-morpho-physiological characterization of moroccan saffron accessions in order to selecting best accessions; (2) in vitro culture via direct and indirect organogenesis and somatic embryogenesis as a means of rapid multiplication of elite saffron accessions; (3) the fine chemical characterization of saffron (hplc, cpg, medium ir, colorimetry coupled with chemometrics) with the aim of determining the quality of the product and guaranteeing its authenticity against fraud caused by its high price; (4) the application of molecular markers (ssr & issr), sequencing and barcoding to elucidate the genetic diversity of moroccan saffron, distinguish it from accessions from other countries and assign it molecular identities; and (5) the development of saffron by-products, in particular through the use of the petals of its flower for the production of bio-dyes currently highly coveted in the textile industry and through their antioxidant and antibacterial effects. keywords:keywords: saffron, crocus sativus l., molecular markers, biotechnologies, bio-dyes. o2. effect of different substrates on growth, development o2. effect of different substrates on growth, development and corms production of saffron (and corms production of saffron (crocus sativuscrocus sativus l.) l.) abdelghani tahiri1*, youssef karra1, naima ait aabd1, qessaoui redouan2, and rachid bouharroud3 1 rnpt unit, national institute of agricultural research– crra, agadir, av. des far, bp. 124, inezgane, morocco; 2 r&d unit, national institute of agricultural research– crra agadir, av. des far, bp. 124, inezgane, morocco; 3 pic unit, national institute of agricultural research– crra agadir, av. des far, bp. 124, inezgane, morocco. *corresponding author: abdelghani. tahiri@inra.ma. abstractabstract saffron (crocus sativus l.), the most expensive spice in the world derived from stigmas, is a geophyte perennial plant from the iridaceae family with underground soft corms. it is a sterile autotriploid (2n=3x=24) and autumn flowering geophyte plant. this plant is propagated by vegetative reproduction through the formation of daughter corms from the mother corm as the flowers are sterile and fail to produce viable seeds. a mother saffron corm produces typically one to four cormlets per season through field cultivation. however, many factors affect the efficient propagation of saffron through traditional practice and the low multiplication rates of daughter corms under natural conditions reduce productivity, thereby restraining the availability of planting material. in order to evaluate the effect of some substrates on saffron growth and corms multiplication, a greenhouse experiment was conducted at the national institute of agricultural research– crra of agadir (morocco). six combinations of substrates, including compost, perlite, biochar, black, and blond peat and soil as control, were used. the main obtained results of trials indicate that saffron growth and corms production depend on the substrate composition. indeed, no significant difference was observed in leaf parameters (length and number). however, the dry weight and the chlorophyll a and b rates were improved by the s3 substrate (perlite/soil/ compost; 2:1:1). whereas the corms weight and the average daughter corms number were improved respectively by the substrates s2 (soil/black peat/blond peat; 2:1:1) and s6 (biochar/black peat/blond peat; 2:1:1) as well as s3. keywords: keywords: saffron, crocus sativus l., corm, substrates, vegetative propagation. o3. effect of fruit thinning on fruit development and fruit o3. effect of fruit thinning on fruit development and fruit size of cactus pear [opuntia ficusopuntia ficus--indica (l.) size of cactus pear [opuntia ficusopuntia ficus--indica (l.) mill.]mill.] siham farhat1§ and mohamed arba2* 1 national office of food products safety and health (onssa), sidi slimane, morocco; 2 plant ecophysiology and cultures of arid zones laboratory, hassan ii institute of agronomy and veterinary medicine, horticultural complex of agadir, morocco. *corresponding author: arbamohamed@yahoo.fr. §presenting author: frht.siham@gmail.com. abstractabstract the aim of this research work was to study the effect of fruit thinning on fruit development and fruit size of cactus pear opuntia ficus-indica (l.) mill. trials were carried out on an adult plantation in a semi-arid area in southern morocco. fruit thinning consisted of reducing the fruit load of cladodes to 6 or 12 fruits per cladode by removing some floral buds or young fruits at the flowering stage. it was practiced in early may on three types of one-year old cladodes (small cladodes, the medium ones and large cladodes). the control cladodes have an average number of 18 fruits per cladode. obtained results showed that fruit thinning improved fruit size at ripening stage. in thinned plants to 6 fruits per cladode and for the three types of cladodes average fruit weight was 160.44 g and average fruit length and diameter was 8.44 and 6.38 cm respectively. while in not thinned plants and for the three types of cladodes, average fruit weight was only 74.53 g and average fruit length and diameter was 6.40 and 4.46 cm respectively. fruit thinning and the type of cladodes and the interaction of the two factors have a significant effect (p ≤ 0,001) on fruit development. on may 10, 2019, the highest right of development was obtained with the combination fruit thinning to six fruits per cladode and large cladodes, with 1.6 cm in fruit length and 1 cm in fruit diameter. however, the lowest rate of fruit development was obtained with the combination of thinned plants and small cladodes (0.5 cm in fruit length and 0.3 cm in fruit diameter). keywords:keywords: 769 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) cactus pear; fruit thinning; fruit development; fruit size. o4. the effect of earthworm-associated bacteria on plant me-o4. the effect of earthworm-associated bacteria on plant metabolism under abiotic stress conditions tabolism under abiotic stress conditions lamia yakkou1*, sofia houida1, serdar bilen2, leyla okyay kaya2, mohammed raouane1, souad amghar1, and abdellatif el harti1 1 reserch team “lumbricidae, improving soil productivity and environment (lapse)”, centre “water, natural resources, environment and sustainable development (cerne2d)”, mohammed v university in rabat, ecole normale supérieure (ens). avenue med belhassan el ouazani. bp 5118, takaddoum-rabat, morocco; 2 soil science and plant nutrition department, faculty of agriculture, atatürk university, 25000, erzurum, turkey. *corresponding an presenting author: lamia.yakkou@ um5s.net.ma abstractabstract soil-level changes have been shown in various investigations to entail synergistic interactions between earthworms and microorganisms. knowing the influence of earthworm coelomic fluid (cf) on plant development and defense, we set out to isolate bacteria from this “immune compartment.” bacteria isolated from aporrectodea molleri were investigated for their effects on maize metabolism under abiotic stress conditions such as alkaline soil and nitrogen, phosphate, and potassium (npk) deficit. a study of metabolomic profiles by gas chromatography coupled to a mass spectrometer (gc-ms) on leaves of plants treated and untreated with bacteria isolated from cf was carried out to reveal potential metabolites that could explain the effect of different treatments on maize under stress conditions (cfb). after 60 days of growth, plants infected with bacteria isolated from the coelomic fluid of the earthworm aporrectodea molleri (cfb1,..., cfb7), as well as those not inoculated (control), were used to extract the lipid metabolites at the leaf level. a total of 114 distinct metabolites have been discovered. fatty acids were the most prevalent class (45 metabolites, 43 %). different metabolomic fingerprints indicate that various biochemical mechanisms were engaged in plant response to biostimulants. metabolic reprogramming induced by bacterial treatments was more evident than in the control group. abiotic stress did not result in a drop in lipid composition and concentration in plants treated with cfb, in contrast to the control, demonstrating the rigidity of defensive mechanisms. compounds associated with tolerance building pathways (glyoxylate and dicarboxylate metabolism, as well as the tricarboxylic acid cycle) accumulated greater in bacterial treatments. keywords:keywords: earthworm, coelomic fluid, bacteria, plant, metabolomic. o5. genetic diversity and population structure of almond o5. genetic diversity and population structure of almond genotypes from eastern morocco assessed by ssr markersgenotypes from eastern morocco assessed by ssr markers souhayla kodad1,2, christina m. müller3, mohammad jawarneh3, annette becker2, ahmed elamrani1, mihamou atika1, and malika abid1,2* 1 equipe agroalimentaire, ‘lapab e’, faculté des sciences, université mohammed premier, oujda, morocco; 2 justus-liebiguniversität, institut für botanik, ag entwicklungsbiologie der pflanzen, heinrich-buff-ring 38, 35392 gießen, germany; 3 justus-ag spezielle botanik, heinrich-buff-ring 38, d-35392 giessen, germany. *corresponding author: malikaabid213@ gmail.com. abstractabstract almond (prunus dulcis miller [d. a. webb] or prunus amygdalus l.) is a major tree nut species and consumed worldwide. morocco has a long-standing presence of domesticated almonds and harbors a secondary genetic diversity hotspot. this genetic diversity requires protection as it is an important resource for future almond breeding. however, detailed information on genetic diverimportant diversity is lacking, even more so about the traditionally grown almonds propagated for centuries from sity seeds (“beldi”) which are representatives of the original cultivar pool. here, we provide a compreseeds comprehensive genetic study of 98 moroccan almond accession including 93 of the beldi type and five hensive non-morrocan cultivars by analyzing 12 simple sequence repeat (ssr) markers with high polymornon-polymorphism information content (pic). we used population structure and principal coordinates analyphism analyses (pcoa), analyzed the molecular variance and described the genetic diversity of the identified ses subpopulations. we identified five subpopulations with little genetic differentiation between the populations, but two subpopulations with high genetic diversity. those subpopulations reprepopulations, represent promising genetic pools and need to be fully protected. they are a major resource for the sent development of novel almond varieties that provide high yields in adverse conditions to establish profitable moroccan breeding programs for sustainable almond agriculture. keywords: keywords: almond genotypes, geographic origin, ssr-genotyping, genetic diversity, genetic clusters, analysis of mogenotypes, molecular variance (amova), principal coordinates analyses (pcoa). concurrent session ii: water and irrigationconcurrent session ii: water and irrigation co-chair: bouchta el moumni, abdelmalek essaadi university, co-chair: bouchta el moumni, abdelmalek essaadi university, moroccomorocco co-chair: hamid el amri, university mohamed v, moroccoco-chair: hamid el amri, university mohamed v, morocco o1. using spatial data in water resources management in o1. using spatial data in water resources management in semi-arid areassemi-arid areas abdelhakim amazirh1* and abdelghani chehbouni1,2 1 center for remote sensing applications (crsa), mohammed vi polytechnic university (um6p), morocco; 2 international water research institute (iwri); mohammed vi polytechnic 770 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 771 university, morocco. *corresponding and presenting author: abdelhakim.amazirh@um6p.ma abstractabstract the question of availability and access to water is undoubtedly one of the major problems that humanity will face in the coming century. water is a vital resource for agriculture, for drinking water supply, for the health of populations, and all life on earth depends on it. water scarcity is likely to be exacerbated soon under the combined effect of the alteration of the hydrological cycle, climate change, and increasing water demand for agriculture, urban and industry (ipcc, 2009). countries characterized by an arid climate in the world, such as morocco, are already experiencing water crises that are getting worse and worse. this is due to several factors that are linked to both human actions “water management” and climate and natural changes. thus, in these countries, agricultural (mainly cereal) production is dependent on the frequency of rainfall. a small variability in rainfall patterns can have dramatic consequences on agricultural yield. consequently, irrigation is the only way to stabilize and improve production. a calculation of crop water requirements is necessary to optimize the management of irrigation volumes. to meet this information, satellite remote sensing has shown a powerful potential for monitoring fluxes and water masses for irrigation management at a large scale. this work presents the role of remote sensing data for water management in the tensift watershed in central morocco. o2. water and irrigation systemso2. water and irrigation systems abdes samed bernoussi* and mina amharref faculty of science and technology, university abdelmalek essaadi, tanger, morocco. abstractabstract to meet a growing demand for agricultural product (fao, 2018) an efficient agricultural practices and intensification of the production are crucial (brümmer, 2006). however, agricultural intensification has an important negative impact on environment, among others: ground and surface water pollution, greenhouse gas emissions (ipcc, 2014), biodiversity loss. also in some situations, the excessive use of irrigation water can have double effects: crop failure and water loss. these negative effects put in danger agricultural production and environmental sustainability. a proper management of agricultural land (plant irrigation, fertilization, etc.) is necessary to ensure food production while minimizing its negative impact on environment and particular protection of water resources. in this paper we present a tool for smart management of the water irrigation using remote sensing data and mathematical algorithms by considering crops as a dynamical systems. keywords:keywords: water, irrigation, remote sensing, dynamical systems. [this work is the result of a research project entitled “al khawarizmi: tool for intelligent management of irrigation water and forest heritage” and funded by mesrsfc, cnrst and add, morocco]. o3. climatic and environmental risks and trends in the medi-o3. climatic and environmental risks and trends in the mediterranean regionterranean region adil salhi* geography and development group, flsh, abdelmalek essaadi university, martil, morocco. *corresponding and presenting author: asalhi@uae.ac.ma. https://orcid.org/00000001-8756-2484. abstractabstract the mediterranean is a climate change hotspot where the rate of warming exceeds the global rate and disruptions in precipitation patterns are predicted. our recent findings confirm this assumption showing a predictive pattern of precipitation likely to lead to prolonged and staggered intra-annual droughts (even in so-called “wet” years) with profound ecological and socio-economic impacts, and repetitive hydroclimatic hazards. the evolution of the mediterranean towards warmer and drier conditions is heading towards an increase of +2 to +4°c on average in 2080s. by 2050, the lesser average estimates are above the paris agreement +1.9°c and the temperature deviation is likely to be wider, and thus heatwaves may be more frequent and harsher. moreover, a climatic-environmental degradation (except in a few dispersed places of relative abundance) is expected to be more stressed by human activity by 2050. consequently, long lean periods are likely to alternate with sporadic intense thunderstorms, which means droughts will be more severe. the latter are the main driver of migration as they have a substantial impact on the local ecosystem and agriculture, including reduced crop growth and yield and loss of livestock. failure to take the issue seriously will exacerbate multi-scale socio-ecological imbalances and the potential spread of social divide, which will intensify with the expected increase in the number of forced migrants arriving in areas of relative abundance. beyond the challenge of ensuring food and water security, governments must improve collaborative resilience measures to meet hydro-agricultural needs and defuse the sociological conflicts. concurrent session iii: health sciencesconcurrent sciencesconcurrent session iii: health sciencesconcurrent sciences co-chair: mohammed el hassouni, mohammed v university, co-chair: mohammed el hassouni, mohammed v university, moroccomorocco co-chair: hassan ghazal, cnrst, moroccoco-chair: hassan ghazal, cnrst, morocco o1. digital health in morocco: integration of new technolo-o1. digital health in morocco: integration of new technologies in access to medical caregies in access to medical care fatima zahra ouassou* ecole supérieure de technologie de laâyoune (e.s.t.l), unia tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 772 versité ibn zohr, agadir, morocco. *corresponding and presenting author : f.ouassou@uiz.ac.ma. abstractabstract the implementation of digital technology in many activities in morocco is fundamental and reinforced by a national plan of digitalization. the access to health care services as an example, during the pandemic times, was oriented to the use of platforms reserved to on-line consultations, the collecting of medical data on these platforms which can lead to many interrogations and problems related to the protection of the personal data and privacy under the moroccan law. medical digital health platforms, under which a lot of moroccan patients during the first wave of covid-19 consulted health practitioners, represent a fertile land to debate about the guarantees of the privacy, the manipulation of the medical or personal data and its impact on the traditional relation between a patient and a health care professional. there is other aspects of the use of new information technologies in health care, but the medical identity accounts escpecially in these platforms and the legal accountability are the main points that will be analyzed in this presentation. keywords:keywords: health care, medical, personal data, transition, digital health, patient, legal accountability, protection of privacy. o2. plant-based ingredients for formulation of healthy snack o2. plant-based ingredients for formulation of healthy snack recipesrecipes mohamed addi1*, reda melhaoui1, nadia houmy1,2, and ahmed elamrani1 1 laboratoire d’amélioration des productions agricoles, (lapabe), equipe agroalimentaire, faculté des sciences, université mohammed i, bp-717, 60000 oujda, morocco; 2 laboratoire de technologie et qualité alimentaire, crrao, inra, bp-428, 60000 oujda, morocco. *corresponding author: m.addi@ump.ac.ma. abstractabstract junk foods are found to be associated with obesity, because they are high in saturated fat, sodium and added sugar, but low in micronutrients and fiber. the obesity epidemic and all its consequences (such as type 2 diabetes, cardiovascular disease, fatty liver disease, and certain types of cancers) affect almost all countries, but its prevalence appears to be low in the population of mediterranean countries due to the mediterranean diet (md). in fact, md is essentially a plant-based diet, with a high consumption of fresh fruits and vegetables, grains and legumes as major sources of fiber and antioxidant compounds. this research involves the study of plant-based food ingredients that are rich in bioactive compounds, have health benefits, especially in child and adolescent nutrition, and play (or have played but no longer) an important role in the recipes and food cultures of mediterranean countries such as morocco. thereby, we choose for snack recipes fresh and healthy ingredient readily available, such as nuts, cereals, legumes, fruits, to formulate healthy, weight loss-friendly snacks compositions. as a first step, considering the availability of ingredients, we have started testing a snake recipe (prototype) based on 5 ingredients, namely: almonds, figs, dates, carob and oranges, which are locally produced and widely consumed in mediterranean countries. these ingredients were analyzed in order to develop novel snack formulations and technologies based on functionally nutritious fruit and vegetable ingredients. keywords:keywords: plant-based ingredients, mediterranean diet, healthy snacks, formulation. o3. the need for a biosocial-agriculture approach to improve o3. the need for a biosocial-agriculture approach to improve maternal and child health nutrition in malimaternal and child health nutrition in mali drissa toure1* , karen stoelzle midden2§ , jack turman, jr3, and rachel redington-noble4 1 division of epidemiology & community health, university of minnesota of public health, minneapolis, mn 55454, usa; 2 school of forestry & horticulture, southern illinois university, carbondale, il 62902, usa; 3 department of social and behavioral sciences, richard m. fairbanks school of public health indiana university, indianapolis, in, usa; 4 department of social and behavioral sciences richard m. fairbanks school of public health and department of pediatrics, school of medicine indiana university, indianapolis, in, usa. *corresponding author: dtoure@umn.edu; §presenting author: dtoure@umn. edu and kmidden@siu.edu. abstractabstract women and children are the most affected during times of food insecurity, stemming from undernourishment and malnutrition that leads to poor lifelong physical and mental health and limited, if any, educational and economic opportunities. the lack of proper nutrients in a diet caused by food insecurity greatly affects a child’s health, development, and often, life. according to unicef 3 nearly half of all deaths of children less than five years of age in mali are attributable to undernutrition. in 2018, this translated to nearly 37,500 death*s of children under five from factors related to not having enough food. as the number of people suffering from hunger and malnutrition increases each year, much of the country also lacks access to a healthy and nutritious diet. according to the world health organization 8, a healthy diet protects against malnutrition in all forms and protects against many non-communicable diseases. a well-balanced diet is crucial for improving maternal and child nutrition, but is often more expensive and unaffordable to many, with the cost exceeding the international poverty line 9. as approximately 49 percent of people residing in mail live below the poverty line 10, accessing a healthy diet may seem unattainable unless a nutrition-sensitive invention is implemented. in the review of available literature, collaboration between agriculture and public health has been shown to positively affect maternal and child health outcomes in prior circuma tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 773 stances. a biosocial approach incorporates the fields of biomedicine, social sciences, and community involvement to improve health outcomes. to address malnutrition in mali, an innovative approach proposes the addition of farmers and agricultural scientists, creating a biosocial-agriculture model to improve maternal and child nutrition and allow for more inclusive planning and implementation strategies. this model presents opportunities for global application. keywords: keywords: biosocial-agriculture, maternal and child health nutrition, food insecurity. agriculture and public health. o4. insight into antidiabetic and antiglycation effects of o4. insight into antidiabetic and antiglycation effects of polyphenols extract from polyphenols extract from schinus terebinthifoliusschinus terebinthifolius raddi in raddi in alloxan-induced diabetic miceebinthifolius micealloxan-induced diabetic miceebinthifolius mice zouaoui zakia1§, reda ben mrid1,2*, najat bouchmaa2,3, imad kabach1, sara el asri1, abdelmounaim laabar4, abdelhamid ennoury1, zoulfa roussi1, nada nhhala1, and mohamed nhiri1 1 laboratory of biochemistry and molecular genetics, faculty of sciences and technologies, university abdelmalek essaadi, bp 416, tangier 90000, morocco; 2 institute of biological sciences (issb-p), mohammed vi polytechnic university (um6p), ben guerir 43150, morocco; 3 team of experimental oncology and natural substances, cellular and molecular immunopharmacology, faculty of science and technology, sultan moulay slimane university, beni-mellal 23000, morocco; 4 laboratory of pharmacology and toxicology, biopharmaceutical and toxicological analysis research team, faculty of medicine and pharmacy, university mohammed v of rabat, morocco. *corresponding author: reda.benmrid@um6p.ma. §presenting author: zakia.zouaoui@etu.uae.ac.ma. abstractabstract diabetes mellitus is considered one of the most prevalent metabolic troubles of the 21st century and is accelerating at a menacing rate worldwide. natural products can be an effective alternative remedy as a treatment. our study aims to analyze the possible therapeutic potential of schinus terebinthifolius raddi leaves (stl), to evaluate their polyphenols’ power on oxidative stress, in vitro hyperglycemia, as well as in vitro and in vivo power glycation in alloxan-induced diabetic mice. after induction of diabetes, a significant increase was observed in the levels of blood glucose, total cholesterol (tc), low-density lipoprotein (ldl), hepatic malondialdehyde (mda), peroxide (h2o2), and the renal advanced glycated end products (age) compared to the control. in addition, a reduction in the level of lipoprotein cholesterol (hdl) is associated with the diminution of the corresponding antioxidant enzymes, such as catalase (cat), glutathione reductase (gr), glutathione peroxidase (gpx), superoxide dismutase (sod), was observed in all diabetic mice. furthermore, treatment with stl (150 and 250 mg/kg) for three weeks significantly decreased blood glucose, tc, ldl, hepatic mda, and h2o2 levels and renal age content of diabetic mice. in contrast, it increased serum hdl levels and reactivated the hepatic antioxidant enzymes in diabetic mice. these results demonstrate that the stl extract may have the capacity to inhibit hyperglycemia induced-diabetes. furthermore, this data suggest that the administration of the extract may be helpful in the prevention of diabetic complications associated with glycation, oxidative stress, and hyperlipidemia. therefore, we conclude that the extract should be evaluated as a candidate for future studies on diabetes mellitus. keywords:keywords: schinus terebinthifolius raddi, alloxan-diabetic mice, anti-glycation, antioxidation, polyphenols, antioxidant enzymes. o5. autosomal dominant intellectual development disor-o5. autosomal dominant intellectual development disorder-6 (mrd6) linked to a novo mutation in the grin2b gene der-6 (mrd6) linked to a novo mutation in the grin2b gene revealed by exome sequencing: a case reportrevealed by exome sequencing: a case report el mouhi hinde1,2,3*, leila bouguenouch1,2, hanane sayel2, said trhanint2, brahim el hejjioui1,2,3, abdelhafid natiq7, youssef ahmadi2,6, and sana chaouki1,5 1 laboratory of biomedical and translational research, faculty of medicine and pharmacy, sidi mohammed ben abdellah university, fes, morocco; 2 unit of medical genetics and oncogenetics, university hospital hassan ii, fes, morocco; 3 engineering science and technology doctoral study center, faculty of sciences and technologies, sidi mohammed ben abdellah university of fez, morocco; 4 research team in genomics and molecular epidemiology of genetic diseases, genomics center of human pathologies, faculty of medicine and pharmacy, mohammed v university in rabat, morocco; 5 department of pediatrics chu hassan ii, fes, morocco; 6 faculty of medicine and pharmacy and dental medicine, sidi mohammed ben abdellah university, fes, morocco; 7 faculty of medicine and pharmacy, mohammed v university, rabat, morocco. *corresponding and presenting author: hinde.elmouhi@usmba.ac.ma. abstractabstract mrd6 is a rare autosomal dominant neurodevelopmental disorder associated with grin2b. it is characterised by psychomotor delay and intellectual disability of varying severity. other features may include seizures, hypotonia, abnormal movements (e.g. dystonia) and autistic features. some patients may have structural malformations of cortical development on brain imaging. the phenotype is highly variable, reflecting a range of neurodevelopmental abnormalities, from mild intellectual disability without seizures to epileptic encephalopathy. to date, fewer than 100 individuals with a grin2brelated neurodevelopmental disorder have been reported. we report on a 3-year-old moroccan child referred to our neuropediatric department for the diagnosis of psychomotor developmental delay, dysmorphia and language absence. given the complex clinical picture of this disease, we performed exome sequencing followed by targeted analysis. a mutation was identified in a known gene for autosomal dominant developmental disorder-6 (mrd6) (omim 613970) or early infantile epileptic encephalopathy 27 (omim 616139) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 774 associated with grin2b. by exome sequencing, we identified a new pathogenic mutation: c.3912c>g (p.tyr1304ter) in the grin2b gene. this study demonstrates the impact of a novel homozygous mutation found to be pathogenic in the grin2b gene, diagnosed by exome sequencing approach, on brain development and function in a moroccan child. keywords: keywords: mrd6, neurodevelopmental disorder, exome sequencing, grin2b, novel mutation, moroccan patient. session iv: animal sciences and zoologysession iv: animal sciences and zoology co-chair: amer abughazaleh, southern illinois university, co-chair: amer abughazaleh, southern illinois university, usausa co-chair: badr benjelloun, inra, moroccoco-chair: badr benjelloun, inra, morocco o1. towards a sustainable management of farm animal ge-o1. towards a sustainable management of farm animal genetic resources: the case of sheep and goatsnetic resources: the case of sheep and goats badr benjelloun1,2*, and françois pompanon2 on behalf of the nextgen consortium3 1 livestock genomics laboratory, regional center of agricultural research tadla, national institute of agricultural research inra, rabat, morocco; 2 univ. grenoble alpes, univ savoie mont blanc, cnrs, leca, f-38000 grenoble, france; 3 https://www.epfl.ch/labs/nextgen/consortium, france. *corresponding and presenting author email: badr.benjelloun@ inra.ma. abstractabstract the sustainability of breeding systems relies first and foremost on the ability of farm animals to remain productive in the context of climate changes. this ability primarily depends on adaptive traits and standing genetic variation in the raised breeds. the understanding of genetic bases of local adaptation and the accurate assessment of genetic diversity are thus key elements when conceiving management programs aiming at developing sustainably farm animals in a country. otherwise, sequencing technologies allow now for an unprecedented access to genotyping animals by sequencing. the future programs of farm animal management have to exploit these technological advances to improve their effectiveness and efficiency. by using a wide framework based on whole genome analyses of moroccan small ruminants, this talk will present outcomes from research by our team aiming at depicting genetic traceability of local breeds as well as genetic bases of local adaption to specific and extreme environments encountered in the country. based on the results we obtained, we will present: (i) genomic bases of local adaptation to eco-climatic constraints in moroccan sheep and goats; (ii) whole genome traceability of the main local breeds raised in morocco; (iii) the global genomic diversity in local sheep and goats compared to their wild relatives and a panel of worldwide ‘cosmopolitan’ breeds; (iv) the evaluation of whole genome genotyping strategies to assess adaptive and neutral genome diversity; and (v) the possible uses of the obtained results combined for implementing wide-scale programs for a sustainable management of farm animals. keywords:keywords: local adaptation, farm animals, genome diversity, genotyping strategies, methylation. o2. effect of adding bromoform to ruminant animal diet on o2. effect of adding bromoform to ruminant animal diet on methane gas emissionmethane gas emission mohamed embaby* department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: mohamed.embaby@siu.edu abstractabstract ruminant’s contribution to global greenhouse gas emissions accounts for 6% of the global emissions. scientists have been investigating different strategies to reduce methane emissions as communities became more aware of the climate change negative impacts on the environment. seaweed is a potential source to reduce methane emission when added to livestock animals’ diet due to its bioactive ingredients mainly bromoform that might work as a hydrogen sink and compete with methanogens. the effect of bromoform on methane gas emission from ruminants was investigated in this paper invitro using batch culture technique. bromoform was added at different inclusion rates of 4%, 2%, 1%, 0.5%, 0.25%, 0.125%, 0.625% per gram of the dry matter intake. results showed that adding bromoform suppressed the production of methane by 99% (p<0.05) compared to control, while no significant effect was observed on dry matter digestibility. further research required to investigate the effect of adding reduced levels of bromoform, and the effect of bromoform on the microbial community, and rumen fermentation characteristics. keywords: keywords: methane, ruminants, seaweed, bromoform. o3. molecular and phylogenetic analysis of h9n2 avian in-o3. molecular and phylogenetic analysis of h9n2 avian influenza viruses isolated from moroccan flocks between 2021 fluenza viruses isolated from moroccan flocks between 2021 and 2022and 2022 oumayma arbani1*, faiçal salamat1, mariette ducatez2, mohammed el houadfi1, and siham fellahi1 1 avian pathology unit, department of veterinary pathology and public health, agronomy and veterinary institute hassan ii, rabat b.p. 6202, morocco; 2 ihap, toulouse university, inrae, envt, 31300 toulouse, france. *corresponding and presenting author: arbanioumayma@iav.ac.ma. abstractabstract over the past two decades, the low pathogenic avian influenza virus h9n2 (lpaiv) has circulated globally in poultry, becoming the most widespread subtype in asia, middle east and africa. despite their low pathogenicity, h9n2 avian influenza viruses have highlighted global awareness, not only because of their heavy economic losses in poultry industry, but a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 775 also due to their high zoonotic infection rates and their potential for a worldwide pandemic. the objective of this work was to monitor and assess the presence of avian influenza virus lpaiv in four different regions of morocco using real-time rt-pcr, and to assess the phylogenetic and molecular characterization of the h9n2 viruses in morocco during the period between september 2021 and june 2022. 176 filed samples were collected from 115 suspect farms of being infected with lpaiv h9n2 virus and from different types of production (94 broilerfarms, 20-layer farms and 1 breeder farm). samples were tested for the presence of the lpai h9n2 genome using real-time rt-pcr. highly positive samples were subjected to virus isolation and amplified by conventional rt-pcr in order to sequence the ha gene for subsequent phylogenetic analysis. the results of the epidemiological survey revealed a global positivity percentage of 24.35% (28/115). the phylogenetic analysis of the ha gene showed that the circulating strains belonged to the g1 lineage showing great similarity (97-98%) to the strains isolated and characterized in 2018 and 2019 (a/chicken/morocco/as14/2018(h9n2)) and cluster with the strains isolated in the middle east and in west and north africa. keywords:keywords: avian influenza, h9n2, molecular biology, phylogenetic analysis, morocco. o4. epidemiological and pathological findings of gizzard o4. epidemiological and pathological findings of gizzard erosion and ulceration syndrome (geus) in moroccan poul-erosion and ulceration syndrome (geus) in moroccan poultry between 2014 and 2022try between 2014 and 2022 ikram ouchhour1*, mourad mastour1, faouzi kichou1, oumayma arbani2, and siham fellahi2 1 histology and pathology unit, department of veterinary pathology and public health, hassan 2nd institute of agronomy and veterinary medicine, b.p. 6202, rabat, morocco; 2 avian pathology unit, department of veterinary pathology and public health, hassan ii institute of agronomy and veterinary medicine, b.p. 6202, rabat, morocco. *corresponding and presenting author: ouchhourikram@iav.ac.ma. abstractabstract gizzard erosion and ulceration syndrome (geus), caused by a fowladenovirus serotype 1 (fadv-1), has spread globally among poultry during the last two decades and has been associated with important economic losses. in this work, a total of 23 flock-cases with geus in morocco from different types of poultry production (broilers, laying hens, breeder chicks, turkeys, and partridge) were assessed between 2014 and 2022. the suspicion of this condition was based on clinical course and mortality which reached 10% in some cases, and on pathological changes detected in dead necropsied birds. macroscopic changes included distended gizzard with fluid or hemorrhagic content, and presence of several small to large foci of erosions and/or ulceration of the chitin layer and the surface mucosa with a variable degree of severity. microscopic lesions were mainly focal to multifocal ulceration and/ or erosions, with cuticle loss, necrosis of surface and glandular epithelial cells and a variable degree of inflammatory cell infiltrate of the lamina propria predominantly lymphoplasmacytic. among the flock-cases investigated, seug was diagnosed in 13 broiler cases aged between 10 and 40 days, 6-layer cases with an age range from 17 to 52 weeks, and one case in meat type breeder chicks aged 10 weeks. moreover, and for the first time, geus was observed in 2 flock cases of turkey poults and 1 case of partridge aged 22-23 days and 5 weeks, respectively. in addition to the above-described microscopic changes, pathognomonic viral basophilic and/or eosinophilic intranuclear inclusion bodies within degenerate epithelial cells were identified in birds from 2 broiler cases and 3-layer cases and hence were attributed to fadv. however, other causes such as mycotoxins and biological amines should not be ruled out hence raised the need for further investigations. indeed, the involvement of fadv in the occurrence of seug cases in poultry in the country is being further assessed by retrospective virological and molecular investigations in order to identify the fadv serotypes involved and eventually the characterization of their pathogenicity. key-keywords: words: gizzard erosion and ulceration syndrome, fowl adenovirus, poultry, epidemiology, pathology, morocco. concurrent session v: environmental sciences and land res-concurrent session v: environmental sciences and land restorationconcurrent restorationtorationconcurrent restoration co-chair: john w. groninger, southern illinois university, usaco-chair: john w. groninger, southern illinois university, usa co-chair: hamid mazouz, university moulay ismail, moroccoco-chair: hamid mazouz, university moulay ismail, morocco o1. research supporting wetlands forest restoration in mid-o1. research supporting wetlands forest restoration in midwestern usawestern usa john w. groninger*, clayton k. nielsen, elizabeth m. hillard, and thanchira suriyamongkol school of forestry and horticulture, southern illinois university, carbondale, il, usa. *corresponding and presenting author: groninge@siu.edu. abstractabstract the mid-20th century saw a rapid and dramatic increase in row crop agriculture at the expense of forested wetlands throughout the mississippi river alluvial valley. however, growing public concern regarding decline of wetlands quality and quantity and changes in agricultural markets contributed to extensive ecosystem restoration, including within the cache river region of illinois. beginning during the early 1990’s, marginal row-cropped lands were reforested using native species and with support from government agencies and non-governmental organizations. presently, many of the locations undergoing reforestation have developed characteristic forest structure and functions associated with natural wetlands forests. initial post-agricultural research focused on identifying barriers to successful reforestation and developing techniques to establish recalcitrant species. emergent and underlying stressors on reforestation were then addressed as a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 776 researchers gained a deeper knowledge of the ecosystem and the role of past human activities on shaping current ecosystem conditions. as forest stands attained broader wildlife habitat functionality, policy makers sought tangible measures for ecosystem restoration success and challenged researchers to identify indicators to guide ecosystem management activities. current research addresses the roles played by specific vegetation communities and structures within the mosaic of remnant and restored forest stands. the purpose of this presentation is to provide an overview of the cache river ecosystem, review restoration research, and discuss implications for policy makers and managers. lessons learned over a career working within a landscape undergoing restoration and how these may apply to other situations will be discussed. i conclude that integrating anticipated management challenges into academic research programs can help inform future ecosystem restoration initiatives to ensure that human, financial, and land resources are efficiently and effectively deployed. keywords: keywords: afforestation, land use change, migratory birds, plant succession, water resources. o2. overview of fertility management in extensive green o2. overview of fertility management in extensive green roof food production systemsroof food production systems karen midden* and s. alan walters school of forestry and horticulture, southern illinois university, carbondale, il, usa. *corresponding author: kmidden@ siu.edu. presenting author: kmidden@siu.edu. abstractabstract the production of healthy vegetables, fruits and herbs on extensive green roofs serves a valuable role in response to food insecurities by providing nutritional produce in urban and often underserved locations. many green roof urban farms have made significant positive impacts on their communities by providing educational opportunities, jobs, and a source of local food. numerous urban farms exist on intensive green roofs which support media depths from 0.2 to 1 m or greater allowing a significantly higher ratio of organic matter in the mix. in comparison, extensive green roofs are those with growing media depths of only 50 to 130 mm that are typically composed of light weight aggregate (which is heat expanded clay) and 4% to 5% organic matter. unlike traditional agriculture, plants on extensive green roofs are grown in a shallow engineered soil to reduce the weight load on the roof. the primary historic and current uses of extensive green roofs are to provide numerous environmental and economic benefits such as storm water management, reducing energy costs, mitigating urban heat effect, increasing ecological biodiversity, and improving biophilic and aesthetic design. plants typically grown in extensive green roofs are generally heat and drought tolerant species, such as sedum spp. that require minimal fertility. in comparison, horticultural food crops require a continued source of plant available nutrients to optimize their yields, challenging extensive green roof food production. there is sparse research regarding fertility system evaluations for edibles grown on extensive green roofs. this presentation will provide a brief overview of green roof farming as well as fertility research conducted on green roofs to maximize their production. o3. fertility management of vegetables grown in a green-o3. fertility management of vegetables grown in a greenroof environmentroof environment walters s. alan*, richard a. little, victoria vogt thomas, and karen midden school of forestry and horticulture, southern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: awalters@siu.edu. abstractabstract urban green roof agriculture provides an alternative growing space to supply fresh produce and herbs to local markets or restaurants, which can also help to address food security concerns in cities. although many vegetables can be grown on extensive green roofs, few studies have evaluated the fertility requirements necessary to optimize productivity. managing green roof substrate fertility is challenging since they tend to have low amounts of organic matter and provide high permeability with low cation-exchange capacity. due to the composition of extensive green roof substrates, supplemental nutrient additions are critical to maximize vegetable crop growth and productivity. therefore, studies were conducted to evaluate different nutrient management systems for ‘sweet hungarian’ peppers (capsicum annuum l.) and various romaine lettuce (lactuca sativa l.) cultivars. results indicated that nutrient management systems are important to optimize both pepper and lettuce production in a stressful rooftop environment. for pepper, multiple, repeated applications are necessary to optimize yields as a complete slowrelease fertilizer provided the greatest leaf chlorophyll content, plant vigor and fruit yields. although two applications were used for other standard conventional (synthetic) and organic fertilizers, these provided lower pepper plant growth and yields. similarly, for lettuce, a complete fertilizer (n-p-k) applied at weekly intervals throughout the growing season provided plant growth and yields comparable to a complete slow-release fertilizer that was applied only once during the growing season (mixed in the media prior to transplant). thus, our research indicates that both longand short-season vegetables require multiple, supplemental fertilizer applications to maintain high nutrient availability throughout the growing season to optimize yields. keywords:keywords: extensive green roofs, lettuce, pepper, rooftop gardens, sustainable food production, urban food security, vegetable. o4. growth, yield and macronutrients requirement of qui-o4. growth, yield and macronutrients requirement of quinoa crop under arid conditions of morocconoa crop under arid conditions of morocco nawal taaime1*, khalil el mejahed1, rachid bouabid2, abdala tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 777 lah oukarroum3, redouane choukr-allah1, and mohamed el gharous1 1 agricultural innovation and technology transfer center, mohammed vi polytechnic university, ben guerir, morocco; 2 department of agronomy, national school of agriculture, meknes; 3 plant stress physiology laboratory, agrobiosciences, mohammed vi polytechnic university, ben guerir, morocco. *corresponding and presenting author: nawal.taaime@ um6p.ma. abstractabstract in the context of climate change, quinoa represents a potential alternative crop for increasing agricultural productivity in arid regions. however, appropriate crop management practices under limited water supply are still poorly documented. quinoa like other cultivated crops, needs sufficient amount of nutrients, especially nitrogen (n), phosphorus (p) and potassium (k) for better growth and higher grain yield. to determine the adequate levels of these nutrients needed and study their effect on quinoa growth and productivity, a field experiment was conducted during two growing seasons at the um6p experimental farm and consisted of a randomized complete block (rcb) design with three replications. the treatments studied represent the combination of four n rates (0, 40, 80 and 120 kg/ha), three p rates (0, 30 and 60 kg p2o5/ha) and three k rates (0, 60 and 120 kg k2o/ha). data on quinoa physiological, nutritional, and production parameters were collected and analyzed. the results showed that optimum rates that gave the highest total biomass (3.9 t/ha) and grain yield (0.76 t/ha) under arid and semi-arid region were 40 kg n/ha, 60 kg p2o5/ha and 120 kg k2o/ha. n, p and k application increased plant height, chlorophyll content index, total biomass, grain yield and 1000 seed weight. among the three macronutrients, n was the most limiting factor, followed by k and p. in terms of nutrients uptake, it was observed that quinoa needed 68 kg n/ha, 18 kg p2o5/ha and 182 kg k2o/ha to produce the optimal yield in this experiment. keywords: keywords: nitrogen, phosphorus, potassium, fertilization, quinoa, um6p experimental farm. session vi: biochemistry & molecular biology session vi: biochemistry & molecular biology co-chair: elmostafa el fahime, cnrst, moroccoco-chair: elmostafa el fahime, cnrst, morocco co-chair: mark byrd, southern illinois university, usaco-chair: mark byrd, southern illinois university, usa o1. biochemistry and chemistry discovery using simulations o1. biochemistry and chemistry discovery using simulations on a quantum computeron a quantum computer mark byrd1*, ali abu-nada2, and lian-ao wu3 1 school of physics and applied physics, southern illinois university, carbondale, usa; 2 faculty of engineering technology and science, higher colleges of technology, abu dhabi, uae; 3 ehu quantum center, university of the basque country upv/ehu and ikerbasque, basque foundation for science, 48011 bilbao, spain. *corresponding and presenting author: mbyrd@siu.edu. abstractabstract chemistry and biochemistry problems are notoriously difficult to simulate using on a typical computer. however, computers that use quantum mechanics for their operation mechanism can, in principle, simulate such problems efficiently. in this presentation, some introductory comments concerning the operation of quantum computers are made before examining how one would use a quantum computer to simulate molecular interactions including chemical and biochemical interactions. the quantum computing industry is expected to grow into a multibillion-dollar industry in the next few years due, in part, to the investments by the pharmaceutical industry. keywords: keywords: biochemical reactions, quantum computing, simulations. o2. effect of extract from the algae o2. effect of extract from the algae ulva lactucaulva lactuca on the alon the alleviation of salt stress in common bean, leviation of salt stress in common bean, phaseolus vulgarisphaseolus vulgaris l. l. nhhala nada1*, reda ben mrid1,2, abdelhamid ennoury1, zoulfa roussi1, zakia zouaoui1, anass kchikich1, and mohamed nhiri1 1 laboratory of biochemistry and molecular genetics, faculty of sciences and technologies of tangier, abdelmalek essaadi university, tetouan, morocco; 2 institute of biological sciences, mohammed vi polytechnic university, ben guerir 43150, morocco. *corresponding and presenting author: nada.nhhala@gmail.com. abstractabstract the use of seaweed extracts has been proven to provide positive effects on the enhancement of crop quality and resistance to abiotic and biotic stresses, due to their content of multiple active components. salinity is one of the major abiotic stressors which limits the yield of major crops. the macro algae ulva lactuca (uc) produced naturally creates a huge problem due to its stranding on the local coast, congestion, decomposition, and release of bad smell. to contribute to the mitigation of this environmental problem and find an appropriate valorization alternative, and since it contains commercially valuable components, such as bioactive compounds, uc algae were chosen as a source of bio-stimulant for experiment purposes on common bean plants (phaseolus vulgaris l.) under salt stress. bio-assay culture experiments of common bean were carried out in the absence and presence of salt stress with two levels (34,2 mm and 68,4 mm) also with and without uc extract treatment using three levels (1%, 3%, and 6%). results showed that uc extract has a great benefit on common bean growth both in the absence and under salt stress conditions especially at 3%. these results proved that uc extract significantly improved the activities of enzymes involved in the activation of the carbon-nitrogen and antia tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 778 oxidant enzymatic system, osmolytes accumulation, and also improved the morphological parameters. thus, we can say that uc extract could be successfully used to overcome the negative effects of salt stressors conditions for common bean plants in areas where irrigation water contains some salt content. keywords: keywords: biostimulants, ulva lactuca algae extract, common beans, enzymatic activities, salt stress. o3. effect of methanolic plant extract against penicillium o3. effect of methanolic plant extract against penicillium digitatumdigitatum salahddine chafiki1,2*, redouan qessaoui1, ahmed boumair1,3, yassine imlil1, naima chabi1,4, abdelmalek mahroug1,3, abdelhadi ajerrar1, abdellah oukarroum2, mohamed alouani3, and rachid bouharroud1 1 regional center of agricultural research of agadir, national institute of agricultural research, rabat, morocco; 2 agrobiosciences department (agbs), mohammed vi polytechnic university (um6p), ben guerir, morocco; 3 laboratory of biotechnologies and valorization of natural resources faculty of sciences agadir, ibn zohr university, agadir, morocco; 4 center of agrobiotechnology and bioengineering, cadi ayyad university (uca), marrakech, morocco. *corresponding and presenting author: salahddine.chafiki@um6p.ma. abstractabstract currently, the world agricultural sector has become an intensive production system. this situation has posed many problems related to the environment and human health and has also led to the development of resistance of pathogens to the chemical pesticides used. among the measures considered to reduce the negative effects of pesticides, there was the emergence of alternative control methods. therefore, the use of biopesticides can be an effective alternative to control fruit diseases. in morocco, the post-harvest diseases present a major constraint for the development of fruit sectors such as citrus which is threatened by the green rot penicillium digitatum. the main objective of this study is to evaluate the effectiveness of methanolic extract of an aromatic and medicinal plant of the genus salvia against penicillium digitatum. this evaluation was carried out by the direct contact method. the results obtained showed that 5mg/ml of this extract inhibited 71% of the mycelial growth of this fungus. this extract can be used as an active ingredient of a biopesticide for the management of this fungus. keywords:keywords: penicillium digitatum, biopesticide, plant extracts o4. chemical characterization, antioxidant activity, and phe-o4. chemical characterization, antioxidant activity, and phenolic compounds of moroccan opuntia dillenii fruits species nolic compounds of moroccan opuntia dillenii fruits species for potential use in food applicationsfor potential use in food applications sofia zazouli1,2* and ahmed jouaiti1 1 laboratory of sustainable development, faculty of science and technology, university sultan moulay slimane, beni mellal, morocco; 2 laboratory of bio-organic an analytical chemistry, faculty of science and technology, university sultan moulay slimane, beni mellal, morocco. *corresponding and presenting author: sofia.zazouli@usms.ma. abstractabstract this research consisted in carrying out a chemical characterization (total betalain, total condensed tannins, total phenols, flavonoid and ascorbic acid contents), antioxidant activity (by 2,2-diphenyl-1-picrylhydrazyl (dpph)), and quantification of some individual phenolic compounds of different morphological part of o. dillenii (seeds, juicy pulp, and peels) collected from essaouira regions of morocco was investigated. the total phenolic content of fruit fractions ranges from 202 to 56 g/ 100 g extract. total flavonoids range from 185 to 11 mg/100g extract. the total of betalains and condensed tannins ranged respectively from 229 to 5 mg/l and 12 to 8 mg/100g extract, while ascorbic acid was concentrated in the juicy pulp with (580 mg/100 g) and absent in the seeds fraction. a great variety of phenolic compounds were identified in the different extracts using hplc-esi-ms, mainly phenolic acids and flavonoids, some of which have been described for the first time. furthermore, the antioxidant results showed that all opuntia dillenii parts exhibited an excellent antioxidant activity ranged from 4.5 to 6.2 μg/ml. in conclusion, the different morphological parts of o. dillenii may serve as a potential source of natural antioxidants for food applications and medicinal functions. keywords:keywords: opuntia dillenii, bioactive compounds, chemical characteristics, phenolic compounds, flavonoids. plenary session vii: health, climate change and food securityplenary session vii: health, climate change and food security co-chair: khalid meksem, southern illinois university, usaco-chair: khalid meksem, southern illinois university, usa co-chair: moulay abdelmajid kassem, fayetteville state uni-co-chair: moulay abdelmajid kassem, fayetteville state university, usaversity, usa o1. impact of changing environment on our health: what o1. impact of changing environment on our health: what environment?environment? srini kaveri1,2* 1 inserm, paris, france; 2 cnrs bureau, new delhi, india. *corresponding and presenting author: srini.kaveri@cnrs.fr abstractabstract over the last century, we are witnessing a steady and significant changes in the diseases pattern all over the world. thanks to antibiotcs and vaccination, we have successfully fought over many infectious diseases. however, we are witessing the emergence of many autoimmune, allergic and cardiovascular and metabolic diseases. a healthy status of the body depends largely on how an intricate and delicate homeostasis of the immune system is maintained, via neutralization of microbes on one hand, and maintaining self-tola tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 779 erance to prevent autoimmunity on the other. any deviation leading to pathology is governed by three important factors: the genetic background of the individual; the bio-molecular interactions and equally importantly, the environment. the environmental factors include not only our immediate surrounding elements such pollutants and natural fauna and flora, but also the emotional and other stress factors. and one another extremely important and interesting environmental factor is our microbial ecosystem. investigating the interaction of microbial ecosystem and the host immune system may allow us to understand the molecular and cellular basis of the pathogenesis of several diseases and may also provide indications for the design of novel therapeutic strategies. o2. pd1-targeted car t cells as a therapy for lymphoma and o2. pd1-targeted car t cells as a therapy for lymphoma and solid tumorssolid tumors amorette barber* department of biological and environmental sciences, longwood university, farmville, va 23909, usa. *corresponding and presenting author: abstractabstract adoptive transfer of tumor-reactive t cells is a promising antitumor therapy for many cancers. while car t cells can successfully treat hematopoietic malignancies, car t cells have not shown success in treating solid tumors. there are many factors that may enhance car t cell efficacy and safety for solid tumor treatment, including addition of costimulatory domains. we developed a novel car (chimeric pd-1, chpd1) that targets the programmed death 1 receptor (pd-1) ligands expressed on many types of solid tumors. we also compared the inclusion of cd28, dap10, 41bb, gitr, icos, or ox40 costimulatory domains in our chpd1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. to determine if this novel car could potentially target a wide variety of tumors, the anti-tumor efficacy of chpd1 t cells against lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. regardless of the costimulatory domain in the car, all of the chpd1 t cells induced similar levels of t cell proliferation and tumor cell lysis. however, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the car. while most of the chpd1 t cell receptor combinations secreted both pro-inflammatory and anti-inflammatory cytokines, chpd1 t cells containing a dap10 costimulatory domain secreted high levels of proinflammatory cytokines but did not secrete a significant amount of anti-inflammatory cytokines. furthermore, t cells expressing chpd1 receptors with a dap10 domain also had the strongest anti-tumor efficacy in vivo. chpd1 t cells did not survive for longer than 14 days in vivo, however treatment with chpd1 t cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. therefore, adoptive transfer of chpd1 t cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. o3. breeding by design for healthy plant products from soy-o3. breeding by design for healthy plant products from soybeanbean khalid meksem* school of agricultural sciences, southern illinois university, carbondale, il, usa. *corresponding and presenting author: meksem@siu.edu. abstractabstract not provided. o4. improving plants specialized metaboliteso4. improving plants specialized metabolites adnane boualem* institute of plant sciences paris-saclay (ips2), inrae, university paris-saclay, cnrs, 91405 orsay, france. *corresponding and presenting author: adnane.boualem@ips2.universiteparis-saclay.fr. abstractabstract not provided. session viii: soil fertility and fertilizerssession viii: soil fertility and fertilizers co-chair: abdelaziz nilahyane, mohammed vi polytechnic co-chair: abdelaziz nilahyane, mohammed vi polytechnic university, moroccouniversity, morocco co-chair: rachid bouharroud, inra, moroccoco-chair: rachid bouharroud, inra, morocco o1. soil coo1. soil co22 flux and active carbon across soil salinity graflux and active carbon across soil salinity gradient have promising implications for revival of arid-saline dient have promising implications for revival of arid-saline soils subjected to forage grass-legume mixturessoils subjected to forage grass-legume mixtures dennis s. ashilenje§, abdelaziz hirich, lamfeddal kouisni, krishna devkota, and abdelaziz nilahyane* african sustainable agriculture research institute (asari), mohammed vi polytechnic university (um6p), laâyoune, morocco. *corresponding author: abdelaziz.nilahyane@um6p. ma. §presenting author: dennis.ashilenje@um6p.ma. abstractabstract soil salinity has devastating effects to forage crop productivity in arid lands of morocco. field experiments in the randomized complete block design was established in three sites with low, medium and high salinity of laayoune, morocco, to test if a 50-50% and 70-30% mixture of alfalfa and blue panicum grass and separately, grass pea and barley can a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 780 spur microbial activities necessary for buildup of soil organic matter and alleviating salinity (ece up to 54.4 ds m-1) compared to monocrops. over a duration of 15 weeks, alfalfa-blue panicum grass mixtures enhanced co2-c flux compared to monocrops regardless of background soil salinity. this behavior was divergent in a way that the 70-30 mixture had the highest flux in extreme soil salinity while the 50-50% mixture dominated in medium to low salinity. near similar effect was witnessed for barley-grass pea mixtures which enhanced soil co2-c flux under low salinity, but grass pea succumbed to excessive salinity stress which severely depressed soil co2c flux. the 70-30 mixture of alfalfa and blue panicum grass slightly enhanced soil active carbon as much as grass monocrop in high salinity and comparable to alfalfa monocrop in medium salinity. there were slight benefits of the 70-30 mixture of grass pea and barley to soil active carbon across soil salinity platforms. in a series of seven events of soil analysis, there was only slight evidence that mixtures could alleviate initially low soil salinity, but moderate to high salinity persisted across the year of crop establishment. it is however emerging that grass-legume mixtures can sustain microbial activities with promising effects to build up of soil organic matter, despite persistent salinity. keywords:keywords: soil salinity, grass-legume mixture, microbial activity, soil co2 flux. o2. cellulose/nanoclay biocomposites coated phosphate o2. cellulose/nanoclay biocomposites coated phosphate fertilizer: novel slow-release fertilizer for reducing phos-fertilizer: novel slow-release fertilizer for reducing phosphorus leaching and promoting plant growthphorus leaching and promoting plant growth ihsane kassem1§, el-houssaine ablouh1, fatima-zahra el bouchtaoui1, soumia boukind1, manal mhada2, salma mouhib2, houssine sehaqui1, and mounir el achaby1* 1 materials science, energy and nanoengineering department (msn), mohammed vi polytechnic university, hay moulay rachid, benguerir, morocco; 2 agrobiosciences department (agbs), mohammed vi polytechnic university, hay moulay rachid, benguerir, 43150, morocco. *corresponding author: mounir.elachaby@um6p.ma. §presenting author: ihsane.kassem@um6p.ma. abstractabstract sustainable phosphorus management (p) is crucial for efficient plant nutrition. polymer coating of highly soluble p fertilizer is an effective technology that can limit the risks of pleaching and/or runoff into water systems, thus, increasing pbioavailability and reducing water pollution. in this context, a novel coated triple superphosphate (tsp) fertilizer is developed using a simple process and green materials based on phosphorylated cellulose, methyl hydroxyethyl cellulose and montmorillonite clay. experimental results showed an improved morphology and hardness of phosphate fertilizer granules after coating as well as a positive impact on the water retention capacity of the soil. p leaching in soil was monitored for 80 days, and a substantial reduction of p leaching up to 80 % was successfully achieved by coated tsp fertilizer. in addition, common bean growth was improved by coated tsp treatments that showed a remarkable effect on leaf parameters and roots architecture of beans with increased yield by 28.5 %, p uptake by 23 %, p use efficiency by 37.9 % and agronomic efficiency by 54.5 %. keywords: keywords: slow-release fertilizers, coated fertilizers, phosphorus leaching, phosphorylated cellulose, montmorillonite. o3. o3. bacillus circulansbacillus circulans tc7 mediated the alleviation of metal tc7 mediated the alleviation of metal stress and the decrease of metal actc7 accumulation in stress and the decrease of metal actc7 accumulation in spinach tissuesspinach tissues sofia houida1*, lamia yakkou1, leyla okyay kaya2, serdar bilen2, mohamed raouane1, abdellatif el harti1, and souad amghar1 1 research team « lumbricidae, improving soil productivity and environment (lapse) », centre : eau, ressources naturelles, environnement et développement durable (cerne2d), ecole normale supérieure (ens), mohammed v university in rabat, 5118 morocco; 2 department of soil science and plant nutrition, faculty of agriculture, atatürk university, erzurum, 25240 turkey. *corresponding and presenting author: sofia_ houida@um5.ac.ma abstractabstract the contamination of agricultural soils with heavy metals is one of the primary causes of numerous ecological and environmental issues. it has altered the microbial community, degraded the soil, reduced plant growth and yield, and entered the food chain. the tolerance of plants to heavy metal stress must be enhanced in order to permit the growth of crops with minimal or no accumulation of heavy metals in edible plant parts, thereby satisfying the rapidly expanding global population’s demand for safe food. in this study, a pot experiment was performed to determine the effects of hms-tolerant pgp-bacteria, bacillus circulans tc7, previously isolated from the chloragogenous tissue of aporrectodea molleri, on the growth and metal accumulation in spinacia oleracea cultivated in an alkaline soil highly contaminated (mg kg-1) with cadmium 7, along with nickel 100, copper 150, zinc 300, and manganese 600. the results indicated that hms stress was significantly alleviated when soil was inoculated with b. circulans tc7 compared to the uninoculated control. indeed, root length increased significantly by 74.5%, whereas shoot length increased substantially by 106.3%. additionally, bacterial treatment improved spinach’s fresh and dry weights by 5.5 and 3.5 times, respectively, and increased the chlorophyll content (a+b) by 75.6%. furthermore, soil inoculation with b. circulans tc7 decreased root uptake of mn, zn, cu, cd, and ni by 43.3%, 49.9%, 62.00%, 22.03%, and 46.3%, respectively. moreover, the bacterial isolate limited the uptake of metals in the edible parts as manganese, cadmium, and nickel concentrations in aerial tissues were reduced by 71.1%, 59.9%, and 44.2%, respectively. analysis of the results revealed that soil inoculation with b. circulans tc7 can stimulate plant growth a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 781 while limiting metal bioaccumulation, indicating its potential use in mitigating hm stress. keywords:keywords: heavy metal stress, arthworm’ isolates, pgp bacteria, spinacia oleracea. o4. exploration of the diversity and function of bacterial o4. exploration of the diversity and function of bacterial community in maize rhizoplane across moroccan agro-sys-community in maize rhizoplane across moroccan agro-systems: towards a microbiome facilitated agriculture in mo-tems: towards a microbiome facilitated agriculture in morocco rocco dounia nkir1§, tessa reid3, brahim benbrik1, meryem haddine1, ibn yasser ammar1, rachid ghani1, ian clark3, tim h. mauchline3, jim harris4, zineb rchiad2, and adnane bargaz1 1 plant and soil microbiome sub-program, agrobiosciences, mohammed vi polytechnic university, ben guerir, morocco, 2 african genome center, mohammed vi polytechnic university, ben guerir, morocco, 3 rothamsted research, sustainable soils and crops, harpenden, uk; 4 cranfield university, school of water, energy and environment, cranfield, uk. *corresponding author: adnane.bargaz@um6p.ma, zineb.rchiad@ um6p.ma. §presenting author: dounia.nkir@um6p.ma. abstractabstract maize (zea mays l.) is one of the most important staple food crops grown worldwide to feed an ever-growing world population. to increase crop production, current agricultural practices rely on unsustainable techniques such as the overapplication of fertilizers. to overcome this challenge, there is a need for developing sustainable approaches to increase the agricultural system’s production. one applied, eco-friendly approach is bacterial consortia that have been reported in many studies for their beneficial plant growth promotion compared to single strains inoculation. the aim of this work is to map the diversity of the bacterial community in the rhizoplane of the maize plants and to select pgprs to construct promising bacterial consortia. despite the low diversity of microbes colonizing the root surface, the pgp activities in this niche are highly dynamic. bacterial isolation was performed from maize rhizoplane soil from 28 agroecological zones of 7 different regions in morocco using a novel consortia-oriented isolation approach that aims to isolate and examine consortia constructed from phosphate solubilizing and nitrogen-fixing bacteria, in addition to bacteria with other pgp activities from the rhizoplane soil. a total of 144 bacteria were isolated with 44 psb, 43 nfb, and 57 other pgpr. genetic fingerprinting using randomly amplified polymorphic dna-generated profiles allowed elimination of duplicates before bacterial identification using 16s sequencing. phylogenetic analysis of 16s rrna sequences revealed the diversity of the isolates with the main dominant genera belonging to bacillus, pseudomonas, and streptomyces that were reported to have an antifungal and antibacterial activities with salt stress tolerance property. moreover, candidate novel species were also identified that should be tested afterwards. the constructed consortia will be further screened for their pgp properties both in vitro and in planta to determine their beneficial plant growth effect on maize plants. thereafter, the most performing consortia will be selected to study their individual strains for better understanding of the functions of promising consortia. keywords: keywords: pcr fingerprinting, pgprs, consortia, nitrogen fixation, phosphate solubilization. o5. organic amendments to restore sandy soils to produce o5. organic amendments to restore sandy soils to produce organic vegetables under arid conditionsorganic vegetables under arid conditions khalid azim1*, mostapha ajrari2, fouad elame1, rachid aboutayeb3, and jamal hallam1 1 integrated crop production research unit, regional center of agricultural research, national institute of agricultural research, agadir, morocco; 2 institut agronomique et vétérinaire hassan ii-complexe horticole agadir, morocco; 3 regional center of agricultural research, national institute of agricultural research, settat, morocco. *corresponding author: khalid.azim@inra.ma abstractabstract to identify the best compost amendments rate in squashgreen beanmelon-pepper cropping system that could maintain better the soil fertility in arid conditions; organic matter, humic fraction, humic acids and micro-nutrient elements were evaluated in sandy soil. this study was conducted at the experimental farm of melk zhar inra-agadir (morocco) between 2008 and 2013 to determine the effect of four annual compost amendment rates (10 t ha-1, 15 ha-1, 20 t ha-1, and 25 t ha-1) on soil fertility of four organic vegetables at different soil depth (0.30 m, 0.50 m, 0.70 m and 0.90 m). the results showed that when 25 t ha-1 of annual compost was applied to the amended horizon, the ph level decreased by 4.83% compared to control. in the other hand, for all depth, the electrical conductivity (ece) increased by 28.1% and 32% at a rate of 15 and 25 t ha-1, respectively. soil organic matter (som) content has obviously increased, reaching a maximum of 1.2% at the amended horizon rate of 25 t ha-1. for all rates at the amended horizon, there was an average increase in the humic fraction (42% of organic carbon), fulvic acids (34%), and humic acids (14.5%). for all organic amendments rates and soil depths, the total nitrogen, available phosphorus, exchangeable cations (magnesium and potassium), and micro-nutrient elements (iron and copper) increased. organic vegetable production has increased by an average of 24% over the control. therefore, in five growing seasons, there has been an increase in production and improvement of sandysoil fertility. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 782 concurrent session ix: plant sciences iiconcurrent session ix: plant sciences ii co-chair: miguel botella, university of malaga, spainco-chair: miguel botella, university of malaga, spain co-chair: mohamed nhiri, university abdelmalek essaadi, co-chair: mohamed nhiri, university abdelmalek essaadi, moroccomorocco o1. genetic diversity of walnut (o1. genetic diversity of walnut (juglans regiajuglans regia l.) genotypes l.) genotypes across middle and high atlas moroccan mountains using across middle and high atlas moroccan mountains using issr markersissr markers ghizlane kabiri§, said bouda*, mohammed elhansali, and abdelmajid haddioui laboratory of biotechnology and valorization of plant genetic resources, faculty of sciences and techniques, sultan moulay slimane university, b.p. 523, beni mellal, morocco. *corresponding author: saidbouda@yahoo.fr. §presenting author: kghizlane84@gmail.com. abstractabstract the genetic diversity and genotypes structure of walnut (juglans regia l.) are essential to understand and manage genetic resources of this species, as well as for further progress in breeding programs. issr markers were used to assess the genetic diversity of 66 individuals’ trees from 11 accessions, representing the main cropping area of walnut belonging to two ranges types of mountains in morocco: middle and high atlas. eleven issr primers rendered a total of 135 bands (91%) with 0.88 polymorphic information content. the utilization of 123 polymorphic bands revealed a high level of genetic variation within and among the examined accessions. the multi-locus values of ht and hs were 0.25 and 0.20, respectively. the amova analysis showed that 71.30% of total genetic variability is accounted within accessions and 28.70% between accessions. this was congruent with the coefficient of genetic differentiation (gst=0.16). bayesian model-based clustering approach identified three gene pools that were not correlated with mountain range type. this is the first application of issr markers for the assessment of genetic diversity in moroccan germplasm of walnut. this information will be useful to define conservation strategies and improvement programs of this species. keywords: keywords: walnut, issr, genetic diversity, differentiation, geographic group, bioclimatic group. o2. in situ production and multiplication of saffron corms o2. in situ production and multiplication of saffron corms ((crocus sativuscrocus sativus l.) in taliouine, morocco l.) in taliouine, morocco karra youssef1* and abdelghani tahiri1 natural resources and local products research unit, agadir regional center of agricultural research, national institute of agricultural research (inra), morocco. *corresponding author: youssef.karra@inra.ma. abstractabstract saffron is a sterile triploid plant that propagates only through the vegetative multiplication of corms, which is an underground stem, it’s an organ of storage and asexual propagation. therefore, the preservation and multiplication of corms is a strategic necessity (in situ conservation), these corms have adapted for several decades in the region of taliouine, the particular origin of production of 95% of moroccan saffron. this study is a monitoring-evaluation of the agro-morpho-physiological behavior of saffron corms from 10 origins of taliouine and taznakht region, over two consecutive years. the planting was in a specific place, platform ifri in taliouine for production and multiplication of corms, it had main objectives: (1) the agronomic characterization of the different clones from different origins (2) the safeguard and conservation of this diversity (3) and the selection and multiplication of performing corms. during this period of monitoring this plant material, we were able to assess the production potential and the agro-morphological variability of these corms from different sources: the observations made led to the identification of two cultivars which group together the corms of different origins in the platform, the first cultivar is made up of corms from provenances belonging to the medium altitude plateau (≈1600m), the 2nd cultivar comes from high altitude mountain areas (≥1900m). these two cultivars have a distinct behavior in terms of the development of replacement corms, this diversity of expression and vegetative development is of great agronomic importance and arouses the valuation of this plant material for new plantations of saffron cultivation. this project is part of an agreement between inra and fimasafran for the production and multiplication of saffron corms. keywords: keywords: saffron, corm, taliouine, multiplication, production, selection. o3. cultivar resistance as a main tool for restoration of cac-o3. cultivar resistance as a main tool for restoration of cactus industry damaged by cochineal tus industry damaged by cochineal dactylopius opuntiaedactylopius opuntiae rachid bouharroud1*, mustapha el bouhssini2, abdelaziz mimouni1, and naima ait aabd1 1 regional center of agricultural research of agadir, national institute of agricultural research, morocco; 2 biodiversity and plant sciences program, agrobiosciences, mohammed vi polytechnic university, ben guerir, morocco. *corresponding and presenting author: rachid.bouharroud@inra.ma. abstractabstract the cactus crop (opuntia sp.) was almost damaged in morocco by the carmine wild cochineal dactylopius opuntiae (cockerell, 1896) (hemiptera: dactylopiidae) since its first report in 2014. cactus species was used for food as fresh fruits or byproducts, as animal feed, land fence and also to prevent erosion and desertification. this cam photosynthesis types plant is the most profitable crops thanks to its strong adaptation to arid and semi-arid climates. the genetic diversity preserved a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 783 in the cactus collection at the inra-agadir from 1999 to 2022 was used as a baseline to find resistant cultivars for rehabilitation of destroyed cactus lands. the 8 identified resistant cultivars to carmine cochineal are described and their roles to restore the cactus industry crops in morocco is showed. the 2 scenarios suggested are tuned based on the land uses as for farming production (cultural practices) or to prevent erosion and desertification in natural areas like mountains. these 8 cultivars namely: bellara (light green flesh), marjana (red flesh), melk zhar, cherratia, aakria (carmin flesh), ghalia and karama (light red) belong to opuntia ficus indica, o. engelmannii, o. robusta and o. dillenii. for each one of these 8 cultivars the resistance mechanism involved, the genetic diversity and specific roles will be discussed. keywords: cactus, resistance, erosion, beekeeping, animal feed. o4. evaluation of agronomic performance of legumes in an o4. evaluation of agronomic performance of legumes in an agroforestry system based on olive trees in the south of the agroforestry system based on olive trees in the south of the mediterranean seamediterranean sea asmae amassaghrou1,2*, karim barkaoui3,4, ahmed bouaziz1, si bennasseur alaoui1, fatemi zain el abidine2, and khalid daoui2 1 hassan ii agronomic and veterinary institute, bp 6202 rabatinstituts10112, rabat, morocco; 2 national institute of agricultural research, regional center of meknes, km10, haj kaddour road, meknes, morocco; 3 cirad, umr absys, f-34398 montpellier, france; 4 absys, univ montpellier, ciheam-iamm, cirad, inrae, institut agro, montpellier, france. *corresponding and presenting author: a.amassaghrou@iav.ac.ma. abstractabstract in olive tree-based agroforestry, trees compete with crops for soil resources, but they can also improve crops growing conditions under drought conditions, mainly by providing shade during the growing season. olive trees provide permanent shade for intercrops with different intensity depending on the season and the orientation of sowing intercrops. in this study, we evaluated three legumes (faba bean, chickpea and lentil) in olive agroforestry in northern morocco. we compared growths in sole crop or intercrop legumes to examine aboveground competitive interactions and pinpoint the likely response mechanisms. we assessed the effect of shade and distance from trees on crop growth, yield components, and final yields. plant height, aboveground biomass, and grain yield were all higher in 2016 than 2017, and lower in agroforestry than sole crop for all the legume species. in average, faba bean had the tallest plants (74.33 cm), the highest biomass (32.30 g.m-2) and yield (17.23 g.m-2 ), while lentil had the shortest plants (32 cm) and the lowest biomass (13.80 g.m-2) and yield (1.18 g.m-2) amongst the three species. under olive trees, faba bean grain yield was highest under non-shaded conditions and declined with increased shade, while lentil recorded the highest yield under the shade, and for chickpea the distance from the trees had no effect on the final grain yield. the number of pods and grains per unit area was the most impacted yield component in both 2016 and 2017. olive trees limited crop growth and caused a significant reduction in grain yield, whatever in 2016 under drought, olive shade had a significant impact on grain number per unit area and grain yield. despite lower crop yields, we show how shade may impact the performance of legumes under olive agroforestry. keywords: chickpea, faba bean, lentil, agroforestry, drought, tree-crop interaction. o5. red fruits in the souss massa regiono5. red fruits in the souss massa region redouan qessaoui*, y. rahal, m. alouani2, s. chafiki1, a. ajerra1, a. tahiri1, n. ait abd1, a. wifaya1, a. mimouni1, and r. bouharroud1 1 regional center of agricultural research of agadir, national institute of agricultural research, avenue ennasr, bp 415 rabat principale, 10090 rabat, morocco; 2 laboratory of biotechnologies and valorization of natural resources faculty of sciences agadir, ibn zohr university, b.p 8106, agadir, morocco. *corresponding and presenting author: redouan.qessaoui@inra.ma. abstractabstract the souss-massa region, thanks to its climatic conditions which allow good and rapid adaptation of crops, has recently become a region producing red fruits, in particular raspberries. in order to obtain a phytosanitary inventory of red fruit cultivation in the souss-massa region, a survey was conducted in the region. the results showed that all agricultural areas in the souss-massa region are currently targeted for the cultivation of red fruits. raspberry, blueberry and blackberry are respectively the most cultivated in the region. raspberry cultivation is most popular in all regions with abundance in ait amira (45%), followed by biougra (31%) and belfaa-massa (21%). while the cultivation of blueberries is widespread in the regions of biougra (30%) and balfaa-massa (15%). regarding the cropping system, it is the most widespread integrated crop in the region (95%). the results showed that the soilless mode is mainly used for blackberry and raspberry. however, the raspberry can be grown in full soil. in terms of production, the yazmin variety is the most productive (900 tons) followed by carmina (500 tons) and adelita (350 tons). then, for the blackberry, the vectoria and laurita varieties showed impressive productivity, reaching 441 and 300 tons respectively. for blueberries, the corina variety showed a high production (722 tons) compared to the others. according to these results, the modified canarian greenhouses are the most used. according to this survey, cladosporiosis and gray mold are the most economically important diseases in terms of the damage caused to the cultivation of red fruits in the soussmassa region. the results of this survey showed that phytosanitary problems due to diseases constitute a priority subject among producers of red fruits in the region of souss-massa. keywords: keywords: red fruits, raspberry, blueberry, blackberry, souss a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 784 massa. concurrent session x: genetics and genomicsconcurrent session x: genetics and genomics co-chair: mohcine bennani mechita, university abdelmalek co-chair: mohcine bennani mechita, university abdelmalek essaadi, tangier, moroccoessaadi, tangier, morocco co-chair: mohammed el hassouni, university abdelmalek es-co-chair: mohammed el hassouni, university abdelmalek essaadi, tangier, moroccosaadi, tangier, morocco o1. qtl and candidate genes that control several seed com-o1. qtl and candidate genes that control several seed composition traits in soybeanposition traits in soybean moulay abdelmajid kassem1* and khalid meksem2 1 plant genomics and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc, usa; 2 school of agricultural sciences, southern illinois university, carbondale, il, usa. *corresponding and presenting author: mkassem@uncfsu.edu. abstractabstract soybean seeds are rich in protein, oil, fatty acids, isoflavone, sugars, amino acids and other beneficial compounds. here, we summarize 8-10 years of findings on genetic mapping of seed composition traits (protein, oil, fatty acids, and sugars contents) using four ril (recombinant inbred line) populations [‘pi 438489b’ by ‘hamilton (pixh, n=50), ‘md 96-5722’ by ‘spencer’ (mdxs, n=92), ‘hamilton’ by ‘spencer’ (hxs, n=93), and ‘forrest’ by ‘williams 82’ (fxw82, n=309) ril populations] and different environments in north carolina and illinois. the new linkage maps are useful tools that will serve the soybean community in genetic mapping of qtl for seed composition and other agronomic traits and in identifying candidate genes controlling these traits. keywords: keywords: soybean, qtl, protein, oil, fatty acids, sugars. o2. deciphering protein and oil biosynthesis pathway using o2. deciphering protein and oil biosynthesis pathway using tilling-by-sequencing+: an effective platform for high-tilling-by-sequencing+: an effective platform for highthroughput gene functional analysis throughput gene functional analysis naoufal lakhssassi*, abdelhalim el baze, and khalid meksem school of agricultural sciences, southern illinois university, carbondale, il, usa. *corresponding and presenting author: lakhssassi.naoufal@siu.edu. abstractabstract reverse genetic approaches have been widely applied to study gene function in different crop species; however, these techniques, including gel-based tilling, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their dna sequence. chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. we used an optimized high-throughput tilling-by-sequencing+ technology, coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. an ethyl methanesulfonate mutagenized population (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. the mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. to test the efficiency of tbys+ in complex genomes, we used soybeans as a model with a focus on several desaturase gene families, gmsacpd, gmfad2, and gmfad3, that are involved in the soybean fatty acid biosynthesis pathway in addition to targeting genes involved in increasing protein content and improving amino acid composition. the use of tbys+ allows the discovery of novel sources of soybean oil traits, including high saturated, monosaturated fatty acids, low polyunsaturated fatty acid contents, in addition to soybean lines with increased protein content while maintaining steady oil. this technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. the obtained soybean mutants can be used in subsequent soybean breeding programs for improved protein and oil composition traits. keywords:keywords: gmfad2; gmfad3; gmsacpd; cogy, tilling by target capture sequencing; tilling-by-sequencing+; fatty acid desaturases; oil biosynthesis pathway, protein content, and amino acid composition. o3. genetics and genomics of fruit color in strawberry o3. genetics and genomics of fruit color in strawberry ((fragariafragaria spp.) spp.) cristina castillejo1, josé f. sánchez-sevilla1,2, iraida amaya1,2* 1 instituto andaluz de investigación y formación agraria y pesquera, (ifapa), centro de málaga, málaga, spain; 2 unidad asociada de i+d+i ifapa-csic biotecnología y mejora en fresa, málaga, spain. *corresponding and presenting author: iraida.amaya@juntadeandalucia.es. abstractabstract fruit color is determined by the accumulation of a variety of secondary metabolites, including carotenoids, anthocyanins and other flavonoids. apart from contributing to fruit attractiveness, these secondary metabolites provide a wide range of health-promoting benefits and are therefore considered to be bio-active constituents. the characteristic red fruit color of strawberries is the result of anthocyanin accumulation. using a geographically diverse sample of diploid and octoploid fragaria species we have shown that differential anthocyanin content could be explained by independent mutations in myb10. in the cultivated octoploid strawberry, famyb10-2 is the dominant homoeolog regulating anthocyanin biosynthesis. as such, mutations in famyb10-2 coding region abolished red color formation. however, some fruits with a wild type famyb10-2 copy do accumulate anthocyanins a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 785 in their epidermis but not in the flesh. we have shown that this white flesh phenotype is associated with the absence of faenspm-2, a cacta-like transposon inserted in famyb10-2 promoter from red-fleshed accessions. natural variants in yellow/white fruit shades are documented in different strawberries, including fragaria chiloensis and f. vesca as i.e. the f. vesca ‘yellow wonder’ cultivar. the genetic control of yellow/white flesh variation was analyzed using an interspecific f2 population segregating for the yellow/white trait derived from the cross of ‘senga sengana’ and f. chiloensis ssp. lucida. quantitative trait loci (qtl) mapping detected a single locus named ‘yellow flesh’ controlling most of the observed variation. white flesh (absence of yellow pigments) was dominant over yellow flesh and fitted the 3:1 expected ratio (p=0.53). the ‘yellow flesh’ locus mapped to a reduced interval where faccd4-b, involved in carotenoid metabolism has been detected. accordingly, yellow fleshed f2 lines showed a downregulation of faccd4-b and significantly higher accumulation of a number of carotenoids, such as lutein, as well as a 2.3-fold increase in total carotenoid concentration. the outcome of these genetic analyses allowed us the development of predictive hrm and kasp markers for targeted breeding and marker-assisted selection of strawberry varieties with improved sensorial and nutritional fruit. this work is funded by agencia estatal de investigación (pid2019-111496rr-i00 / aei / 10.13039/501100011033) and paidi p18-rt-4856 (junta de andalucía, feder funds). keywords: keywords: strawberry, anthocyanins, carotenoids, qtl, marker-assisted selection. o4. qtl and candidate genes for seed tocopherol content o4. qtl and candidate genes for seed tocopherol content in ‘forrest’ by ‘williams 82’ recombio4. recombinant inbred in ‘forrest’ by ‘williams 82’ recombio4. recombinant inbred line (ril) population of soybeanline (ril) population of soybean dounya knizia1,2§, jiazheng yuan3, naoufal lakhssassi1, abdelhalim el baze1, mallory cullen1, tri vuong4, hamid mazouz2, henry t. nguyen4, my abdelmajid kassem3, and khalid meksem1* 1 school of agricultural sciences, southern illinois university, carbondale, il 62901, usa; 2 laboratoire de biotechnologies & valorisation des bio-ressources (biovar), department de biologie, faculté des sciences, université moulay ismail, meknès 50000, morocco; 3 plant genomics and biotechnology laboratory, department of biological and forensic sciences, fayetteville state university, fayetteville, nc 28301, usa; 4 division of plant science and technology, university of missouri, columbia, mo 65211, usa. *correspondence: meksem@siu. edu. §presenting author: dounya.knizia@siu.edu. abstractabstract soybean seeds are rich in secondary metabolites which are beneficial for human health, including tocopherols. tocopherols play an important role in human and animal nutrition thanks to their antioxidant activity. in this study, the ‘forrest’ by ‘williams 82’ (f×w82) recombinant inbred line (ril) population (n = 306) was used to map quantitative trait loci (qtl) for seed α-tocopherol, β-tocopherol, δ -tocopherol, γ-tocopherol, and total tocopherol contents in carbondale, il over two years. also, the identification of the candidate genes involved in soybean tocopherols biosynthetic pathway was performed. a total of 32 qtl controlling various seed tocopherol contents have been identified and mapped on chrs. 1, 2, 5, 6, 7, 8, 9, 10, 12, 13, 16, 17, and 20. one major and novel qtl was identified on chr. 6 with an r2 of 27.8, 9.9, and 6.9 for δ-tocopherol, α-tocopherol, and total tocopherol content, respectively. reverse blast analysis of the genes that were identified in arabidopsis allowed the identification of 37 genes involved in soybean tocopherol pathway, among which 11 were located close to the identified qtls. the identified tocopherol seed qtls and candidate genes will be beneficial in breeding programs to develop soybean cultivars with high tocopherol contents. keywords: soybean, qtl, seed, tocopherols, forrest, williams82. o5. mapping qtl for soybean seed protein and amino acids o5. mapping qtl for soybean seed protein and amino acids in the forrest x williams82 ril populationin the forrest x williams82 ril population jiazheng yuan1*, nacer bellaloui2, my abdelmajid kassem1, dounya knizia3,6, tri vuong4, naoufal lakhssasi3, mariola usovsky4, qijian song5, sonia viera1, layla rashid1, frances betts1, teresa register1, earl williams1, hamid mazouz6, henry t. nguyen4, and khalid meksem3,6 1 plant genomics and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc, usa; 2 usda, agriculture research service, crop genetics research unit, 141 experiment station road, stoneville, ms 38776, usa; 3 school of agricultural sciences, southern illinois university, carbondale, il 62901, usa; 4 division of plant science and technology, university of missouri, columbia, mo 65201, usa; 5 soybean genomics and improvement laboratory, usda-ars, beltsville, md 20705, usa; 6 laboratoire de biotechnologies & valorisation des bio-ressources (biovar), faculté des sciences, department de biology, université moulay ismail, meknès, morocco. *corresponding and presenting author: jyuan@uncfsu.edu. abstractabstract soybean [glycine max (l.) merr.] is an important crop in the america, and a total of 95.2 million metric tons of soybean seeds have been harvested each year with the value of approximately 31 billion dollars. soybean seeds are the major protein source for animal feed. however, soybean seeds contain limiting amount of some amino acids including cysteine (cys), lysine (lys), methionine (met), and threonine (thr), among them, lys, met, and thr are essential amino acids (eaas). the supplement with the synthetic amino acids in animal feed is expensive. therefore, the investigation of amino acid dynamics and reveal the genetic contribution of amino acid contents in soybean will help us to develop elite soybean lines with the enriched certain amino acids. the objective of this study was to map the quantitative trait loci a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 786 (qtl) associated with protein and amino acid contents in the recombinant inbred population derived from forrest × williams 82 (fxw82) within two diverse environments, spring lake, nc in 2018 and carbondale, il in 2020, respectively. in this undergraduate centered research, a total of 63 qtl in spring lake and 42 qtl in carbondale, respectively were identified (lod>2) for protein and amino acid contents using composite interval mapping (cim) method in fxw82 ril population, which was genotyped by soybean 6k illumina infinium beadchips. the range of phenotypic variation explained by the qtl was varying from 3.3153% in the dataset. the significant snps adjacent to the qtl can be converted into the kasp based pcr assays for marker-assisted selection. the genetic information uncovered in our study will be useful to enhance soybean improvement with better nutritional value. session xi: plant products and bioactive compoundssession xi: plant products and bioactive compounds co-chair: abdelfettah maouni, abdelmalek essaadi univer-co-chair: abdelfettah maouni, abdelmalek essaadi university, moroccosity, morocco co-chair: katelyn kesheimer, auburn university, usaco-chair: katelyn kesheimer, auburn university, usa o1. bioactive compounds, antioxidant activity and fatty ac-o1. bioactive compounds, antioxidant activity and fatty acids composition of fruits of ten moroccan pomegranate cul-ids composition of fruits of ten moroccan pomegranate cultivars (tivars (punica granatumpunica granatum l.) l.) sarah loukhmas1,2*, meriem outaki3, ebrahim kerak1, and hasnaâ harrak2 1 hassan ii casablanca university, faculty of science and techniques, laboratory of virology, microbiology, quality and biotechnology/ etb. p.o. box 146 yasmina city, 20650 mohammedia, morocco; 2 agro-food technology and quality laboratory, plant improvement and quality research unit, regional center of agricultural research of marrakesh, national institute of agricultural research, avenue ennasr, bp 415 rabat principale, 10090 rabat, morocco; 3 laboratory of applied chemistry and environment, faculty of sciences and technologies, university hassan i, p.o. box 577, settat, morocco. *corresponding and presenting author: s.loukh@ gmail.com. abstractabstract the pomegranate fruit is gaining worldwide increasing interest for its consumption. it is mainly consumed fresh or as juice. this interest in pomegranate is due to its nutritional and therapeutic benefits. in this context, this study aimed to evaluate the characteristics of fruit juice and seed oil of ten pomegranate cultivars grown in the center of morocco. mineral composition, organic acids, total polyphenols, anthocyanins content and antioxidant activity were determined in pomegranate juices. while fatty acid composition and antioxidant activity were determined in pomegranate seed oils. the results showed interesting characteristics for the studied cultivars. for the pomegranate juice, mineral composition showed that iodine was the most abundant macroelement, followed by phosphorus and sulphur. iron and aluminum were the main microelements. the most dominant organic acid was citric acid with concentrations ranging from 0.11 to 2.92 g/100 ml for the sour cultivar “lhamdha”. oxalic acid was mostly detected in sweet cultivars with concentrations ranging from 0.04 to 0.10 g/100 ml. the cultivar “lhamdha” presented the highest total polyphenol concentration with 116.83 mg gallic acid equivalent/100 g followed by the sweet cultivars “sefri of beni meskine”, “lahmer” and “marrakchia”. the highest total anthocyanin content was registered for cultivar “lahmer” (which means red) and “marrakchia” with 20.41 and 21.28 mg/100 ml, respectively. the sour cultivar “lhamdha” and the sweet cultivar “sefri of beni meskine” presented the highest antioxidant activity with 111.24 and 105.99 mg acid ascorbic equivalent/100 g, respectively. pomegranate seed oil presented high contents of polyunsaturated fatty acids (pufas) exceeding 89%. the major fatty acid was punicic acid, which represented more than 80% of fatty acids, while other fatty acids such as linoleic, oleic, and palmitic acids are minor. oils also showed high antioxidant activity (inhibitory concentration ic50: 0.69-1.80 mg/ml). this study highlighted the nutritional and functional potential of the studied cultivars and the importance of their valorization, especially in food and pharmaceutical industries. keywords:keywords: morocco, punica granatum, fruit juice, seed oil, bioactive compounds, antioxidant activity, fatty acids. o2. anticancer activity and characterization of secondary o2. anticancer activity and characterization of secondary metabolite compound from dichloromethane fraction of metabolite compound from dichloromethane fraction of methane methane zizyphus lotuszizyphus lotus sofia zazouli1,2*, mohammed chigr2, patrícia a. b. ramos3,4, daniela rosa5,6, maria m. castro5, ahmed jouaiti1, maria f. duarte5,6, sónia a. o. santos3, and armando j. d. silvestre3 1 laboratory of sustainable development; 2 laboratory of bio-organic an analytical chemistry, faculty of science and technology, university sultan, moulay slimane, beni-mellal 23000, morocco; 3 ciceco-aveiro institute of materials, department of chemistry, campus de santiago, university of aveiro, 3810-193 aveiro, portugal; 4 laqv requimte, department of chemistry, campus de santiago, university of aveiro, 3810-193 aveiro, portugal; 5 alentejo biotechnology center for agriculture and agro-food (cebal), polytechnic institute of beja (ipbeja), 7801-908 beja, portugal; 6 mediterranean institute for agriculture, environment and development— med, cebal, 7081-908 beja, portugal; *corresponding and presenting author: sofia.zazouli@usms.ma. abstractabstract the present work investigated the trimethylsilylation dichloromethane extract of different morphological parts of zizyphus lotus (seeds, pulp, leaves, and root barks) by gc-ms and evaluation of their anti-tumor activity and the mechanism of action. in this study, 123 compounds were detected and quantified, from which fatty acids (455-8501 mg/kg dw) and a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 787 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) triterpenic acid (248-10230 mg/kg dw) are the major families of lipophilic components present in z. lotus, followed by sterols, long-chain aliphatic alcohols, aldehydes, monoglycerides, aromatic compounds among other. moreover, z. lotus lipophilic extract of root barks demonstrated higher inhibitory effects against human tumor cell lines of mda-mb-231, mcf7, and hepg2. while, the exposing mda-mb-231 cell for 48h, to the respective ic50 concentration of root barks lipophilic extract, showed its ability to stop cell migration, arrest cell cycle at g2, and processes apoptosis as well as the down regulation of the pi3k/akt pathway. in summary, z. lotus has the potential to be exploited as a source of bioactive compounds that can be applied in the formulations of different ingredients for the food and pharmaceutical industry. key-keywords: words: zizyphus lotus, lipophilic fraction, triterpenic acid, anti-cancer activity, apoptosis, cell migration, cell cycle. o3. sorghum (o3. sorghum (sorghum bicolor sorghum bicolor (l.) is a potent medicinal food (l.) is a potent medicinal food as an antioxidant, antiglycation, and anti-diabetic as an antioxidant, antiglycation, and anti-diabetic ben el mahdi nora1*, laurent lemée2, and mohamed nhiri1 1 laboratory of biochemistry and molecular genetics, faculty of sciences and technologies, university abdelmalek essaadi, bp 416, 90000 tangier, morocco; 2 institut de chimie des milieux et matériaux de poitiers (ic2mp cnrs umr 7285), université de poitiers, 4 rue michel brunet, tsa 51106, 86073 poitiers cedex 9, france. *corresponding and presenting author: benelmahdinora24@gmail.com. abstractabstract advanced glycation end products (ages) generated from glycation might cause diseases such as diabetes. antioxidants are essential in protecting humans against infections and degenerative diseases due to their ability to neutralize ages. sorghum (sorghum bicolor l.) is an ancient plant in the mediterranean basin known for multiple end-uses such as food and a source of bioactive compounds that could be used for medical applications. the present research deals with phytochemical screening and the antioxidant properties of both aqueous and ethanolic extracts of seeds from five sorghum ecotypes. additionally, antidiabetic inhibitory activities (α‐amylase and α-glucosidase and inhibitory potential on ages formation have been investigated using the ethanolic extract of the five sorghum ecotypes. the ethanolic extract exhibited a high phenolic and flavonoid content. furthermore, phenolic composition analysis using lc-ms/ms demonstrated the presence of attractive major compounds. the mineral composition using both icp-ms and icp-oes of all ecotypes also showed the valuables potential of the fivesorghum ecotype as a therapeutic food. the ethanolic extract of sorghum seeds also showed high inhibitory effects on the production of amadori products and ages from albumin in the presence of fructose and a significant inhibitory effect against α-amylase and α-glucosidase. these results suggest the possibility of using sorghum seeds as a source of natural bioactive foods to prevent glycation-associated complications in diabetes and investigate their possible combination effect with conventional drugs of preclinical use. keywords: keywords: sorghum bicolor l.; bioactive compounds; antioxidant effect; antidiabetic activity; antiglycation potential. o4. the effect of moroccan seaweeds from mediterranean o4. the effect of moroccan seaweeds from mediterranean coast (coast (halopteris scopariahalopteris scoparia, , gracilaria duraduragracilaria duradura, and , and enter-entermorpha compressamorpha compressa) on ) on medicago sativamedicago sativa plant growth plant growth oumaima ouala1*, soukaina el amrani zerrifi1, el mahdi redouane1, richard mugani1, asmae aknaf2, ouafa cherifi1, fatima el khalloufi1,3, and brahim oudra1 1 water, biodiversity and climate change laboratory. phycology, biotechnology and environmental toxicology research unit, faculty of sciences semlalia marrakech, cadi ayyad university, p.o. box 2390, 40000 marrakech, morocco; 2 observatoire de la lagune de marchica de nador et région limitrophes (labo.olman-rl), faculté pluridisciplinaire de nador, université mohamed premier, bp 300, 62700, selouane, nador, maroc; 3 polydisciplinary faculty of khouribga (fpk), university sultan moulay slimane of beni-mellal, p.b. 145, 25000 khouribga, morocco. *corresponding and presenting author: oumaima.ouala@ced.uca.ma. abstractabstract in agriculture, the use of chemical fertilizers and pesticides causes negative effects to the environment and human health. therefore, choosing a sustainable agricultural model has become a challenge for agriculture and food security in order to reduce the use of these chemical products. in fact, organic farming has experienced a great development. hence the interest in natural products of natural origin, such as plants and algae. the main objective of this study was to evaluate the biostimulant effect of extracts of a brown alga (halopteris scoparia), a red alga (gracilaria dura) and a green alga (entermorpha compressa) on germination and growth of medicago sativa plant. for this purpose, four treatments with the macroalgal extracts (25%, 50%, 75% and 100%) were tested. then, the determination of the germination rate and the evaluation of the growth of the plant through physiological and biochemical parameters was carried out on the obtained plant biomasses. the results obtained revealed that the treatment of plants by 25%, 50% and 75% of aqueous extracts of halopteris scoparia, gracilaria dura and entermorpha compressa, induced a significant increase in the germination rate and an improvement of the vegetative biomass growth (dry weight). in addition, the application of theses algae extracts studied resulted in an increase in the content of mineral elements and photosynthetic pigments (chlorophyll and carotenoids). in light of the results obtained, it was concluded that the macroalgal extracts studied could be suitable for promoting plant development. furthermore, they can be an alternative way to develop sustainable agricultural practices that respect the environment. keywords: keywords: a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 788 halopteris scoparia, gracilaria dura, entermorpha compressa, seaweeds, biofertilization, biostimulation, medicago sativa, growth, mineral elements, pigment contents. concurrent session xii: soil sciences and soil resilienceconcurrent session xii: soil sciences and soil resilience co-chair: eric brevik, southern illinois university, usaco-chair: eric brevik, southern illinois university, usa co-chair: rachid moussadek, moroccoco-chair: rachid moussadek, morocco o1. eastern red cedar as a bioenergy crop to improve soil o1. eastern red cedar as a bioenergy crop to improve soil quality in the great plains, usaquality in the great plains, usa thomas j. sauer1, ken wacha2, eric c. brevik3*, and diomides zamora4 1 usda-ars, national laboratory for agriculture and the environment, ames, ia, usa; 2 usda-ars, national soil erosion research laboratory, west lafayette, in, usa; 3 college of agricultural, life, and physical sciences, southern illinois university, carbondale, il, usa; 4 usda, national institute of food and agriculture, kansas city, mo, usa. *corresponding and presenting author: eric.brevik@siu.edu. abstractabstract eastern red cedar (erc, juniperus virginiana l.) is commonly found in tree windbreaks in the semi-arid great plains of the central usa. erc has strong potential as a bioenergy feedstock due to its physical and chemical characteristics. this study sought to assess the sustainability of erc bioenergy plantings by evaluating carbon sequestration and selected soil quality parameters in existing erc stands across the northern great plains. nine sites were selected in five states with mean annual precipitation (map) that ranged from 446 to 999 mm and mean annual temperature (mat) that ranged from 4.4 to 10.0 °c. infiltration was measured using the twin ring technique at nine locations in each tree planting and adjacent agricultural field (crop, pasture, or hay) at each site. after infiltration measurements were made, a 4.8 cm-diameter soil core was collected to a depth of 30 cm inside one infiltration ring to collect samples to determine bulk density, texture, ph, aggregate stability, soil organic carbon (soc), and nitrogen (n). penetration resistance was measured to 30 cm depth in 2.5 cm increments in the other infiltration ring. infiltration rate and depth to 2 mpa penetration resistance averaged 55% and 20.3% greater, respectively, under tree cover. estimated tree aboveground biomass c and soc were both strongly correlated with map. tree aboveground c stocks were estimated to increase 2.05 mg ha-1 yr-1 on average. soc stocks under tree cover were 16.8% greater than in the adjacent agricultural land use. erc planting for bioenergy feedstock production is likely to increase soil carbon sequestration and improve soil physical and chemical quality, especially for degraded soils or soils with marginal crop production potential. these same erc plantings may also provide enhanced income potential from areas that currently generate little to no income. key-keywords: words: windbreak; agroforestry; bioenergy feedstock; carbon sequestration; marginal soils. o2. soil-human health-nexus: with focus on dimensions of o2. soil-human health-nexus: with focus on dimensions of soil-water-plant-microbes and humansoil-water-plant-microbes and human hassan el-ramady1,2§, józsef prokisch2, tamer elsakhawy3, alaa el-dein omara3, megahed amer4, and eric c. brevik5* 1 soil and water dept., faculty of agriculture, kafrelsheikh university, 33516 kafr el-sheikh, egypt; 2 institute of animal science, biotechnology and nature conservation, faculty of agricultural and food sciences and environmental management, university of debrecen, 138 böszörményi street, 4032 debrecen, hungary; 3 agriculture microbiology department, soil, water and environment research institute (sweri), sakha agricultural research station, agriculture research center, 33717 kafr el-sheikh, egypt; 4 soil improvement and conservation dept., water and environment research institute, agricultural research center, giza, egypt; 5 college of agricultural, life, and physical sciences, southern illinois university, carbondale, il 62901 usa. *corresponding author: eric.brevik@siu.edu. §presenting author: hassan el-ramady; hassan.elramady@agr.kfs.edu.eg. abstractabstract there are a number of strong relationships between soil and human health, both direct and indirect. the soil system is essential for life due to its influence in the provision of clean water, healthy edible foods, feed, fiber, shelter, and fuel. soil also serves as a repository for components of human culture. the soil-human health nexus should be studied with focus on some crucial dimensions including soil and its components water, plants, and microbes. water is an essential component of soil, and transports many elements that may be in nutrient form for or present at toxic levels for plant production. soil water, or the soil solution, represents the theater of interactions for many soil processes. many nexuses are created among the soil components including different aspects of the soil-water-plant-human nexus (swph). the components of this nexus have many combinations such as soil-human, soil-plant, soil-water, soil-water-human, soil-plant-human, etc. nexuses. the ultimate goal of this study is to add to knowledge of the swph nexus in a way that incorporates art and culture into our scientific understanding of some of today’s major global challenges. although the soil and its relationship to human health was originally noted several centuries ago, each of the water, plant, and microbe components in soil have many direct and/or indirect impacts on human health. several open questions concerning each component still need to be answered and will be discussed. keywords: keywords: soil system, microflora, microfauna, soil-water-plant-human nexus. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 789 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) o3. status of macronutrients in selected valencia orange or-o3. status of macronutrients in selected valencia orange orchards in the gharb region of moroccochards in the gharb region of morocco abail zhor1*, rania brital1,2, mohamed ibriz2, rachid aboutayeb1, and hamid benyahia1 1 national institute of agricultural research, avenue ennasr, bp 415 rabat principale, 10090 rabat, morocco; 2 faculty of science, ibn tofail university, kenitra, morocco. *corresponding and presenting author: zhor.abail@inra.ma. abstractabstract valencia orange is one of the principal varieties grown in morocco. in this study, we conducted a field survey to assess the status of essential macronutrients (n, p, k, ca and mg) in valencia orange orchards located in the gharb region, the second largest citrus growing area in morocco. soil (0-20 cm) and leaf samples were collected from 20 valencia late orange orchards. soils of the investigated orchards were medium to heavy in texture. their content in organic matter varied from 0.6 to 3.1 %, ph ranged from 7.8 to 8.7, ec was between 0.2 and 0.7 ds m-1, and total caco3 content varied widely from 0.2 to 25%. their content of total nitrogen was in the typical range (0.5 to 1.6 g kg-1) reported for citrus orchards in morocco. these soils had low to optimum levels in available p (7.5-59.1 mg kg-1) and optimum to high levels in available k (143-540 mg kg-1). available ca levels were extremely high in all soils (5.3-9.6 g kg-1), while mg levels were very low (4.640.6 mg kg-1). leaf analysis, in accordance with soil analysis, showed that all orchards were deficient in mg and most of them had suboptimum levels of n (6.8-21.1 g kg-1), optimum to high levels of p (0.9 − 2.2 g kg-1), k (2.8 − 9.0 g kg-1) and ca (46 − 96.6 g kg-1). generally, the result of this study showed an unsatisfactory nutrient management of the studied valencia orchards, particularly for mg, n and p whose fertilization should be carefully implemented to support optimum yields. keywords:keywords: valencia orange, citrus, leaf analysis, soil analysis, macronutrient, gharb. o4. estimating actual abundance of european sousliks: uav o4. estimating actual abundance of european sousliks: uav imagery, pixel based imaging, and random forest classifica-imagery, pixel based imaging, and random forest classification for counting surface burrow openings and gpr for iden-tion for counting surface burrow openings and gpr for identifying sub random subsurface burrowstifying sub random subsurface burrows csongor i. gedeon1*, mátyás árvai1, gábor szatmári1,3, eric c. brevik2, tünde takáts1, zsófia a. kovács1, and jános mészáros1 1 department of soil mapping and environmental informatics, institute for soil sciences, herman ottóút 15., budapest-1022, hungary; 2 college of agricultural, life, and physical sciences, southern illinois university, carbondale, il, 62901-4403, usa; 3 department of physical geography and geoinformatics, faculty of science and technology, university of debrecen, egyetem tér 1, debrecen-4032, hungary. *corresponding and presenting author: gedeon.csongor@atk.hu. abstractabstract soil quality is a compound characteristic that cannot be measured directly. if functions provided by soils are diminished, then quality is decreased. burrowing mammals frequently play a crucial role in the functioning of grassland ecosystems, maintaining their diversity, functions, services, and high quality. however, the non-destructive estimation of burrowers’ population size, spatial and temporal population dynamics is a challenge for conservation. determining the number of burrow openings and mounds would be a good proxy for estimating actual population sizes when one individual occupies one burrow system and if the ratio of openings per burrow system was known. first, we present an imagery-based method to identify and count animals’ burrows semi-automatically by combining remotely recorded rgb images, pixel-based imagery and random forest classification. then we show the sketch of a pilot study where we aimed at identifying and locating artificial burrows with gpr on a natural grassland similar to a ground squirrel or mole-rat habitat. field images were collected for four colonies, then combined and processed by histogram matching and spectral band normalization to improve the spectral distinction between the categories burrow, soil, tree, and grass. accuracy index of classification for burrow kappa (κ) was 95 (precision) and 90% (sensitivity) respectively. a 10-time bootstrapping of the final model resulted in coefficients of variation (cv%) of burrow κ for sensitivity and precision lower than 5%, moreover cv% values were not significantly different between those scores. the consistency of classification and balanced precision and sensitivity confirmed the applicability of this approach. our approach provides an accurate, user-friendly and relatively simple approach to count the number of burrow openings, estimate population abundance and delineate the areas of occupancy non-invasively. for the gpr survey we used a mala system with 160 and 750mhz shielded antennas to identify and locate artificial burrows at different depths from 10 to 165cm. we sketch out the experiment and a few results of the analyses of the radar profiles with special reference to depth limits of gpr surveys and visual representations of air-filled burrows on radar profiles. keywords:keywords: random-forest; ground squirrel; image processing; ground penetrating radar. session xiii: plant sciences iiisession xiii: plant sciences iii co-chair: mohammed amine seghrini, university ibn zohr, co-chair: mohammed amine seghrini, university ibn zohr, moroccomorocco co-chair: nicole gauthier, university of kentucky, usaco-chair: nicole gauthier, university of kentucky, usa o1. peripheral membrane proteins modulate stress tolerance o1. peripheral membrane proteins modulate stress tolerance by safeguarding cellulose synthasesby safeguarding cellulose synthases christopher kesten1,2, álvaro garcía-moreno3, vítor amorimsilva3, alexandra menna2, araceli g. castillo4, francisco percio3, laia armengot5, noemi ruiz-lopez3, yvon jaillais5, clara sánchez-rodríguez2, and miguel a botella3* a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 790 1 department of biology, eth zurich, 8092, zurich, switzerland; 2 department for plant and environmental sciences, university of copenhagen, 1871, frederiksberg c, denmark; 3 instituto de hortofruticultura subtropical mediterránea, universidad de málaga-consejo superior de investigaciones científicas (ihsm-uma-csic), dept. biología molecular bioquímica, campus de teatinos, málaga e-29071, spain; 4 instituto de hortofruticultura subtropical y mediterránea, universidad de málaga-consejo superior de investigaciones científicas (ihsmuma-csic), dept. biología celular, genética y fisiología, campus de teatinos, málaga e29071, spain; 5 laboratoire reproduction et développement des plantes, université de lyon, ens de lyon, cnrs, inrae, f-69342, lyon, france. *corresponding and presenting author: mabotella@ uma.es. abstractabstract cellulose, the main load-bearing component of plant cell walls, constitutes a quarter of all organic material on earth. this remarkable amount of cellulose is synthesized by cellulose synthase complexes (csc). csc propels itself forward through the plasma membrane with its own catalytic activity using cortical microtubules as steering devices. four components of the csc machinery have been identified, cesas, korrigan, csi1, and ccs, the last members of the complex identified in 2015. we have identified the tetratricopeptide thioredoxin-like (ttl) proteins as new members of the cellulose synthase complex (csc) and describe their unique and hitherto unknown dynamic association with the csc under cellulose-deficient conditions. ttls are essential for maintaining cellulose synthesis under high salinity conditions, establishing a stress-resilient cortical microtubule array, and stabilizing cscs at the plasma membrane. to fulfill these functions, ttls interact with cellulose synthase1 (cesa1) and engage with cortical microtubules to promote their polymerization. we propose that ttls function as bridges connecting stress perception with dynamic regulation of cellulose biosynthesis at the plasma membrane. keywords: keywords: abiotic stress tolerance, cellulose biosynthesis, cell wall signaling. o2. breeding programs in argane tree: microsporogenesis o2. breeding programs in argane tree: microsporogenesis study and cytogenetic approachstudy and cytogenetic approach ait aabd naima*, abdelghani tahiri, redouan qessaoui, abdelaziz mimouni, and rachid bouharroud national institute of agronomic research, regional center of agronomic research of agadir, p.o. box 124, avenue des far, inezgane, agadir 86350, morocco. *corresponding and presenting author: naima.aitaabd@inra.ma. abstractabstract the cross-pollination is often insufficient to obtain a desirable fruit yield in the absence of compatibility between the orchard’s argane trees. proper pollination design is therefore essential to ensure a supply of compatible pollen. in this study, improvement programs in the argane tree can be facilitated by integrating knowledge of the cytogenetic and microsporogenesis studies. so, during cytological analysis of argane tree meiosis, unreduced pollen has been observed, in parallel with this study, a study on parent chromosomal numbers detects a chromosomal diversity. evaluations of fertility and sterility levels in mature pollen were done and strongly depend on both of these tools used. good cross compatibility has been correlated with aberrant microsporogenesis and variation in number of chromosomes. the cross-compatibility rate depends on cross associations and it varies from 0 to 84 %. for argane tree, it was observed, for the first time, that compatible pollinizers have pollen effects on the weight, size and volume of the fruit and not on the ripening period of the fruits. keywords:keywords: argane tree, breeding program, pollinizers, self-incompatible, chromosomal diversity, compatibility, unreduced pollen. o3. the efficacy of pheromone traps in controlling tomato o3. the efficacy of pheromone traps in controlling tomato leafminer, leafminer, tuta absolutatuta absoluta sabbahi rachid1* and khalil azzaoui2 1 higher school of technology, quartier 25 mars, p.o. box 3007, laayoune, morocco; and laboratory of plant biotechnology biotecv, faculty of science, university of ibn zohr, p.o. box 8106, hay dakhla, agadir, morocco; 2 laboratory of applied chemistry and environment lcae, faculty of science, first mohammed university, po box. 717, 60 000, oujda, morocco. *corresponding and presenting author: r.sabbahi@uiz. ac.ma. abstractabstract the development of new operational techniques for monitoring adult populations of tomato leafminer, tuta absoluta (meyrick) (lepidoptera: gelechiidae) is regarded as a pressing need for surveillance and control of this major pest of tomato crops. trap design is an important component of a monitoring or mass trapping system, along with the semiochemical attractant. here we present the results of an experiment carried out in tomato farms to assess the efficacy of a newly designed pheromone trap (i.e., sticky trap) in capturing t. absoluta adults and compare it to traditional pheromone traps. analysis of variance demonstrated significant differences in the number of captures between the three traps. the mean (± se) number of captures/trap/week in sticky traps was 70.44 ± 4.57, significantly higher than those captured in delta traps (55.94 ± 4.77) and water pan traps (18.63 ± 1.49). the results showed that the newly designed pheromone trap is a promising solution to lessen t. absoluta populations and thereby protecting tomato crops from infestations. keywords:keywords: mass trapping, monitoring, sex pheromone, tomato, trap type, tuta absoluta. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 791 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) o4. assessment of east moroccan almond germplasm and o4. assessment of east moroccan almond germplasm and selection of promising accessions based on screw-pressed selection of promising accessions based on screw-pressed seed oil quality and its potential for micropropagationseed oil quality and its potential for micropropagation kodad souhayla*, reda melhaoui, hana serghini-caid, ahmed el amrani, malika abid, and aatika mihamou laboratory of agricultural production improvement, biotechnology and environment (lapabe), faculty of sciences, mohammed premier university, oujda, morocco. *corresponding author: souhaila.kodad@gmail.com. abstractabstract the almond (prunus dulcis mill.) is one of the most important nut crops worldwide and it is largely planted under different environmental conditions. the present research aimed to study the traditional almond trees plantations based on seedlings in three sites in eastern morocco (rislane, bsara, and ainsfa), which represents an important gene pool, both for breeding programs and selection of superior genotypes adapted to moroccan conditions. unfortunately, those trees are generally abandoned or replaced, leading to genetic erosion and regression of indigenous populations of this species, while they have an ability to adapt to the most varied pedoclimatic conditions. thus, finding and characterizing old genotypes for the conservation of biodiversity is of interest to the diversity of crops in these almond groves. the objective of this study is to assess the quality of screw-pressed seed oil based on tocopherol content (tc), acid value (av), peroxide value (pv), and oxidative stability (os). 20 genotypes were selected according to the highest shelling rates of their kernels from more than 150 “beldi” almond genotype grown in eastern morocco, with a shelling percentage ranging from 24 to 32%. the highest value of the major homolog α-tocopherol was equal to 555,88 mg/kg using a screw press extraction at a temperature of 100 c°. genotypes singled out as the most promising for breeding and commercial growing were multiplied by in vitro tissue culture techniques. the results showed multiple shoot initiation using thidiazuron (tdz) at the concentration of 1mg/l, singled out as the most effective for in vitro proliferation of the “beldi” ecotype. while the most significant rooting rate (60.41 % ± 0.81) was achieved with 1 mg / l of indole-3-butyric acid (iba) incorporated into ms½ medium. plantlets were successfully adapted to the in vivo conditions, and they were grown vigorously in the greenhouse without any morphological abnormalities. keywords: keywords: almond, biochemical characterization, oil quality, tocopherol content, in vitro tissue culture. session xiva: studies in the usasession xiva: studies in the usa co-chair: moulay abdelmajid kassem, fayetteville state uni-co-chair: moulay abdelmajid kassem, fayetteville state university, usaversity, usa co-chair: khalid meksem, southern illinois university, usaco-chair: khalid meksem, southern illinois university, usa o1. attending the graduate school at southern illinois uni-o1. attending the graduate school at southern illinois universityversity dounya knizia* school of agricultural sciences, southern illinois university, carbondale, il, usa. *corresponding and presenting author: dounya.knizia@siu.edu. abstractabstract to attend graduate school at southern illinois university, the presentation will describe the following steps that a student will follow to facilitate the application process: (1) take your toefl test and score of at least 80%, (2) translate your diplomas and transcripts into the english system by doing a course-by-course evaluation, (3) write your research and personal statements and get them ready for submission, (4) contact a professor (supervisor) who is working in the field of your interest, (5) ask for at least three letters of recommendations (lors) to be sent by your references upon request, (6) apply online at: https://siu.edu/apply-now/ (it takes about one to two months to get an answer about your application), (7) once your application is accepted, apply for the visa on https://www.ustraveldocs.com/ma/ma-niv-visaapply.asp (complete ds-160 form, pay the visa fee), (8) pay the i-901 sevis fee ($350), (9) book your air ticket, travel earlier than the start of the semester, and attend the grad school orientation, (10) start looking for lodging before flying to the us, (11) bring with you enough funds to pay for your living expenses for at least 2 months ($3,000) until you get your first paycheck, and (11) keep your supervisor (major professor) always informed by email about each step. o2. undergraduate and graduate studies in the usao2. undergraduate and graduate studies in the usa moulay abdelmajid kassem* plant genomics and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc 28301, usa. *corresponding and presenting author: mkassem@uncfsu.edu. abstractabstract usa has many of the world’s most prestigious colleges, universities, institutes, and institutions of higher education. these institutions of higher education are well funded by us government and educate millions of students from us and many parts of the world. in addition, usa has several national labs funded and run by the dept. of defense (dod), national a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 792 institute of health, and several other agencies. therefore, thousands of international students come to study in the us and earn their degrees. here, we summarize the process of attending us institutions of higher education step-by-step at the undergraduate and graduate levels. opportunities and challenges of living in the us as an international student will also be discussed. keywords: keywords: undergraduate, graduate, higher education, scholarships, usa. session xv: student oral presentations isession xv: student oral presentations i co-chair: naoufal lakhssassi, southern illinois university, co-chair: naoufal lakhssassi, southern illinois university, usausa co-chair: fatima zahra ouasso, university ibn zohr, moroccoco-chair: fatima zahra ouasso, university ibn zohr, morocco o1. biocontrol potential of phosphate-solubilizing rhizobac-o1. biocontrol potential of phosphate-solubilizing rhizobacteria isolated from astragalus armatus rhizosphere against teria isolated from astragalus armatus rhizosphere against fusarium oxysporumfusarium oxysporum mounia bakkali bouarrakia*, anas elyemlahi, ouiam elgaliou, mounir hassani zerrouk, amin laglaoui, mohammed bakkali, and abdelhay arakrak biotechnology and biomolecular engineering research team, fstt, abdelmalek essadi university, tetouan, morocco. *corresponding and presenting author: mounia.bkkl@gmail.com. abstractabstract plant growth promoting rhizobacteria (pgpr) are defined as root-colonizing bacteria that positively affect plant growth through direct or indirect mechanisms. with the aim to select beneficial bacteria exhibiting several plant growths promoting (pgp) traits, 102 bacteria were isolated from rhizospheric soil of astragalus armatus growing wildly in northeastern region of morocco, of which 40 bacteria were tri-calcium phosphate solubilizers (psb). based on solubilization halos diameter, ten bacterial strains were selected to explore more activities. indeed, all tested bacteria were siderophores producers except r29 strain. only two bacteria, r23 and r32, were positive for indole acetic acid synthesis. for ammonia, all isolates were able to produce it, while 50% of isolates secrete hydrogen cyanide. additionally, bacteria ability to secrete lytic enzymes was evaluated. all isolates were amylase and cellulase producers, only two bacteria, r29 and r101 secreted protease, while for urease activity, only r44 strain was negative. all test strains were assessed by tri-calcium phosphate solubilizing quantitative assay in pvk liquid medium. the obtained concentrations were between 212.93 mg l-1 and 265.28 mg l-1. this solubilization was accompanied by a ph decrease of the medium from 7 to 3.86. fusarium oxysporum was selected to evaluate the antifungal activity of the selected strains due to it high pathogenicity to wide variety of plants. as a result, they could inhibit the fungal growth with significant different percentages. the evaluated strains in this study could be used as biocontrol agents to improve agricultural yield and productivity. keywords: keywords: pgpr, psb, fusarium oxysporum, astragamus armatus. o2. effect of provenance on yield and secondary metabolite o2. effect of provenance on yield and secondary metabolite content of extracts from content of extracts from juniperus thuriferajuniperus thurifera in morocco in morocco khalid benhssaine1*, jamal aabdousse2, nora salim2, ilias oussif2, mohamed elhabty2, m’hamed ramchoun3, younes abbas1, and hicham berrougui1 1 polyvalent team in research and development, department of biology and geology, polydisciplinary faculty of beni mellal, sulatn moulay slimane university beni mellal, bp 592, 23000 beni mellal, morocco; 2 environmental, ecological and agro-industrial engineering laboratory, department of life sciences, faculty of sciences and techniques, sulatn moulay slimane university beni mellal, bp 523, 23000 beni mellal, morocco; 3 biotechnology and sustainable development of natural resources team, department of biology and geology, polydisciplinary faculty of beni mellal, sulatn moulay slimane university beni mellal, bp592, 23000 beni mellal, morocco. *corresponding and presenting author: benkhalid78@gmail. com. abstractabstract juniperus thurifera is a dioecious tree belonging to the cupressaceae family. it has important ecological, socio-economic, floristic and cultural interests. it is endemic to the western mediterranean basin to which belongs morocco. it is among the moroccan species less valued and less preserved. its stands do not cease that has degraded vigorously. it is distributed in areas with semi-arid and sub-humid bioclimates with cold to very cold variants in the middle atlas, the eastern anti-atlas and the eastern and central high atlas and on altitudes ranging from 1800 m to 3300 m and on all types of substrates. in traditional medicine, the essential oils of juniper have been used as abortifacient and regulator of menstruation, while the wood tar is used as veterinary remedy. the objective of the present work is to explore the effect of provenance on the yield, phenolic compounds, flavonoids and tannins contents and antioxidant activity of the extracts of the leaves of this species. the results obtained show a significant variability between the samples coming from three different biogeographic zones: the eastern, central and western high atlas. this allowed us to highlight the effect of the origin on the production of secondary metabolics. in fact, we found that the population of eastern high atlas has a good yield in methanolic extracts (9.41% ±0.59) and that the population of el haouz is the richest in polyphenols (191.30±4.27), as well as it has endowed an important reducing power (ic50 = 0.98 ± 0.05 mg/ml). these results can be exploited as bioindicators in any program of valorization and conservation of moroccan juniper thurifer. keywords: juniperus thurifera, provenance, extract, yield, compound, morocco. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 793 o4. sustainable soybean [o4. sustainable soybean [glycine max glycine max (l.) merr.] production (l.) merr.] production through the use of rhizobium inoculation and different through the use of rhizobium inoculation and different sources of phosphate fertilizer in ghanasources of phosphate fertilizer in ghana alfred balenor buernor1*, muhammad rabiu kabiru1, noura bechtaoui1, edwin k. akley2, michael asante2, and martin jemo1 1 agrobioscience program, university mohammed vi polytechnic, benguerir, morocco; 2 council for scientific and industrial research-savanna agricultural research institute (sari), tamale, p. o. box tl 52, ghana. *corresponding and presenting author: alfred.balenor@um6p.ma. abstractabstract the relevance of biological nitrogen nutrition of soybean is recognized worldwide but inoculation with rhizobium (rhz) shows variable results, and the benefit needs to be validated under current crop production practices. also, we wanted to elucidate the effect of phosphate fertilizer source in combination with inoculants. a total of 9 field experiments in three different soil types namely, planosols, plinthosols, and acrisols were established in northern ghana. the experimental design was a split-plot arranged in a randomized complete block with four replications. the main plot was assigned to different phosphorus sources; control, triple superphosphate (tsp), and 50% tsp + 50% rock phosphate (rp), and the subplots to nitrogen sources; control, rhizobium phaseoli dsm 30137 (s1), bradyrhizobium japonicum dsm 30140 (s2), bradyrhizobium japonicum, usda 110 (sarifix) and urea. tsp + rhizobium application gave the best yield across all three soil types. the average yield due to inoculation only was 1.4t ha-1 while the average for the control was 0.6 t ha-1 representing an average relative yield increase of 138.6%. treatments with rhizobium inoculant produced heavier nodules than their respective controls. sarifix together with tsp gave the heaviest dry biomass of about 47% relative increase compared to the control. these results demonstrate the potential of improving soybean nodulation, growth, and seed yield profitability using rhizobia and p fertilizer in ghana. the strain s2 showed good prospects under all the soil types, further studies will be needed to validate our findings in other to incorporate this strain into the sustainable soybean farming systems in ghana. keywords: keywords: soybean, phosphate fertilizer, rhizobium, yield, sustainability. o5. a moroccan comparative study of urban landscapes o5. a moroccan comparative study of urban landscapes and farming lands in bird species: evidence from morocco and farming lands in bird species: evidence from morocco (northwest africa)(northwest africa) ikram douini1*, mohamed mounir1, el mostafa benka1, mohamed dakki2, and soumaya hammada1 1 agro-industrial and medical biotechnology laboratory, faculty of sciences and technology, sultan moulay slimane university of beni mellal, morocco; 2 geo-biodiversity and a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) o3. actinobacteria-based biofertilizer improve cereal plant o3. actinobacteria-based biofertilizer improve cereal plant growth and nutrient acquisition under rock phosphate/po-growth and nutrient acquisition under rock phosphate/potassium fertilizationtassium fertilization kenza boubekri1,2§, abdoulaye soumare1, ilham mardad1, karim lyamlouli1, yedir ouhdouch1,2, mohamed hafidi1,2*, and lamfeddal kouisni1,3 1 agrobiosciences program, mohammed vi polytechnic university (um6p), benguerir, 43150, morocco; 2 laboratory of microbial biotechnologies agrosciences and environment (biomage), cnrst labelled research unit n°4, faculty of sciences semlalia, cadi ayyad university, po box 2390, marrakech, morocco; 3 african sustainable agriculture research institute (asari), mohammed vi polytechnic university (um6p), laayoune, morocco. *corresponding author: hafidi@uca. ac.ma. §presenting author: kenza.boubekri@um6p.ma. abstractabstract soil fertility and plant nutrition require an adequate management of essential macronutrients such as potassium (k) and phosphorus (p), which are mandatory for plant development, crop yield and quality. although abundant in soils, the availability of p and k is too low to meet plant’s need as they occur mostly in insoluble forms. therefore, exploiting soil p and k mineral reserve by using beneficial microorganisms as biofertilizer can improve their availability and, consequently, crops yield in a sustainable way. in this context, greenhouse experiments were conducted to evaluate the capacity of 9 actinobacteria strains (p13-p14-p15-p161-p17-p18-bc3-bc10 and bc11) belonging to streptomyces and nocardiopsis to solubilize p and k rocks. results showed that rock p and k combined with these selected actinobacteria strains consistently increased shoot/root dry weights, yields, and enhanced soil mineral availability, suggesting their potential use as biofertilizer. this positive relationship emphasizes the potential importance of managing p/k solubilizing actinobacteria for improving both p and k acquisition by cereal crops. the most performing strains were p18, bc3, bc10, and bc11 since they have a broad spectrum of rp and rk solubilization as well as the capacity to produce a plethora of beneficial substances such as iaa, hcn, ammonia, and siderophores. moreover, inoculation with these strains improved soil enzymatic activities such as acid and alkaline phosphatase in plant rhizosphere. moreover, the mechanism of root colonization was also studied. in fact, in-vitro tests and scanning electron microscopy analysis revealed that the elite actinobacteria strains not only colonized effectively the roots of cereals plants by also formed a strong biofilm along epidermal tissues which highlight the positive interaction between plants and microorganism. these findings showed that the selected strains are promising candidates for the implementation of an efficient biofertilization strategy and a sustainable solution to valorize low reactive rp and rk in agriculture. keywords:keywords: actinobacteria, phosphate & potassium rock, biofertilizers, wheat, maize, greenhouse. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 794 natural patrimony laboratory, scientific institute, university of mohammed v, rabat 10106, morocco. *corresponding and presenting author: ikram.douini@gmail.com. abstractabstract urban expansion leads to modifications of habitat features, organization, and resources. bird assemblages are known to respond by escaping destructive changes and adapting to sustainable ones. in this study, we investigated for the first time the avian diversity and its variation following the rural-urban gradient in beni mellal (morocco) from 2018 to 2021. we used the line-transect method and multivariate analysis to demonstrate the selection of breeding habitats. our result revealed a total of 84 species divided into resident breeders (64.28%), passage migrants (17.85%), breeding migrants (26.19%), winter visitors (32.18%) and accidental visitors (1.19%). two globally vulnerable species counting the european turtle dove streptopelia turtur and the european goldfinch carduelis carduelis were recorded. breeding populations were concentrated in green spaces (9 species) located in the urban zone, compared with farmlands (7 species), peri-urban (3 species), and rural areas (3 species). therefore, these results reverse the hypothesis that rural and farming lands are more species-rich because of a higher population size. this is due to the abundance of breeding and foraging resources in urban green spaces compared to arid lands surrounding cities in this north african area. furthermore, our study provides a new opportunity for comparative studies of avian diversity in morocco and northwest africa. keywords: keywords: avian diversity, richness, rural-urban gradient, beni mellal. o6. ethnobotanical survey on traditional uses of saffron by-o6. ethnobotanical survey on traditional uses of saffron byproducts by local people from taliouine center and regions products by local people from taliouine center and regions in moroccoin morocco zineb khadfy1*, s.m jadouali1,3, hajar atifi1, rachid mamouni1, youssef karra2, and abdelghani tahiri2 1 team of materials catalysis and valorization of natural resources, faculty of sciences, ibn zohr university, agadir, morocco; 2 research unit natural resources and terroirs products ur rn&pdt-inra-crra, agadir, morocco; 3 laboratory of biotechnology, bioresources and bioinformatics, est khenifra, université sultan moulay sliman, khénifra, morocco. *corresponding authors: khadfyzineb@gmail.com. abstractabstract crocus sativus (saffron) is common in morocco, especially in the taliouine regions due to their multiple climatic and altitude conditions. the aim of our study is to assess the state of the traditional uses of saffron by-products by local populations in taliouine and regions. an ethnobotanical survey was conducted with 42 local people and in six communities (taliouine center, tassousfi, sidi hssaine, taouyalte, askaouen). the information was obtained from traditional farmers and people in different villages through interviews using structured questionnaires. this study was performed in august 2022. as a result, 64% of the population of taliouine and regions do not valorize saffron by-products, while 36% of respondents report that these by-products are being exploited, in which 16% who work in cooperatives valorizing local products including saffron, presented use of these biological residues in various areas. cosmetic uses showed the highest percentage (38%), followed by food uses (26%) and then carpet and tablecloth dyeing (26%). the petals (50%) and stamens (42%) were the main used parts. saffron byproducts were mostly used in powder form (69%), followed by maceration form (19%). this survey is the first survey on the traditional uses of saffron by-products by the local population of taliouine and its regions in morocco. among the many respondents interviewed, more than 66% of the local population is uneducated, resulting in a low proportion of saffron by-products uses in the six communities. keywords: keywords: ethnobotanical survey, saffron by-products, farmers, traditional uses, taliouine. concurent session xvi: student oral presentations iiconcurent session xvi: student oral presentations ii co-chair: karen midden, southern illinois university, usaco-chair: karen midden, southern illinois university, usa co-chair: mohamed addi, university mohamed i, moroccoco-chair: mohamed addi, university mohamed i, morocco o1. morphological and physiological analysis of o1. morphological and physiological analysis of botrytis ci-botrytis cinerea nerea field isolates from vineyardfield isolates from vineyard faical aoujil1,2§, majida hafidi1, hiba elyahyaoui1,2 , abdellatif benbouazza2, and khaoula habbadi2* 1 laboratory of plant biotechnology and bio-resources valorization, faculty of sciences, moulay ismail university, meknes 50000, morocco; 2 laboratory of phyto-bacteriology and biocontrol, plant protection unit-national institute of agronomic research inra, meknes 50000, morocco. *corresponding author: khaoula.habbadi@inra.ma. §presenting author: faical. aoujil@edu.umi.ac.ma. abstractabstract botrytis cinerea, causing gray mold rot, is the most important necrotrophic fungal pathogen of grapes. it is responsible of significant economic damage in vineyards worldwide. despite the considerable losses caused by this disease, studies related to evaluate the diversity of b. cinerea populations on grapes in morocco are restricted to very few published reports. our study has been conducted to analyze the phenotypic diversity, the aggressiveness and the fungicide resistance of botrytis cinerea isolated from grapevine in different regions of morocco. the isolates were collected from 7 vineyards in two regions of morocco. seventy-five isolates were obtained and purified by monospore isolation. all the isolates were identified as b. cinerea based on morphological features including mycelia and conidiophores. botrytis cinerea strains collected showed a considerable morphological variability. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 795 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 72% of the population collected, developed a sclerotial morphology, while 28% belonged to mycelial type. the aggressiveness of isolates was tested on vitis vinifera leaves of two grapevine cultivars. all tested isolates were virulent against grape leaves, with different levels of aggressiveness. we use the natural resistance of botrytis pseudocinerea against fenhexamid as a phenotypic marker. six isolates from the collection exhibited a high level of resistance to this fungicide, suggesting that it was botrytis pseudocinerea. keywords: keywords: botrytis cinerea, botrytis pseudocinerea, variability, aggressiveness. o2. preliminary results of chilling and heat requirements o2. preliminary results of chilling and heat requirements estimation of several olive (estimation of several olive (olea europaeaolea europaea l.) cultivars: a stal.) cultivars: a statistical and experimental approachtistical and experimental approach omar abou-saaid1,2,3*, hayat zaher1, sara oulbi1, magalie delalande3, ahmed el bakkali6, adnane el yaacoubi5, cherkaoui el modafar2, and bouchaib khadari3,4 1 inra, ur amélioration des plantes, marrakech, morocco; 2 université cadi ayyad, centre d’agrobiotechnologie et bioingénierie , unité de recherche labellisée cnrst (url-cnrst 05), marrakech, morocco; 3 agap, université de montpellier, cirad, inra, montpellier supagro, montpellier, france; 4 conservatoire botanique national méditerranéen de porquerolles (cbnmed), umr agap, montpellier, france; 5 university sultan moulay slimane, école supérieur de technologie khenifra, khenifra, morocco; 6 inra, ur amélioration des plantes et conservation des ressources phytogénétiques, meknès, morocco. *corresponding and presenting author: omar.abousaaid@gamil.com. abstractabstract in the global warming context, knowledge of the dormancychilling requirement of olive tree has become a key consideration in orchard planning and management, as well as for breeding programs aiming to select new adapted cultivars. identifying the period for chilling by pinpointing the exact start and end dates of olive cultivars is still difficult and ultimately based on the researchers’ subjective judgment. how could we define chilling and heat periods for olive cultivars? here, we examine this question by combining statistical and experimental approaches. we applied partial least squares (pls) regression using blooming data collected on 16 cultivars in the worldwide collection of marrakech during six years. to determine the time of dormancy breaking for these cultivars, we applied the tabuenca test under controlled growth conditions. dates of dormancy breaking delineated by tabuenca tests were consistent with data derived from pls outputs for 7 cultivars, while the two approaches show different dates of budburst for the other cultivars. our investigations highlight the importance to combine statistical and experimental approaches to accurately define chilling and heat phases. key-keywords: words: olea europaea l., global warming, phenology, pls regression, chilling and heat requirements, tabuenca test. o3. mitigation of drought and phosphorus deficiency stress-o3. mitigation of drought and phosphorus deficiency stresses in intercropped wheat and faba bean plants through rhi-es in intercropped wheat and faba bean plants through rhizobium-pgpr-based consortia inoculationzobium-pgpr-based consortia inoculation bouchra benmrid1§, hicham oukfi1, said cheto1,2, ammar ibn yassar1, meryem haddine1, joerg geistlinger3, youssef zeroual4, lamfeddal kouisni1, cherki ghoulam1,2*, and adnane bargaz1 1 mohammed vi polytechnic university, agrobiosciences, plant-microbe interaction, benguerir, morocco; 2 center of agrobiotechnology & bioengineering, research unit labeled cnrst, fst, cadi ayyad university, marrakech, morocco, 3 anhalt university of applied sciences, bernbourg, germany; 4 ocp-innovation, jorf lasfar el jadida, morocco. *corresponding author: ghoulam.cherki.@um6p.ma. §presenting author: bouchra.benmrid@um6p.ma. abstractabstract drought and phosphorus (p) deficiency are among the major challenges facing current agriculture. phosphorus availability depends on soil water status, both of which have a negative impact on plant productivity. farming practices like intercropping showed positive facilitative interactions which may contribute to enhancing plants nutritional status, like p, under water deficit condition. thus, we hypothesized that the application of multibacterial inoculants, presenting some main growth promoting traits, constitutes a promising strategy to improve the performance of associated crops in intercropping system under stress combining drought and p deficiency. thus, we conducted a greenhouse experiment to evaluate the performance of three rhizobia-containing consortia and single rhizobia inoculation on the growth of intercropped and sole-cropped wheat (triticum durum) and faba bean (vicia faba) plants under p-deficient well-watered (rock phosphate (rp), 80 % field capacity (fc)), or drought pdeficient (rp, 40 % fc) treatments versus a positive control (monoammonium di-phosphate (map), 80% fc). the results showed that consortium 4 (c4) is more likely adapted to well-watered p-deficient conditions. indeed, shoot dry weight (sdw) of intercropped wheat and faba bean reached up to 4.7 g. 4 plants-1 and 14.35 g. plants-1, respectively. inoculated faba bean roots were well-nodulated under both well-watered (p deficient) and combined stress conditions compared to non-inoculated controls, with c4 inducing the highest nodule dry weight (ndw) under well-watered and drought stressed conditions, for both faba bean sole crop and intercrop respectively. additionally, a significant, variable response of the rhizobia-containing consortia was noticed for the morphophysiological root traits, shoot inorganic p content (pi), and acid phosphatase (apase activity) compared to non-inoculated rp fertilized controls. additionally, aboveground physiological parameters, notably chlorophyll content, chlorophyll fluorescence, and plant leaf area were also improved following consortia inoculation, compared to the negative controls, whatever the cropping pattern or water rea tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 796 gime. the results indicate that the inoculation of intercropped wheat and faba bean with mixed bacterial inoculants may be a promising solution to enhance crops growth under drought and p-deficiency stresses. keywords:keywords: consortia, drought, faba bean, intercropping, phosphate, rhizobacteria, wheat. o4. does phosphogypsum application affect salts, nutrients, o4. does phosphogypsum application affect salts, nutrients, and trace elements displacement from saline soils?and trace elements displacement from saline soils? outbakat mbarka1§, redouane choukr-allah1, mohamed el gharous1, kamal el omari2, aziz soulaimani1, and khalil el mejahed1* 1 agricultural innovation and technology transfer center (aittc), mohammed vi polytechnic university, benguerir, morocco; 2 ocp s.a., sustainability & green industrial development (sgid), casablanca, morocco. *corresponding author: khalil.elmejahed@um6p.ma. §presenting author: mbarka.outbakat@um6p.ma. abstractabstract salinity and sodicity are the most agricultural challenges in arid and semi-arid regions. a pot experiment was undertaken, to evaluate the effect of phosphogypsum (pg) and gypsum (g), to remove salts, nutrients and trace elements in leached water from saline and saline-sodic soils. in order to determine the efficiency and safety of these amendments, as an affordable strategy, for overcoming salinity and sodicity stress. the pg at 0, 15, 30 and 45 t/ ha and g at 15 t/ha were mixed with the upper 9 cm soil in the pot before being leached. the soils were collected from sed el masjoune and sidi el mokhtar areas of morocco with ece of 140.6 ms/cm and 11.7 ms/cm respectively. the highest doses of pg (≥30 t/ha) removed significant amount of salts and nutrients. calcium sulfate supplies calcium ions to replace salt ions (sodium, especially). the replaced salts are leached from the soil. the pg was more efficient compared to g in terms of salts leaching. quantities of trace elements in the leachate, for most analyzed elements, were below the recommended limits of drinking and irrigation water. because the experiment’s alkaline conditions (basic water and soil) reduce the solubility and mobility of trace elements. the amendment application did not affect saturation index (si) of the main minerals. keywords:keywords: soil salinity and sodicity, phosphogypsum, gypsum, leached water, plant nutrients, trace elements, saturation index. o5. effect of pgprs and water-hope on potato growtho5. effect of pgprs and water-hope on potato growth el allaoui nadia1,2§, allal douira2, abdellatif benbouazza1, el hassan achbani1, and khaoula habbadi1* 1 laboratory of phytobacteriology and biological control, urpp-inra-meknes, morocco; 2 plant, animal and agro-industrial production lab, department of life and earth sciences, faculty of sciences, kenitra, morocco. *corresponding author: khaoula.habbadi@inra.ma. §presenting author: nadiaelallaoui5@gmail.com. abstractabstract the potato is a plant of the nightshade family which is cultivated for its tubers. this reserve organ is a staple food for many populations in the world and more specifically in morocco. however the potato can be the target of different diseases and pathogens which decrease its growth and consequently its yield, in order to fight or minimize biologically against these, there are bacteria which are beneficial to health and growth plants, and which have the ability to colonize the roots intensely which are the pgprs (plant growth promoting rhizobacteria). the objective of this work is to study the effect of two bacterial strains (s1=gaj222 and s2=gab111) on potato growth by adding water-hope as a water-retaining substrate under form of two different doses (d1=3g and d2=1,5g), and this carrying out two field trials on two sites ain-taoujtate and douiyat and over two successive years 2020-2021. on the other hand, the gaj222 strains showed an efficiency of 75% on the growth of the potato in the presence of water-hope with the second dose of 1,5g withe 75%. key-keywords:words: potato, growth, pgprs, water-hope. o6. genome wide association study of hessian fly resis-o6. genome wide association study of hessian fly resistance in bread wheat (tance in bread wheat (triticum aestivumtriticum aestivum l.) l.) imseg imane1,2*, el gataa zakaria, samir karima, el bouhssini mustapha, and wuletaw tadesse 1 international center for agricultural research in the dry areas (icarda), p. o. box 6299, rabat, morocco; 2 ecology and environment laboratory, faculty of sciences, university hassan ii, casablanca, morocco. *corresponding and presenting author: abstractabstract hessian fly (hf), mayetiola destructor (say), is an important pest of wheat, causing huge yield losses every year. in morocco, this insect causes 32–36% wheat yield losses during dry years. the present study was carried out on 210 bread wheat (triticum aestivum l.) genotypes from the international center for agricultural research in dry areas (icarda) to identify the genomic region and marker-trait associations (mta) responsible for the hessian fly resistance and putative genes associated with this trait. the screening was carried out in the growth chamber using a population of the hessian fly mayetiola destructor (say), collected from chaouia ouardigha. the 210 genotypes tested showed various degrees of resistance to the hessian fly; there were eighty-six accessions with high levels of resistance to the hessian fly. genome-wide association studies (gwas) using a mixed linear model (mlm) identified 20 significant marker-trait associations (mtas) at p < 0.001 associated with hessian fly resistance. the highest number of mta (10) was recorded in the a sub-genome, followed by b with 9 (mta). whereas 1 mta was found with an unknown chromosome position, the most significant marker is ax-95143016 located in chromosome a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 797 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 3b with a lod of 10,23453. after mapping the 20 significant markers associated with hessian fly resistance, there were 14 potential candidate genes found. this study found highly resistant genotypes and significant markers that could be used in wheat breeding programs in the future. keywords:keywords: hessian fly, gwas , mta , resistance , breeding , candidate genes. session i: hemp and cannabis productionsession i: hemp and cannabis production co-chair: karla gage, southern illinois university, usaco-chair: karla gage, southern illinois university, usa co-chair: jose leme, southern illinois university, usaco-chair: jose leme, southern illinois university, usa o1. effect of biostimulants on cannabis productivity and soil o1. effect of biostimulants on cannabis productivity and soil microbial activity under outdoor conditionsmicrobial activity under outdoor conditions jose leme* school of forestry and horticulture, sothern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: jose.leme@siu.edu. abstractabstract the illegal status of cannabis (cannabis sativa l.) post-world war ii resulted in a lack of research on agricultural practices. however, there is a resurgence of interest in cannabis due to diverse uses such as a rich source of cellulosic/woody fiber and construction uses, seed oil, bioenergy, and pharmaceutical properties. the fact that few agricultural products are certified for cannabis leaves producers with scarce alternatives to facilitate management and increase productivity. organic certified plant biostimulants may help cannabis producers increase crop yield and quality. in 2019 and 2020 we investigated the individual and combined effects of two biofertilizers (manure tea and bioinoculant) and one humic acid (ha) product on cannabis biochemical and physiological parameters and soil co2 evolution under outdoor conditions. our hypothesis was that ha would increase the microbial activity in the biofertilizers and synergy of both compounds would promote better plant performance and stimulate soil microbial activity. in 2020, the individual and combined application of biofertilizers and ha increased cannabis height, chlorophyll content, photosynthetic efficiency, aboveground biomass, and bucked biomass by 105, 52, 43, 122 and 117%, respectively. impacts were greater under suboptimal growing conditions caused by planting delay experienced in 2020. in 2019, planting date occurred in-between the most favorable period and chlorophyll content and photosynthetic efficiency were the only parameters influenced by the application of biostimulants. the discrepancies between the two growing seasons reinforce the evidence of other studies that biostimulants efficacy is maximized under stress conditions. this study could not conclusively confirm that the combined use of biofertilizer + ha is a superior practice since affected plant parameters did not differ from application of the compounds singly. similarly, only one biofertilizer + ha treatment increased soil microbial activity. more research is needed to oral presentations abstractsoral presentations abstracts cannavis & hemp sciences & entrepreneur-cannavis & hemp sciences & entrepreneurship day wednesday, december 14, 2022ship day wednesday, december 14, 2022 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 798 define optimum rates and combinations of biofertilizer and stimulants for cannabis. o2. fusarium species threaten postharvest cannabiso2. fusarium species threaten postharvest cannabis nicole gauthier*, d szarka, h smith, e dixon, m munir, and m rahnama department of plant pathology, university of kentucky, lexington, ky, usa. *corresponding author: ngauthier@uky.edu abstractabstract as hemp becomes established as a commodity in the us, continued cultivation results in pathogen build up and increased disease severity. this also demands a greater understanding of the pathogens that affect the consumable portions such as flowers and grain. several fusarium species that are known to produce mycotoxins have been confirmed pathogenic on hemp in kentucky. several of the resulting toxins are regulated in grains used for human and animal consumption (don, niv, t-2), therefore fusarium management is critical. determining which fusarium species infect hemp is the first step to producing safe material. while several studies are ongoing regarding field disease, there have been no studies regarding stored hemp. postharvest material can remain in barns or supersacks for months or years before processing. harvested and stored floral material for production of cannabidiol (cbd) were collected from 17 field sites across kentucky from 2019 and 2020 harvests. material was screened using a fusarium-selective medium and dna sequencing. at least 13 different species were confirmed, including known mycotoxin-producers f. equiseti, f. graminearum, f. incarnatum, and f. sporotrichioides. additional research is essential to determine pathogenicity of these species and whether they can produce toxins dangerous for humans and animals. such information is crucial to determine how to store hemp, manage infected material, and promote successful production of hemp products. keywords: keywords: cannabis, hemp, fusarium, mycotoxin, postharvest, storage. o3. hemp and cannabis insect pest managemento3. hemp and cannabis insect pest management katelyn kesheimer* department of entomology & plant pathology, auburn university, usa. *corresponding and presenting author: kak0083@auburn.edu abstractabstract not provided o4. weed and crop interactions in cannabis productiono4. weed and crop interactions in cannabis production karla gage* department of plant, soil and agricultural systems, sothern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: kgage@siu.edu. abstractabstract there is rapid evolution of herbicide resistance in agronomic weeds in us cropping systems. this phenomenon represents the extreme selection pressure caused by a reliance upon herbicides for weed control. therefore, there is a need for alternative methods of weed control to incorporate into current production systems. integrated weed management (iwm) is the concept of diversifying weed management to include cultural, mechanical, biological, and chemical weed control tactics in combination. the cannabis sativa l. plant (hemp, <0.03 % tetrahydrocannabinol) could be used in a iwm program in a cultural control tactic, through the practice of crop rotation with maize (zea mays l.) and soybean (glycine max l.) in us cropping systems. federal legislation passed in 2014 allows a new possibility for hemp to be used for fiber and seed production in the us, and hemp has the potential to become a valuable rotational crop if commodity markets develop. several characteristics of the cannabis plant may enhance value as a rotational crop. our work suggests that cannabis plants produce potentially allelopathic phytochemicals that may suppress the emergence and growth of economically damaging weeds. allelochemicals may provide value in certain forms of iwm approaches, such as chaff lining, a practice where weed seeds are deposited into crop residue as weeds and crops are harvested at the end of season. our work also suggests that cannabis can be a competitive crop in some production situations, where yields were not reduced with heavy weed competition. however, cannabis for fiber and seed is still a new, potential crop in the us and basic knowledge regarding agronomic production is lacking. poor agronomic practices that reduce the early establishment of a cannabis crop in the field will negatively impact the value of the crop for weed suppression. our research has also documented some agronomic challenges, such as negative impacts of heavy spring rainfalls and soils that have high levels of silt and clay. there is much work to be done to develop cannabis as a fiber and seed crop in the midwestern us. o5. the problem with ppfd: advances in cannabis lighting o5. the problem with ppfd: advances in cannabis lighting technology and terminologytechnology and terminology brian gandy* southern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: brian.gandy@valoya. com. abstractabstract cannabis cultivation technology and horticultural lighting technologies have been driven by the explosion of growth in the industry. the legalization of cannabis in the united states a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 799 and other markets along with the evolution of chip and optics technological efficiencies and engineering are combined to offer many new avenues of exploration. light emitting diode (led)-based lighting technology provides the opportunity to apply wavelengths that target photosynthesis in the photosynthetically active radiation (par) range of 400 to 700 nm. in addition, we can now target the extended par (epar) range from ~300 to– 780 nm, exploring the morphological and physiological effects of ultraviolet (uv) and far-red (fr or nir) ranges. led lighting has been touted for its improvements in efficiency, which on the surface are true. however, the primary benefit (and challenge) to this shift in technology is the de-coupling of heat and light energies to the canopy from the lighting source. the efficiency gains and distance to canopy improvements require more nuanced control of the other environmental parameters to reap the benefits. in particular, the full understanding and utilization of the vapor pressure deficit (vpd) is needed, as well as understanding and automating the quality of light to gain desired yields and results. terminology for horticultural lighting is an amalgam of radiometric lighting, typically used in to define human perception along with agronomic terminology used to define energy delivered to field crops like corn and soybeans, directly from the sun (i.e. inverse square law and daily light integral (dli) respectively). historically, crops grown in controlled environments had a much smaller stature and much more 2-dimensional canopy to design and apply lighting applications toward. the exceptions are vining crops like cucumber and tomato that often utilize inter-canopy lighting and can be grown on their 2-dimensional vertical planes. the morphology of cannabis as a tall, bushy, and flowering plant with a dense canopy provides a unique challenge, and opportunity to further our understanding of the physics, application, and terminology used to specify and optimize horticultural lighting in controlled environments. session ii: hemp and cannabis programs initiatives in mo-session ii: hemp and cannabis programs initiatives in moroccorocco co-chair: noureddine el aouad, abdelmalek essaadi univer-co-chair: noureddine el aouad, abdelmalek essaadi university, moroccosity, morocco co-chair: mouad chentouf, inra, moroccoco-chair: mouad chentouf, inra, morocco o1. inra hemp and cannabis initiativeo1. inra hemp and cannabis initiative mouad chentouf* and foaouzi bekkaoui inra, morocco. *corresponding and presenting author: abstractabstract not provided. o2. abdelmalek essaadi university’s hemp and cannabis ini-o2. abdelmalek essaadi university’s hemp and cannabis initiativetiative noureddine el aouad* polydisciplinary faculty of larache. university abdelmalek essaadi, larache, morocco. *corresponding and presenting author: n.elaouad@uae.ac.ma abstractabstract not provided. o3. new approach for the screening for modulatory activi-o3. new approach for the screening for modulatory activities of polymerization depolymerization of tubulins by can-ties of polymerization depolymerization of tubulins by cannabinoidsnabinoids anissi jaouad1*, khalid sendide2, mohamed el hassouni3, and salim bounou1 1 school of engineering biomedtech, university eumromed of fes, fes, morocco; 2 laboratoty of biotechnology, school of science and engineering, al akhawayn university, av. hassan ii, p. o box 104-ifrane; 3 laboratoire de biotechnologie, faculté des sciences dhar el mehrez, université sidi mohamed ben abdellah, fés, morocco. *corresponding and presenting author: j.anissi@ueuromed.org. abstractabstract cannabinoids have been recognized as possessing anticancer properties by inducing cell cycle arrest. this paper aims to assess the hypothesis that tubulins/microtubules are among the targets of cannabinoids in the anti-proliferation process. twenty-two cannabinoids were isolated from the aerial parts of c. sativa using a green process for extraction and purification. their structures were characterized by detailed spectrometric and spectroscopic analysis. subsequently, we carried out an in vitro screening of the modulation effect of cannabinoids on purified tubulins polymerization and depolymerization with ic50 values in the range of 1.5 μmol/l, and constant rates in average of 0.19 μm/s paclitaxel equivalent. the results show that several cannabinoids exhibit remarkable modulation activities of tubulin polymerization/depolymerization with a constant rate exceeding those observed with the standard paclitaxel. besides, results obtained provided a structure activity relationship that would be used to design new anticancer agent. keywords:keywords: cannabinoids, tubulin polymerization, tubulin depolymerization. o4. genetic identification, chemical and biological investiga-o4. genetic identification, chemical and biological investigation of moroccan tion of moroccan cannabis sativacannabis sativa l. seeds l. seeds amira metouekel1§, elmostfa el fahime2*, saïd el kazzouli1, and nabil el brahmi1 1 euromed research center, euromed faculty of pharmacy, euromed university of fes (uemf), meknes road, fez 30000, morocco; 2 centre national de la recherche scientifique et technique (cnrst), angle avenues des far et allal el fassi, hay ryad, rabat 10102, morocco. *corresponding author: melfahime@gmail.com. §presenting author: a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 800 abstractabstract cannabis contains an interesting group of natural metabolites that apply their actions by binding to specific receptors in different part of the human body such as the central nervous system, peripheral tissues and particularly the immune system. recently, the interest on its main active secondary metabolites as therapeutic alternatives has grown. for their applications as pharmaceutical and medicinal purposes and according to the major national problem of cannabis’ genetic identification, we conducted an in-depth genetic-chemical study on various moroccan cannabis samples. in this communication, we will present a part of our study carried out on ten varieties of cannabis seeds originating from the north morocco. a botanical, morphological, and biomolecular identification as well as, the total chemical composition studies using different analytical techniques. finally, the biological properties of different seed extracted were also assessed. keywords: keywords: cannabis, seed, molecular identification, chemical characterization, biological properties. o5. efficient removal of eriochrome black-t dye using acti-o5. efficient removal of eriochrome black-t dye using activated carbon of waste hemp (vated carbon of waste hemp (cannabis sativabiscannabis sativabis l.) grown in l.) grown in northern morocco enhanced by new mathematical modelsnorthern morocco enhanced by new mathematical models fouad el mansouri1,2*, joaquim esteves da silva2, and jamal brigui1 1 research team: materials, environment and sustainable development (medd), faculty of sciences and techniques of tangier, university abdelmalek assaadi, bp 416 tangier 90000, morocco; 2 chemistry research unit (ciqup), dgaot, faculty of sciences of university of porto, r. campo alegre 697, 4169-007 porto, portugal. *corresponding and presenting author: fouad.elmansouri@etu.uae.ac.ma. abstractabstract in this study, the adsorption behavior of eriochrome black t (ebt) on waste hemp activated carbon (whac) was investigated. the surface of the whac was modified by h3po4 acid treatment. the surface and structural characterization of the adsorbents was carried out using fourier transform infrared spectroscopy (ftir) and scanning electron microscopy (sem) analysis. the effect of influential adsorption parameters (ph, contact time, dosage, and initial concentration) on the adsorption of ebt onto whac was examined in batch experiments, some adsorption parameters such as ph, concentration and dose were improved by new mathematical models. the adsorption behavior of ebt on the surfaces of whac was evaluated by applying different isotherm models (langmuir, freundlich, temkin and dubinin radushkevich) on equilibrium data. the adsorption kinetics was studied by using pseudo-first-order, pseudo-second-order, elovich and intraparticle models on the model. adsorption followed the pseudo-second-order rate kinetics. the maximum removal of ebt was found to be 44-62.08% by whac at ph= 7, adsorbent dose of 10-70 mg, contact time of 3 h and initial dye concentration of 10 mg.l-1. the maximum adsorption capacities were 14.025 mg.g-1 obtained by calculated according to the langmuir model, while the maximum removal efficiency was obtained at 70 mg equal to 62.08% for the whac. regarding the adsorption process is physical on the monolayer and multilayer. keywords:keywords: cannabis waste; isotherms; eriochrome black t; biosorbent, mathematical models. o5. um6p initiative on cannabis sativa valorizationo5. um6p initiative on cannabis sativa valorization mohamed hafidi1, lamfeddal kouisni2, mohamed el gharous3, khalil el mejahed3, loubna el fels1, rachid bouharroud4, redouan qessaoui4, aziza tangi3, khadija nasraoui3, mohamed louay metougui3, adil mazar2, abdelaziz nilahyane2§, youssef el kharrassi2, and amine ezzariai2* 1 cadi ayyad university, marrakech, morocco; 2 mohammed vi polytechnic university, african sustainable agriculture research institute (asari), laayoune, morocco; 3 mohammed vi polytechnic university, african innovation and technology transfer center (aittc), benguerir, morocco; 4 inra agadir, morocco. *corresponding author: amine.ezzariai@um6p.ma. §presenting author: abdelaziz.nilahyane@um6p.ma. abstractabstract moroccan agriculture challenges require developing biomasses valorization with high added value. cannabis sativa l is characterized by several properties that encourage the valorization of this plant in several areas, such as agriculture and medicine. hence, cannabival project aims to develop scientific and strategic solutions for the valorization of cannabis through advanced biochemical extraction methods and innovative bioprocesses, for promising applications. the final products that will be developed through cannabival project will be tested in many experimental farms (laâyoune, benguerir, agadir). this integrated approach will provide an original roadmap for the valorization of cannabis sativa l and its derivatives in morocco. keywords: keywords: cannabis sativa l, valorization, biochemical extraction, bioprocesses. o5. legalization of cannabis in morocco: valorization of o5. legalization of cannabis in morocco: valorization of hemp seeds for oil extraction and their incorporation into hemp seeds for oil extraction and their incorporation into poultry feedpoultry feed elamrani ahmed1*, y taafi1,3, y rbah1, a allay1, c benkirane1, a far-hat2, e azeroual3, k belhaj1,4, f mansouri, r melhaoui1, m addi1, m abid1, a mihamou1, and caid h serghini1 1 equipe agroalimentaire, lapabe, faculté des sciences, université mohammed premier, oujda, morocco; 2 agence nationale de plantes médicinales & aromatiques, taounate, morocco; 3 institut royal d’elevage, fouarat, kenitra, morocco; 4 laboratoire de management de l’agriculture durable, est sidi bennour, université chouaib doukkali, el jadida, morocco. *corresponding and presenting author: ahmed.elamrani@ a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 801 gmail.com abstractabstract in may 2021, the moroccan parliament adopted law no. 1321 (1) on the decriminalization and legalization of cannabis farming for medical, cosmetic and industrial use. cannabis agribusiness mainly values the leaf parts and flowering tops of the plant; thus, it generates many co-products such as woody stems, branches and cannabis seeds. this opens a research field aimed at minimizing and efficiently managing this issue to support the concept of zero waste. this collaborative work (between ump-oujda, anpma-taounate and irtsef kenitra), focus on the chemical characterization of cannabis seeds, hemp seed oil and the agro-industrial valorization of whole seeds and cakes (residues from the extraction of oil from hemp seeds), by incorporating them into poultry feed. for this reason, the chemical composition and lipid profile of cannabis seeds of a local ecotype (beldia) and a hybrid variety (critical), from four regions of northern morocco, were analyzed. results registered show that biochemical composition of analyzed hemp seeds vary as follows: protein (19.25 24.18%), fiber (26.40 37.40%, ash (3.72 5.39%), phenols (134.57 199.90 mg/100 g) and flavonoids (39.40 69.54 mg/100 g. in addition, oil recovery from hemp seed ranges from 26.42 to 35.19 g%, its tocopherols content varies between 376.46 and 796.06 mg/kg oil, with -tocopherol as the dominant isoform. “rancimat test” for oil oxidation stability at 100°c ranged from 9.73 to 15.42h. the fatty acid profiles of hemp seed oils analyzed by cpg-fid show richness in unsaturated fatty acids (ufa: 87.30 to 88.96%) the main ufa are: linoleic acid (48.26 55.39%) followed by oleic acid (14.75 22.49%) and -linolenic acid (13.27-16,55 %). moreover, optimal conditions extraction allows achieving the maximum tpc (53.65 mg gae per g extract) with higher antioxidant activities. the major phenolic coumpounds identified by hplc-dad/esi-ms2 are hydroxycinnamic acid amides and lignanamides, while n-trans-caffeoyltyramine, cannabisin a, and cannabisin b might contribute strongly to the potent antioxidant activity of hempseed extracts. these results are in line with what is reported in the literature (2, 3) and they are currently used for the industrial valorization of hemp seeds. for instance, the incorporation of (whole or ground) hemp seeds as an ingredient in the formulation of feed for laying hens with a view to produce omega 3-eggs. as well, hemp seed oils and bioactive molecules extracted from cakes are intended for use cosmetics or for medical and therapeutic purposes. keywords:keywords: hemp seeds oil, oxidation stability, ufa, tocopherols, phenols layer hen, omega 3-eggs. session iii. hemp, cannabis, and entrpreneurshipsession iii. hemp, cannabis, and entrpreneurship co-chair: khalid meksem, southern illinois university, usaco-chair: khalid meksem, southern illinois university, usa o1. a pre-scientific expertise, validating moroccan landrace o1. a pre-scientific expertise, validating moroccan landrace hemp for architectural science and lightweight compositeshemp for architectural science and lightweight composites monika brümmer* coopérative adrar nouh, morocco and cannatektum habitat and materials science, s.l., spain. corresponding and presenting author: info@cannabric.com and firtagourth@gmail. com. abstractabstract energy-efficient building materials are increasingly attracting material science and architecture in the past decades. novel formulations are based on the use of residues from agriculture to produce carbon storing insulation materials and lightweight bio aggregate concretes, replacing materials from non-renewable and energy intense synthetic and mineral resources. in addition, the valorization of agricultural products to develop into premade building materials might be a novel challenge of employment in remote rural regions, like the moroccan upper central rif. the use of plant-based materials in the energetic retrofit of vernacular housings and rurally inspired, bio constructive ways of building, might be an additional venture for socio-economic development and valorization of architectural heritage. this paper reports on the expertise of employing stem materials of moroccan landrace hemp, treated as a waste product of cannabinoid farming, in the preparation of lightweight composites and other building materials. the construction of two houses, in benguerir (south morocco) and issaguen (nord morocco), has shown that moroccan beldiya hemp stem materials can be used and perform similar like industrial hemp building products and allow versatile solutions in architecture. the stem materials have been used untreaded or processed locally into hemp aggregates (hurds) and hemp wool. the hurds were used in self bearing and load bearing hemp concretes whereas the wool was used in hemp fiber concretes, masonry mortar reinforcement and hemp fiber composite. the present work emphasizes some fundamental parameters of hemp raw materials and composites and seeks to provide a useful tool for future research into sustainable building materials from moroccan landrace hemp, and more efficient recycling of the waste produced by human activities. keywords:keywords: moroccan landrace hemp, beldiya hemp, hemp concrete, hemp lightweight composite, hemp stem materials, hemp hurd, hemp fiber, hemp-built architecture, basis of sustainable design. o2. hemp (o2. hemp (cannabis sativacannabis sativa l.) seed and its cake inclusion in l.) seed and its cake inclusion in animal nutritionanimal nutrition belhaj kamal1*, larb allaii1, yassine taaifi2, farid mansouri2, embarek azeroual3, hana serghini-caid2, and ahmed elamrani2 1 laboratoire de management de l’agriculture durable, est sidi bennour, université chouaib doukkali, el jadida, morocco; 2 equipe agroalimentaire, laboratoire ‘lapab e’, faculté des sciences, université mohammed premier, oujda, morocco; 3 institut royal d’elevage (irtse-f), fouarat, kenitra, morocco. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 802 *corresponding and presenting author: belhaj.kamal90@ gmail.com. abstractabstract hemp (cannabis sativa l.) has great potential as a useful plant. the hempseeds are an important source of several essential nutrients particularly the polyunsaturated fatty acids n-3 and digestible protein. in the last decades, the consumers have become more aware and demand healthy foods exempt from synthetic origin substances. the fatty acid (fa) profile of animal products reflects both the fa of tissues and those of ingested lipids. the omega-6/omega-3 ratio remains the main criterion for evaluating the fat quality of foodstuffs and should be lower than the 4-value recommended by the who/fao. currently, this ratio turns out to be 10 to 30 times more omega-6 than omega-3. these findings indicate a deficiency in polyunsaturated fatty acids (pufa) n-3. in response to the concerns of the health organizations and health-conscious consumers, research in meat and egg production has focused on the diminution of the omega-6/omega-3 ratio, by enhancing the content of pufa n−3. animal products are important sources of essential amino acids, some vitamins, and oligo-elements, but their lipid composition is often criticized. the enrichment of animal products with n-3 pufas remains a sustainable solution for a healthy diet. furthermore, the use of phytogenic feed additives is becoming an attractive and sustainable strategy to improve the lipid quality and functional properties of animal products. the hemp seed and its cake represent a main source of bioactive compounds such as amino acids, phenols, flavonoids, tocopherols, minerals and particularly polyunsaturated fatty acids n-3.these characteristics imply its potential use as a phytogenic ingredient or as an additive in animal feed with health-promoting properties. in the present review, we will present the chemical and nutritional values of hempseed and cake. in addition, the beneficial properties of hempseed and cake in poultry and ruminants ‘nutrition will be discussed. keywords:keywords: polyunsaturated fatty acids, omega 3, hempseed, eggs, meat. o3. transformation, valorization, and commercialization of o3. transformation, valorization, and commercialization of cannabis productscannabis products aziz makhlouf* bio cannat cooperative, douar beni yabta, bab berred, chefchaouen, morocco. *corresponding and presenting author: biocannat@gmail.com abstractabstract bio cannat is a cooperative created in 2021 on the initiative of a young moroccan, son of a farmer from the chefchaouen region, more precisely from the municipality of bab berred. the cooperative has developed with an emphasis on finding the opportunities that cannabis offers for morocco. it has thus shown the potential that moroccan cannabis can offer to farmers in the region and to the socio-economic development of the rif. this work of a year and a half was distinguished by the receipt of authorization number 1 in morocco for the processing, marketing and export of cannabis and its products. today, the journey continues with the establishment and construction of the first cannabis processing unit in the province of chefchaouen. o4. broad access to safe, reliable, and sustainable cannabis o4. broad access to safe, reliable, and sustainable cannabis derived productsderived products brett goldman* the ocan group, usa. *corresponding and presenting author: abstract abstract not provided. o5. challenges and opportunities with opening a hemp fi-o5. challenges and opportunities with opening a hemp fiber processing centerber processing center patrick van meter* midwest natural fiber, columbia, mo, usa. *corresponding and presenting author: patrick@midwestnaturalfiber.com. abstractabstract not provided. session iv: regulatory policies of hemp and cannabis pro-session iv: regulatory policies of hemp and cannabis production and compliancesduction and compliances co-chair: khalid meksem, southern illinois university, usaco-chair: khalid meksem, southern illinois university, usa o1. the rif and the kif beldiya: past, present, and futureo1. the rif and the kif beldiya: past, present, and future abdellatif adebibe* coopérative adrar nouh (sustainable socio-economic of the high rif region), douar assegzaf, issaguen 32302, al hoceima, morocco. *corresponding and presenting author: boujmildev@gmail.com abstractabstract indigenous beldiya hemp was cultivated for centuries in morocco’s historic hemp farming area of the central rif and was historically used in very versatile ways in morocco, without legal concerns. the difficulties came in recent decades with the consequences of prohibitive laws. parallel to the legal ban, farmers had no choice but to continue with their usual activity and have been victims of cannabis traffickers and repression. we have militated to seek legal alternatives and participated in the dynamization of a legalization process. we a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 803 also were promoting the creation of alternative rural socioeconomies with hemp. the new law means a mayor challenge for the berber population of the historic farming area, it is necessary that we get support in this transition period to be able to act in favour of the development of a region that still is very poor in infrastructures. the solutions, projects and hemp processing industries have to part from the heart of the affected areas, be implanted there, and then spread to other new hemp farming areas in morocco. o2. navigating the evolving complex cannabis legislative/o2. navigating the evolving complex cannabis legislative/ regulatory challengesregulatory challenges steve bevan* the ocan group, usa. *corresponding and presenting author: abstractabstract not provided. o3. foundation of cannabis unified standardso3. foundation of cannabis unified standards lezli engelking* focus [focus: the international cannabis health & safety organization (www.focusstandards.org)], usa. *corresponding and presenting author: abstract abstract not provided. o4. cannabis: innovation and challengeso4. cannabis: innovation and challenges myriam lahlou-filali* and abdelmoumen mahly* pharma 5, morocco abstract abstract not provided. session xivb: academic exchange programs between mo-session xivb: academic exchange programs between morocco and usarocco and usa co-chair: moulay abdelmajid kassem, fayetteville state uni-co-chair: moulay abdelmajid kassem, fayetteville state university, usaversity, usa co-chair: khalid meksem, southern illinois university, usaco-chair: khalid meksem, southern illinois university, usa o1. educational and cultural exchange programs between o1. educational and cultural exchange programs between morocco and the usa, the fulbright programsmorocco and the usa, the fulbright programs hafsa el bastami* (deputy executive director) and meryem hammam* (community engagement officer) the moroccan american commission for educational and cultural exchange (macece), rabat, morocco. abstractabstract several educational and cultural exchange programs between morocco and the usa will be discussed including the fulbright-national archives heritage science fellowship, fulbright-national archives heritage science fellowship, fulbright study grant, the fulbright joint-supervision program, fulbright post-doctoral research grant, fulbright fulbright scholar in-residence (sir) program, fulbright (flta) program, fulbright distinguished awards in teaching (dai) program, teachers of critical languages program (tclp), hubert h. humphrey fellowship program, fulbright u.s. student research program, fulbright teaching assistant program, and other programs relevant to the audience. o2. attending us universities: graduate school applicationo2. attending us universities: graduate school application khalid meksem* department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: meksem@siu.edu. abstractabstract about 800,000 to 1 million international students are enrolled in varuous degrees and certificates in us institutions of higher education. therefore, many students worldwide intend and plan to pursue their studies in the usa. here, we will discuss how to apply to the graduate school in the us in a step-by-step basis, show you how to access and apply online as well as how to apply to scholarships and teaching assistantships. session xvii: student oral presentations iiisession xvii: student oral presentations iii co-chair: noureddine el aouad, univ. abdelmalek essaadi, co-chair: noureddine el aouad, univ. abdelmalek essaadi, moroccomorocco co-chair: malika abid, university mohamed i, moroccoco-chair: malika abid, university mohamed i, morocco o1. olive oil quality characteristics of olive orchards (o1. olive oil quality characteristics of olive orchards (olea olea europaeaeuropaea var. europaea l.) in a semiarid continental zone: var. europaea l.) in a semiarid continental zone: case of the beni mellal-khenifra region in moroccocase of the beni mellal-khenifra region in morocco elhabty mohamed1*, jamal aabdousse1, zehor ait yacine1, khalid benhssain2, aziz hasib1, and abdelhaq boundi3 1 environmental, ecological and agro-industrial engineering laboratory, department of life sciences, faculty of sciences and techniques, sulatn moulay slimane university beni mellal, bp 523, 23000 beni mellal, morocco; 2 polyvalent team in research and development, department of biology and geology, polydisciplinary faculty of beni mellal, sulatn moulay slimane university beni mellal, bp 592, 23000 beni mellal, moa tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 804 rocco ; 3 regional office of agricultural development of tadla morocco. *corresponding and presenting author: mohamed. elhabty@usms.ma. abstractabstract the physical and chemical analysis of the quality of olive oil trees (olea europaea var. europaea l.) in the years 2017-2019 period in a continental environment with a semi-arid climate, beni mellal-khenifra lands, located in central morocco, revealed territorial specifications. four homogeneous agricultural territorial units were identified. the result showed the plain with great hydraulic irrigation, the pluviometry plain with private irrigation pumping, the piedmont zone (alias “dir” zone), and the medium mountain zone. these spatial units showed significant differences in the values of the essential parameters of the quality of the olive oil. the percentage free acidity content was 0.96, 1.76, 1.19, and 1.31, peroxide index (with meq/o2/kg) were 7.38, 6.34, 10.00, 7. 09, the two specific extinctions under ultraviolet radiation at wavelength 232 nm showed 0.94, 1.34, 0.79 and 0.74, respectively. the wavelength at 270 nm showed non-significant differences in the values of 0.08, 0.09, 0.15, and 0.08. it was found that the agricultural territorial unit “the plain of great hydraulic irrigation” presented significantly the best quality of olive oil. it was the nearly equal frequency of the class of extra virgin oil (50.02%) and the class of virgin oil (49.48 %). the performance of the area is explained by the fertility of the soils, the judicious choice of the plant material, the relatively young age of the olive orchards, the suitable levels of technicality of the local olive growers, and the respect of the good practices of hygiene by the crushing units with improved modes within the studied area. keywords: beni mellal khenifra morocco region’s, olive oil quality, olive orchard, physicalchemical, semi-arid climate. o2. technological characterization and exopolysaccharide o2. technological characterization and exopolysaccharide production by lactococcus lactis strains isolated raw milk of production by lactococcus lactis strains isolated raw milk of cows in eastern moroccocows in eastern morocco nora hamdaoui1,2*, mohamed mouncif2, and mustapha meziane1 1 laboratory for the improvement of agricultural production, biotechnology, and the environment, department of biology, faculty of sciences, university mohammed i, 60000 oujda, morocco; 2 process engineering and food technologies departement, institute of agronomy and veterinary medicine (iav-hassan ii) bp 6202 rabat, morocco. *corresponding and presenting author: n.hamdaoui@ump.ac.ma. abstractabstract raw milk contains many essential nutrients and is characterized by a rich and diverse lactic microflora. lactic acid bacteria (lab) are natural and important microorganisms used in various forms in the field of health and in industrial food fermentations. their metabolites are generally regarded as safe. lab can be used in foods as probiotics and preservatives that prevent numerous disorders caused by oxidation in the host. they also play an important role in the manufacture of cheese and various fermented dairy products and contribute to texture, flavor and the production of aromatic compounds, the aim of the present research was the identification and investigation of technological attributes of lactococcus lactis and focused on dextran production test, extraction and quantification of (eps), lipolytic and proteolytic abilities, thickening, texturizing, coagulating and acidifying properties by 24 strains of lactic acid bacteria (lab) genus lactococcus lactis isolated from raw cow milk from eastern morocco. the search for eps production was based on two types of tests: qualitative tests (on modified mrs agar, mse medium, rhutenium red medium, chinese ink, and chalmers medium, skimmed milk) . and quantitative tests (modified mrs broth). the results of the evaluation of the technological aptitudes of the lactococcus lactis strains indicate a significant acidifying power (39°d to 88°d), high viscosity, and important proteolytic power (8mm and 25 mm). the strains lc 10, and lc 26 remain the most highly productive of exopolysaccharide (esp). lc 1, lc 10 lc 11 lc 12 lc 24 lc 26 and lc 27 strains, by their thickening capacity can be classified as thickening food additives. thus the result shows the composition of the culture medium (carbon) influences the production of eps. the strains are selected for their ability to acidify milk and form flavors and produce exopolysaccharides, three parameters crucial to their utility as dairy starters. all strains presented good technological potential. the lc.lactis isolated from raw cow milk can be used in the dairy-product industry. keywords: raw milk, lactococcus lactis, acidity, proteolytic , viscosity , eps. o3. beneficial role of exogenous silicon and phosphate-solu-o3. beneficial role of exogenous silicon and phosphate-solubilizing bacillus subtilis on yield and antioxidant metabolism bilizing bacillus subtilis on yield and antioxidant metabolism in chickpea (in chickpea (cicer arietinumcicer arietinum l.) under low phosphorus availl.) under low phosphorus availabilityability habiba kamal1§, mohammed mouradi1, cherki ghoulam2, and mohamed farissi1* 1 unit of biotechnology & sustainable development of natural resource, sultan moulay slimane university, polydisciplinary faculty of beni–mellal, morocco; 2 centre of agrobiotechnology & bioengineering, research unit labeled cnrst, cadi ayyad university, marrakech, morocco. *correspondence: farissimohamed@gmail.com/mohamed.farissi@usms.ac.ma. §presenting author: kamalhabiba20@gmail.com. abstractabstract low phosphorus (p) availability is a limiting factor of plant growth in many agricultural regions. to remedy this problem, farmers use chemical p fertilizers extensively, although these are a serious menace to the environment. the use of biofertilizers-biostimulants such as silicon (si) and p-solua tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 805 bilizing bacteria (psb) have been emerged as a promising way to improve plant p nutrition. in this context, the present study aimed at investigating the synergistic effects of si treatment and phosphate-solubilizing bacilus subtilis inoculum on plant growth, p nutrition, oxidative stress markers and antioxidant response in moroccan chickpea zahour (za) variety under low p availability. results revealed a significant reduction in dry biomass, plant height, leaf number, and area under low p conditions. p deficiency also altered p nutrition and chlorophyll (chl) content. however, p-deficient chickpea plants treated with si or inoculated with psb strain showed higher plant growth, chl content, and remarkably the effect was more important when si was applied together with psb strain. moreover, the simultaneous application of si and psb strain to the p-deficient chickpea plants improved p content in both the shoots and roots. the content of malonyldialdehyd and hydrogen peroxide and the level of electrolyte leakage were significantly increased in p-deficient chickpea, while were significantly decreased after simultaneous application of si and psb inoculum. this reduction was correlated with a significant increase in both enzymatic and non-enzymatic antioxidant systems including superoxide dismutase, polyphenol and flavonoid contents. our findings suggest that the combined application of si and phosphate-solubilizing bacilus subtilis could be a promoting strategy to mitigate the damages of low-p availability on chickpea growth and yield. keywords: keywords: chickpea, silicon, phosphorus deficiency, psb, antoxidant metabolism. o4. antioxidant activity and chemical characteristics of wild o4. antioxidant activity and chemical characteristics of wild prickly pear seed oil grown in the wild in northeastern mo-prickly pear seed oil grown in the wild in northeastern moroccorocco marhri ahmed1*, kamal belhaj1,3, reda melhaoui1, mehdi boumediene1, aziz tikent1, aatika mihamou1, hana serghinicaid1, ahmed elamrani1, christophe hano2, malika abid1, and mohamed addi1 1 laboratory for agricultural productions improvement, biotechnology and environment (lapabe), faculty of sciences, university mohammed first, oujda, morocco; 2 ligneous and field crops biology laboratory, inra usc1328, orleans university, cedex 2, 45067 orléans, france; 3 laboratory of sustainable agriculture management, higher school of technology sidi bennour, university chouaib doukkali, street jabran khalil jabran bp 299-24000 el jadida, morocco. *corresponding author: ahmed.marhri@ump.ac.ma. abstractabstract the great usefulness of the species opuntia ficus indica makes it a very important species in the cactus family. the aim of this work is to initiate a study on the quality of prickly pear oil produced in the area of chouihia located in the north of eastern morocco. due to the high price of the oil, the sector of the seeds is the best yield. hence, nopal cactus industry is active and expanding rapidly. to assess the quality of the studying oil, the quality index, oxidative stability, flavonoid, total phenolic, chlorophyll and carotenoid content of prickly pear seed oil were analyzed. the samples were obtained from wild trees during the harvesting season 2020. the results showed that the acidity and peroxide index were 1.11% and 4.44 meq o2/kg, respectively. while the total phenolic and flavonoid contents in these seed oils were 260.07 mg/kg and 65.99 mg/kg, respectively. in addition, the results show a carotenoid content of 0.61 mg/kg and a total chlorophyll content of 1.52 mg/kg, and an oxidative stability of 17.31 hours. keywords:keywords: prickly pear, chemical characteristics, oil, opuntia ficus india, eastern morocco. o5. validation of a composite of epigenetic biomarkers for o5. validation of a composite of epigenetic biomarkers for screening, early diagnosis and precise prognosis of colorec-screening, early diagnosis and precise prognosis of colorectal cancertal cancer omayma mazouji* and hicham mansour ges-lcm2e, fpn, mohamed first university, oujda, morocco. *corresponding and presenting author: omazouji@gmail. com. abstractabstract colorectal cancer (crc) is one of the major health concerns and a leading cause of carcinogenic death worldwide. according to statistics from globocan, crc is ranked the second one in the aspect of mortality and the third one in terms of incidence with an estimated number of more than 1.9 million new cases in 2020. in the clinical field, the management of crc is based on the use of solid biopsy as a golden standard for the determination of crc status. in this project we identified the methylation sites in the dna extracted from patients with crc at stages i and ii. a dna bio-bank of 100 samples from patients with crc, 25 in stage i, 25 in stage ii and 50 normal autologous tissues has already been collected. the dnas from these patients were extracted and quantified. the next generation sequencing tool was used to target 84mb of the human genome containing 3.7 million of cpgs. these cpgs are present mainly in the cpg islands, found in the 20,000 annotated human genes and theirs promoters, as well as in all referenced regulatory sequences. between 8 and 10 gb of data were generated per patient in order to have at least 100x of sequencing coverage. a bioinformatics and statistical analysis were carried out in order to determine the relevant methylation sites. these sites have been identified on six genes: kcna1, kcna6, col4a2, gdf7, arhgef7 & h4c9. real-time pcr validation of these sites is in progress. these genes are able to: 1/ discriminate between tumor and normal condition, 2/ detect the sites which induce (driver sites) the tumor and 3/ determine the prognosis of affected patients. this work aims to validate the methylation sites obtained in the previous study, determine the statistical power of the relevant methylated sites and offer a reliable and less expensive molecular tool for the early detection and prognosis of crc patients. finding the methylation sites that contribute to the a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 806 early development of crc is critical and essential not only for early cancer detection, but also for collecting information and providing strategies for colorectal cancer therapy. key-keywords: words: colorectal cancer, biomarkers, methylation, diagnosis, prognosis, screening. o6. optimization of phenolic compounds extraction from o6. optimization of phenolic compounds extraction from hemp (cannabis sativa l.) seeds using simplex lattice mix-hemp (cannabis sativa l.) seeds using simplex lattice mixture design and hplc-dad/esi-ms2 analysisture design and hplc-dad/esi-ms2 analysis chaymae benkirane1*, abdessamad ben moumen1, marie-laure fauconnier2, yassine taaifi1, youssef rbah1, allay aymane1, kamal belhaj1, farid mansouri1,3, malika abid1, hana serghini caid1, and ahmed elamrani1 1 laboratory of agricultural productions improvement, biotechnology and environment, faculty of sciences, mohammed i university, oujda, morocco; 2 laboratory of chemistry of natural molecules, gembloux agro-bio tech, university ofliège, passage des déportés, 2,5030 gembloux, belgium; 3 saseflaboratory, higher school ofeducation and training, mohammed i university, oujda, morocco. *corresponding and presenting author: chaymae.1.benkirane@gmail.com. abstractabstract hemp (cannabis sativa l.) is an annual plant of the cannabaceae family. it is cultivated for industrial, therapeutic, recreational, and nutritional purposes. hemp seeds are a good source of protein, oil, fiber, and bioactive compounds. in this study, the extraction of phenolic compounds from defatted hempseeds was optimized using a simplex lattice mixture design with three solvents (water, methanol, and acetone). the total phenolic content (tpc) was evaluated by folinciocalteu’s method. the results showed that the binary acetonewater mixture in equal proportions is the optimal combination to achieve the maximum tpc (53.65 mg gae per g of extract). the phenolic profile analysis of defatted hempseeds showed the predominance of hydroxycinnamic acid amides and lignanamides, especially n-trans-caffeoyltyramine, cannabisin a, and cannabisin b. in addition, hplc-dad/esi-ms2 analysis allowed visualizing the effect of each solvent mixture on the relative extracted amount of each identified phenolic compound. this study encourages the use of defatted hemp seeds as a source of bioactive compounds with added value for pharmaceutical and cosmetic applications. keywords: keywords: hemp, bioactive compounds, tpc, mixture design, hplcdad/esi-ms2 analysis. session xvii: student oral presentations ivsession xvii: student oral presentations iv co-chair: abdelghani tahiri, university ibn zohr, moroccoco-chair: abdelghani tahiri, university ibn zohr, morocco co-chair: karen midden, southern illinois university, usaco-chair: karen midden, southern illinois university, usa o1. dynamics of soil nitrates in a plot under onion cultiva-o1. dynamics of soil nitrates in a plot under onion cultivation in the saiss basintion in the saiss basin nessah chaimae1,2*, abdellah el hmaidi3, el faleh el mâti2, and bouhafa karima1 1 national institute of agronomic research, soil, plant, and water laboratory, meknes, morocco; 2 department of geology faculty of sciences, moulay ismail university, geosciences: geodynamics and georesources, meknes, morocco; 3 department of geology faculty of sciences, moulay ismail university, laboratory of geo-engineering and environment, meknes, morocco. *corresponding and presenting author: nessah.chaimae@gmail.com. abstractabstract morocco has always made the development of the agricultural sector a strategic choice. this sector is faced with the challenge of the intensive use of nitrogen fertilizers which threaten the quality of agricultural soils.this study is part of the research related to this issue. it aims to establish the spatiotemporal variation of soil nitrate concentration in an onion plot. a nitrogen fertilization trial was carried out in the “douyet” experimental station of the regional center of agronomic research of meknes. the experimental design adopted is complete random blocks. six nitrogen treatments (0, 90, 135, 180, 225 and 270kg n/ha) were tested. soil samples were taken at five depths (0-20 cm, 20-40 cm, 40-60cm, 60-80cm and 80-100cm) for their nitrate content. the monitoring shows a richness in nitric elements. the direct impact of nitrogen application is more felt after the herbaceous growth stage, and it seems to have more of a marked impact at shallow depths, this root zone could lose no3 at the time of bulbing when the crop rapidly absorbs nitrogen.the results show that for each of the rates, the soil measured concentrations decrease gradually with time. the assimilation of nitrogen by the plant during the crop cycle may explain this result. the difference in soil no3 concentrations is clearly noted between the 0, 90 and 135kg/ha rates and the 180, 225 and 270kg/ha rates. a significant difference in soil nitrate levels over time was observed. however, no significant difference was observed between the concentrations measured at different depths for the different n application rates. keywords:keywords: nitrogen fertilization, onion, soil nitrate dynamic, saiss basin, morocco. o2. forest composition effect on carbon stock potential and o2. forest composition effect on carbon stock potential and other soil physico-chemical properties in the oulmes central other soil physico-chemical properties in the oulmes central plateauplateau ikraoun hicham1*, mohamed el mderssa2, laila nasiri1, and jamal ibijbijen1 1 environment and valorization of microbial and plant resources lab, department of biological sciences, faculty of sciences, moulay ismail univesity. meknes, morroco; 2 polydisciplinary faculty, soltane moulay slimane university, beni mellal, morroco. *corresponding and presenting author: hichamenfi40@gmail.com. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 807 abstractabstract the climate change repercussions are more than ever recognized and felt by humanity and are no longer to be denied. these changes, which are essentially due to the increase of greenhouse gases (ghg) in the atmosphere, are manifested by the recurrence of extreme events such as drought, floods and pandemics. this increase in ghgs is due to human activity through the combustion of fossil carbon by industry and transportation. nevertheless, the planet earth has always had sinks able to store a large part of the ghgs, represented essentially by co2, but at different potentials. among these sinks we note the forest ecosystem, which captures co2, in the form of organic carbon, at different levels, namely the aerial biomass, the root biomass and the soil. the present study is a contribution to assess the effect of forest composition on soil carbon storage potential and others physico-chemical properties. the study took place in oulmes central plateau which hosts forest formations based on cork oak, green oak and thuja. the stock of organic carbon of the soil were operated on the organo-mineral layer above 30 cm depth. these soil samples were subjected to conventional physical and chemical analyses. the results of the analyses show a clear effect of the forest composition on the organic carbon stock in the soil. indeed, this stock is high when the forest composition of the woody species is in its pure state (pure thuja strata [47.91 t ha-1]) and decreases in the presence of mixtures with other woody species, especially in the presence of hardwood/softwood mixtures (thuja and holm oak strata [6.71 t ha-1]). these results are considered very useful for a rational management in the framework of forest management, by recommending mono-specific reforestation of forest species promoting a great potential of organic carbon stock in the soil, and thus of reduction of greenhouse gases and mitigation of the effects of climate change. keywords:keywords: climate change, greenhouse gases, soil organic carbon, forest ecosystem, oulmes central plateau. o3. concentrations of arsenic, cadmium, mercury, and lead o3. concentrations of arsenic, cadmium, mercury, and lead in sardina pilchardus from the three moroccan mediterra-in sardina pilchardus from the three moroccan mediterranean coasts nean coasts khaoula kasmi1*, kamal belhaj2, and abdelhafid chafi1 1 laboratory for agricultural productions improvement, biotechnology and environment, faculty of sciences, university mohammed i, oujda, morocco; 2 laboratory of sustainable agriculture management, higher school of technology sidi bennour, university chouaib doukkali, street jabran khalil jabran bp 299-24000 el jadida, morocco. *corresponding and presenting author: kasmikhaoula77@gmail.com. abstractabstract in morocco, maritime fishing is a main component of the national economy given its geographical position and its long coastline. the total moroccan production of fishery products is around 1,376,420 tons with a contribution of 2.5% of the national gdp. heavy metals are a severe threat because of their toxicity, long persistence, bioaccumulation, and biomagnification in the food chain. this investigation deals with human health risk assessment of metal contamination through the consumption of sardina pilchardus (walbaum, 1792) as the most consumed fish species in morocco. the aim of this study is to assess the health risks associated with the consumption of sardine by assessing the contamination level of mercury (hg), lead (pb), arsenic (as), cadmium (cd) in the muscle of this species. three hundred sixty (n = 360) specimens of sardine sampled between december 2020 and december 2021 at 3 mediterranean coast sites beni ensar, cap-de l’eau and alhoceïma in the northern region of morocco. the results show that the as and hg were influenced by geographical area and season (p<0.05), while the lead and cadmium were not affected (p>0.05). the highest values were recorded in winter season (p<0.05). the lowest contamination’s levels were found in spring season. geographically, the al-hoceïma specimens present the highest heavy metals content (p<0.05). according to the standard of world health organization, we conclude that the consumption of s. pilchardus from the moroccan mediterranean coast was not likely to have an adverse effect on human health. keywords:keywords: sardina pilchardus, moroccan coast, toxicity, heavy metals. o4. distribution of earthworms in a o4. distribution of earthworms in a valencia orangevalencia orange orchard orchard as affected by distance to trees and soil propertiesas affected by distance to trees and soil properties benmrich ahmed mansour1,2*, zhor abail1, rania brital1,2, mohammed ibriz2, hamid benyahia1, and tarik essafryouy1 1 national institute of agronomic research, regional center of kénitra, laboratory of fertility and soil ecology, route de sidi yahia du gharb, km9, kénitra, morocco; 2 faculty of science, ibn tofail university, kenitra, morocco. *corresponding and presenting author: ahmedsourbenmrich@gmail.com. abstractabstract earthworms are an important component of the soil macrofauna that plays a significant role in maintaining soil fertility and plant productivity in various agroecosystems. however, these organisms are still poorly studied in moroccan agricultural soils. in this study, we examined the effect of distance to tree and soil properties on earthworm distribution in a valencia orange orchard. ten points were chosen randomly, five were close to the tree (1 m) and five points were about 2.5m away from the tree. in each point, we collected soil samples and earthworms by handsorting from a pit of 0.16m² and 25 cm depth. the soil samples were analyzed for ph, electrical conductivity and moisture. the earthworms were preserved in an ethanol solution (80%) and brought to the laboratory for identification and biomass measurement. the results, obtained so far, showed no significant difference in earthworm abundance and biomass between distance the tree. moreover, we did not found any significant correlation between a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 808 earthworms and the measured soil properties. further investigations should be conducted with larger sample size on multiple orchards to better understand earthworm distribution in citrus orchads in morocco. keywords: keywords: earthworm, soil, citrus, orange, spatial distribution. o5. assessment of performance of five rootstocks under iron o5. assessment of performance of five rootstocks under iron and lime stressand lime stress rania brital1,2*, zhor abail1, ahmed mansour benmrich, mohamed ibriz2, rachid aboutayeb1, hamid benyahia, and tarik es-safryouy 1 national institute of agricultural research, avenue ennasr, bp 415 rabat principale, 10090 rabat, morocco; 2 faculty of science, ibn tofail university, kenitra, morocco. *corresponding and presenting author: raniabrital@gmail.com. abstractabstract rootstocks play a crucial role in the mineral nutrition of citrus crops and can have a strong influence on their uptake of iron. iron deficiency is, indeed, a serious concern for citrus production in mediterranean countries, including morocco, where soil alkalinity and calcareousness reduce the bioavailability of iron to crop uptake. in this study, we examined the effect of iron and lime stress on the performance of five rootstocks, increasingly used in moroccan commercial orchards as substituent to sour orange rootstock. the experiment was conducted on a greenhouse, in pots containing sandy soil planted with one of the five rootstocks (carrizo citrange, citrumelo, poncirus trifoliaite, macrophylla, volkameriana) and subjected to one of three treatments (control, lime stress, lime stress + iron stress) replicated 10 times in a completely randomized bloc design. we induced lime stress by the addition of caco3 to the nutrient solution used in irrigation, while iron stress implied the non-addition of iron to the nutrient solution. the results, obtained so far, showed a significant difference between rootstocks in response to the applied treatments. citrumelo rootstock was the most affected by lime and iron stress, followed by poncirus trifoliaite, carrizo citrange, volkameriana and macrophylla. ongoing measurement of iron concentration in plant tissue will help us better determining the joined effect of lime and iron on rootstock performance. keywords: keywords: citrus, rootstocks, soil, calcareous, lime, iron. o6. prevalence, risk factors and multidrug resistance profile o6. prevalence, risk factors and multidrug resistance profile of gram-negative bacteria recovered from patients admitted of gram-negative bacteria recovered from patients admitted to beni mellal regional hospitalto beni mellal regional hospital dihmane asmaa1*, abouddihaj barguigua1, rafik aniba1, habiba raqraq1, kaotar nayme2, and mohammed timinouni2 1 team of biotechnology and sustainable development of natural resources, polydisciplinary faculty, sultan moulay slimane university, beni mellal, morocco; 2 molecular bacteriology laboratory, pasteur institute of morocco, casablanca, morocco. *corresponding and presenting author: asmaa.dihmanefpb@usms.ac.ma. abstractabstract the aim of this study was to determine the burden of multi-drug resistance, the production of extended-spectrum β-lactamases (esbls) and carbapenemase in gram-negative bacilli recovered from patients admitted to beni mellal regional hospital and risk factors associated with mdr infection. a total of 75 gram-negative bacilli were recovered from oct-2021 to oct-2022, among which escherichia coli were the predominant isolates followed by klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aerogenosa, serratia marcescens and pantoea agglomerans. the highest percentage of antibiotic resistance was noted against ampicillin (84%) followed by nalidixic acid (66.7%) and ciprofloxacin (62.7%). moreover, the isolates showed better sensitivity towards chloramphenicol and carbapenem drugs. out of 75 isolates, 61.3% were multi drug resistant. nearly 34.7 % and 22.7 % of isolates were producers of esbl and carbapenemase, respectively. previously hospitalization, previous antibiotherapy, patient admitted to surgical ward, patients with malignancy and patients with anemia, urinary tract infection and urinary tract catheter was found as significant risk factors. the emergence of mdr bacteria in our hospital specifically isolates producing esbl and cabapenemase is highly alarming. keywords:keywords: gram negative, mdr, esbl, carbapenemase, risk factor. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 809 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) p01. genome wide association study (gwas) of grain yield p01. genome wide association study (gwas) of grain yield and yield related traits in spring bread wheat (and yield related traits in spring bread wheat (triticum aes-triticum aestivumtivum l.) under drought and heat conditions in three local.) under drought and heat conditions in three locationstions zakaria el gataa1,2*, samira el hanafi1, fadil basheer1, zakaria kehel1, yassin bouhouch1,3, khalil el messoadi1,4, kenza eddakir1, nawel ladraa1, karima samir2, and wuletaw tadesse1 1 biodiversity and crop improvement program, icarda, agdal, rabat 10080, morocco; 2 faculty of sciences ben m’sick, university hassan ii of casablanca, morocco; 3 faculty of sciences of meknes, moulay ismail university, morocco; 4 faculty of sciences kenitra, ibn tofail university, morocco. *correspondence: elgataa.zakaria@gmail.com. abstractabstract abiotic stress, especially drought and heat, affects cereal yields and wheat production worldwide, more particularly in west and south asia, north africa, and sub-saharan africa. the present study was carried out on 229 spring bread wheat (triticum aestivum l.) genotypes from the international center for agricultural research in dry areas (icarda) to identify the genomic region and marker-trait associations (mta) responsible for drought and heat tolerance. the study was carried out in three different locations, merchouch station (morocco), sids station (egypt), and wadmadani station (sudan) over a period of two years (2018 and 2019). a genome-wide association study (gwas) was performed by using the mixed linear model (mlm) and 13,698 dartseq markers were used for genotyping. linkage disequilibrium revealed that 1914 pairs of markers mapped on the b sub-genome, followed by d and a sub-genomes with 1745 and 1726 paired markers, respectively. a total of 342 mtas (p < 0.05) and 78 genes were identified in the three environments, of which 134 mtas were recorded at sids station and 128 and 80 mtas at wadmadani station and merchouch station. the markers 822842, 196565, and 753901 were significantly correlated with grain yield under drought, heat, and yield potential stations in morocco, sudan, and egypt, respectively. the markers and candidate genes identified in this study have the potential for markerassisted selection to develop high yielding wheat genotypes with resistance to heat and drought stresses. keywords: keywords: gwas, mta, drought stress, heat stress, wheat. p02. p02. calendula officinalis calendula officinalis l. and l. and ammi visnagaammi visnaga l.: phytol.: phytochemical tests, total polyphenols, and tol.: total flavonoids chemical tests, total polyphenols, and tol.: total flavonoids and ftir analysisand ftir analysis benabderrahmane asmae1,2*, majid atmani1, faouzi errachidi, and saadia belmalha2 1 functional ecology and environmental engineering laboratory, faculty of technical sciences, sidi mohamed benabdellah university, fez, morocco; 2 plant protection and environment department, national school of agriculture, meknes, morocco; *corresponding and presenting author: asmaebenabd@gmail.com. abstractabstract the moroccan flora contains many plant species that have long been used as aromatic and medicinal plants. calendula officinalis l., a member of the asteraceae family and ammi visnaga l., a member of the apiaceae family both belong to this rich flora. these two herbaceous annual medicinal plants are observed in the mediterranean region and have many benefits. thus, this study demonstrates the richness in biological compounds of the aqueous extracts from the two plants. in addition, a study of an organic extract with potential therapeutic properties and a comparison of the chemical compositions by ftir were conducted. the two plants were collected in two localities of the region of meknes then dried and crushed to prepare the extracts. two types of aqueous extracts were made: the infused and the decocted and an organic extract (methanolic). the aqueous extract was used to investigate some chemical compounds using standard tests and both extracts were used for total polyphenols and total flavonoids. ftir analysis was performed using the powder obtained by grinding. phytochemical screening showed a richness of both plants in terpenoids, alkaloids, flavonoids and coumarins. there is a total absence of saponosides in both plants. in comparing the two types of aqueous extracts, we observed that the infused ones are much more concentrated in polyphenols than the decocted ones. the comparative analysis of the aqueous and organic extracts shows a higher polyphenol content in the methanolic extract for both plants. flavonoids are more numerous in the infusion. the powders analyzed by ftir reveal the existence of a variety of chemical compounds and showed characteristic absorption bands in approximately the same wavelength regions. this abundance of active ingredients gives these plants remarkable properties that could justify its multiple therapeutic indications and its use in traditional medicine. keywordskeywords: calendula officinalis l., ammi visnaga l., phytochemical screening, ftir. p03. predict the threshold wind erosion velocity of cultivat-p03. predict the threshold wind erosion velocity of cultivated organic soils in montérégie (quebec, canada)ed organic soils in montérégie (quebec, canada) kanga idé soumaila*, jean caron, and andrés felipe silva dimate département des sols et génie agroalimentaire, université laval, québec, canada. *corresponding and presenting author: soumaila.kanga-ide.1@ulaval.ca & soumkanga@gmail. com. poster presentation abstractsposter presentation abstracts tuesday & wednesday tuesday & wednesday december 13-14, 2022december 13-14, 2022 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 810 abstractabstract the aim of this research is to evaluate the necessary wind velocities to initiate wind erosion based on the field measured characteristics of the organic soil (particle diameter, bulk density, gravimetric water content) and of the measured aerodynamic roughness length. organic soils are very vulnerable to wind erosion. when dry and exposed to wind erosivity, organic soils are likely to lose large amounts of soil. as such, threshold friction velocities in air dry and wet conditions as well as bare or plant covered were computed to find the required friction velocities in these soil conditions. the variation of the threshold friction velocity as a function of soil particle diameters, bulk density and soil cover was determined. the results suggest that the threshold friction velocity averages 0.14 m/s for air dry soil, goes up to 2.62 m/s for wet (100 % to 203 % of gravimetric water content) soil condition and reaches1.88 m/s for soil covered by vegetation or plant residues. the threshold friction velocity in the dry soil condition increases with increasing soil particle diameter and bulk density. the threshold friction velocity in covered soil condition is found to increase with the aerodynamic roughness length. indeed, under wet and covered soil conditions, the threshold friction velocity increases up to 13 to 18 times compared to the threshold friction velocity for a bare air-dry soil on average. keywords:keywords: organic soil, wind erosion, friction threshold velocity, bulk density, soil particle diameter. p04. effect of inclusion of hemp seeds in the diet of laying p04. effect of inclusion of hemp seeds in the diet of laying hens on liver fatty acids profile, tocopherols, and choles-hens on liver fatty acids profile, tocopherols, and cholesterol contentsterol contents youssef rbah1*, aymane allay1, yassine taaifi1, chaymae benkirane1, kamal belhaj2, hana serghini caid1, reda melhaoui1, and ahmed elamrani1 1 laboratory of improvement of agricultural production, biotechnology, and environment, faculty of science, mohammed first university, oujda, morocco; 2 laboratory of management of sustainable agriculture, est sidi bennour, university chouaib doukkali, el jadida, morocco. *corresponding and presenting author: yrbah50@gmail.com. abstractabstract hemp seeds (hs) are a good source of protein, fat, fiber, and other micronutrients (vitamins and minerals), they can be used as poultry feed. currently, the low availability and high cost of most oilseeds commonly used in poultry feed are pushing feed producers to find alternative ingredients. hs known for its richness of omega-3 fatty acids especially, alpha-linoleic acid (ala: c18 :3n-3), could be an excellent oilseed ingredient for poultry feed formulation. thus, this study focuses on the evaluation of the alteration of fatty acid profiles, cholesterol, and tocopherol levels in the liver of laying hens fed with different doses of hs. one hundred and eight (n=108) 22-week-old laying hens (lohmann’s classic brown), were divided into 3 batches of 36 laying hens, with 6 repetitions of 6 hens each, and received a formulated poultry feed containing 0%-hs (control),10%-hs or 30%-hs. the results demonstrate a significant decrease (p<0.001) in the amount of saturated fatty acids (sfas) between the 30-hs treatment and the control however, diets containing hs show a substantial increase (p<0.001) in n-3 and n-6 pufas and a significant decrease (p<0.001) in the n-6/n-3 ratio. the content of cholesterol decreased significantly (p<0.05) in the 10%-hs treatment compared to the control. concerning tocopherol content and in parallel with the hs incorporation rate, a significant increase (p<0.001) is noted. for the amount of α, γtocopherols, a significant rise (p<0.001) it is observed for the 30%-hs treatment. the results indicated that hs supplementation up to 30% has interesting effects and is beneficial for the lipid profile of the liver, mainly n-3 fatty acids and the n-6/n-3 ratio, cholesterol concentration; and tocopherol content. keywords:keywords: hemp seed, layer hen, diet, fatty acid, n-3, n-6/n-3 ratio, cholesterol, α, γ-tocopherol. p05. physico-chemical characterization of a rehabilitated p05. physico-chemical characterization of a rehabilitated soil mine based on phosphate industry by-products and sew-soil mine based on phosphate industry by-products and sewage sludgeage sludge donatien gueable1§, mohamed hafidi2,3, mohamed el gharous1, and khalil el mejahed1* 1 mohammed vi polytechnic university, agricultural innovation and technology transfer center (aittc), ben guerir 43150, morocco; 2 laboratory of microbial biotechnologies, agrosciences and environment (biomage), cadi ayyad university, marrakesh 40000: labelleld research unit n°4 cnrst, morocco; 3 mohammed vi polytechnic university, agrobiosciences (agbs) department, ben guerir 43150, morocco. *corresponding author: khalil.elmejahed@um6p.ma. §presenting author: donatien.gueable@um6p.ma. abstractabstract reclamation of mining sites and valorization of phosphate by-product are at the heart of ocp strategies [1]. mixtures of these by-products along with sludge can improve soil physical and chemical properties and therefore contribute to sustain revegetation of mine sites. thus, the recent studies of [2], related pots trial showed that mixture of 65% pg, 30% bl and 5% bs was the best substrate when compared to other mixtures. the objective of this study was to evaluate soil physicochemical parameters in the rhizosphere of six species planted on substrate under field conditions. the study was carried out on 1.15 hectares with the above substrate. the experimental layout consists of randomized complete bloc design with 6 replicates and six tree species: argan, eucalyptus, pistachio, false pepper, carob and olive. the physical and chemical parameters such as particle size, electrical conductivity, ph, cec and heavy metals contents, were determined using appropriate analytical techniques. the results showed that the texture was sandy-silt. soils ph (7.74-7.90) were slightly alkaline. cona tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 811 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) ductivity ranged from 3.14 to 3.30 ds/cm. the organic matter was low with values varied between 1.20% and 1.23%. cec was high with values between 40.50 and 45.80 cmol. kg-1. the heavy metal contents of the rhizosphere samples of the different species showed that the mean values of cd, pb, zn, ni, cr and cu ranged from 4.30 to 4.66 mg.kg-1, 3.80 to 4.39 mg.kg-1, 52.52 to 58.02 mg.kg-1, 9.48 to 10.30 mg.kg-1, 34.03 to 37.09 mg.kg-1, 6.22 to 7.51 mg.kg-1, respectively. comparison of the heavy metal contents with the standard guidelines for agriculture indicated that cd (5 mg.kg-1), pb (100 mg.kg-1), zn (300 mg.kg-1), ni (100 mg.kg-1), cr (100 mg.kg-1) and cu (100 mg.kg-1) are below the standard guidelines [3]. in our experimental conditions, the 65% pg, 30% bl and 5% bs substrate used in this study contributes to soil properties improvement without generating or constituting any hazardous pollution t for the environment. keywords: keywords: phosphate industry, phosphogypsum, phosphate sludge, sewage sludge, rehabilitation, mining soils. p06. effect of hemp seed incorporation in the diet of laying p06. effect of hemp seed incorporation in the diet of laying hens on laying performance and physical quality traits of hens on laying performance and physical quality traits of eggseggs yassine taaifi1*, kamal belhaj2, youssef rbah1, melhaoui reda1, embarek azeroual3, farid mansouri1, ahmed elamrani1, and hana caid serghini1 1 equipe agroalimentaire, laboratoire ‘lapabe’, faculté des sciences, université mohammed premier, oujda, morocco; 2 laboratoire de management de l’agriculture durable, est sidi bennour, université chouaib doukkali, el jadida, morocco; 3 institut royal des techniciens spécialisés en elevage (irtse), fouarat, kenitra, morocco. *corresponding and presenting author: taaifi.yassine@ump.ac.ma. abstractabstract hemp seeds could be a valuable alternative to improve and to reduce the cost of imported feeding stuff for animals, mainly for poultry. in fact, hemp seeds, which are considered a by-product of cannabiculture, have a low commercial value; however, they have many advantageous nutritional values in terms of lipid profile. the final objective of the research is to promote hemp seeds by incorporation into the poultry diet. the present study was carried out to evaluate the effect of the diets based on various incorporation rates of hemp seeds on the laying performance and physical quality characteristics of the eggs. ninety-six (n= 96) lohmann brown classic laying hens were randomly assigned to 3 feed treatments and a control group: each treatment was repeated 6 times (4 hens/replicates). sampling was performed after 30 weeks of breeding (laying peak). no significant differences in laying performance were observed between the groups examined at this stage of production. egg weight, length, and diameter decreased with increasing hempseed incorporation rate (p<0.001). in addition, the yolk height was influenced by the hempseed rate (p<0.05), while the albumen height was not affected (p>0.05). high levels of hemp seed incorporation had a negative effect on the physical quality characteristics of the eggs, however, egg laying performance was not affected (p>0.05). on the basis of this finding, we can conclude, that at small incorporation rates, hemp seeds could be used in poultry feeding as an alternative constituent to partially replace high-cost imported ingredients such as corn and soybeans. keywords:keywords: hempseed, laying hens, laying performance, eggs physical traits. p07. faisibility study of producing unpasteurized liquid egg p07. faisibility study of producing unpasteurized liquid egg white with enhanced functional properties: safety and tech-white with enhanced functional properties: safety and technological assessmentsnological assessments kmichou ilham1*, triqui réda2, and mazouzi mohamed1 1 ensem engineering institute, hassan ii university, casablanca, morocco; 2 agronomic and veterinary institute hassan ii, madinat al irfane b.p., 6202, rabat, morocco. *corresponding and presenting author: ilhamkmichou@gmail.com abstractabstract pasteurization allows to preserve the salubrity of liquid egg white by destroying the pathogenic microorganisms. on the other hand, the pasteurization to has effects on the functional properties of the egg products. compared to manually separated raw egg white, mechanically separated pasteurized liquid egg white reduces the volume and stability of the foam. it is for, in morocco, pasteurized liquid egg white is not well accepted by the intended users. the present study was to evaluate the possibility of producing liquid egg white without the necessity of pasteurization and to evaluate the functional qualities of such a product. the study was realized in an approved egg product plant. the tests were done under real conditions of production. thus, and through several tests, the microbiological stability of liquid egg white not pasteurized was evaluated both in relation to the flora of alteration and the pathogenic flora. the results obtained indicated that a liquid egg white improved in terms of functional characteristics can be attained without the need for pasteurization. the microbiological evaluations agreed with a safe product. keywords: keywords: raw liquid egg whites, pasteurization, microbiological quality, functional properties, safety, salmonella. p08. effect of provenance on phenolic compounds of p08. effect of provenance on phenolic compounds of arbu-arbutus unedotus unedo in morocco in morocco ilias oussif1*, nadya wahid1, jamal aabdousse1, and nora salim1,2 1 environmental, ecological and agro-industrial engineering laboratory, department of life sciences, faculty of science and technology, sultan moulay slimane university, beni-mellal, morocco; 2 industrial and surface engineering laboratory, department of life sciences, faculty of science and technology, sultan moulay slimane university, beni-mellal, morocco. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 812 *corresponding and presenting author ilias.oussif@gmail. com. abstractabstract aromatic and medicinal plants (map) contain a multitude of compounds involved in the enzymatic and physiological reactions of the organism. these compounds implicated in the physiology of plants have a positive impact on human health. among these compounds are the phenolic compounds differentially synthesized by plants at the level of different organs and this according to the vegetative state of the plant. currently, phenolic compounds are the subject of several research works because of their benefits on human health thanks to their virtues and their various biological activities. in this context, the present study aims to determine the content of phenolic compounds in a medicinal plant, and we have chosen the strawberry tree as a case study. this plant, which knew an evaluation of the nutritive value of its fruit in opposition to its leafy part that remains limited and fragmentary in morocco. in addition, we also aim to evaluate the effect of provenance on the composition and phenolic compound content of the leaf extracts of the said species. in order to cover the entire range of the arbutus in morocco, samples were collected in different biogeographic regions of morocco where this species thrives (pre-rif, western rif, central plateau, middle atlas and high atlas). the collected leaves are dried at room temperature, protected from light and humidity, and then reduced to powder. the extraction is carried out by maceration in methanol to obtain dry extracts for the determination of phenolic compounds. the content of total polyphenols and flavonoids was determined by the colorimetric method using the visible spectrophotometer. the results obtained show that there is a significant variability between the populations studied in terms of total polyphenol and flavonoid content of leaf extracts. thus, analysis of variance (anova) and principal component analysis (pca) show that the provenance of the plant material could be the cause of this intraand inter-population variability in phenolic compound content of arbutus leaves extracts. this variability seems to be correlated with the variation in environmental conditions such as climate, soil, orography, etc. keywords:keywords: arbtus unedo, phenolic compounds, provenance, climate, morocco. p09. screening of microorganisms in the rhiosphere of the p09. screening of microorganisms in the rhiosphere of the grapevine, and study of the antagonistic activity towards grapevine, and study of the antagonistic activity towards al-allorhizobium vitis lorhizobium vitis and and botrytis cinereabotrytis cinerea for the implementation for the implementation of a biological control methodof a biological control method yahyaoui hiba1,2§, majida hafidi1,2, faiçal aoujil1,2, khaoula habbadi1*, and abdelatif benbouaza1 1 centre de recherche regional, inra, meknes, morocco; 2 faculty of sciences, university moulay ismail, meknes, morocco. *corresponding author: khaoula.habbadi@inra.ma. §presenting author: hibayahyaoui065@gmail.com. abstractabstract bacteria form complex and dynamic associations with plants that range from mutually beneficial to commensal or pathogenic and play key roles in soil quality, host productivity, and host health through direct or indirect mechanisms, such as mineralizing soil organic matter, activating plant defense mechanisms, and even producing antibiotics against phytopathogens. viticulture represents one of the most important agricultural domains in the world in terms of cultivated surface and financial profitability and occupies an important place in moroccan agriculture. however, crop diseases take heavy toll on viticulture; it is subject to various biotic threats, notably crown gall and grey rot caused by allorhizobium vitis and botrytis cinerea respectively. agricultural practices are getting innovative with advancement in science and technology. out of several management options of disease control potential use of microbial consortia immerge as a new ecofriendly, sustainable approach. synthetic biology has generated many examples of what microbes can do and what we can learn from them when they are creatively engineered in the laboratory environment. microbial consortia help to increase tolerance toward several stresses such as drought, salinity, heavy metal, and biotic stress . the aim is to better understand the role of the soil and root microbiome, in the context of , discovering the composition of a microbial consortium to defend the vine against these biotic and abiotic stresses. for this purpose, isolations from the rhizosphere and the roots of the vine were carried out and the morphologically different colonies were tested for their antagonistic activity against allorhizoium vitis and botrytis cinerea. these strains were tested for their compatibility with each other, in order to form a microbial consortium capable of defending the vine against these pathogens and which could be marketed as a biopesticide. keywords:keywords: viticulture, allorhizobium vitis, botrytis cinerea, biocontrol, microbial consortia. p10. nutritional quality of feeds used for fattening local kids p10. nutritional quality of feeds used for fattening local kids in the moroccan western rif regionin the moroccan western rif region asma al rharad1,2*, bouassab abderrahman2, and ayadi mohammed1 1 ur production animale. crra, inra, tanger, morocco; 2 fst, university abdelmalek essaadi, tanger, morocco. *corresponding and presenting author: alrharad.asma@gmail.com. abstractabstract this study is a contribution to evaluate nutritional quality of feeds resources used for fattening local goat kids of extensive livestock farms in northern moroccan region. for this purpose, samples were analyzed for dry matter, mineral matter, nitrogenous matter, ether extract and fibers content. the analyses were also interested in the digestibility by using two methods: enzymatic and in vitro. the protein value (pdi) and energy value (uf) of the samples were calculated to evaluate a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 813 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) the nutritional value of goat kids feeding. the result showed that the mineral content varies between 96 and 17 g/kg dm, the highest content is observed in the pelleted feed. most of the species are characterized by low fat contents, however the energetic species are characterized by important fat contents (25 to 48 g/kg dm). for protein content, the species with high content are represented by vetches, faba beans and bitter vetch (279, 237 and 264 g/kg dm respectively). for the ndf content varied between extreme values of 661.85 g/kg dm for barley and 512.35g/kg dm for bitter vetch with an average content of 600.54 g/kg dm. the lignocellulosic fraction adf, varied between 160.21 and 79.20 g/kg dm. the lignin adl content was an average of 25.40 g/kg dm. for the energy value (ufl and ufv), the results record an average of 0.90/kg dm for ufl and 0.87/kg dm for ufv. the pdins have an overall average of 110.93 g/kg dm while the pdies have an average of 103.49 g/kg dm. the nutritional quality of the concentrated feeds used in the fattening of goat kids is now well known, it will allow a better optimization of the use of the available fodder resources and a good feeding of the kids in the northern region of morocco. keywords:keywords: concentrated feeds, chemical composition, digestibility, pdi, uf. p11. hydroponic green fodder: an alternative solution for p11. hydroponic green fodder: an alternative solution for feeding livestock in arid and semi-arid areasfeeding livestock in arid and semi-arid areas othmane elghalmi¹,²*, karima samir¹, and mohamed el koudrim² ¹ laboratory of ecology and environment, department of biological sciences, university hassan ii, faculty of sciences ben m’sick, casablanca, morocco; ² regional center of agricultural research, inra, settat, morocco. *presenting author: othmaneelghalmi@gmail.com. abstractabstract in arid and semi-arid areas, the animal production subsystem is an essential component of the production of agricultural holdings, however several constraints hinder the development of the sector, which are mentioned: the low and irregular rainfall resulting in fluctuations in the food supply of the livestock resulting in high feed prices, the small area reserved for fodder crops and thus the weakening of the livelihoods of millions of farmers. taking climate change into account, as a structural phenomenon affecting morocco like many other countries with semi-arid or arid climates, the question of the resilience of production systems to climate change is at the heart of the morocco green plan. the pmv’s approach to mitigate, adapt and prevent its effects and impact is based on the deployment of innovative and sustainable techniques and practices. the adoption of the technique of green hydroponic fodder, will thus allow the breeders to be able to produce large quantities of good quality fodder all year round on reduced areas. the objective of this study is to rely on hydroponic green fodder as an alternative solution for the sustainable feeding of livestock in arid and semi-arid areas, by studying and monitoring the growth and morphological and physiological characteristics of a hydroponic crop with a mixture of cereals (barley, triticale, forage peas) grown alone and in a mixture. crop growth was monitored, and several measurements were made, measuring morphological characteristics: germination rate, biomass, root and leaf length, and physiological characteristics: nitrogen content, mineral matter, dry matter, fiber content. keykeywords: words: green fodder, hydroponics, barley, feed peas, triticale, livestock feed. p12. grapevine microbiome: a biological control method p12. grapevine microbiome: a biological control method against biotic stressesagainst biotic stresses yahyaoui hiba1,2§; majida hafidi2; aoujil faiçal1,2; benbouaza abdelatif1; and habbadi khaoula1* 1 laboratory of phyto-bacteriology and biocontrol, plant protection unit, national institute of agronomic research inra, meknes 50000, morocco; 2 laboratoire de biotechnologie végétale et valorization des bioo-resource, faculty of science meknes, university moulay ismail, meknes 50000, morocco. *corresponding author: khaoula.habbadi@inra. ma. §presenting author: hibayahyaoui065@gmail.com. abstractabstract viticulture represents one of the most important crops in the world in terms of cultivated surface, and socioeconomic impact and occupies an important role in moroccan agriculture. however, crop diseases take a heavy toll on viticulture; it is subject to various biotic threats. soil microbial communities represent the greatest reservoir of biological diversity. an increasing body of evidence also signifies the importance of this plant microbiome, which consists of the entire complex of rhizosphere-associated microbes, their genetic elements, and their interactions, in determining plant health. the collective genome of this microbial community is much larger than that of the plant and is referred to as the plant’s second genome. it is from these various microbial populations that the idea of selecting a microbial consortium containing compatible, beneficial, and antagonistic strains against the various plant pathogens has immerged. the application of microbial consortium consisting of efficient strains for biological control is a superior technique compared to the application of individual microbes for managing plant diseases. moreover, the application of microbes in a consortium may improve the efficacy, reliability, and consistency of the microbes under diverse soil and environmental conditions. compatible microbial strains that have no suppressive effect on other microbial strains, in the consortium may have an enhanced impact on plant growth promotion or disease suppression. this microbial consortium triggers several mechanisms of action including competition for resources, boosting systemically the defensive capacity of the plant, and the creation of anti-microbial metabolites. keywords: viticulture, biocontrol, microbial a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 814 consortium. p13. optimization of roasting process of almonds intended p13. optimization of roasting process of almonds intended for preparation of healthy, weight-loss-friendly snacksfor preparation of healthy, weight-loss-friendly snacks nadia houmy1,2*, farid mansouri1, kamal belhaj1,3, reda melhaoui1, youssef rbah1, mohammed addi1, and ahmed elamrani1 1 laboratoire d’amélioration des productions agricoles, (lapabe), equipe agroalimentaire, faculté des sciences, université mohammed i, bp-717, 60000 oujda, morocco; 2 laboratoire de technologie et qualité alimentaire, crrao, inra, bp-428, 60000 oujda, morocco; 3 laboratoire de management de l’agriculture durable, école supérieure de technologie, sidi bennour, université chouaib doukkali, route ben maachou, 24000, el jadida morocco. *corresponding and presenting author: houmy.nadia@gmail.com abstractabstract due to the drought resistance of almond trees, the almond industry is becoming a lever for development in the semiarid regions of eastern morocco. according to *dra-oriental, young almond plantations contribute to the performance of the sector, where the production of shelled almonds continues to increase, (13,000 tons in 2008 to 30,000 tons in 2020), and a better post-harvest recovery of almonds is noticed. indeed, several agri-food cooperatives have been created and various transformation processes for the valorization of almonds in almond products with high added value have emerged (e.g.: marzipan, almond oil for food and cosmetic use). this study focuses on the optimization of the roasting process of almonds in order to improve their nutritional quality. whole or split roasted almonds are used with cereals and other ingredients in the formulation of new healthy and weight loss -friendly snacks (healthy bars snacks). we will present here the results of the method for optimizing the almonds torrefaction. indeed, response surface methodology (rsm) was used to determine the optimal roasting conditions. twenty experiments were randomly selected using a central composite design (ccd) with two-controlled factors. almonds were roasted at five temperatures (112, 120, 140, 160 and 168 °c) and for five duration times (7, 10, 17.5, 25 and 28 min). mathematical models have shown that roasting conditions significantly affected response variables (p < 0.001), namely color, browning index, sweet, acid bitter and roasted tastes, hardness, and peroxide index. the optimized results showed that the best responses were reached when the roasting time was 17.5 min and roasting temperature was 140°c. the roasting process at these conditions produced the most acceptable almonds by consumers. keywords: keywords: almond roasting, nutritional quality, healthy bars snacks. p14. incidence of codling moth (p14. incidence of codling moth (c. pomonellac. pomonella l.) in apple l.) in apple tree cultivars according to its voltinism and several physi-tree cultivars according to its voltinism and several physicochemical traits of fruitscochemical traits of fruits dina akroute1,2§, rachid benkirane2, khaoula habbadi1, ahmed elbakkali1, rachid razouk1, younes ogad1, abdellatif benbouazza1, and salma el iraqui el houssaini1* 1 regional center of agricultural research of meknes, national institute of agricultural research, av. annasr, 10000 rabat, morocco; 2 university ibn toufail, faculty of sciences, laboratory of plant, animal and agro-industry productions, b.p 242, kenitra, morocco. *corresponding author: salma.eliraqui@inra.ma. §presenting author: dinakroute@gmail.com. abstractabstract codling moth, c. pomonella l., is known as one of the main pests of pome fruits (apple and pear) and walnuts worldwide. several control methods have been performed to decrease its incidence, however, it still represents a threat to the production and remains unsuccessfully managed. the current study has been conducted to highlight potential correlations between the codling moth damage and the physicochemical traits of fruits according to the voltinism of the insect through a growing season. the voltinism was determined and thirteen well-known apple cultivars were investigated between june and july, for codling moth damage as well as for physicochemical fruits characterization. results showed highly significant differences between varieties in terms of codling moth infestation. the incidence of the insect varied according to the maturity stage of the fruits; the medium maturing varieties were the most attacked among the studied varieties, namely galaxy and obro gala. moreover, the firmness and sugar content seemed to be involved in codling moth attraction towards certain varieties. the preference to these two parameters changed among the insect’s generations. keywords: keywords: apple cultivars, c. pomonella, fruits physicochemical traits, voltinism. p15. mitigating global warming through the recovery of p15. mitigating global warming through the recovery of organic wasteorganic waste eliadini sara1§, abderrazak rfaki1,2, and mohamed merzouki1* 1 bioengineering laboratory, faculty of sciences and technology, sultan moulay slimane university, beni-mellal, morocco; 2 national center for scientific and technical research (cnrst), rabat, morocco. *corresponding author: m.merzouki@gmail.com. §presenting author: saraeliadini@ gmail.com. abstractabstract over the past decade, the amount of organic waste has increased worldwide. this biomass is underestimated despite its richness in biomolecules of interest that can be transa tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 815 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) formed into several types of biological products such as biostimulants, biofertilizers, anti-aging, nutraceuticals, and pharmaceutical drugs. the non-valorization of these co-products of plant or animal origin is the main cause of greenhouse gases (ghg) responsible for climate change and its harmful effects on agriculture, human health, and the environment. the scientific community encourages the recovery of this waste by producing natural agricultural inputs such as biofertilizers, biopesticides, and biostimulants, to satisfy the food security of the population and minimize polluting waste. care must be taken to recover this abandoned organic biomass rich in biomolecules of interest. indeed, the challenge that appears from this proposed valuation is to find the most eco-efficient technique that preserves the environment and improves economic profitability within the framework of a circular bioeconomy.the main aim of this communication is to discuss and interpret the different opportunities for the recovery of organic waste. additionally, various valuation limits are revealed and discussed, to which we offer recommendations to minimize the adverse effects of climate change to ensure food security. keywords:keywords: recovery, organic waste, valorization, biomolecules, circular economy, climate change, food security. p16. antioxidants from p16. antioxidants from rubus idaeusrubus idaeus l. cv maravilla leaves: l. cv maravilla leaves: ability to inhibit the formation of glycation end products and ability to inhibit the formation of glycation end products and key hyperglycemia enzymeskey hyperglycemia enzymes el asri sara1*, reda ben mrid1,2, imad kabachi1, zakia zouaoui1, abdelhamid ennoury1, zoulfa roussi1, nada nhhala1, and mohammed nhiri1 1 laboratory of biochemistry and molecular genetics, faculty of sciences and technologies of tangier, abdelmalek essaadi university, tetouan, morocco; 2 institute of biological sciences, mohammed vi polytechnic university, ben guerir 43150. *corresponding and presenting author: saraelasri40@gmail. com. abstractabstract as a by-product of red raspberry production, rubus idaeus l. cultivar maravilla leaves, which are well-known for their antioxidant-rich fruits, are discarded as trash. this study intends to determine for the first time the potential inhibitory activity of key enzymes related to hyperglycemia, αglucosidase and α-amylase, as well as the protein glycation inhibitory ability of this plant methanolic extract, in order to discover natural remedies. the results demonstrated that the leaves had a high levels of phenolic compounds (167.2 mg gae/g dw) and a flavonoid content of 17.08 mg qe/g dw. furthermore, the leaves showed a significant antioxidant potential in various tests, including dpph free radical scavenging (ic50= 0.03 mg/ml) and 2,2’-azino-bis(3ethylbenzothiazoline6-sulphonic acid) scavenging (ic50= 0. 049 mg/ml), ferrous ion chelation (ic50= 1.28 mg/ml), and ferric iron reducing power (266.03 mg/ml). moreover, the antidiabetic assays revealed that the extract could considerably reduce the activity of both α-glucosidase and αamylase enzymes. an albumin/ fructose glycation model was also used to assess the extract’s protein glycation inhibitory ability. various biochemical indicators, such as fructosamine and protein carbonyl group, were used to estimate glycation inhibition, and the formation of advanced glycation end products (ages) was determined using a fluorometer. the present investigation confirmed that rubus idaeus cv maravilla leaves could be a prospective plant, as replacements for synthetic drugs, for diabetes treatment and glycation-related diabetic complications prevention. keywords: keywords: rubus idaeus leaves extract, antioxidant activity, alpha-glucosidase, alpha-amylase, glycation, ages. p17. pre-incubation of rhizobia strains with hesperetin affect p17. pre-incubation of rhizobia strains with hesperetin affect vicia fabavicia faba growth and nodulation under salt stress condigrowth and nodulation under salt stress conditionstions benidire lounba1,2,3*, k. oufdou1, and m. göttfert3 1 laboratory of microbial biotechnologies, agrosciences, and environment (biomage) faculty of sciences semlalia, cadi ayyad univerity, marrakech, morocco; 2 higher school of technology laayoune, ibn zohr university morocco; 3 institute of genetics, university of technology, dresden, germany. *corresponding and presenting author: l.benidire@uiz.ac.ma. abstractabstract associated with appropriate crop and soil management, inoculation of legumes with rhizobacterial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. plant-rhizobial symbiosis are subjected to osmotic stress imposed by high levels of nacl that is frequently found in arid and semi-arid lands. poor nodulation and n2 fixation of legumes, which can lead to substantial loss of yield, has been attributed to negative effect of abiotic stress in the signal exchange process between the plant-rhizobial symbiosis. it well known that successful symbiotic interactions are complex and require the regulation and function of multiple genes/gene families in both partners. indeed, flavonoids (e.g., hesperetin) play a crucial role as signal molecules in promoting the formation of nodules by symbiotic bacteria. this study examines the effect of inoculation with preinduced rhizobia by hesperetin on v. faba plant growth and nodulation under salt stress conditions. three rhizobia strains (rhof4, rhof6 and rhof53), which have a different tolerance to salinity, were used to inoculate faba bean plants grown under controlled environmental conditions. germinated seeds were inoculated with rhizobial strains preinduced with 10 μm hesperetin, at different levels of salt stress (0 and 70 mm). rhizobia grown without signal molecules were used as control inoculums. data collected from this experiment showed that saline treatment inhibits plant growth of faba bean. pretreatment of the bacteria with hesperetin had no stimulatory effect on nodulation. however, it seems to stimulate plant growth by increasing shoot and root bioa tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 816 mass in v. faba inoculated with the tolerant strain rhof53. keywords: keywords: faba bean (vicia faba), rhizobia, salt stress, flavonoid, hesperetin. p18. variability of phenological traits of mediterranean olive p18. variability of phenological traits of mediterranean olive ((olea europaeolea europae l.) cultivars from the world olive germplasm l.) cultivars from the world olive germplasm bank of marrakechbank of marrakech omar abou-saaid1,2,3*, bouchaib khadari3,4, magalie delalande3, abdelmajid moukhli1, adnane el yaacoubi5, cherkaoui el modafar2, and hayat zaher1 1 inra, ur amélioration des plantes, marrakech, morocco; 2 université cadi ayyad, centre d’agrobiotechnologie et bioingénierie , unité de recherche labellisée cnrst (url-cnrst 05), marrakech, morocco; 3 agap, université de montpellier, cirad, inra, montpellier supagro, montpellier, france; 4 conservatoire botanique national méditerranéen de porquerolles (cbnmed), umr agap, montpellier, france; 5 university sultan moulay slimane, école supérieur de technologie khenifra, khenifra, morocco. *corresponding and presenting author: omar.abousaaid@gamil.com abstractabstract flowering phenology is an adaptive trait controlled by two major mechanisms: the chilling requirements (cr) necessary to start growth and floral development when the risk of frost decreases and the heat requirements (hr) for the development of flowering stages. future warming predicted in the mediterranean region may reduce winter chill and increase heat. therefore, the characterization of germplasm collections constitutes a cornerstone for the selection of cultivars with low cr and the orientation of breeding programs towards accurate climate change mitigation strategies. phenological records for six years from 2014 to 2019 of 331 identified cultivars were used for assessment of olive phenology and the estimation of cr and hr of all cultivars. full flowering dates (ffd) were significantly affected by genotype, year, and their interaction. ffd cultivar characterization showed early flowering cultivars and late-flowering cultivars ranging between day of the year (doy) 115 to doy 132. partial least squares (pls) estimation of chilling and heat phases revealed high variability between cultivars for their cr computed using the dynamic model. however, limited inter-cultivar variability for heat requirement hr was observed. our findings suggest that the temperature variation during the chilling and the forcing periods has a great influence on the ffd of olive cultivars. these results provide a strong portfolio to be explored in the framework of adaptation to climate change. keywords: keywords: olea europaea l., climate change, phenology, pls regression, chilling and heat requirements. p19. biofilm formation among carbapenemase-producing p19. biofilm formation among carbapenemase-producing enterobacterales isolated from patients at mohammed vi enterobacterales isolated from patients at mohammed vi university hospital centeruniversity hospital center dilagui ilham1,2*, souad loqman1,2, asmae lamrani hanchi asmae1,2, kawtar nayme3, mohammed timinouni3, and nabila sorra1,2 1 laboratoire de lutte contre les maladies infectieuses, faculty of medicine and pharmacy, university cadi ayyad, bd prince my abdellah, marrakesh, morocco; 2 laboratory of microbiology-virology of ar-razi hospital, mohammed vi university hospital center, av ibn sina amerchich, bp 2360, marrakesh, morocco; 3 laboratory of molecular bacteriology, pasteur institute, casablanca, morocco. *corresponding and presenting author: ilhamdilagui@gmail.com. abstractabstract the prevalence of carbapenemase-producing enterobacterales (epcs) infections is increasing worldwide. these infections are often a source of therapeutic impasses, leading to increased mortality and morbidity in infected patients. the emergence of this type of resistance as well as the property of biofilm formation contribute significantly to the wide diffusion of multidrug-resistant enterobacterales in the hospital environment. the main objective of this work was to detect in vitro biofilm formation in 50 clinical strains of enterobacterales resistant to carbapenems by producing metallo-betalactamases. the study is conducted on 195 carbapenem-resistant enterobacterales isolated from patients hospitalized at the mohammed vi university hospital centre of marrakech, between the period 01 january to 31 december 2018. the bacterial identification as well as the antibiogram of the strains were done according to the standard procedures of medical microbiology. the search for carbapenem resistance genes (oxa-48, imp, ndm, vim, and kpc) was performed by phenotypic methods and subsequently confirmed by pcr. quantification of biofilm formation was measured by the tissue culture plate method. during the study period, among 195 carbapenem-resistant enterobacterales, 50 strains were ndm carbapenemase producers. the coexistence of the blandm and blaoxa-48 genes was detected in 8 strains. bacterial identification showed a dominance of enterobacter cloacae (54%), followed by klebsiella pneumoniae (28%), escherichia coli (12%), and serratia marcescens (6%). all 50 strains had high levels of resistance to imipenem, ciprofloxacin, and gentamicin. more than 50% of the strains were categorized as strong biofilm formers. the study underlines the importance of implementing surveillance methods for epcs and their biofilm-forming capacity, in order to control their diffusion in the hospital environment. keywords:keywords: enterobacterlaes, carbapenemases, resistance, biofilm formation. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 817 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) p20. inoculation with rhizobacteria consortia for mitigating p20. inoculation with rhizobacteria consortia for mitigating the effect of stress combining water and phosphorus defi-the effect of stress combining water and phosphorus deficiency in intercropped faba bean and wheatciency in intercropped faba bean and wheat said cheto1,2*, imane chamkhi1, bouchra benmrid1, ammar ibn yasser1, joerg geistlinger3, youssef zeroual4, lamfeddal kouisni1, adnane bargaz1, and cherki ghoulam1,2 1 mohammed vi polytechnic university, agrobiosciences, plant-microbe interaction labora-tory, benguerir, morocco; 2 center of agrobiotechnology & bioengineering, research unit labeled cnrst, fst, cadi ayyad university, marrakech, morocco; 3 anhalt university of applied sciences, bernburg, germany; 4 situation innovation, ocp group, morocco. *corresponding author email: said.cheto@um6p.ma. abstractabstract drought and phosphorus (p) shortage are among the most prevailing stresses throughout the world especially in arid and semiarid areas. thereby, they are hampering crop production, quality, and energy. in moroccan agrosystems, faba bean and wheat crops are submitted to the stress combining water deficit and p limitation that adversely affect their productions. inocula-tion with osmotolerant p solubilizing bacteria (psb) under adapted cropping system could con-tribute a lot in mitigating the negative effects of this combined stress. thus, our study aims to assess, under greenhouse, the impact of stress combining water deficit and p limitation on faba bean-rhizobia symbiosis and intercropped wheat and the role of inoculation withbacterial con-sortia gathering rhizobia and non-antagonistic plant growth promoting rhizobacteria (pgpr) to alleviate this impact and improve plant growth under such conditions. two vicia faba varieties aguadulce (ag) and reina mora (rm), and one triticum durum variety karim (k), grown as sole crop or intercropped in pot culture, were inoculated with two rhizobacterial consortia and submitted to. the sandy substrate was added either with rock phosphate (rp) (p deficient) or with monoammonium phosphate fertilizer (p sufficient) and plants were inoculated with two rhizobacterial consortia c1 and c2, each one contains one rhizobia strain and two psb identi-fied during the previous works of bioas team of fst at cadi ayyad university. plants were then submitted to water deficit based on 40% of substrate field capacity (fc) versus 80% fc for the control plants. the results demonstrated that the inoculation with both consortia im-proved plant biomass, leaf water parameters, membrane stability, and phytase and phosphatase activity under p limitation especially for ag variety inoculated with c2 compared to rm varie-ty. inoculation and the intercropping system improved plant biomass, leaf water parameters, membrane stability, and phytase and phosphatase activity for wheat plants compared to sole cropped ones under all stress conditions. the results confirmed that consortium c2 was more performing and could be used as inoculum for enhancing faba bean and wheat production un-der the combined stress of water deficit and p limitation. keywords: keywords: drought, phosphorus, intercropping, pgpr, triticum durum, vicia faba. p21. p21. cistus monspeliensiscistus monspeliensis extract as a prospective biostimuextract as a prospective biostimulant for enhancing antioxidant defense system in sorghum lant for enhancing antioxidant defense system in sorghum plant under cadmium stressplant under cadmium stress zoulfa roussi1§, reda ben mrid1,2, abdelhamid ennoury1, sara el asri1, nada nhhala1, zakia zouaoui1, azzouz krid3, and mohamed nhiri1* 1 laboratory of biochemistry and molecular genetics, faculty of sciences and technologies of tangier, abdelmalek essaadi university, tetouan, morocco; 2 institute of biological sciences (issb-p), mohammed vi polytechnic university (um6p), 43150 ben-guerir, morocco; 3 research team : environmental technology, biotechnology, and valorization of bio-resources, faculty of science and techniques of al hoceima, abdelmalek essaadi university, bp 34, ajdir 32003, al hoceima, morocco. *corresponding author: med.nhiri@gmail.com. §presenting author: zoulfa.roussi@etu.uae.ac.ma. abstractabstract sorghum (sorghum bicolor l.) is a species known for accumulating high quantities of cadmium (cd), which can damage physiological and metabolic functions, impede growth, and reduce yield. maintaining sorghum’s production, therefore, requires enhancing its tolerance to the toxic effects of cd. in this study, we investigate the effects of cistus monspeliensis extract (cme) on cd stress tolerance in sorghum. sorghum plants exposed to cd (200 μm) showed a decrease in their growth, biomass, and chlorophyll content as well as increased signs of oxidative stress compared to unstressed ones. however, cme supplementation (5 mg/l, 20 mg/l, and 60 mg/l) to the stressed plants reversed the detrimental effect of cd and elevated biomass and pigment content. cme also reduced superoxide ions (o -) accumulation and boosted antioxidant enzyme activities (superoxide dismutase (sod), glutathione peroxidase (gpx), glutathione reductase (gr), and glutathione -stransferase (gst)). cme, therefore, appears to improve cd stress tolerance by upregulating antioxidant defense enzymes and decreasing ros production, thus leading to a better growth rate. keywords:keywords: cistus monspeliensis extract, cadmium stress, sorghum bicolor, antioxidant enzymes. p22. impact of innovation platforms on the dissemination of p22. impact of innovation platforms on the dissemination of biotechnological innovation for the date palms’ sustainable biotechnological innovation for the date palms’ sustainable production: case of tafilalet palm groves (southeast of mo-production: case of tafilalet palm groves (southeast of morocco)rocco) hamriri kaoutar1,2*, larbi aziz3, ali abidar2, mouhamadou moustapha kane2, rachid bouamri3, and majid atmani1 1 department of biology, laboratory of functional ecology and environmental engineering, faculty of science and technology, sidi mohamed ben abdellah university, fez, b.p. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 818 2202 –route d’imouzzer, fès, morocco; 2 department of rural development engineering, national school of agriculture, meknes, 10 km., route haj kaddour, b.p. s/40, meknès 50001, morocco; 3 department of plant and environment protection, national school of agriculture, meknes, morocco, 10 km., route haj kaddour, b.p. s/40, meknès 50001, morocco. *corresponding and presenting author: kaoutar.hamriri@usmba. ac.ma. abstractabstract the sustainability of date palm production, in tafilalet’s palm groves, is threatened by many constraints related to the dryland’s severe environment, climate change, and improper human activities. biotechnological innovations are new agricultural research discoveries increasingly used to improve agricultural sustainability. such as compost, which has proved its benefits in facing date palm production constraints, improving its productivity, and enhancing soil health. using linear approaches has proven their ineffectiveness to disseminate innovations’ advantages to small producers. as an alternative, innovation platforms (ips) are a participatory approach based on a multi-stakeholder alliance for disseminating innovations. the purpose of this article is to determine ips’ impacts on the adoption and dissemination process and to evaluate compost impacts on the date palm production system. two types of investigation tools were conducted on 47 ips’ members. a survey was conducted on 33 producers’ members of ips, and a semi-structured interview was destined for 14 institutional actors. data were analysed using factorial analysis, content analysis, and communication network analysis. the results show that ips are a new organizational innovation impacting positively on date palm social systems. they create powerful collective learning through their strong dynamism and interaction. the producers who adopted compost are characterized by a high level of education, take a responsible position in gie, have a large social network, interact with the research team and other producers, engage and participate in ips’ activities, search for agricultural news, and have the ability to accept change and develop their skills. compost can improve soil’s water-holding capacity, increase yield, and reduce expenses by decreasing the need for water, fertilizers, and phytosanitary treatments. keywords:keywords: innovation platforms, date palm’s sustainable production, tafilalet’s palm groves, biotechnological innovation, compost, adoption and dissemination process. p23. drivers of post-harvest aflatoxin contamination: evi-p23. drivers of post-harvest aflatoxin contamination: evidence gathered from knowledge disparities and field sur-dence gathered from knowledge disparities and field surveys of maize farmers in the rift-valley region of kenyaveys of maize farmers in the rift-valley region of kenya grace gachara1,2,5§, rashid suleiman1, sara el kadili3, essaid ait barka4, beatrice kilima1, and rachid lahlali5* 1 department of food sciences and agro-processing, school of engineering and technology, sokoine university of agriculture, p. o. box 3006, morogoro, tanzania; 2 southern africa centre of excellence for infectious diseases (sacids), sacids foundation for one health, sokoine university of agriculture, p.o. box 3019, morogoro, tanzania; 3 department of animal production, ecole nationale d’agriculture de meknès, route haj kaddour, bp. s/40, 50001 meknes, morocco; 4 unité de recherche résistance induite et bio-protection des plantes-ea 4707, université de reims champagne-ardenne, 51100 reims, france; 5 department of plant protection, phytopathology unit, ecole nationale d’agriculture de meknès, km10, rte haj kaddour, bp s/40, meknès, morocco. §presenting author: grace.gachara@sacids.org. *corresponding author: rlahlali@ enameknes.ac.ma. abstractabstract maize-dependent populations in kenya are continually exposed to aflatoxin poisoning owing to their regular consumption of this dietetic cereal. this study assessed awareness levels, knowledge disparities and perceptions regarding aflatoxin contamination at the post-harvest phase among farmers in the rift-valley region of kenya. households were randomly selected using a geographical positioning system (gps) overlay of the agro-ecological zones within uasin gishu and elgeyo marakwet counties. face-to-face interviews were conducted in 212 smallholder and large-scale farms. the study documented the demographic profiles of farmers, knowledge, awareness and perceptions of aflatoxin contamination using a pre-designed structured questionnaire. most farmers were familiar with aflatoxins and the adverse effects they present to health (61.32%). almost all the farmers (94.37%) were aware of storage molds and food spoilage fungi. however, few farmers adopted good post-harvest practices (phps) such as avoiding premature harvests (49.8%), using well-ventilated storage spaces (44.6%), grain sorting (30.5%), proper drying of maize (17.8%) and using hermetic bags for storage (30.5%). intensified farmer education is required to train farmers on good phps to protect their maize from aflatoxigenic fungi and aflatoxin accumulation. keywords:keywords: stored maize; aflatoxins; hybrid-6 series maize cultivars; food security; rift-valley; kenya. p24. soil amendments improve quinoa tolerance level to p24. soil amendments improve quinoa tolerance level to high salinity: agrophysiological analysishigh salinity: agrophysiological analysis mohammad redouane bourhim1,2*, cheto said1,2, hirich abdelaziz2, qaddoury ahmed1, and ghoulam cherki1,2 1 center of agrobiotechnology and bioengineering, cnrst labeled research unit, fst, cadi ayyad university, marrakech, morocco; 2 african sustainable agriculture research institute, mohammed vi polytechnic university, benguerir, morocco. *corresponding and presenting author: medred1. mrb@gmail.com. abstractabstract salinity stress is among the most severe abiotic stresses that a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 819 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) cause many disruptions to soil and crop productivity. therefore, in order to deal with this problematic, we rely on cultivating alternative crops that can tolerate salinity stress, such as quinoa (chenopodium quinoa). despite of its tolerance to salinity, quinoa performance could be affected under high salinity levels. our study aimed at the assessment of the effects of some seeds treatments and soil amendment to improve quinoa tolerance under high salinity level. three quinoa varieties (puno, icba-q5 and titicaca) were grown in the greenhouse in pots containing soil with five amendments; biochar “bc”, compost “cp”, black soldier insect frass “if”, cow manure “fb” and phosphogypsum “pg”, with a negative control “t (-)”and a positive control “t (+)” with no amendment, the plants were irrigated after 20 days after the seed sowing with saline water at 16 ds/m of nacl except the “t (+)” that was irrigated with distilled water. the results showed that salinity stress induced negative impact on the quinoa plants for all the tested agrophysiological parameters in the three varieties compared to their positive controls “t (+)”. however, most of these parameters were significantly enhanced by the application of soil amendments compared to the negative controls “t (-)”. for instance, in puno variety biomass was especially increased by more than 90%, for the total nitrogen and phosphorus amount by more than 300% with “bc” amendment application. yet, for titicaca, the most important improvement was noted in the potassium amount where it was 606% better with “pg” amendment compared to the negative control. besides, icab-q5 amended with bc showed an improvement of 222% for the leaves area, of 219% for yield, and of 170% for nitrate reductase activity. nevertheless, differences between varieties were noticed with puno and titicaca presenting the highest performances particularly when amended with if, bc and pg. keywords:keywords: quinoa (chenopodium quinoa), salinity stress, tolerance to salinity, soil amendments, biochar, compost, black soldier insect frass, cow manure, phosphogypsum. p25. epidemiological profile and clinical characteristics of p25. epidemiological profile and clinical characteristics of nasopharyngeal cancer in northern morocconasopharyngeal cancer in northern morocco ayman reffai1,3*, mohamed mesmoudi2, touria derkaoui1, naima nourouti ghailani1, amina barakat1, nabila sellal2, parag mallick3, and mohcine bennani mechita1 1 biomedical genomics and oncogenetics research laboratory, biology department, faculty of science and technology of tangier (fstt), abdelmalek essaadi university (uae), tangier 90000, morocco; 2 regional center of oncology “ahmed ben zayed al nahyan center of cancer treatment”, chu tangier 90000, morocco; 3 canary center for cancer early detection, radiology department, school of medicine, stanford university, ca 94304, usa. *corresponding and presenting author: aymanreffai96@gmail.com. abstractabstract nasopharyngeal cancer is a complex disease distinguished from other cancers of the head and neck by its risk factors and geographic distribution mainly affecting the asian and north african populations including morocco. the aim of this study was to determine the epidemiological and clinical characteristics of nasopharyngeal cancer in northern morocco. one hundred twenty-nine (129) patients diagnosed with nasopharyngeal cancer and followed at the regional center of oncology of tangier between 2017 and 2019 were included in this study. epidemiological and clinical data was collected using technical sheets and analyzed using statistical package for the social sciences (spss) software. the survival analysis was performed using the kaplan meier method. nasopharyngeal cancer (npc) represented 5% of all cases of cancer with a median age of 50 and a remarkable sex disparity counting 65.9% of men (sex ratio= 1.93 m/f). the most affected age group was 40-54 years (41.1%). cervical mass caused by lymphadenopathy was the most frequent symptom in our cohort. 96.12% of patients had undifferentiated nasopharyngeal carcinomas (ucnt) which is the most common histological type of this type of cancer. most patients (82.2%) had an advanced stage of npc at diagnosis including 5.4% of metastatic cases. 81.4% of patients received radiotherapy combined with chemotherapy preceded in 54.3% of cases by induction chemotherapy. the overall survival (os) at 5 years was 86.8% for all patients. this latter was significantly influenced by disease staging. these findings show that nasopharyngeal cancer is very common in northern morocco as is the case in other endemic areas with a late declaration problem. these results converge with the literature data and show the necessity of further studies regarding this type of cancer for better understanding and a better diagnosis. keywords:keywords: nasopharyngeal cancer, oncology, epidemiology, clinicopathology, prevalence, therapy, survival, morocco, northern morocco. p26. gut microbiota: a strong correlation with colorectal p26. gut microbiota: a strong correlation with colorectal cancercancer el moussaoui seham, derkaoui touria*, michab soukayna, amina barakat, naima ghailani nourouti, and mohcine bennani mechita biomedical genomics and oncogenetics research laboratory, faculty of sciences and techniques of tangier, abdelmalek essaâdi university, morocco. *corresponding and presenting author: derkaoui.touria.22@gmail.com. abstractabstract colorectal cancer (crc) is ranked among the most common cancers worldwide, it is defined as a pathology that develops from the cells that line the inner wall of the colon or rectum. it represents the second leading cause of death by cancer of both sexes. in morocco, its incidence ranks third for all genders. the human gut microbiota is the sum of non-pathogenic microorganisms called commensals, it’s an organ composed of billions of bacteria, viruses, yeasts and fungi that reside mainly in the distal colon. the microbiota plays an essential a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 820 role in the metabolism of the host and at the same time it maintains a symbiotic relationship with the latter. this microbiota can be sometimes unbalanced, it is intestinal dysbiosis, the results of a decrease and/or an increase of certain types of bacteria. in other words, it is the modification of the composition of the gut microbiota. several studies have highlighted the correlation between colorectal cancer and the gut microbiota. indeed, the alteration and the dysfunction of this microbiota triggers and facilitates the carcinogenesis process, by inducing inflammation and disturbing the permeability of the intestinal wall. the new discoveries of the composition, the functions of the gut microbiota and the way in which it intervenes in carcinogenesis, can today consider the microbiota as an excellent tool for the management of colorectal cancer. keywords: keywords: gut microbiota, colorectal cancer, dysbiosis. p27. antifungal effect of some plants on p27. antifungal effect of some plants on fusarium oxyspo-fusarium oxysporumrum f. sp. albedinis (foa), the causal agent of bayoud of date f. sp. albedinis (foa), the causal agent of bayoud of date palmpalm ben-amar houria*, eimad dine tariq bouhlali, and adil essarioui national institute of agronomic research, regional center of errachidia, morocco. *corresponding and presenting author: houriabenamar@hotmail.fr. abstractabstract “bayoud”, a vascular disease caused by fusarium oxysporum f. sp. albedinis (foa), is the main threat to date palm in morocco. nine plants (atriplex canescens, atriplex lentiformis, teucrium polium, chrysopogon zizanioides, thymus vulgaris, foeniculum vulgare, retama monosperma, opuntia ficus indica, and artimesia herba alba) were grounded and added to gzapeck culture medium at 2 doses each, c1 (5g/l) and c2 (10g/l). the resulting medium was filtered and used to evaluate plant fungitoxic and/or fungistatic effects on foa by measuring its spore germination and mycelia. spore germination inhibition ranged between 41% to 69% at dose c1, and from 37% to 92% at dose c2. the three plants with the greatest effects on spore germination, thymus vulgaris, atriplex canescens, and atriplex lentiformis, showed also strong inhibitory effects against mycelial growth, ranging from 16% to 47% for dose c1 and from 20% to 68% for dose c2. these results highlight the potential of plants to produce bioactive compounds and open new research paths for their use in the management of “bayoud” disease of date palm. keywords: keywords: bayoud, bioactive compounds, date palm. p28. in vitro and in vivo effect of salts to control postharvest p28. in vitro and in vivo effect of salts to control postharvest citrus blue mold disease caused by citrus blue mold disease caused by penicillium italicumpenicillium italicum hamrani meriem1,2*, lamyaa zelmat2, rabea ziri2, and mohammed el guilli1 1 plant pathology and postharvest quality laboratory, regional center of agricultural research of kenitra, national institute of agricultural research, kenitra, morocco; 2 plant, animal and agro-industrial production laboratory, university ibn tofail, kenitra, morocco. *corresponding and presenting author: meriem.hamrani@uit.ac.ma. abstractabstract blue mold caused by penicillium italicum is among the most economically impactful post-harvest diseases of citrus fruit. chemical control is the most employed method used to fight this disease around the world. however, prolonged and excessive application of fungicides adversely impacts the environment and human health. the use of alternative approaches is considered a safe and more sustainable strategy to reduce gradually fungicide utilization. the present study aimed to evaluate the in vitro and in vivo antifungal activity of some salts including, potassium sorbate, sodium benzoate, sodium tetraborate and sodium bicarbonate against p. italicum. in in vitro experiments, the solutions of salts were added to the pda medium to obtain the final concentrations of 0, 500, 1000, 1500 and 2000 ppm. the in vivo curative activity of salts to control citrus blue mold was evaluated on ‘valencia late’ oranges artificially inoculated with p. italicum. fruits were dipped, after 24 h of inoculation, in 0, 20 g/l and 40 g/l salt solutions for 2 min. results showed that the potassium sorbate reduced the p. italicum mycelial growth by 45 % at 500 ppm, with a ci50 = 2.75 ppm.the complete inhibition of p. italicum was observed with sodium bicarbonate and sodium tetraborate at 1000 ppm and 2000 ppm, respectively. the in vivo findings showed significant antifungal activity of potassium sorbate, sodium benzoate and sodium tetraborate.they totally inhibited (100 %) the development of blue mold on ‘valencia late’ oranges at a low concentration of 20 g/l. its effectiveness was similar to that obtained by the conventional fungicide “imazalil” used as positive control. keywords:keywords: citrus, post-harvest diseases, penicillium italicum, blue mold, salts, antifungal activity. p29. genetic diversity and population structure of isolates of p29. genetic diversity and population structure of isolates of alternariaalternaria spp., the causal agent of the postharvest diseases spp., the causal agent of the postharvest diseases in citrus fruits in moroccoin citrus fruits in morocco zelmat lamyaa1,2*, hamrani meriem1,2, ibriz mohammed2, and el guilli mohammed1 1 plant pathology and postharvest quality laboratory, regional center of agricultural research of kenitra, national institute of agricultural research, kenitra, morocco; 2 plant, animal and agro-industrial production laboratory, ibn tofail university, kenitra, morocco. *corresponding and presenting author: lamyaa_zelmat@hotmail.com. abstractabstract alternaria alternata is one of the most important fungi causa tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 821 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) ing various diseases on citrus worldwide. in morocco, alternaria black rot (abr) and alternaria brown spot (abs) are two major diseases causing serious losses in commercial cultivars of citrus. the aim of the present work was to study the genetic diversity and the population structure of isolates belonging to sect. alternaria obtained from infected citrus fruits, collected from seven provinces at different locations in morocco (markets, packinghouses, and orchards). forty-five isolates were analyzed by sequence-related amplified polymorphism (srap) markers, and cluster analysis of dna fragments was performed using upgma method and jaccard coefficient. cluster analysis revealed that isolates were classified in four distinct groups. amova revealed also a large extent of variation within sect. alternaria isolates (99%). the results demonstrate that no correlation was found among srap pattern, host, and geographical origin of these isolates. population structure analyses showed that the alternaria isolates from the same collection origin had almost a similar level of admixture. keywords:keywords: citrus, alternaria alternata, post-harvest diseases, genetic diversity, population structure. p30. weeds associated with the arganery ecosystem of souss p30. weeds associated with the arganery ecosystem of souss valley: case of the rasmouka region (tiznit)valley: case of the rasmouka region (tiznit) bouamair ahmed1,2*, m. alouani2, r. qessaoui2, a. ajerrar1, n. ait aabd1, a. tahiri1, a. mimouni1, and r. bouharroud1 1 centre régional de la recherche agronomique d’agadir, morocco; 2 laboratoire de biotechnologie et de valorisation des ressources naturelles, ibn zohr university, morocco. *corresponding and presenting author: a.bouamair@gmail.com. abstractabstract weeds are major constraints on crop production in general. the success of argan orchard under the national arganiculture program depends on the control of alias linked to bioaggressors and weeds. the identification of the most important weed species may allow an understanding to address their interactions with argan orchards and prevent their negative effects of competition in water and fertilizers. our study aimed to investigate the distribution and diversity of weeds in selected argan orchard and surrounding forest in argan biosphere. to explore the weed flora, a floristic inventory was carried out in rasmouka, tiznit region during the month of march 2021. a total of 19 species belonging to 19 genera of 14 botanical families were recorded. asteraceae was the dominant family with 4 species, amarranthaceae (2 species) and poaceaes (2 species). however, the most abundant family in argan orchard is plantaginaceae with a single species (plantago arenaria) while the amarranthaceae family (chenopodiastrum murale) is the most represented under argan trees of neighboring forest. it should be noted that no species belonging to the fabaceae family was observed in the bordering forest. similarly, no species belonging to the 4 families rubiaceae, boraginaceae, caryophyllaceae and brassicaceae was identified in the argan orchard. keywords:keywords: argan tree, weeds, diversity, abundance, species richness. p31. tomato in the souss massa regionp31. tomato in the souss massa region redouan qessaoui1, s. chafiki1, n. chabbi1, a. ajerra1, h. lahmyed1 , a. tahiri1, n. ait abd1, a. wifaya1, m. alouani2, and r. bouharroud1 1 regional center of agricultural research of agadir, national institute of agricultural research, avenue ennasr, bp 415 rabat principale, 10090 rabat, morocco; 2 laboratory of biotechnologies and valorization of natural resources faculty of sciences, ibn zohr university, b.p 8106, agadir, morocco. *corresponding and presenting author: redouan.qessaoui@ inra.ma. abstractabstract agriculture in morocco is a strategic sector for the social and economic development of the country. the green morocco plan (pmv) and the generation green strategy are two major programs whose main objectives are to increase productivity, promote and encourage new technologies in agriculture. currently, the market gardening sector, of which tomatoes represent 84% play an important socio-economic role in morocco. it is an essential source of employment and a lever for foreign currency entry. the objective of this study is to make an inventory on the phytosanitary plan of tomato cultivation in the souss massa region. the results obtained show that four types of tomato are the most used in the souss region: round tomato, elongated round tomato, round cherry tomato and elongated cherry tomato and several types of varieties are adopted for each type of tomato. calvi is the variety most used for the round tomato with a percentage of use exceeding 35%, followed by the pristyla and pitenza variety with 20%. for the elongated round tomato, three varieties are more used, marcus, adriano and papales with 30, 35 and 35% of use respectively. angelle is the most used variety for the elongated cherry tomato, likewise creativo and genio for the round cherry tomato with 75 and 25% use respectively. the survey results also show that maxifort-type rootstock is the most used for the majority of tomato varieties in the souss massa region (20 to 100% depending on the tomato variety), followed by arazi and superpro rootstock. open-ground cultivation is dominant in the souss region by (67%). however, market gardeners are increasingly moving towards soilless crops with a rate of (33%). the most recorded yield is around 201 to 240 t/ha. indeed, 80% of tomato exports are recorded by varieties of cherry tomatoes. despite this significant tomato production, the latter is subject to attacks due to diseases and pests. tomato leaf mold and gray mold cause very remarkable economic damage. farmers in the region rely heavily on a conventional system. indeed, more than twelve active molecules have been used to combat this disease. keywords: keywords: tomato, tomato leaf mold, gray mold, souss massa region. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 822 p32. effects of organic fertilization and intercropping of po-p32. effects of organic fertilization and intercropping of potato (tato (solanum tuberosumsolanum tuberosum) with faba bean () with faba bean (vicia fabavicia faba) on mi-) on microbial load and biomasscrobial load and biomass aziza irhza1,2§, laila nassiri1, jamal ibijbijen1, fouad rachidi2, and ghizlane echchgadda2* 1 department of biology, faculty of sciences, moulay ismail university, meknes, morocco; 2 department of plant protection and environnement, ecology and environnement unit, national school of agriculture, meknes, morocco. *corresponding author : gechchgadda@gmail.coma; §presenting author: aziza.irhza@gmail.com. abstractabstract microorganisms play a very important role in nutrient cycling in agriculture and can lead to enhance soil quality and improve crop production. thus, there is a huge need to identify some suitable management practices which contribute to increases in soil microbial load and biomass. intercropping can achieve sustainable agricultural development by increasing plant diversity. organic fertilization can contribute to improve microbial biomass and diversity due to the availability of organic substrates that can be used by microorganisms as an energy and carbon substrate. potato (solanum tuberosum) is one of the most cultivated crops in morocco, but its continuous cultivation in monoculture systems represents the greater factor deteriorating soil organic matter. in the aim to break this rule, we investigated the effects of potato monoculture and potato intercropping system (potato/faba bean) under and without organic fertilization on microbial characteristics such as microbial load and biomass. the treatments are: t1 (potato sole crop), t2 (potato + organic fertilization), t3 (potato + faba bean), t4 (potato+ faba bean+ organic fertilization). the results showed that the microbial load evolved over time in all treatments, it was abundant in the intercropping model compared to the potato sole crop. organic fertilization and intercropping increased the microbial load in terms of bacteria and fungi. soil microbial biomass plays a significant role in soils, and it is often used as an early indicator of change in soil quality. we found that intercropping increased the microbial biomass, however it was strongly impacted by treatment t1 (potato sole crop). keywords:keywords: intercropping, monoculture, microbial load, microbial biomass, organic fertilization. p33. antibiofilmogram: an innovative test for personalized p33. antibiofilmogram: an innovative test for personalized treatment of pathogenic biofilm-producing bacteriatreatment of pathogenic biofilm-producing bacteria aniba rafik*, abouddihaj barguigua, asmaa dihmane, and habiba raqraq team of biotechnology and sustainable development of natural resources, polydisciplinary faculty, sultan moulay slimane university, beni mellal, morocco. *corresponding and presenting author: rafikaniba1466@gmail.com. abstractabstract pathogenic bacteria are characterized by their ability to adhere to a surface and form a protective biofilm, highly tolerant to antimicrobial agents. in routine medical microbiology, the classical techniques of antibiogram: agar diffusion method or liquid microdilution method are adapted to the evaluation of the activity of antibiotics on planktonic bacteria. the minimum inhibitory concentrations and the resulting classification of strains (resistant or sensitive) allow, in most cases, to choose an effective antibiotic therapy for an acute infection. however, we have seen that in chronic infections where a biofilm is present, bacteria are able to tolerate very high concentrations of antibiotics, which can lead to therapeutic failures, despite the administration of an antibiotic therapy classified as effective by the classical methods of antibiogram. the latter are no longer systematically predictive of therapeutic success. indeed, the methods currently used routinely in clinical microbiology do not take into account the sessile forms potentially present in a biofilm. knowing that 65% of human infections seem to involve the formation of a biofilm, it appears necessary today that the approaches chosen to predict the activity of antibiotics in vivo integrate the “biofilm” dimension of bacterial phenotypes found in an infectious context. we are therefore talking about a new generation of tests that can be described as antibiofilmogram. the realization of antibiofilmogram on clinical isolates has made it possible to highlight phenomena of inhibition and induction of biofilm formation. more precisely, aminoglycosides are able to delay bacterial adhesion. conversely, the β-lactam family has the ability to stimulate early adhesion of microorganisms. the clinical relevance of the antibiofilmogram is therefore confirmed by its ability to detect the early initiation of bacterial adhesion, to select the molecules that inhibit it and to rule out those that may induce it. combined with traditional antibiotic susceptibility tests, its application can be used to refine therapeutic strategies for the treatment and monitoring of bacterial infections. keywords:keywords: antibiofilmogram, antibiogram, bacterial infections, antibiotic therapy. p34. prevalence and antibacterial resistance of uropatho-p34. prevalence and antibacterial resistance of uropathogenic staphylococci, casablanca, moroccogenic staphylococci, casablanca, morocco aniba rafik1,4*, barguigua abouddihaj1, dihmane asmaa1, momen ghizlane2, zahoor muhammad3, nayme kaotar4, and timinouni mohammed4 1 team of biotechnology and sustainable development of natural resources, polydisciplinary faculty, sultan moulay slimane university, beni mellal, morocco; 2 laboratory of microbiology, biotechnology, pharmacology and environment, faculty of science ain chock, casablanca, morocco; 3 department of biochemistry, university of malakand at chakdala, dir lower khyber pakh-tunkhwa, pakistan; 4 molecular bacteriology laboratory, pasteur institute of morocco, casablanca, morocco. *correspondence: rafikaniba1466@gmail.com. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 823 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) abstractabstract the purpose of this research is to evaluate the resistance profile of uropathogenic staphylococci bacteria isolated from medical analysis laboratories, casablanca, morocco. in this retrospective cross-sectional research, from january 1, 2017 to december 30, 2020, a total of 4374 patients attended medical analysis laboratories for cytobacteriological examination of the urine. the culture was carried out according to the usual techniques in medical microbiology, and the antibiogram was performed according to the guidelines proposed by the antibiogram committee of the french society of microbiology (ca-sfm). data were grouped as percentages, and presented as tables and figures using ibm spss statistics 26 (ibm, armonk, ny, usa). during our research, we discovered eighteen percent of cases of urinary tract infection (utis). utis were more frequent in females 483 (63%) than male 289 (37%). the prevalence of uropathogenic staphylococci bacteria was 37.61%. coagulase-negative staphylococcal (cons) was more prevalent (92.68%). s. saprophyticus was the most frequently isolated cons (39.47%). several strains of s. aureus were also reported (7.32%). the proportion of methicillinresistant s. aureus (mrsa) among the isolates was observed in sixty-six percent. all s. aureus isolates were susceptible to aminoglycosides, quinolones, erythromycin, linezolid, cotrimoxazole, and vancomycin. also, s. saprophyticus isolates were highly resistant to fusidic acid (60%), but 66,66% of the s. heomolyticus isolates were resistant to kanamycin and tobramycin. for s. heomolyticus, s. hominis, and s. warneri we also demonstrated 100% sensitivity to linezolid, cotrimoxazole, and vancomycin. the frequency of mrsa was found to be rather high in this research study. there are additional isolates that are resistant to other antibiotics that have been studied, such as vancomycin, linezolid and cotrimoxazole. the findings have significant implications for the quality of patient treatment in both settings, including antibiotic selection and infection control procedures, as well as the need for more research. keywords: urinary tract infection, s. aureus, coagulase-negative staphylococcal, antibiotics resistance. p35. crop association and agroforestry approach for the re-p35. crop association and agroforestry approach for the resilience and sustainability of argan farmingsilience and sustainability of argan farming karra youssef1*, hallam jamal1, elame fouad2, tahiri abdelghani1, and wifaya ahmed2 1 natural resources and local products research unit, agadir regional center of agricultural research, national institute of agricultural research (inra), morocco; 2 integrated crop production research unit, regional center of agricultural research, national institute of agricultural research, agadir, morocco. *corresponding author: youssef.karra@inra.ma. abstractabstract in the arid and semi-arid areas, exploitation of water and plants calls attention in terms of the ecological, social and economic balance. climate change and drought accentuate the sensitivity to degradation as is the case for the ecosystems of argan forest in southwestern morocco where biodiversity is subjected to human pressure and grazing. consequently, cultivation of argan tree has become an essential niche for the safeguard and competitiveness of this region. however, the monoculture of the argan crop raises the question of the sustainability and health of the plants. an agroecological practice has proved to be a solution to remedy this risk like the association of crops in agroforestry approach, which could be an alternative to reduce the vulnerability of the argan farming. thus, our study consists in evaluating the adaptation of this production system, using some aromatic and medicinal plants as associated crops. the monitoring includes growth and performance of young plants of the argan tree and associated plants as well as the beneficial impact on water and soil conservation. in general, the combination of crops show beneficial effects and conserves the water state of the soil as long as the critical point of competition for light and soil nutrients is not attained, this is the reason why lowdensity planting is recommended for the argan farming as part of this agro-ecological approach. the association of the argan with underlying crops has a positive impact on the stability of the soil structure and the upgrading of its fertility, this production model demonstrates an agro-ecological option to enhance marginal areas and thus contribute to soil conservation, carbon sequestration and rehabilitation of degraded lands. finally, on the economic level, the rehabilitation of degraded lands has enabled rights holders to benefit remarkably, to enrich their harvesting schedule and at the same time to contribute to the mitigation of the impact of climate change. keywords: keywords: agroforestry, crop association, argan farming. p36. modeling using machine learning of resistance to scald p36. modeling using machine learning of resistance to scald ((rhynchosporium communerhynchosporium commune) in two specific barley genetic ) in two specific barley genetic resources subsetsresources subsets houda hiddar1,2*, sajid rehman2, berhane lakew3, ramesh pal singh verma2,4, muamar al jaboobi2, adil moulakat1,2, zakaria kehel2, abdelkarim filali maltouf1, michael baum2, and ahmed amri2 1 laboratory of microbiology and molecular biology, faculty of sciences, biobio research center, university mohammed v, rabat, morocco; 2 international center for agricultural research in the dry areas (icarda), biodiversity and crop improvement, rabat, 10010, morocco; 3 ethiopian institute of agricultural research, holetta, ethiopia; 4 icar-indian institute of wheat and barley research, karnal, haryana, 132001, india. *corresponding and presenting author: hiddar.houda@ gmail.com. abstractabstract barley production worldwide is limited by several abiotic and biotic stresses and breeding of highly productive and adapta tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 824 ed varieties is key to overcome these challenges. leaf scald, caused by rhynchosporium commune is a major disease of barley that requires the identification of novel sources of resistance. in this study two subsets of genebank accessions were used: one extracted from the reference set developed within the generation challenge program (gcp) with 191 accessions, and the other with 101 accessions selected using the filtering approach of the focused identification of germplasm strategy (figs). these subsets were evaluated for resistance to scald at the seedling stage under controlled conditions using two moroccan isolates, and at the adult plant stage in ethiopia and morocco. the results showed that both gcp and figs subsets were able to identify sources of resistance to leaf scald at both plant growth stages. in addition, the test of independence and goodness of fit showed that figs filtering approach was able to capture higher percentages of resistant accessions compared to gcp subset at the seedling stage against two moroccan scald isolates, and at the adult plant stage against four field populations of morocco and ethiopia, with the exception of holetta nursery 2017. furthermore, four machine learning models were tuned on training sets to predict scald reactions on the test sets based on diverse metrics (accuracy, specificity, and kappa). all models efficiently identified resistant accessions with specificities higher than 0.88 but showed different performances between isolates at the seedling and to field populations at the adult plant stage. the findings of our study will help in fine tuning figs approach using machine learning for the selection of best bet subsets for resistance to scald disease from the large number of genebank accessions. keywords: keywords: machine learning, rhynchosporium commune, scald, barley, genetic resources. p37. diversity and abundance of breeding birds, habitat and p37. diversity and abundance of breeding birds, habitat and nesting-support use: a relevant case in fez, morocconesting-support use: a relevant case in fez, morocco ikram douini1*, mohamed mounir1, el mostafa benka1, mohamed dakki2, and soumaya hammada1 1 agro-industrial and medical biotechnology laboratory, faculty of sciences and technology, sultan moulay slimane university of beni mellal, morocco; 2 geo-biodiversity and natural patrimony laboratory, scientific institute, university of mohammed v, rabat 10106, morocco. *corresponding author’s email: ikram.douini@gmail.com abstractabstract urban areas may impact the richness of avian species. the abundance and diversity of urban landscapes might offer breeding habitats and nesting resources for urban-adapted species. in our study, we investigated the breeding birds in urban landscapes of fez historical city (morocco), the habitat uses counting breeding habitats and nesting resources, and predicting factors. we used the line-transect method and we searched nests of birds on threes and cavities of historical walls in four habitats counting green gardens, old city walls, urban farms, and urban forests. a total of 109 nests of 13 breeding species, 12 resident-breeders, and one migrantbreeder were observed. most nests were recorded in cavities (50 nests), olea europaea (17), citrus aurantium (15), bambusa vulgaris (11), and eucalyptus globulus (7). olea oleaster, cupressus sp. hosted only 3 nests each, populus sp. and washingtonia filifera hosted only one nest each. most nests were recorded in habitats rich in nesting trees and close to water sources. on the contrary, the number of nests declined while the habitats increased surface and distance to the urban center. our data revealed the diversity, habitat use, and nesting support of urban breeding bird communities in morocco and southern slope of the western palearctic. future urban plans must integrate measures to provide suitable breeding resources such as cavities of old walls and diversity of green areas for urban birds to enhance their breeding performances, thus promoting the well-being of the population via increasing biodiversity. keywords:keywords: breeding birds, nests, habitat use, nesting trees, urban landscapes, urbanization. p38. cadmium tolerance of (p38. cadmium tolerance of (averrhoa carambolaaverrhoa carambola) and its po-) and its potential use for remediating cd polluted environmentstential use for remediating cd polluted environments luted el mellouki meryem1*, a. boulaarbah1,2, and f. kebede1 1 center of excellence for soil and africa research in africa, agrobiosciences, mohammed vi polytechnique – university lot 660, hay moulay rachidben guerir, morocco; 2 université cadi-ayyad, faculté des sciences et techniques marrakech, laboratoire bioressources et sécurité sanitaire des aliments, bp 549, m-40000, guéliz, marrakech, morocco abstractabstract carambola (averrhoa carambola), often known as star fruit, is a high-biomass and fast-growing tropical evergreen perennial tree that has spread widely throughout the tropical and subtropical regions of the world. in its tropical habitat, the carambola tree blooms nearly year-round. this tree has potential for cd phytoextraction, it can accumulate considerable amounts of cd in its shoots (about 500 mg kg−1, on a dry weight basis) without significant reduction in biomass when grown in solutions containing 5 mg cd l−1 for 3 months. rootspecific uptake of the symplastic component and root-todust translocation of cd in a. carambola is a lot more efficient compared to other non-cd accumulating species, contributing to its high ability for cd accumulation. the phytoextraction of cadmium by the carambola tree is an ideal solution for the decontamination of soils in cd, it has been proven that 50% of the cd can be extracted throughout 13 years in the soil slightly contaminated in cd. carambola is not significantly affected by zn, whereas zn transport is strongly inhibited by cd. this implies that cd accumulation in this species is mediated at least partially by zn transporters, which seem to have a greater affinity for cd than for zn. these mainly indicate that a. carambola has developed remarkable physiological mechanisms to accumulate cd at a high level. which a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 825 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) qualifies it as one of the best plants for cd phytoextraction currently discovered. keywords:keywords: averrhoa carambola; perennial tree; tropical-subtropical tree; cd hyperaccumulation; cd phytoextraction. p39. plant growth promoting potential of p39. plant growth promoting potential of bacillus subtilisbacillus subtilis isoisolated from lated from ziziphus lotusziziphus lotus oulkhir fatimaezzahra* and abdelmounaaim allaoui coalition center for innovation and prevention of epidemics in morocco (cipem), mohammed vi polytechnic university, benguerir, morocco. *corresponding and presenting author: fatimaezahra.oulkhir@um6p.ma. abstractabstract plant growth under different biotic and abiotic stresses is enhanced by the presence of plant microbiota. the latter plays an important role in the production of secondary metabolites that promote plant growth. among this microbiota we distinguish endophytes, plant beneficial bacteria that thrive inside plants and can improve plant growth under normal and stress conditions. the objective of this work is to characterize the beneficial effect of endophytic bacteria isolated from the root of the wild jujube (ziziphus lotus l.), with the aim of selecting bacteria possessing a maximum of phyto-beneficial functions. for this, we first isolated endophytes then performed a set of in vitro tests namely: p, k and zn solubilization, phytohormones production such as iaa, siderophores production, atmospheric nitrogen fixation, nacl tolerance. the identification of these strains was achieved by amplifying the gene encoding rrna 16s. this study allowed the selection of the most efficient bacterial strains, namely: bacillus, pseudomonas, and arthrobacters which have different profiles whose strain ed24 has shown a high performance. it solubilizes 108 mg/l of tcp, 7.71mg/l potassium, produces iaa, siderophore, ammonia, and cellulase, tolerates 6% nacl, also resistant to ampicillin, kanamycin, and spectinomycin. based on these results, we studied the inoculation effect of this bacterium on the germination and growth of wheat seeds, where it showed a significant difference, with a germination rate of 66.66 % compared to a negative control of 26.667%. as perspectives, we aim to study the root colonization and penetration of the de24 bacteria using gfp reporter gene. keywords:keywords: wild jujube, endophytic bacterial, antibiotic resistance, phosphate and zinc solubilization, soil fertilization. p40. the preservation of green spaces: a means to improve p40. the preservation of green spaces: a means to improve biodiversity in moroccobiodiversity in morocco chkird fadoua*, ahmed maghri, azzouz boukroute, abdelbasset berrichi, and nour-eddine kouddane laboratory for the improvement of agricultural production, biotechnology and environment, department of biology, faculty of sciences, mohammed premier university, oujda, morocco. *corresponding author: chkird.fadoua@ump.ac.ma. abstractabstract the diversity of morocco’s landscape is illustrated by its green spaces, which include multiple types such as parks, gardens, square,... each one more characteristic than the other. they assure to every city a continuity and proximity of the natural element, a reduction of the oppression of the constructed spaces and the improvement of the quality of the living environment and the urban landscape of the city. they are truly green lungs in the heart of the city. the objective of the work is to analyze green spaces according to a scientific approach in order to establish an inventory in terms of green space development. the method adapted for this study is based on a data analysis that relies on field observation, maps, satellite images, determination of certain factors, etc. with the help of software; all this information is based on archival sources, and cartographic tools. we studied the different types of green spaces, based on the one hand on the landscape analysis and on the other hand on the process of determining certain factors. the green space is constructed according to precise rules of perspective and composition in order to understand the different architectural, natural and landscape elements that characterize the space (lines, reliefs...). these elements will structure the design and their systemic action will lead to the definition of the garden type. this approach aims to bring an in-depth reflection on the green component, aspires to reposition it and capitalize the moroccan experience in this regard, as part of the preservation and enhancement of national green heritage. keywords: keywords: biodiversity, green heritage, green space, landscape analysis, morocco, parks. p41. evaluation of the food deficit of livestock goats in the p41. evaluation of the food deficit of livestock goats in the region of chefchaouenregion of chefchaouen asma al rharad1,2*, bouassab abderrahman2, acherkouk mohammed1, and ayadi mohammed1 1 ur production animale. crra, inra, tanger, morocco; 2 fst, université abdelmalek essaadi, tanger, morocco. *corresponding and presenting author: alrharad.asma@gmail.com. abstractabstract this study contributes to the evaluation of the feed deficit of the goat herd in the northern region of morocco. for a first test, the study of the food deficit of goats in pasture was carried out by collecting data on the diet adopted by three different breeders of the northern regions in cold period and which is based essentially on the pastoral plants of the region, we note the breeders of the region kalaa, bouhala and chrafat and who have herds of goats composed of goats, billy goats and goat kids. in a second test, a collection of data from three breeders in the same regions above having a main activity which is fattening goat kids. the study of the feed deficit of billy goats showed that their diet is deficient in 15% in uf and 32% in pdi. for goats showed more deficit, where it has provided high protein intake that is 118.48% (71%), cera tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 826 tainly, for a deficit of 29%. and in terms of energy, their diet provided an intake of 1.08 in uf and a deficit of 35%. for the kids on pasture, the protein intake is 85% and for uf is 0.46 uf. and for the doeling, there protein intakes were high. for the fattening kids, the inputs provided made it possible to ensure an input of 42.03 g/day in protein value, and 55% of deficit in uf. the feed provided to the goats on extensive farms assured a good portion of their needs. the feeding of the fattening kids provided a high protein value compared to the energy value and the deficit is higher in uf than in pdi. keywords: keywords: food deficit, goat, diet, uf, pdi. p42. history and inconsistencies within the rhizobiaceae p42. history and inconsistencies within the rhizobiaceae family taxonfamily taxon battas nizar1,2*, khaoula habbadi1, abdellatif benbouazza1, khalid azim3, and majida hafidi2 1 moulay ismail university, faculty of sciences meknes, laboratory of biotechnology and bio-resource valorization, faculty of sciences, meknes, morocco; 2 national institute of agronomic research, regional center of agronomic research of meknes inra-crra meknes, plant protection research unit, km 10, haj kaddour road, bp 578 (vn) 50 000, meknes, morocco; 3 national institute of agronomic research, regional center of agronomic research of agadir inra-crra agadir, integrated production of cultures research unit, b.p.124. inzegane, 80350, morocco. *corresponding author: battasnizar@ gmail.com. abstractabstract the rhizobiaceae family is one of the most inconsistent bacteria family due to the varied behavior of its species that interact with plants; while it contains multiple plant growth promoting rhizobacteria (pgpr) such as rhizobium leguminosarum (frank, 1889) which conducts symbiosis with the leguminoseae family, it also contains non-symbiotic pathogens such as agrobacterium tumefaciens, a specie that is also referred to as rhizobium radiobacter depending on the author, putting both a pgpr and a pathogen on the same genus. and while phenotypically speaking, both bacteria exhibit different behavior, genetically, they’re significantly similar since both the symbiosis and the pathogenicity is linked to different plasmids (young et al, 2001). but for some species such as ensifer (also called synorhizobium), the symbiosis is linked to the dna. since several rhizobiaceae species do not exhibit robust phylogenetic positions, it has caused controversy in taxonomic studies. currently, the rhizobiaceae family contains 25 genera, of which 4 are not validly published, 5 being a synonymous name with another genera within the rhizobiaceae family, 3 having a single entry research, with 8 valid genera having an interaction with plants rhizobium, neorhizobium, pararhizobium, agrobacterium, allorhizobium, ensifer, shinella, and ciceribacter. keywords:keywords: rhizobiaceae, taxonomy, pgpr, phytopathogens. p43. potential effects of p solubilizing bacteria in improving p43. potential effects of p solubilizing bacteria in improving wheat tolerance to cadmium stresswheat tolerance to cadmium stress saidi rym§, ibn yasser ammar, wissal elhaissoufi, meryem haddine, rachid ghani, and adnane bargaz* plant and soil microbiome sub-program, agrobiosciences, mohammed vi polytechnic university, ben guerir, morocco. *corresponding author: adnane.bargaz@um6p.ma. §presenting author: rym.saidi@um6p.ma. abstractabstract heavy metals phytotoxicity is one of the biggest abiotic threats that affects negatively cropping systems. among these, cadmium (cd) is the most abundant contaminant in soils that is known to impair plant growth and affects human health. with this in mind, the objective of this study was to examine the effect of applying cd-tolerant phosphate solubilizing bacteria (psb) to stimulate the growth and resistance of cd-stressed plants, while enhancing phosphorus (p) bioavailabililty. in our experiment, three isolates b8 (bacillus sp.), b12 (bacillus sp.) and b31 (rahnella sp.) were selected based their cd-tolerance potential (showing a minimum inhibitory concentration (mic) of 300 ppm cdcl2 (cd [300])), along with others plant growth promoting features. to determine their effect on wheat growth under cd stress, seeds were inoculated with these isolates individually (b8, b12 and b31) or in the form of consortium and grown under controlled conditions (in-vitro and inplanta) using three levels of cd (0 ppm = cd [0], 50 ppm = cd [50] and 100 ppm = cd [100]). based on the obtained results, isolate b31 belonging to the genus rahnella sp. significantly increased plant physiological parameters such as stomatal conductance (sc) and the chlorophyll content index (cci).moreover, a significant increase was reported in the plants’ morphological traits namely length, and spike length at 100 ppm of cd. the isolate b31 was found to induce a relatively high cd tolerance capacity compared to the remaining isolates by increasing plant dry weight and root morphological. on the other hand, the three isolates b12, b31 and the consortium showed a high solubilization of p in the 75-days old roots at 100 ppm of cd compared to the control with the isolate b31 showing the highest results. our findings suggest that isolate b31 may have a positive impact on stimulating plants growth under cd-stressed conditions. keywords: keywords: cadmium, wheat, rhizobacteria, phosphate, phosphate solubilizing bacteria, solubilization, tolerance. p44. behaviour, production potential and capacity for carbon p44. behaviour, production potential and capacity for carbon sequestration by different species of reforestation pine in sequestration by different species of reforestation pine in the western rif (northern morocco)the western rif (northern morocco) el aich nabila1,3*, bakhiyi belghazali2, mohamed el mderssa4, laila nassiri3, and jamal ibijbijen3 1 department of water and forests; sale, morocco; 2 national forestry school of engineers, salé, morocco; 3 environment and valorization of microbial and plant resources unit, faca tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 827 a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) ulty of sciences, moulay ismail university meknes, morocco; 4 sultan moulay slimane university, polydisciplinary faculty of béni mellal, morocco. *corresponding and presenting author: nabilaelaichenfi@gmail.com. abstractabstract the need for wood is steadily growing. consequently, the areas of artificial plantations continue worldwide to colonize forest lands at the expense of natural forest areas. today 7% of all forest lands are occupied by artificial plantations. hence the importance of their study both on ecological and economical aspects. artificial plantations based on softwood species are increasingly widespread, due to their rapid growth and easy success. nevertheless, they are very vulnerable to pathogenic agents and fire. in the western rif (northern morocco), the most used coniferous species to reforestation are [aleppo pine (pinus halepensis), maritime pine (pinus pinaster) and monterey pine (pinus radiata)]. these species are usually planted for three principal roles that can be summarised in wood production, soil and infrastructures protection, or recreation. in all cases, trees purify the air through the process of photosynthesis, thereby limiting the concentration of greenhouse gas by their potential of carbon sequestration. in the current global context marked by climate change, the present study aims to measure the contribution of the species above mentioned to mitigate the impact of climate change. therefore, we are going to deal with two main axis relating to (1) identification of the potential of wood production in order to put forward silvicultural practices, adapted to each of the three species, according to the type of stations identified, (2) quantification of carbon sequestration by the different strata of the stands (tree, shrub, herbaceous, soil), their contribution and role in limiting greenhouse gas emissions. for this purpose, stratified sampling will be used. the stratification criteria being the age of plantation, and type of substrate for each forest specie. the distribution of the sampling plots will be carried out randomly. the field and laboratory phases will afford valuable data for (1) identification of suitable environments for optimal growth of the three pines, (2) estimation of their production potential, and (3) assessment of carbon sequestration. keywords: keywords: maritime pine, radiata pine, aleppo pine, western rif, carbon sequestration. p45. effect of sulla flexuosa (p45. effect of sulla flexuosa (hedysarum flexuosumhedysarum flexuosum) on the ) on the milk production and fatty acid profile) of milk from beni milk production and fatty acid profile) of milk from beni arouss goats in northern morocco arouss goats in northern morocco soumaya boukrouh1,2*, jean-luc hornick1, nassim moula1, ali noutfia2, claire avril3, jean-françois cabaraux1, and mouad chentouf2 1 department of veterinary management of animal resources, farah, ivt, faculty of veterinary medicine, university of liège, belgium; 2 regional agricultural research centre, inra, tangier, morocco; 3 heph condorcet, agronomy category, belgium. *corresponding and presenting author: s.boukrouh@uliege.be. abstractabstract sulla flexuosa is a spontaneous and non-cultivated legume from the northern region of morocco. it is conducted on rainfed conditions and can be considered as an interesting alternative source of protein especially in the climate change conditions. in the perspective of its cultivation and use in animal feed, the objective of the present work was to evaluate the effect of its incorporation in the diet of goats on the production and fatty acid (fa) profile of milk. thirty beni arouss goats were divided into three groups and fed an isoenergetic and isoproteinic diet of either 70% sulla flexuosa hay (sf70), 35% sulla flexuosa hay and 22.5% alfalfa hay (sf35), or 50% alfalfa hay (co). the three groups received 30% of concentrates based on oat and barley grains. for three months, milk production was sampled weekly to analyze milk production and quality parameters. as a result, sulla flexuosa incorporation did not impact milk production. compared with co group, mufa, lcfa, pufa, n-9, n-6, and n-3 ratios increased while mcfa and sfa contents decreased in the sf70 group. in the sf35 group, only lcfa and mufa were increased compared to co group. moreover, sf70 diet decreased mufa/ pufa and increased pufa/sfa compared to co diet. in addition to its environmental and nutritional effects, sf enhanced health promoting indexes as its incorporation increased proportionally epa+dha. we presume that sulla flexuosa hay has positive effects on the fa profile of goat milk which will consequently positively impact consumers health. keywords: keywords: hedysarum flexuosum; hay; goat; milk production; milk quality; fatty acid. p46. effect of modified atmosphere packaging on the preser-p46. effect of modified atmosphere packaging on the preservation of strawberry and extension of its shelf lifevation of strawberry and extension of its shelf life yousra zantar1,3*, notfia younes2, amin laglaoui1, zantar said3, and mounir hassani zerrouk1 1 équipe de recherche en biotechnologies & génie des biomolécules, faculté des sciences et techniques, university abdelmalek essaadi, tanger, morocco; 2 division scientifique, inra, rabat, morocco; 3 unité de recherche sur les techniques nucléaires, l’environnement et la qualité, centre régional de recherche agronomique, inra, tanger, morocco. *corresponding and presenting author: der_zantar@hotmail. com. abstractabstract strawberry is a highly perishable fruit, due to respiration, weight loss and fungus. it is an excellent source of vitamin c, phenolic compounds. the sugars contained in strawberries promote not only the development of microorganisms but also the rotting of the fruit. the physiological and physical deterioration of the strawberry increases if the method of conservation is not controlled. the shelf life of this product, when a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 828 fresh, is limited to 1 or 2 days at room temperature. the shelflife of fresh strawberry is inversely proportional to its respiration rate. the method commonly used for preservation is for extending the shelf is low temperature. the impact of modified atmosphere packaging (map) with low oxygen & high carbon dioxide in combination with low temperature on the preservation of strawberry” palmarita variety “ was studied. the aim of the present work was to evaluate the integrated effects of map and low temperature investigate the change of quality of strawberry : ph, acidity, mass loss, decay, color, firmness, total soluble solids (tss), acidity, polyphenol, anthocyanin, microbiological quality and color, , etc. in order to establish an optimum preservation technology for strawberry. the impact of modified atmosphere packaging (map) was studied with a stable low oxygen content ( 2,5 % ) and a variation in carbon dioxide ( 5% to 20%) in combination with nitrogen on the quality parameters of palmarita strawberries. for this purpose, polyethylene terephthalate trays were used to pack the strawberries under four different initial gas compositions p1( co2, 5%),, p2(co2,10%,), p3(co2,15%,) and p4(co2, 20%,) the physico-chemical and microbiological analysis were monitored during a storage period of 12 days at a refrigerated temperature (4°c). the results showed that the map packaging conditions significantly prevented weight loss, rotting rate and improved the appearance of the strawberries, and inhibited the growth of total aerobic mesophilic flora during storage compared to the fruits packaged with ambient air that were used as reference. the map at 15% of co2 technique was found to significantly reduce weight loss, improve appearance, control rot of strawberries, and extend storage time. keywords: strawberry, modified atmosphere, quality, conservation, shelf life. p47. synergistic impacts of endophytic bacteria bacillus sub-p47. synergistic impacts of endophytic bacteria bacillus subtilis bera 71 and biochar to alleviate salinity stress in maizetilis bera 71 and biochar to alleviate salinity stress in maize abeer hashem1,2, khalid f. almutairi3, and elsayed fathi abd_allah3* 1 botany and microbiology department, college of science, king saud university, p.o. box. 2460, riyadh 11451, saudi arabia; 2 mycology and plant disease survey department, plant pathology research institute, arc, giza 12511, egypt; 3 plant production department, college of food and agricultural sciences, king saud university, p.o. box. 2460, riyadh 11451, saudi arabia. *corresponding and presenting author: eabdallah@ksu.edu.sa. abstractabstract the efficiency of salinity-tolerant endophytic bacteria (bacillus subtilis, bera 71) and biochar (bc) in maize growing in salt marsh clay soils was investigated in the current study. the endophytic bacterium exhibited plant growth stimulation and had a synergistic impact on plant growth promotion with bc in a pot experiment. both alone and in combination, these therapies alleviated the adverse impact of salt stress, resulting in improved stress metabolism and defense responses. the combined treatments alleviated the adverse impacts of salinity by suppressing oxidative stress (h2o2) and stimulating enzymatic and non-enzymatic antioxidant activities (sod, apx, cat, pod, gr, gst, dhar, mdhar, gsh, asc, proline, gb), as well as protecting cell membrane integrity (mda, electrolyte leakage). our current study provided physiological evidence provide fresh insights on the use of combined treatments (separately and/ or in combination) as a viable and environmentally benign strategy for alleviating the adverse impact of salt stress in maize. keykeywords: words: maize, salt stress, bacillus subtilis (bera 71), biochar, antioxidant, non-enzymatic antioxidant activities. p48. polyphenolic characterization and evaluation of anti-p48. polyphenolic characterization and evaluation of antioxidant activity and antimicrobial activity of caper (oxidant activity and antimicrobial activity of caper (capparis capparis spinosaspinosa) cultivars) cultivars meriem el harfi, ghizlane kabiri§, hassan latrache, hajar rizki, and hafida hanine* laboratory of industrial engineering and surface engineering, faculty of science and techniques, sultan moulay slimane university, beni mellal, morocco. *corresponding author: h.hanine@usms.ma. §presenting author: kghizlane84@ gmail.com abstractabstract currently, the research interest is focused on the study of natural antioxidant molecules, which are related to the prevention of various diseases. the present study aims to evaluate the phenolic potential, the antioxidant activity and the antimicrobial activity against ten reference pathogenic bacteria of methanolic extracts from ten moroccan caper (capparis spinosa) cultivars (v0, v1, v2, v3, v5, v6, v7, v8, v9 and vr). the results showed an interesting antioxidant activity, especially for cultivars v9, v0, vr and v2, which were registered the lowest values of ic50. however, these values are weak compared to the dpph radical scavenging capacity of ascorbic acid with ic50 = 0.12 mg / ml. in addition, the findings revealed that the caper cultivars fruits contain significant levels of polyphenols mainly for the cultivars v0, v6 and v5, whereas the cultivar v1 showed the lowest content. regarding the antimicrobial activity, it showed the efficiency of the extracts against most of the strains. the pseudomonas aeruginosa showed greatest sensitivity to the all extracts excluding v0, in contrast to salmonella sp which remained resistant to extracts except v8. for the other extracts, the bacteria reacted differently. these results reveal the richness of moroccan caper in bioactive compounds providing a very important antioxidant and bacterial activity that could provide a wide range of applications. keywords:keywords: capparis spinosa; polyphenols; antioxidant activity; antimicrobial activity. p49. seed protein and oil contents of the soybean mutant, p49. seed protein and oil contents of the soybean mutant, fm6-847, in north carolina, usafm6-847, in north carolina, usa safiah kassem1,2, aeisha mcclelland1, vanessa yepez1, yuan jiazheng1, naoufal lakhssasi3, brandon murphy1, devonta hooker1, brown barbara1, khalid meksem3, and my abdelmajid kassem1* 1 plant genomics and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc, usa; 2 wake tech community college, raleigh, nc, usa; 3 department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: mkassem@uncfsu.edu. abstractabstract in soybean [glycine max (l.) merr.], protein and oil contents traits are polygenic in nature and depend on growth and environmental conditions, and genotype. fm6-847 is a high yielding mutant developed by ethyl methanesulfonate (ems) mutagenesis at southern illinois university from cultivar ‘forrest’ (meksem’s lab). we aim at comparing fm6-847’s seed protein and oil contents with those of three usda reference lines (ld00-2817, ld06-7620, and ld07-3395) grown in a field in north carolina. fm6-847 and the reference lines seeds have been sown in a field in spring lake, nc and seed, protein and oil contents as well as several yield components have been quantified. the results showed that the mutant’s 100-seed weight (100-sw) was significantly higher (p<0.056) than 100-sw of the three reference lines. likewise, the mutant’s protein content was significantly higher (p<0.09) than the protein contents of the reference lines. however, the mutant’s plant height (ph), pod number (pn), seed number (sn), total seed weight (tsw), and oil contents were not significantly higher than the reference lines with p values of (p<0.18), (p<0.11), (p<0.1), and (p<0.22), respectively. the mutant fm6-847 generated higher protein content and seed weight but lower oil content and is a stable line; therefore, it will increase yield and profit benefitting farmers not only in nc but also in other parts of the us and the world. keywords:keywords: soybean, mutant fm6-847, seed yield, protein, oil, plant height, pod number, total seed weight. a tla s jo ur na l o f b io lo gy is sn 2 15 891 51 . p ub lis he d by a tla s pu bl is hi ng , l p (w w w .a tla spu bl is hi ng .o rg ) 829 p50. correlation between root traits and seed protein and p50. correlation between root traits and seed protein and oil contents in soybean grown under drought stressoil contents in soybean grown under drought stress ambrocio zenis1, khalid meksem2, and my abdelmajid kassem1* 1 plant genetics, genomics, and biotechnology lab, dept. of biological sciences, fayetteville state university, fayetteville, nc 28301, usa; 2 school of agricultural sciences, southern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: mkassem@uncfsu.edu. abstractabstract the root system is very important for plants including soybean because it receives water and minerals from the soil so that the plant can photosynthesize, grow, and increase its yield. the objective of this study was to grow the ‘md 96-5722’ by ‘spencer’ recombinant inbred line (ril) population (n=86) in the greenhouse under normal (group i) and drought stress (group ii) conditions, and compare the root length (rl), root surface area (rsa), average root diam¬eter (ard), and average root volume (arv) in the two groups of plants. the results showed that there is a huge variation in these traits among the parents ‘md 96-5722’ and ‘spencer’, and among their rils. for group i plants, the rl of parents and rils ranged from 20.67 cm to 2,327.88 cm; the rsa ranged from 4.57 cm2 to 1,176.79 cm2; the ard ranged from 0.38 mm to 4.04 mm; and the arv ranged from 0.08 cm3 to 47.34 cm3. for group ii plants, the rl of parents and rils ranged from 15.70 cm to 3,562.42 cm; the rsa ranged from 4.15 cm2 to 829.72 cm2; the ard ranged from 0.24 mm to 5.74 mm; and the arv ranged from 0.03 cm3 to 23.67 cm3. group i plants have higher means of rl [572.58 cm vs. 537.33 cm], rsa [201.20 cm2 vs. 165.50 cm2], ard [3.96 mm vs. 1.45 mm], and arv [6.31 cm3 vs. 5.61 cm3] compared to group ii plants which demonstrates that drought-stressed plants have reduced overall plant growth and development. however, statistically, these differences were not significant; therefore, further studies with several replicates should be conducted both in the greenhouse and the field in order to determine the effects of drought stress on the ‘md 96-5722’ by ‘spencer’ rils. keywords: keywords: root length, root surface area, average root diameter, average root volume, md 96-5722, spencer. layout 1 a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) over-expression, purification, and characterization of plant legst01, the mammalian gst-omega homolog that interacts with letpx1 from a tomato cdna library khaled sabarna1,2* and firas fohely1 1 faculty of allied medical sciences, palestine ahliya university (pau), jabal daher, p.o. box 1041 bethlehem, palestine; 2 mediterranean agronomic institute of chania (maich), alsylio agrokepiou, p.o. box 85, gr-73100, chania, crete, greece atlas journal of biology, 2020, pp. 730-740 https://doi.org/10.5147/ajb.vi0.221 abstract the study focuses on an in vivo gstomega homologue (pcrt7/tpxii intb4) over-expression, purification and characterization. experiments purport to characterize the antioxidant activity of the letpx1, the interacting glutathione s-transferases bi-gst/gpx, legst-t1, t2, t3, t4, t5 and the mammalian inhibitor of apoptosis bcl-2. upon specific expression, the proteins exerted, differential protective effects in yeast cells treated with lethal doses of the prooxidants hydrogen peroxide, t-butyl hydroperoxide, and cumene hydroperoxide. the antioxidant activity of letpx1 was highest against the cumene hydroperoxide. the overexpressing gst (omega) homologue tpxintb4 (baier and dietz, 1999) which share a considerable homology of the mammalian gst-omega1. in conclusion, the work shows that yeast parental strains are extremely sensitive to very low concentrations (0.2mm) of cumenehydroperoxide. however, after applying the different antioxidants; it appears that the smallest concentrations t to be tolerated. keywords: letpx1, tpxintb4, over-expression, purification, cdna library, gst (omega) homologue, hydrogen peroxide, tbutyl hydroperoxide, and cumene hydroperoxide, antioxidant activity. received: september 9, 2020 / accepted: november 17, 2020 ________________________________________ *corresponding author: firas@paluniv.edu.ps this is an open access article distributed under the terms of the creative commons attribution license (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 730 introduction oxidative stress reproduces different range of systemic appearance of reactive oxygen species (ros). living systems capability to freely detoxify the reactive intermediates or to restore the subsequent harm. disorders in the standard redox state of cells can lead to toxic properties through the creation of peroxides and free radicals that damage all mechanisms of the cell, including macromolecules synthesis, and particularly dna strand breaks. the destruction is mostly unintended and begins by reactive oxygen species (ros) generated, e.g. o2− (superoxide radical), oh (hydroxyl radical) and h2o2 (hydrogen peroxide) (kala, 2015) additionally, (ros) act as cellular messengers in redox signaling. therefore, oxidative stress can bring some disturbances in regular machineries of cellular signaling. production of reactive oxygen species is a predominantly damaging feature of oxidative stress. these active oxygen species consist of free radicals and peroxides. some of the less reactive of these species (such as superoxide) can be converted by oxidoreduction reactions with transition metals or other redox cycling compounds (including quinones) into more aggressive radical species that can cause extensive cellular damage (valko et al., 2005). most long-term effects are caused by damage to dna (evans et al., 2004). dna harm, made by ionizing radiation is comparable to oxidative stress, and these injuries have been concerned in cancer, aging and apoptosis. the vast majority of the oxygen-derived species are formed by typical laerobic metabolism. regular cellular resistance mechanisms suppress most of them. restoration of oxidative damages to dna is non-stop, mostly synchronized with non-renewable or a new induced injury. in the severe ranks of oxidative stress that cause necrosis, the damage causes atp reduction, inhibiting organized apoptotic death and causing the cell to just collapse (lelli et al., 1998). an obvious result of aerobic metabolism is the creation of (ros). in plants, ros’s are always made by the expected leak of electrons on o2 from the electron transference actions of mitochondria, chloroplasts, and plasma membranes or as a byproduct of several metabolic pathways localized in different cellular parts (del rio, 2006; blokhina and fagerstedt, 2010; heyno et al., 2011; foyer et al., 1997). environmental stresses such a metal toxicity, chilling, salinity, drought, and ultraviolet radiation -b radiation in addition to pathogens attack lead to greater generation of ros in plants due to disturbance of cellular homeostasis (sharma et al., 2012; hu et al., 2008; mishra et al., 2011; apel and hirt 2004). the vast majority of ros’s are very damaging to plants at extraordinary concentrations. after the level of ros go beyond the defense mechanisms, a cell is considered in a state of “oxidative stress.” the greater manufacture of ros through environmental stresses can posture a risk to cells by triggering oxidation of proteins, damage to nucleic acids, enzyme inhibition, peroxidation of lipids, activation of apoptosis leading to death of the cells (verma and dubey, 2003; meriga et al., 2004; maheshwari and dubey, 2009). 731 regardless of their damaging role, ros are well-defined as second messengers in a diversity of cellular processes including tolerance to environmental stresses (desikan, 2001; yan et al., 2007). as a result of the multifunctional roles of ros, it is essential for the cells to regulate the level of ros firmly to escape any oxidative damage and not to remove them totally. scavenging or clearing of additional ros is attained by a well-organizedant oxidative system including the non-enzymatic in addition to enzymatic antioxidants (noctor and foyer, 1998). the enzymic antioxidants contain catalase (cat), superoxide dismutase (sod), enzymes of ascorbate-glutahione (asagsh) cycle such as ascorbate peroxidase (apx), monodehydroascorbate reductase (mdhar), dehydroascorbate reductase (dhar), and glutathione reductase (gr) (. ascorbate (asa), glutathione (gsh),guaiacol peroxidase (gpx), carotenoids, tocopherols, and phenolics (noctor and foyer, 1998) work as potent nonenzymic antioxidants inside the cell. many scientists have reported that activities of various enzymes of the antioxidant protection system in plants to fight oxidative stress prompted by several environmental stresses. conservation of a high antioxidant ability to scavenge the deadly ros has been associated to enlarged tolerance of plants to previously mentioned environmental stresses. substantial development has been achieved in improving stress-induced oxidative stress tolerance in plants by developed transgenic lines with improved levels of antioxidants (faize et al., 2011). immediate expression of many antioxidant enzymes has been exposed to be more active than single or double expression for emerging transgenic plants with improved tolerance to multiple environmental stresses (lee et al., 2007). the newly characterized in vivo gstomega homologue (pcrt7/tpxii intb4) appeared to be a promising antioxidant against oxidative stress and ros systematic production in yeast. genetic background strains: escherihia coli, strain bl21(de3)plyss (invitrogen) one shot competent cells, this strain contains the lamda de3 lysogen that carries the gene for t7 rna polymerase under the control of the lacuv5 promoter. iptg is required to induce expression of the t7 promoter. plasmids: plant gst (omega) pcrt7/tpxintb4 primers for gst (omega) homologue: tpxintb4 ‘5-gaa ttc atg gct act cca agt gta caa 3’ tpxintb4‘5-ctc gag cgc caa ata ttt ttt ctt ata taa-3’ ‘5atc cag cct gac tgg ctg aa -3’ a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) 732 material and methods sds-page electrophoresis sodium dodecyl sulphate (sds) is an anionic detergent, which denature protein by wrapping around, the polypeptide backbone and sds binds to proteins fairly specifically. in by doing sds confers a negative charge to the polypeptide. in denaturing sds-page separations therefore, migration is determined by molecular weight. the resolving gel mixture was poured to the two glass plate sandwiches. this was immediately overlaid with water-saturated n-butanol to exclude oxygen from the surface. after polymerisation, the n-butanol was drained off and the gel surface rinsed with distilled water. stacking gel should be overlaid on the resolving gel up to the brim of the glass plate sandwich. are should be taken to avoid trapping any air bubble. a 10slot comb was inserted such that approximately ten 30μl wells were left on the stacking gel. the monomer was then left to polymerise. the polymerised gel slab-glass plates sandwich was removed from the casting stand and transferred to the buffer chamber, properly covered in buffer before the combs were carefully pulled out. samples were then heated in boiling water for 7 minutes to denature the proteins then micro-centrifuged for 1 min at 12, 000 rpm to avoid protein streaking during electrophoresis due to cell debris. using a prot/elec loading tip, desired amounts of the protein samples or pre-stained sdspage molecular weight standard markers were loaded into each lane. the gel run at a constant current at 40ma per gel until the dye front reached the end of the gel. gel staining/distaining coomasoie brilliant blue staining solution was used to stain gels not made for western transfer and sypro® ruby protein gel stain (regular marker). gels were stained for 60 minutes and then destined in distaining solution till bands were clearly visible. all gels were then photographed. western blotting and antibody labeling of gst (omega) western blotting involves transfer of electrophoretically separated components from a gel to solid support such as a nitro-cellulose, nylon and the hybond pvdf membrane and probing with antibodies that react specifically with antigenic epitopes of specific sequence or tag of amino acids displayed by the target protein. western blotting is extremely useful for identification and quantitation of a specific protein in complex mixture of proteins. transfer efficiency however depends on many factors including gel concentration, thickness, protein size, shape and net charge and thus critical analysis for each individual case is of utmost importance before engaging on the transfer process. in general, power supply recommendations for wet western transfer apparatus are not supposed to exceed 350ma and temperatures of the unit should remain below10°c for 1-hour transfer. the gst -omega protein was expressed as x-press epitope tagged protein for antibody screening. wet western transfer two sheets of 3mm filter papers and one piece of the hybond pvdf membrane were cut to the size of the gel or a little bit bigger. the 3mm filter membranes were then soaked in transfer buffer and the hybond pvdf membrane was activated in 100% methanol for 2 minutes then transferred into transfer buffer for 2-3 minutes. the gel was removed from the glass-plate sandwich, the stacking gel portion carefully removed and then equilibrated in transfer buffer for 5 minutes. two gels were transferred at a time. the 3mm paper, hybond pvdf membrane and the gel were then assembled on the semidry transfer sandwich in the following order, gel holder cassette, fiber pad, 3mm paper, the gel, membrane, 3mm paper, fiber pad, all air bubbles between membrane, gel and filter paper were removed by gently rolling a test tube over the surface after each individual addition of filter paper. assemble the sandwich is assembled on the electrode module in a way that the membrane will be proximal to the cathode, in the buffer tank. carefully, the unit was connected to the power supply and the transfer run under a constant current of 350ma for 60 minutes. sterile bar for distributing cool, and clean forceps was always used when handling membrane. blocking and antibody labeling at the end of the transfer period, the unit was disassembled and the membrane briefly washed in 1xtbs to remove excess methanol and buffer reagents. it was then placed in a heat-sealable plastic bag with 10% non-fat dried milk blocking solution in 1xtbs at the rate of 0.1 ml of blocking solution per square centimeter of membrane to mask potential nonspecific binding sites. air bubbles were carefully removed as much as possible before the bag was sealed and left to gently shake for 3 hours at room temperature. the membrane was then washed twice in 0.1% tween/1xtbs for 5 minutes and incubated with the primary antibody (anti x-press) in 1% non-fat milk/1xtbs for at least three hours at room temperature or overnight at 4 °c. in prior to the addition of the secondary antibody (hrp anti mouse), membrane was extensively washed with several quick changes of 0.1% tween20/1xtbs. after 90 minutes in secondary antibody with shaking at room temperature, the membrane was extensively washed with quick changes of 0.1% tween20/1xtbs for 1 hour. developing the blots the blots were developed using the peroxidase (hrp)-mediated chemiluminescent detection reagents (ecltm amer a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) 733 sham). the detection solution was prepared by mixing equal volumes of solution 1 with solution 2 to yield sufficient volume to cover the membrane. the blot was then removed from the detection solution and excess solution was removed. the blot was enclosed in a plastic wrap while avoiding applying excess pressure as this could cause high backgrounds. the blot was placed, protein side up, into a film cassette making sure that no detection solution leaked out into the cassette. a fluorescence sticker was placed on one side of the cassette for orientation purposes of the developed film. in the dark room, the blot was exposed on an autoradiography film (kodak) and the film cassette was tightly closed eliminating any possibility of light penetration for 1 minute. the film was then processed for 3-5 minutes in developing solution and then transferred to the fixing solution with a brief wash in water in between. the film was left in fixing solution till all background was faded away. optimization of signal print intensity on the film was done by immediate preparation of additional exposures based on conclusions from the signals obtained initially. colony blot (the qia expressionist) the colony blot is used for the identification of clones expressing a 6xhis tagged protein, using the procedure anti his antibody or ni-nta conjugate. in many cases the antibody specific for the target protein was not available, as a result of over washing to avoiding non-specific signals. small scale induction positive colonies from the colony blot test were picked from the lb agar plates and inoculated into 5ml lb medium containing 100g/ml ampicillin. cultures were grown at 37°c with shaking until optical density was 0.5. adding a final concentration of 0.5mm iptg induced expression of the protein. a duplicate set of the cultures was grown without induction, to serve as the un-induced control. the cultures were then grown for a further 6-h at 30 °c. at the end of the induction, 1ml sample was collected from each culture and centrifuged at 13,000 rpm for 2 min. the supernatant was discarded and the pellet was re-suspended in 100µl of 2x sab. the samples were stored at -20°c until used. large scale induction 500-ml lb medium was prepared into 1-litre erlenmeyer flasks, then autoclaved under standard conditions, then supplemented with 100μg/ampicillin and 34µg/ml chloramphenicol final concentration. cooling to around 37°c. 5 ml of an overnight e. coli culture containing pcrt7/tpxintb4 (plant gst-omega) was added and then incubated at 37°c with orbital shaking (~ 250 rpm). culture was allowed to grow until (od600 between 0.5-0.7). induction was started by adding 0.5miptg from the 20% stock to the 500 ml culture to a final concentration of 0.2%. the cultures were then grown for an additional 9 hours at 30 °c. bacterial cells were pelted by centrifugation at 4 °c in a sorvall® rc 24, gs-3 rotor (dupont) for 7minutes in large screw cup bottles. pellets were re-suspended in lysis buffer for purification and dialysis. at this stage cells may keep in –80 °c, or you can keep the pelt at – 20 °c before re-suspending it with the lysis buffer. purification and dialysis of recombinant plant gst –omega homologue it is broadly recommended to empirically establish optimal conditions with small-scale cultures before purification on a large scale is attempted; since the culture conditions and the induction affect the production of the recombinant protein. furthermore, each protein has its specificity interim of expression. as a result, the protocol for purification of gst (omega) as 6xhis-tagged proteins by ni-nta affinity chromatography of native soluble proteins described in the qia expressionist tm handbook (1999) was employed with some modifications to increase the rate of purification. while the pellet of the induced cells chilled on ice, they were re-suspended with lysis buffer 5-ml/1gram pellet, 01mm pmsf was added to a final concentration of the lysate, 0.8 mg lysozyme / 1ml lysate was added as well. the mixture was gently shaken for 20 min at room temperature, after sonication the lysate for 1015 min in ice, (the sonication and the pelleting should be repeated if the lyate is not clear enough). the lysate was centrifuged at 10,000 rpm for 30 minutes at 4°c supernatant was collected and immediately placed on ice. a 1-ml aliquot was collected from the supernatant and saved at 4°c for sds-page and western blot if necessary. the remainder of the supernatant was mixed with 2 ml of prewashed 50% ni-nta slurry per 500 ml of cleared culture lysate and the mixture incubated gently shaking at 4°c for 1 hour. washing of the ni-nta agarose beads slurry was done by adding 2 ml of well mixed 50% ni-nta to a falcon tube and centrifuging at 2,500rpm for 3 minutes, discarding the supernatant, then re-suspending the beads in equal volumes of lysis buffer and centrifuging again. the ni-nta-lysate mixture was then loaded on an empty 10 ml ploy-prep chromatography column to collect the flow-through collected and saved for sds-page and western blot analysis the rest was fast frozen by liquid nitrogen and kept in –80 °c. washing was done with 6 column volumes of wash buffer supplemented with 0.1% tx-100 and the fractions kept for analysis on sds-page and western blot, the rest were fast frozen and kept in –80°c. elution of the bound 6x-his tagged-protein in 500-µl elution buffers for four times in different microfuge tube. an aliquot was taken from each elution for sds-page, western blot, protein concentration, the remainder were stored with the same volume glycerol for fplc and enzyme activity analysis. to eliminate imidazole and the other detergent from the purified a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) 734 gst (omega), dialysis was performed. the protein was loaded into slide-a-lyzer cassettes or dialysis bags if the volume of the protein stock is relatively high. the sample was applied on the dialysis cassettes using a syringe needle, and the cassette was subsequently immersed in continuously mixed 650ml-dialysis buffer. the buffer was changed twice and sample was dialyzed overnight. estimation of protein concentration total protein concentration for all samples was estimated by the method of bradford (1976). this assay is based on the observation that the color of coomassie brilliant blue g-250 (biorad) in dilute acid solution changes proportionally as the dye binds to protein. this is accompanied by a shift in absorbance maximum from 465 nm to 595 nm. standard samples of 0-25µg of bsa were prepared as dilutions of a bsa stock solution of 1 µg/µl to a total volume of 800 ml of distilled deionized water each. seven standards with concentrations of 0, 1, 2, 4, 8, 16, and 25 µg were prepared. to each, 200 ml of the bio-rad dye were added to make a final volume of 1 ml, well mixed and left to stand for at least 15 minutes and then their absorbance at 595 nm was measured on a diode array spectrophotometer (hewlett packard 8452a). small aliquots of the collected samples with unknown protein concentration were prepared by (e.g., 10ml from each sample), mixed with 200 ml of the bradford dye and distilled h2oto final volume 1ml. after calculating the specific concentration (mg per unit volume). normalized concentration of proteins was mixed with sds-page loading buffer. once you have this, then it’s easy to calculate how many micro-liters from each sample will give you a specified number of milligrams which you can set to be equal for all sample. this way you will have loaded the same amount of total protein (µg) per well. gel filtration chromatography (size-exclusion) (akta fplc) gel-filtration chromatography separates proteins on the base of size. it measures the relative rates of passage through a molecular sieve (polysaccharide gel in the shape of spherical beads). the technique is ideal for the final polishing steps in purification when the sample volume has been reduced. buffer conditions are varied to suit the sample type or requirements for farther purification, for tpx and gst -omega we used a double filtrated 0.1m nah2po4 ph 7.5, and with 20mm gsh and 0.1m dtt depending of the on the purpose of the run. protein re-concentration (centricon) y-10 commercial centrifuging filter devices were used for the concentration and purification of biological samples up to volume 0.5and 2 ml. dehydro-ascorbate reductase (dar) activity assay the ahr activity of gsto1 was measured in standard buffer ph7, 1mmgsh, 0.1mm bis-dehydroascorbate (bisdha), by measuring the change in the absorbability of bidha at standard nm, using an extinction co-efficient of 14700 m-1 cm-1. results for small scale induction four 5-ml cultures of the above transformed cells were prepared, two of which were induced by 0.5mm iptg while the other two were grown un-induced. 6 hours after the addition of the inducer, the cells were harvested, lysed and the cells extracts analyzed by sds-page. the presence of legsto1 was confirmed by western blotting using the commercially available anti x-press primary antibody (kampranis et al. 2000). this recognizes an epitope, which is present in the 6xhis fusion protein, situated between the tag and the gsto1 polypeptide. for large-scale production of legsto. western blotting of the purified protein shows two bands of molecular weight close to 35 kda. the predicted size of legsto1 (including a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) 45 kda figure 1. deletion of plant gst-omega homologue expression from bacterial extracts using anti x press epitope anti bodies. g14 g15 g16 g17 g18 g19 g20 g21 figure 2. anti x press labeled western blot purified gst (omega) elution 2 fractions from left to right g14 → g21. 35 kda 735 the fused tag) is approximately 31kda. higher apparent size on sds-page gels indicates that this protein exhibits anomalous mobility in these conditions. gst omega (gsto1) original construct were sequenced, analyzed by specific restriction enzymes (data not shown) the recombinant human gsto1 was also found to have anomalous mobility on sdspage gels.(kampranis et al. 2000). the presence of two distinct bands on western blots is probably due to the protolytic cleavage of the c-terminus of the fusion protein during protein induction or purification (figure 2). dehydro-ascorbate reductase (dar) activity the human homologue of legsto1 has shown to exhibit thiol-transferase and dehydro-ascorbate reductase (dar) activity in this paper. we tested legsto1 for its ability to catalyze the reduction of dehydro-ascorbate. the specific activity of the purified recombinant plant-enzyme was found to be 0.059 u/mg, in good agreement with the activity of the human enzyme, which was reported (22) to be 0.16 u/mg (figure 3). legsto1 was isolated as an inter-actor to letpx, by the means of a yeast two-hybrid assay (13). to study the molecular details of this interaction, recombinant purified proteins were used for in vitro experiments. letpx was found to interact with gsh, since its presence could protect the protein from trypsin degradation. this protection could be reversed by the inclusion of legsto1 in the reaction, suggesting an interaction between legsto1 and letpx. the interaction between these two proteins was investigated further using gel-filtration chromatography. letpx was found to be a strong dimer in solution, having a complex molecular weight of ~45 kda. this dimer was stable both at reducing (dtt) and high ionic strength conditions. legsto1 was found to give rise to two peaks, one representing the monomer and one representing the dimer. when the protein contained in the fraction corresponding to the monomeric form was reapplied to the column, again two peaks were observed, suggesting the existence of a monomer dimmer equilibrium for legsto1. the monomer: dimer ratio was only fractionally dependent on dtt, indicating an interaction not significantly dependent on the presence of inter-subunit disulfide bonds (data not shown). when the two proteins were mixed together, no additional peak indicating hetero-tetramer or any other multisubunit association was observed. the inability of the two proteins to show a stable interaction in these experiments suggests the existence of a transient interaction between the two molecules. this is not stable enough to support the appearance of a clear peak in gel-filtration. letpx belongs to the family of type c plant peroxiredoxin: these proteins reduce hydrogen peroxide or alkylhydroperoxides in the expense of an intersubunit disulfide bond. this bond is then reduced by thioredoxin, which transfers the oxidation equivalents to nadph through nadph-dependent thioredoxin reductase. when letpx is isolated from bacteria and run on sds-page gels in the absence of β-mercaptoethanol, two distinct bands are evident (figure 2). one corresponding to the mono-meric form and one to the di-meric. the dimer has its active site cysteines linked with a disulfide bond. this is a result of oxidation of the enzyme during purification. addition of dtt or βmercaptoethanol results in the disappearance of the dimer band. when letpx is incubated with gsh and analyzed by non-reducing sds-page, the dimmer band disappears. it is clear that the observed interaction of letpx with gsh re a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) 1 2 3 4. 5. 6. 7. 8 figure 3. titration of gsh to investigate the effect of gsh on the removal of the dimerized tpx band. the lanes from left to right 1) 0.088mg/ml tpx +1mm gsh, 2) 0.088mg/ml tpx +0.6mm gsh, 3) 0.088mg/ml tpx + 0.3mm gsh, 4) 0.088mg/ml tpx + 0.1mm gsh, 5) 0.088mg/ml tpx + 0.06 mm gsh, 6) 0.088mg/ml tpx + 0.03 mm gsh, 7) 0.088mg/ml tpx + 0.01mm gsh, 8) 0.088mg/ml tpx . gsh 0 gsh 0 1 2 3 4 5 6 7 8 figure 4. titration of the oxidized form of gsh (gssg) to investigate the effect of gssg on the removal of the dimerized tpx. the lanes from left to right 1) 0.088mg/ml tpx, 2) 0.088mg/ml tpx + 0.005mm gssg, 3) 0.088mg/mltpx + 0.015mm gssg, 4) 0.088mg/ml tpx+ 0.03mm gssg, 5) 0.088mg/ml tpx + 0.05 mm gssg, 6) 0.088mg/ml tpx + 0.15 mm gssg, 7) 0.088mg/ml tpx + 0.3 mm gssg 8) 0.088mg/ml tpx + 6μl 0.5mm gssg. 736 sults in the reduction of the inter-molecular disulfide bond. in figure.3 the effect of increasing concentrations of gsh on disulfide bond formation is studied. at concentrations higher than 0.03 mm, gsh completely abolishes disulfide bond (see figure.3). oxidized glutathione (gssg) failed to break the disulfide bond of tpx at any concentration (figure 4). when increasing concentrations of legsto1 and directly incubated with letpx, breakage of the inter-subunit (see figure 5). disulfide of the letpx was also observed with a concomitant formation of legsto1 high order crosslinked species. this suggests that the interaction of letpx with legsto1 leads to the reduction of the former in expense of a disulfide bond on the latter (figure 6). assessing the potential of the gst-omega homology to inhibit bax lethality as shown in figure 7, expression of bi-gst/gpx, bcl2 and human gst-gst-omega1 inhibit bax lethality. expression of the plant homologous protein does not inhibit the lethality phenotype caused by bax in contrast with all the other characterized legst’s that are at least partial inhibitors of the bax phenotype. to assess the ability of the letpx1 and the legst interacting proteins to function antioxidant molecules, we evaluated their ability to enhance survival to oxidative stress induced cell death by h2o2 (see figure 8) and the lipid soluble organic hydro peroxides, tert-butyl hydroperoxide (t a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) 1 2 3 4 5 6 7 8. 9 10 figure 5. investigating the dimerization of tpx in the presence of highly purified gst-omega and the effect of 20mm gsh and 0.1mmdtt. the lanes from left to right 1) 0.088mg/ml tpx, 2) 0.088mg/ml tpx + 0.028mg/ml gst-omega, 3) 0.088mg/ml tpx + 0.07mg/ml gst-omega, 4) 0.088mg/ml tpx +0.098 gst-omega, 5) 0.088mg/ml tpx +0.098mg/ml gst-omega +0.01mm dtt, 6) 0.088mg/ml tpx + 0.8mm gsh, 7) 0.088mg/ml tpx +0.028mg/ml gst-omega + 0.8mmgsh, 8) 0.088mg/ml tpx +0.07mg/ml gst-omega+ 0.8mm gsh, 9) 0.088mg/ml tpx + 0.098 gst-omega+ 0.8mm gsh, 10) 0.088mg/ml tpx + 0.098mg/ml gst-omega+ 0.8mm gsh+ 0.01mmdtt. 1 2 3 4 5 6 7 8 9 figure 6. tpx and gst-omega dimerization in the presence of ddt. lanes from left to right 1) 0.07mg/ml gst-omega, 2) 0.07mg/ml gstomega + 0.088mg/ml tpx 3) 0.07mg/ml gst-omega +0.01mmdtt), 4) 0.07mg/ml gst-omega+ 1mm gsh 5) 0.07mg/ml gst-omega + 1mm gsh + 0.088mg/ml tpx, 6) 0.088mg/ml tpx, (7) 0.042 mg/ml gst-omega +0.088mg/ml tpx, 8) 0.084mg/ml gst-omega + 0.088mg/ml tpx, 9) 0.161mg/ml gst-omega + 0.088mg/ml tpx.0.098mg/ml gst-omega+ 0.8mm gsh+ 0.01mmdtt. 0 100 200 300 400 500 600 700 800 900 0 3 6 9 12 15 21 24 27 30 33 36 39 45 48 n o c e l l s ! "# #" $% & time (h) human and plant gst-omega growth curve pgilda/bax+4-6/bcl-2 pgilda/bax+4-6/mtc19 pgilda/bax+pjg4-6/gsto-1 pgilda/bax+tpxintb4 pggilda/bax+pjg4-6/bi-gst egy48 figure 7. expression of different antioxidant proteins partially inhibit bax lethality in yeast and restore the mitochondrial membrane potential δψm. egy48 cells expressing the lexa-bax protein with each of the different antioxidants were grown in liquid cultures containing galactose as carbon source to induce proteinexpression. 737 a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) figure 8. survival assays of egy48 yeast cells against hydrogen peroxide. freshly grown egy48, δyap, and by4741 parental strains on galraff/cm against 1n h2o2 lethality on ypd plates. figure 9. survival assays of egy48 yeast cells against tert-butyl hydroperoxide. freshly grown egy48, δyap, and by4741 parental on galraff/cm strains against 100mm t-booh lethality on ypd plates. figure 10. survival assays of egy48 yeast cells against cumenehydroperoxide. freshly grown egy48, δyap, and by4741 parental strains on gal-raff/cm show completely negative growth against 100mm cumenehydroperoxide on ypd plates. figure 11. the legsts, letpx1 and bcl-2 exhibit differential protective effect against h2o2 freshly grown egy48 cells expressing legsts, letpx1 and bcl-2 were diluted to od600 and treated with 4.5mm h2o2. figure 12. the legsts, letpx1 and bcl-2 exhibit differential protective effect against t-booh freshly grown egy48 cells expressing legsts, letpx1 and bcl-2 were diluted to od600 and treated with 1.25mm t-booh. 0 20 40 60 80 100 120 ! " # $ % "! % su rv iva l concentration h2o2 /d10-2 egy48 !yap by4741 0 20 40 60 80 100 120 0 0.5 0.75 1 1.25 1.5 % su rvi va l concentration t-booh/ d10-3 egy48 !yap by4741 0 20 40 60 80 100 120 0 0.5 0.75 1 1.25 1.5 % su rvi va l concentration t-booh d10-2 egy48 !yap by4741 0 20 40 60 80 100 120 egy48 pyes legst-t 1 legst-t 2 legst-t 3 legst-t 4 legst-t 5 bi-g st tpxii bcl-2 4.5 mm h2o2/d 10-2 control treated 0 20 40 60 80 100 120 egy48 pyes leg st-t1 leg st-t2 leg st-t3 leg st-t4 leg st-t5 bi-g st tpxii bcl-2 1.25 mm t-booh/d10-2 control treated figure 13. the legsts, letpx1 and bcl-2 exhibit differential protective effect against chp freshly grown egy48 cells expressing legsts, letpx1 and bcl-2 were diluted to od600 and treated with 0.2mm chp, letpx1 and bcl-2 were diluted to od600 and treated with 1.25mm t-booh. 0 20 40 60 80 100 120 egy48 pyes leg st-t1 leg st-t2 leg st-t3 leg st-t4 leg st-t5 bi-g st tpxii bcl-2 0.2 mm cumene hydroperoxide/d10-2 control treated 738 booh) (see figure 9) and cumene hydroperoxide (chp). (see figure 10). survival assays were initially established to determine the minimum concentration and length of exposure to prooxidants that kills at least 90% cells for three yeast strains. as shown in figure 11, expression of the legst proteins protected yeast cells from h2o2 –induced oxidative stress to varying degrees ranging from complete absence of protection for legst-t1 and legst-t4 to very high levels from legst-t2 (45%) and legst-t3 (51%). bi-gst/gpx was the third most protective protein among the legst proteins conferring 31% survival to the cells. tpxii (32%) show almost the same activity of bi-gst, meanwhile bcl-2 (6.6%) appeared as a non-active antioxidant against 4.5 mm h2o2. the behavior of the proteins against the organic hydroperoxide was dramatically different from that against h2o2. all the tested proteins were more potent in inhibiting cell death the lipid soluble hydro-peroxides. the most potent legst inhibitors were legst-t5 (80% survival for t-booh and chp) and bi-gst/gpx (approximately 75 for t-booh and chp) (figure 12). legst-t1 and legst-t4 also exhibited antioxidant activity. in the case of legst-t4 its protective effect reached 38% for chp treatment. unexpectedly, bcl-2 and tpxii turned to be very active against the organic hydroperoxide (approximately 57% for bcl-2 against chp, and 72% for tpxii against t-booh) (figure 13). in the case of the antioxidant behavior of the legst molecules, it is interesting to note that even though they belong to the same family of type iii (tau) gsts, they exhibit widely varying capacities with different pro-oxidant compounds, suggesting a specialization inside plant cells to cope with ros and environmental injury (figure 14). conclusion the implementing of the two-hybrid genomic screen afforded us with 26 proteins (kampranis et al., 2000) that interact with letpx1.our focus was on the over-expression and characterization of gstomega homologue legsto (baier and dietz, 1999) due to its significant biological and medical effects. dissimilar to other gst omega’s classes it appears to have an active site cysteine that can form a disulfide bond with glutathione (kampranis et al., 2000). the biochemical properties of these proteins were analyzed in vitro. legsto1 was found to have dehydro-ascorbate reductase activity like its human equivalent. remarkably, legsto1 was found to be capable of reducingan inter-subunit disulfide present in the oxidized form of letpx1. the plant gsto1 homologue was also tested for its capability to prevent the bax lethal phenotype in yeast. the plant homologue in difference with its mammalian counterpart did not suppress the bax phenotype. a characterization for the capability of letpx1 and gstomega to terminate bax lethality, characterization was performed as well. expression of the plant homologous to gsto1 protein does not avoid the lethality phenotype created by bax, in difference with the human gst-omega that is an inhibitor of the bax phenotype. it is well wellknown that bax protein is assumed to decline the intracellular levels of entire glutathione, reduce the mitochondrial potential and adjust organelle utility by confining to the outer mitochondrial membrane and creating an ion passage. so as to estimate the protecting consequence conferred by the recently isolated letpx1, bcl-2, and the le gsts proteins to cells under oxidative stress, the aptitude of cells to survive was evaluated by establishment of in vivo survival analyzes. hydrogen a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) figure 14. the activity of each antioxidant toward the lethality of 0.2mm cumenehydroperoxide, 4.5mmh2o2, and 1.25mmt-booh prooxidants is variable. 0 20 40 60 80 100 egy48 pyes leg st-t1 leg st-t2 leg st-t3 leg st-t4 leg st-t5 bi-g st tpxii bcl-2 antioxidant survival assays 0.2mmch 4.5mmh2o2 1.25mmt-booh 739 a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) peroxide, tert-butyl hydroperoxide, and cumene hydroperoxide prooxidants afford reactive oxygen types that can react with a multiplicity of biomolecules, changing or blocking their biological exploit. the focuses of the used prooxidants characterize the slight concentration and interval of exposure that causes above 95% death in egy48 control cells. in these tests, it was exposed that expression of the legst proteins protected yeast cells from h2o2-induced oxidative stress to variable amounts fluctuating from whole nonappearance of protection for legst-t1 and gst-t4 to actual high altitudes from legst-t2 and legst-t3. tpxii and bi-gst display almost a similar activity; in the meantime, blc-2 seemed not to be vigorous antioxidant. for the lipid soluble hydroperoxide, the most powerful legst inhibitors were legst-t5 and bi-gst/gpx. tpxii and bcl-2 exhibited important antioxidant activity against organic hydroperoxide. gst-omega homologue interactor, was expressed in e. coli and purified for additional characterization. purified le gsto1 displayed two separate bands of approximately 35 kda on sds-page gels and western blots. the two distinct bands possibly result of protcolytic cleavage of this protein through expression in bacteria. like its human complement, legsto1 exhibited dehydro ascorbate reductase activity. the precise activity of the tomato enzyme was 0,059 u/mg, which is comparable to the activity of the humanoid enzyme. for the gel-filtration experiments, legsto1 was probably shaped to be both in the monomeric and dimeric arrangement. this proposes that the tomato enzyme is pathetic dimer in solution in dissimilarity to the human enzyme, which is found mainly in the dimer form. gel-filtration experimentations unsuccessful to display any solid link between legsto1 and letpx1. thus, the contact between these two proteins that was noticed in the yeast two-hybrid assay is possibly a temporary. however, this interaction looks to be physiologically related, since legsto1 is capable of reducing the oxidized form of letpx1, as is demonstrated by the reduction of an inter subunit disulfide bridge in letpx1 by the action of legsto1. all the tested antioxidants even for the weakest. (legstt1(31.65%), and legst-t4 (37.93%) which show great deal of toxicity for yeast cells with h2o2 and t-booh. similarly, bcl-2 shows no activity toward h2o2 whereas it shows a considerable activity toward t-booh, and one of the most active antioxidants against cumene hydroperoxide (see figure 14). these results strongly indicate the high specificity of each antioxidant to the same substrate, in other words, more than one pathway or mechanism could be available for the same antioxidant independent adaptation to the generated ros. many factors could be interacting, such as the intensity of the ros, the mechanism of oxygen activation, the structure of yeast membranes or organelles, and the presence of some enzymes or factors that could suppress one pathway and initiate the other. this indicates that it is not the hydroperoxide moiety, that induces the adaptive response, and also that the yeast cells had specifically adapted to loom. considerable work needed to be done in this direction to elucidate these mechanisms and the protein complex interactions responsible for them. conflict of interest the authors declare no conflict of interest in preparing this article, the authors also declare that this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. acknowledgments the authors would like to sincerely thank all the parties that collaborate in this work. references apel k and h hirt (2004) reactive oxygen species: metabolism, oxidative stress, and signal transduction. annual review of plant biology 55: 373-399. https://doi.org/10.1146/annurev.arplant.55.031903.141701. baier m and kj dietz (1999) protective function of chloroplast 2cysteine peroxiredoxin in photosynthesis. evidence from transgenic arabidopsis. plant physiology 119: 1407-1414. http://doi.org/10.1104/pp.119.4.1407. blokhina o and kv fagerstedt (2010) oxidative metabolism, ros and no under oxygen deprivation. plant physiology and biochemistry 48 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of antioxidant enzymes in growing rice plants. plant science 164 (4): 645-655. http://doi.org/10.1016/s0168-9452(03)00022-0. yan j, n tsuichihara, t etoh, and s iwai (2007) reactive oxygen species and nitric oxide are involved in aba inhibition of stomatal opening. plant, cell and environment 30 (10): 13201325. http://doi.org/10.1111/j.1365-3040.2007.01711.x. 740 a tl as j o u rn al o f b io lo g y (a jb ) is s n 2 1 5 8 -9 1 5 1 . p u b li sh ed b y a tl as p u b li sh in g, l l c ( w w w .a tl as -p u b li sh in g. o rg ) atlas journal of biology 2018, pp. 422–545 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) proceedings of the fourth international american moroccan agricultural sciences conference amas conference iv, may 9-11, 2018, meknes, morocco my abdelmajid kassem1*, alan walters2, karen midden2, and khalid meksem2 1 plant genetics, genomics, and biotechnology lab, dept. of biological sciences, fayetteville state university, fayetteville, nc 28301, usa; 2 dept. of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901-4415, usa received: may 12, 2018 / accepted: june 9, 2018 __________________________________________________ * corresponding author: mkassem@uncfsu.edu 422 abstract the international american moroccan agricultural sciences conference (amas conference; www.amas-conference.org) is an international conference organized by the high council of moroccan american scholars and academics (hc-masa; www.hc-masa.org) in collaboration with various universities and research institutes in morocco. amas iv was held on may 9-11, 2018 in meknes, morocco. the current proceedings summarizes abstracts from 73 oral presentations and 168 posters that were presented during amas conference iv. keywords: amas conference, hc-masa, agricultural sciences. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. session i: plant health & production i (plenary session) co-chair: david lightfoot, southern illinois university, usa co-chair: ikram blilou, kaust, saudi arabia o1. detection of qtl underlying seed composition traits in soybean [glycine max (l.) merr.] masum akond1, jiazheng yuan1, shiming liu2, stella k. kantartzi2, khalid meksem2, nacer bellaloui3, david a. lightfoot2, and my abdelmajid kassem1*. oral presentations abstracts wednesday & thursday may 9 & 10, 2018 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 1 plant genomics and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc 28301, usa; 2 department of plant, soil and agricultural systems, southern illinois university, carbondale, il 62901, usa; 3 usda-ars, crop genetics research unit, 141 experiment station road, p.o. box 345, stoneville, ms 38776, usa. *corresponding and presenting author: mkassem@ uncfsu.edu. abstract improving seed composition and quality including protein, oil, and beneficial fatty and amino acids contents is an important goal of soybean farmers and breeders. this study’s aim was to use the ‘hamilton’ by ‘spencer’ recombinant inbred line (ril) population (hxs, n=93) and map quantitative trait loci (qtl) for protein, oil, fatty and amino acids contents. the genetic linkage map was created using 5,376 single nucleotide polymorphism (snp). a total of 13 qtl for the traits studies have mapped on 4 chromosomes (chr) of the soybean genome. three major qtl have been mapped to a 7–13 cm region on chr6. one major qtl for oil content (qoil001; lod=39.47, r2=76%) that explained approximately 76% of the total variation in oil content in this population; one major qtl for ala (qala001; lod=50.24, r2=74%) that explained approximately 74% of the total variation in ala content in this population; and one major qtl for palmitic acid content (qpal001; lod=2.71, r2=21%) that explained approximately 21% of palmitic acid content in this population. these firm qtls cover the approach for soybean seed protein, amino acids, oil, and fatty acids contents breeding by marker-assisted selection. keywords: soybean; snp linkage map; qtl; ril; protein, oil; fatty acids; ‘hamilton’; ‘spencer’. o2. integrative approach toward revealing and understanding complexity of root system architecture in date palm ikram blilou* plant cell and developmental biology, biological and environmental science and engineering, king abdullah university of science and technology, thuwal, saudi arabia. *corresponding and presenting author: ikram.blilou@kaust.edu.sa. abstract the evolution from the primordial aquatic organisms to vascular terrestrial plants has been accompanied by increasing complexity in the structure and functions of their vegetative and reproductive organs. plants have undergone dramatic changes in their root systems to adapt to terrestrial life. the development of complex diverse root architectures gave plants the advantage ability to colonize new and particularly arid and dry environments. date palm phoenix dactylifera fruits are known for their high nutritive, economic and social values. in arid and semi-arid areas, it plays an important role in affecting the microclimate by creating a microsystem allowing desert farming. understanding the properties of growth and development in date palm is an essential step towards gaining insights as to how plants have evolved their strategies to cope with changes in their surrounding and survive in challenged habitats like the desert. to unravel the underlying mechanisms of date palm adaptation to desert conditions we conducted a detailed analysis of date palm anatomy during different stages of development from germination to adult plants. using the art of state imaging technologies, we unraveled new developmental mechanisms in date palm occurring during germination, plant growth and development. microct xray imaging technology combined with high resolution microscopy revealed that date palm roots bear structures that have not been previously described. some of these structures are conserved only among desert palm species. in addition, a comparative studies of date palm cultivars originated from different geographical habitat, tunisia, uae and ksa and having distinct levels of tolerance to soil salinity revealed substantial differences in root system architecture. o3. enhanced nutritional quality by glutamate synthesized by transgenes improves crop growth, herbicide tolerance and reduced fungal toxin contamination. david a. lightfoot*. department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: ga4082@siu.edu. 423 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) abstract maize (zea mays l.) and other crop plants have altered transcript and metabolic profiles caused by in planta expression of the bacterial glutamate dehydrogenase (gdh; ec 1.4.1.2), encoded by a modified gdha. labeling experiments showed glutamate produced in the cytoplasm was increased 2 fold, but only by 5% in the chloroplast. the change in glutamate concentration in the cytoplasmic pool has profound effects on metabolism and homeostasis. the metabolic changes resulted in phenotypic changes that included increases in; mean plant biomass production in dry soils; nutritional value; tolerance to the herbicide phosphinothricin; and tolerance to both severe and mild water deficit improved nue and wue. crosses to waxy, flinty and brown-midrib maize were made. comparisons of transgenic and non-transgenic maize showed a 11% increase in wue and 9% increase in nue across a range of water delivery rates. resistance to rotting necrotrophs including carcinogenic aspergillus flavus and fusarium virguliforme was noted. sporulation of a. flavus was inhibited. the abundance of 747 fungal and 395 maize grain transcripts were altered suggesting the gdh maize was not supporting normal fungal growth because of metabolic and compositional alterations. cancer incidences due to toxin contamination can potentially be reduced by 50% by gdh. better nutrition will lead to less pollution. o4. the rose genome and beyond; understanding rose domestication and the mechanisms underlying major traits. mohammed bendahmane*. ecole normale supérieure (inra cnrs lyon1 ensl), lyon, france. *corresponding and presenting author: mohammed.bendahmane@ens-lyon.fr. abstract roses are of high symbolic value and have great cultural importance in many societies worldwide. they hold high cultural and economic importance as ornamental plants and for the perfume industry. the rose is well suited to be an original model organism for woody ornamental species as it has a relatively small genome size (560mbp) and it has a short life cycle for a perennial woody plant. during centuries, generations of rose breeders had fastidiously selected the showy and desirable traits of rosa species based on keen and meticulous observation. several traits, involving mainly floral quality (ie. recurrent flowering, flower form and double flower formation, scent biosynthesis, …), are of high economic importance, but the molecular and genetic mechanisms controlling these traits remain poorly understood1. during the past years, we generated a number of molecular and biotechnology tools such as reproducible genetic transformation as well as a database that provides useful information on rosa sp. genome structure and expressed genes with thorough annotation and an overview of expression patterns for transcripts with good accuracy1-3. these tools allowed us to discover the molecular mechanisms controlling the doubleflower formation4 and the biosynthesis of major scent molecules, the terpenes5. recently, we used single molecule real-time sequencing and an original meta-assembly approach to obtain a very high-quality assembly -at the chromosome levelfor the rose cultivar rosa chinensis, known to have extensively participated in rose breeding and the creation of modern roses6. resequencing of the genome of 14 major genotypes that contributed to rose domestication, along with genome diversity analyses, highlighted the mosaic origin of the genome of modern rose hybrids that combines the growth vigor traits of european species and recurrent blooming of chinese species6. expert gene annotations along with gene expression data permitted the reconstruction of gene regulatory pathways associated with major rose traits, and allowed to describe epigenetic variation landscapes along the rose genome6. reconstruction of regulatory and secondary metabolism pathways involved in scent and flower color, validated by biochemical and molecular analyses, allows us to propose models of interconnected regulation of flower color and scent compounds6. the data also provide indication on why roses evolved alternative routes to produce scent compounds, such as terpenes, in the petals. together, these resources provide a solid foundation for understanding the mechanisms governing rose traits and their diversity and will accelerate improvement in roses, rosaceae and ornamentals. recent advances will be presented and discussed. 424 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 425 selected references 1. bendahmane et al., (2013) genetics and genomics of flower initiation and development in roses. j exp bot 64(4):847-57. 2. dubois et al., (2012) transcriptome database resource and gene expression atlas for the rose. bmc genomics 13:638. 3. dubois et al., (2011) genomic approach to study floral development genes in rosa sp. plos one 6: e28455. 4. dubois et al., (2010). tinkering with the c-function: a molecular frame for the selection of double flowers in cultivated roses. plos one 5: e9288 5. magnard et al., (2015) biosynthesis of monoterpene scent compounds in roses. science 349: 81-3. 6. raymond et al., the rosa genome provides new insights into the domestication of modern roses. nature genetics (in press). o5. valorization of moroccan saffron through scientific research serghini mohammed amine*, k. lagram, m. ben el caid, l.h. atyane, m. lachheb, l. salaka, and s. el merzougui laboratory of biotechnology and valorization of natural ressources, faculty of sciences, ibn zohr university, agadir, morocco. *corresponding and presenting author: maserghini@live.fr. abstract saffron is the most expensive spice in the world. also called ‘red gold’, this spice is derived from the dried red stigmas of crocus sativus l. it is well appreciated for its color (crocin), taste (picrocrocin) and aroma (safranal). it has medicinal properties at low doses. recently, a growing interest focuses on its components which may have an anti-carcinogenic effect. world saffron production amounts to some 320 tonnes/ year. the main producing countries are: iran (94%), greece (2.2%), morocco (1.5%), india (1%), spain (0.5%) and other countries (0.7%). morocco is the largest african producer of saffron with an annual turnover of 90.000.000 dh. saffron cultivation is an important economic activity in the mountainous regions of taliouine (taroudant province) and taznakht (ouarzazate province). morocco is renowned for the good quality of its saffron, which is a flagship local product and which strongly contributes to the income of local people. improving saffron production is possible through the use of good quality plant material, but also by controlling good cultural practices and post-harvest processes. the conference will focuse on the agro-morpho-physiological stidies of saffron, it’s molecular and chemical chararization and the application of tissue culture to this species. the agro-biological characterization was applied to different provenances of saffron of high, medium and low altitude and was carried out simultaneously in the original production site for each provenance, in a site in taliouine where all the accessions were grouped and in one site with a micro-climate and altitude completely different from the original sites. preliminary information on the agro-morphometric variability of moroccan saffron under different planting sites, allowed us to make a first selection of accessions and planting conditions that show a clear superiority in the production of corms, leaves, flowers and qualitative performance. the simple sequence repeat markers were used to evaluate the genetic variability in different accessions of moroccan saffron and saffron from other countries and some of them were able to trace diversity in these accessions and assign a molecular identity. the chemical study of saffron focused on the quality of spices by evaluating the content of its three main metabolites. it also aimed to control the authenticity and to distinguish different geographical origins of this spice using uv-vis, hplc, gc, ir and colorimetric technics. different methods related to stigma drying, storage and preservation were compared. the auto-triploid nature of saffron creates problems for sexual reproduction and only vegetative propagation is possible. this propagation carries the risk of disease transmission and the regeneration rate of daughter corms under natural conditions is relatively low. this slow vegetative propagation constitutes significant bottlenecks for the development and improvement of saffron culture and in vitro micropropagation is used in order to produce large quantities of the propagating healthy material in saffron. this conference will outline the major advances of in vitro culture in direct and indirect organogenesis and somatic embryogenesis of saffron. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 426 o6. evaluation of ems induced genetic variability in quantitative traits in rapeseed (brassica napus l.) souhail channaoui1,2, mustapha labhilili3, mohamed el fechtali1, hamid mazouz2, and abdelghani nabloussi1* 1 research unit of plant breeding and plant genetic resources conservation, national institute of agricultural research, regional agricultural research center of meknes, morocco; 2 laboratory of plant biotechnology and molecular biology, department of biology, faculty of science, university moulay ismail, morocco; 3 research unit of plant biotechnology, national institute of agricultural research, regional agricultural research center of rabat, morocco. *corresponding and presenting author: abdelghani.nabloussi@gmail. com. abstract genetic variability is a prerequisite feature of any crop-breeding program and chemical mutagenesis has become a proven way of creating new variations within a crop germplasm. seeds of brassica napus l. (variety ‘inra-czh2’) were treated by ethyl methyl sulphonate (ems), with 1, 1.2, 1.4 and 1.6% doses for 6, 7 and 14 hours, and were planted to obtain the m1 plants and then the m2 plants. the objective was to evaluate the novel induced variability observed for some important traits, such as days to flowering, days to maturity, plant height, number of branches/plant, number of pods/plant, number of seeds/pod, pod length, pod diameter and 1000-seed weight. data were recorded on 10 plants taken randomly from m2 populations derived from each treatment (dose by duration), along with control plants (wild type). a large variability was observed and ems treatment had a significant effect on all the traits studied. compared to control plants, genotypes coming from seeds treated with low ems doses were earlier to flowering and maturity. also, seeds treatment by low ems doses during short time allowed obtaining some plants with increased number of pods/plant compared to the control. among the treatment levels used in the present research, 1% ems for 6 hours was found to be the most effective in improving earliness and seed yield. this latter was mainly improved due to the increase of the number of pods/plant. keywords: rapeseed, induced variability, ems treatment, earliness, seed yield. o7. antioxidant and anti-cancer activities of anacyclus pyrethrum from morocco hakima bahri1,2*, abdeslem el idrissi2, salvatore rotondo2, bouchra tazi3, samira oulahboub1,3, and chaymae benkirane1,3 1 laboratory of genetic resources and plant improvement, ecole nationale d’agriculture de meknès, bps 40, meknès, morocco; 2 laboratory of neurosciences, medical technology program, department of biology, college of staten island, city university of new york, new york, 10314, usa; 3 laboratory of chemistry, department of basic sciences, ecole nationale d’agriculture de meknès, bps 40, meknès, morocco. *corresponding and presenting author: hbahri@enameknes.ac.ma. abstract over the ages, man has relied on plants to attend to many health problems and plants. nowadays, a renewed interest in natural products has emerged in order to find safe and effective alternatives to the established chemical drugs. research efforts target plants as an inexhaustible source of naturally occuring bioactive compounds that proved useful in the treatment of many human ilements, including cancer, cardiovascular diseases and inflammatory diseases. this work aims to investigate the antioxydant and anti-cancer activities of anacyclus pyrethrum an indigenous spontaneous medicinal plant commonly prized for its roots. pyrethrum root samples were collected from the mid-atlas region. plant extracts were prepared and antioxidant activity was measured by dpph (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging method and total polyphenol and total flavonoid contents using the folin–ciocalteu and the aluminum chloride colorimetric methods respectively. we assessed anti-cancer activity against two different cell lines, hela (human cervical cancer cell line) and n2a (mouse neuroblastoma cell line) using the atp assay. cell lines were seeded in a 96 well plate at the concentration of 104 cells/well, and plant extracts were added at the concentrations 200, 150, 100, 50, 25 and 12,5 µg/ml. cell viability was asa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 427 sessed by measuring atp content by luminescence using celltiter-glo® reagent. the results obtained show that total polyphenol content ranged from 435 to 721 gae µg/100 mg of extract and total flavonoid from 166 to 721 qe µg/100 mg of extract. pyrethrum roots exhibited an important antioxidant activity with ic50 values ranging from 3.3 to 3,8µg/ ml compared to 3.1 µg/ml for ascorbic acid used as the reference antioxidant. furthermore, the cytotoxicity assay revealed a strong anti-cancer activity of pyrethrum root extracts against the two cancer cell lines used. these findings highlight the interest of anacyclus pyrethrum roots as an important source of polyphenols with potent antioxidant activity and with high cyto-toxicity effect against cancer. keywords: anacyclus pyrethrum, polyphenol content, flavonoid content, antioxidant activity, anti-cancer activity. o8. preliminary results on flowering phenology of the world olive germplasm bank of marrakech zaher hayat1*, moukhli abdelmajid1, khayat meriem1, boutzankad tfarah1,2, gaboun fatima1, sikaoui lhassane1, el modafar c.2, and khadari bouchaib3 1 institut national de la recherche agronomique, crra de marrakech, bp 533 menara, 40000 marrakech, morocco; 2 université cadi ayyad, faculté des sciences et techniques, guéliz, 40000 marrakech, morocco; 3 inra, umr 1334 amélioration génétique et adaptation des plantes (agap), 34398 montpellier, france. *corresponding and presenting author : hayatzaher@yahoo.fr. abstract a set of 591 olive accessions from the world olive collection was planted in marrakech and characterized in 2014, 2015 and 2016 for flowering phenology according to the bbch scale (the time of inflorescence buds start to swell, growth and development of inflorescences, flower opening, full flowering, full petal fall, end of flowering, fruit set). degree days of flowering stages 51 to 69 were higher in 2015 compared to 2014, but the highest flowering period was observed in 2016. a significant positive correlation was detected between all flowering stages except for stage 51, which is negatively correlated with flowering period estimated by the difference between the time of stage 69 and stage 61. variance analysis showed effect of cultivar and year on all flowering stages. stage 51 is a key stage of olive’s trees phenology; its early observation is correlated with long flowering period. flowering period observations show correlation between a short flowering period and the increase of temperature expressed by the sum of degree-days. [(t° max + t° min)/2 – 12.5°c] needed to observe stage 51. varieties with late stage 51 need more time to satisfy their minimum chilling requirements and cumulate more degreedays before reaching the stage. in southern mediterranean countries, we recommend selection of varieties with low chilling requirement (early stage 51) for better adaptation to xeric conditions in the summer period especially with the risk of global warming which is more and more considered. for future study we will focus on the estimation of the minimum chilling requirements to observe stage 51. keywords: olive, flowering, phenology, bbch scale, core, marrakech, world, collection. chilling requirement, selection. concurrent session ii: animal health & production co-chair: karen jones, southern illinois university, usa co-chair: mohammed bouslikhane, iav, morocco o1. first isolation and characterization of fowl adenovirus from a field outbreak of inclusion body hepatitis among moroccan broiler chickens. samira abghour1*, fatima tahiri1, khalil zro2, mohammed mouahid3, jaouad berrada4, and faouzi kichou4. 1 division of pharmacy and veterinary inputs, onssa, rabat, morocco; 2 biopharma, rabat, morocco; 3 mouahid’s veterinary clinic, temara, morocco; 4 hassan ii institute of agronomy and veterinary medicine, b.p. 6206, rabat, morocco. *corresponding and presenting author: abghour.samira@yahoo.fr. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 428 abstract inclusion body hepatitis (ibh) is an acute disease, mainly occurring in young broiler chicken (3-7 weeks old) and caused by several serotypes of fowl adenovirus (fadv). a very few studies were conducted in morocco based only on gross and histopathological examinations. in the present work, an outbreak of ibh in broiler chickens have been investigated through histological examination of affected birds, isolation of virus via chicken embryofibroblasts cell (cef) and detection by conventional pcr based on hexon gene. histopathology examination revealed large basophilic intranuclear inclusion bodies within hepatocytes. the fadv was isolated in chicken embryo fibroblast cell culturesinoculated with tissue homogenate of livers collected from broiler chickens with ibh. the cytopathic effects detected were in the form of rounding, clumping and detachment of cells. cell culture supernatant from second and third passages of isolateas well as the liver homogenate were subjected to pcr amplification using primer pair hexf1/hexr1, the specific product of 800 bp was detected in all samples tested. these results confirmed the implication of fadv in ibh among moroccan chicken flocks. furthermore, in order to determine what genotype and serotype involved in this moroccan outbreak, the fadv isolate is now being further characterized genetically using phylogenetic analysis based on the nucleotide sequences of the pcr product and the results will be discussed and presented. keywords: inclusion body hepatitis, fowl adenovirus, isolation, pcr, genotype, broiler chickens. o2. a comparative analysis of the economic and water use efficiencies of camel and other species in the oases’ farming systems. mohamed taher srairi1*, salah mansour1, mohamed benidir2, mohammed bengoumi3 and véronique alary4. 1 hassan ii agronomy and veterinary medicine institute, department of animal production and biotechnology, p.o. box 6202, 10101, madinate al irfane, rabat, morocco; 2 regional office of agricultural development in ouarzazate, morocco; 3 sub regional office of the food and agriculture organization, tunis; 4 international center for agricultural research for development (cirad), montpellier, france, currently based at the international center of agricultural research in dry areas (icarda), rabat, morocco. *corresponding and presenting author: mt.srairi@iav.ac.ma. abstract multiple constraints, such as an increased demographic pressure and the competition for scarce water resources, are significantly affecting farming systems in the oases. the combination of these limiting factors impacts the efficiency of inputs’ uses and hampers the incomes from the agricultural activities. livestock has always been a component of the oasis farming systems, covering a wide range of functions: transportation, soil preservation, income generation through meat and milk, draft, and saving. faced to the recent changes, this study aimed to characterize the roles and contribution of different livestock species (camel, cattle and small ruminants goats and sheep -) on the overall performances of the oasis farming systems. to do so, twelve farms illustrating four types of livestock systems previously identified (i.e. three farms per type) were selected: (i) multi species livestock rearing (sheep and cattle as well as sedentary goats of the drâa breed), (ii) dairy cattle based activities, (iii) sheep intensification with an important contribution of purchased feed and (iv) range land (camel and goats) systems. the study was undertaken in the drâa valley (south east morocco), a typical oasis context, with an average annual rainfall level not exceeding 110 mm and summer temperatures often above 45°c. the valley consists in a series of seven palm groves along a distance of 200 km following the drâa river and covering a total arable land area of 26,000 ha an annual follow-up was performed in each farm with a set of surveys coupled to field observations. inputs’ uses (water, labor, fertilizers, etc.) and incomes from crops and livestock in the study sample were determined. off farm revenues were also characterized. within each farm, we calculated common agricultural efficiency indicators to assess the efficiency use of the most critical production factors: land, labor, water and capital. the results demonstrated that efficient oasis farming systems rely on the crops/ livestock association. thereby, while providing selfconsumed food products, livestock intensified farming systems (d’man prolific sheep and dairy cattle with off-farm feed resources) allow an increase in crops’ yields and their incomes. this is particularly obvious a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 429 for date palm trees, which benefit from the surplus irrigation of the underlying alfalfa. in parallel, in specific contexts of the oases where the intensification of agriculture is impossible (for instance in downstream areas, with scarce groundwater or saline water) camels remain the main source of income, adding value to the vast pastoral areas and to the by-products of crops (wastes of dates, wheat bran and straw, etc.) and inducing very limited pressure on water resources. altogether, the results emphasize the crucial roles of camel in areas with limited assets (water, land and even capital), and suggest that such a species might be of significant interest in the near future, in case of exacerbated water scarcity, as it might happen with climate change scenarios. keywords: camel, dairy cattle, farms’ incomes, farming systems, oasis, sheep, water use efficiency. o3. bovine tuberculosis and paratuberculosis coexistence in dairy cattle in morocco meryem el mrini1*, jaouad berrada1, faouzi kichou1, beatriz romero2, ana balseiro morales3, christian gortázar4 and mohammed bouslikhane1 1 department of veterinary pathology and public health, hassan ii institute of agronomy and veterinary medicine, rabat, morocco; 2 centro de vigilancia sanitaria veterinaria (visavet), universidad complutense, madrid, spain; 3 centro de biotecnología animal-serida gijón asturias spain; 4 sabio irec universidad de castilla – la mancha, ciudad real, spain. *corresponding and presenting author: meryem.pathologiste@gmail.com. abstract bovine tuberculosis (tb) is a major endemic zoonosis in morocco, with 33% of dairy cattle farms infected countrywide with mycobacteria of mycobacterium tuberculosis complex (mtc) (fao, 2004). according to economic and public health impacts of the disease, eradication of bovine tuberculosis is an important issue in morocco. public animal health autorithy recognises the in vivo single and comparative intradermal tuberculin (idt) test as the official diagnosis assays for bovine tuberculosis field diagnosis. the in vitro interfero-gamma (ifn-γ) assay employment may be authorized to maximise detection of early infected cattle. field diagnosis results interpretations are affected by the coexistence of bovine tuberculosis and other mycobacterial infections like bovine paratuberculosis (ptb) or infection by environmental mycobacteria. we aimed to investigate the coexistence of tb and ptb in dairy cattle farms in morocco. the carcasses of 227 slaughtered idt-positive cows originating from six dairy cattle farms were thoroughly examined and tissue samples were collected from lymph nodes without tb-characteristic macroscopic changes (retropharyngeal, mediastinal, prescapular, mesenteric and iliac lymph nodes) and from ileo-caecal valve and terminal ileon. one batch of fresh tissues was stored at -20°c for isolation and identification of mycobacteria from mycobacterium tuberculosis complex (mtc) by spoligotyping and pcr and mycobacterium avium subsp paratuberculois (map) by pcr. a second batch of bovine paratuberculosis suspected samples was fixed in 10% of neutral buffered formalin for histopathological examination and immunoperoxidase immunohisto-chemistry (ihc) confirmation. preliminary results of isolation and identification yielded 24 mtc isolates by pcr and spoligotyping resulted in having six spoligotypes of mycobacterium bovis (sb0120,0121,0125,0265, 0869 and 1167). map isolation was confirmed by pcr in 3 cattle farms. immuno-peroxidase ihc proved map presence in paraffin embedded tissues in a fourth cattle farm. tb and ptb coexistence in cows was diagnosed in two studied dairy cattle farms, thus providing for the first time the evidence of tb and ptb coexistence among moroccan dairy cattle.keywords: bovine, tuberculosis, paratuberculosis, dairy cattle. o4. effect of the incorporation of the argan press cake detoxified on antioxidant activity of goat milk. nazha lakram1,2*, ikram mercha2, rachid kabbour2, el. haj el maadoudi2, abdalah el housni2, mariam naciri1. 1 laboratory of biodiversity, ecology and genome, faculty of sciences, university mohammed v of rabat, avenue ibn batoutab.p1014 rabat, morocco; 2 laboratory of food technology urpaf, national institute for agricultural research, rabat, morocco. *corresponding and presenting author: nazha.lakram@ gmail.com. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 430 abstract goat milk is rich in various physiologically functional components, including proteins, vitamins (such as vitamins e and c), flavonoïds, and carotenoids with antioxidant properties. therefore, goat milk is considered to possess high antioxidant activity that resists oxidative stability and highly protects consumers from exposure to oxidative stress, which is an important characteristic of numerous acute and chronic diseases. this study aims to investigate the effect of the inclusion of the detoxified press cake from the argan tree in animal feeding on antioxidant activity of goat milk collected from meskala region (essaouira_ morocco). the feeding trails were conducted in the farm using 18 goats of the alpine breed. they were divided in three lots of 6 goats each. the first lot received the detoxified argan press cake (da), the second lot received the non-detoxified argan press cake (nda) and the third receives the concentrate (c). the total phenol compounds content in the milk extracts was determined by spectrometry according to the folin-ciocalteu procedure and calculated as gallic acid equivalents (gae). thus, the flavonoïd content in the milk extracts was determined spectrometrically, using a method based on the formation of a complex flavonoïd-aluminum and calculated as quercetin equivalents (qe). the antioxidant activity was evaluated through scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (dpph) free radical. the results showed a significant difference (p <0.05) of the antioxidant capacity of goat milk for the lot (da) compared to the lots (nda) and (c). similarly, the polyphenol and flavonoid content were higher in lot (da) and (nda) than in lot (c). keyswords: goat milk, argan press cake, polyphenol, flavonoïd, antioxidant activity. o5. estimation of milk yield traits of holstein cattle reared at semi arid climate. abdeslem talbi* and said el madidi§. laboratory of biotechnology and valorization of natural resources, faculty of science, bp 8 106, university ibn zohr, agadir, morocco. *presenting author: talbi_abdslem@yahoo.fr. §corresponding author: s.elmadidi@uiz.ac.ma. abstract the purpose of this study is to analyze the official milk control data between 2008 and 2012 of a cattle herd consisting of 6307 holstein cows. these cows are raised in 118 private farms of dairy cattle which are members of the agricultural cooperative copag. these farms are based in the irrigated perimeter of souss-massa exactly in southwestern of morocco. the climate of this region is characterized by a semi-arid to arid climate with average temperatures variable between 19°c in winter and 27°c in summer and can reach 49°c in late spring and summer with the arrival of warm sub-saharan winds. rainfall is weak and irregular with an annual average of 180 and 280 mm per year. the production parameters were analyzed using a general linear model (glm) including fixed effects: calving season, calving year, lactation number and calving age. the averages of production recorded by this holstein herd are 6578.30 ± 1619.06 kg for the 305 days-milk yield, 7034.88 ± 1842 kg for total milk yield, 3.70 ± 0.53 % for standard fat content (fc) and 325.10±42.96 days for lactation length. the analysis of variance of production parameters depending on non-genetic factors demonstrated very highly significant effects of the calving year and calving season (p<0.001) for all the parameters of production studied, with high production during the cold seasons (autumn and winter) comparing to hot seasons (spring and summer). the lactation number has a very highly significant influence (p<0.001) for all parameters except the fat content whose effect id non-significant (p>0.05). whereas, the effect of calving age was non-significant (p>0.05) for these parameters of production. the production of this herd has shown a great variation between animals, which reveals the heterogeneity of this holstein herd. the great sensitivity of cows to seasonal and interannual variations shows the important effect of heat stress caused by high temperatures in hot seasons. keywords: holstein cattle, milk yield, non-genetic factors, semi arid climate. o6. prevalence of campylobacter spp. in industry broilers from the area of marrakech-safi, morocco. raja asmai1*, reda triqui1, hakim karib1, khadija essoucratti2, brahim bouchrif3, and rachid gradess3. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 431 1 department of pathology and veterinary public health, agronomic and veterinary institute hassan ii, rabat; 2 department of biology, ain chock faculty of sciences, hassan ii university, casablanca; 3food microbiology laboratory pasteur institute of morocco, casablanca, morocco. *corresponding and presenting author: rajaevet@gmail.com. abstract in morocco, the incidence of human campylobacteriosis has not been evaluated so far. prevalence studies are a prerequisite for any risk assessment studies. the current investigation aimed at studying the prevalence of campylobacter spp. in farm broilers in the marrakech-safi area of morocco. this was carried out in the period of july-october 2017. 90 samples of cloacal swabs were collected from broilers in the 8 provinces of the study area. this represented 30 farms with an average of 3 broilers per farm. isolation and identification of campylobacter spp. were performed according to a moroccan standard methods and recommendations of the oie and who (nm o8.o.135/ standard nm iso/ts 10272-3, 2013). thermotolerant campylobacter species (c. jejuni and c .coli) were isolated in 73,34 % of the farms investigated. our results revealed the presence and the probable spread of these two species in the broiler farms within the 8 provinces of the study area. such results are in agreement of an increasing risk of broiler carcass contamination at the slaughter level, along with the possible hazard transfer to consumers under circumstances of either undercooking or cross-contamination at the preparation level. keywords: thermotolerant campylobacter, cloacal swabs, prevalence; broiler industry. o7. epidemiological investigations of small ruminant abortions in the regions of khemisset and el hajeb. faouzi kichou*, fatima ezzahra belhassan, mohammed bouslikhane, ahlam kadiri, and jaouad berrada. département de pathologie et santé publique vétérinaires, iav hassan ii, rabat, morocco. *corresponding and presenting author: f.kichou@iav.ac.ma. abstract abortions in small ruminants deserve particular attention because of their heavy economical burden. the main objective of this study was to evaluate the impact of abortions in small ruminants in two regions of morocco (khémisset and el hajeb regions) and to determine the seroprevalence of main abortive infections (brucellosis, chlamydophilosis, q fever, toxoplasmosis and neosporosis) and their involvement in some abortion outbreaks. sero-epidemiological survey was carried out in 57 flocks (36 sheep flocks, 14 herds of goats and 7 mixed herds) originating from 4 rural counties (had lghoualem, ait ichou, ait bourzouine and ait hraz allah). data about herd structure, reproductive performances and abortion events were collected. in parallel, a total of 433 blood samples were taken from 329 sheep and 104 goats with or without history of abortion. harvested sera were examined for brucellosis using rose bengal test (trb) and then subjected to elisa in order to search for chlamydophilosis, q fever, toxoplasmosis, and neosporosis. results revealed abortion rates ranging from 1.5-10% in sheep and from 0.8-7.3% in goats in the county of had-laghoualem, from 8-11.2% in sheep and from 5.8%-44.4% in goats in the county of ait ichou, from 0.8%-10% in sheep and from 5-8% in goats in ait-bourzouine, and abortion rates ranging from 0.5-5% in sheep in the county of ait-hrazallah. among the examined 57 flocks/herds, 47.3% were positive for toxoplasmosis with a seropositivity rates (sr) of 21.2% in sheep and 2% in goats, 40.3% tested positive for chlamydophilosis with a sr of 9.4% in sheep and 36.5% in goats, 45.6% were positive for q fever with an sr of 10% in sheep and 8.6% in goats, and 21% were tested positive to neosporosis with a sr of 4% in sheep. no flock was found to be serologically brucellosis positive. these results reflect the relative importance and implication of these abortive infections in the occurrence of abortions and the need for confirmation by direct examinations. furthermore, the present work documents for the first time in morocco the serological evidence of infection with neospora caninum in sheep, which deserves further investigations. keywords: abortion, small ruminants, soats, sheep, khémisset, el hajeb, brucellosis, chlamydophilosis, q fever, toxoplasmosis, neosporosis, serology. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 432 o8. spring and autumnal hormonal variations of ouled djellal ewes in algeria azdinia zidane1* and sarra metlef2 1 laboratory of natural bio-resources, department of biology, faculty of nature and life sciences, hassiba benbouali university of chlef, po box 151, (02000) chlef, algeria; 2 laboratory of natural bio-resources, department of nutrition and food sciences, faculty of nature and life sciences, hassiba benbouali university of chlef, po box 151, 02000 chlef, algeria. *corresponding and presenting author: azdinia. zidane@yahoo.fr. abstract the photoperiodism represents the capacity of plants and animals to measure the duration of day ; this process implies certain biological calender which allows the bodies to determin the convenient moment of the year to make a commitment in appropriate seasonal adaptations. during the annual cycle, animals are often exposed to a range of gradually increasing and decreasing photoperiods ; these seasonal annual variations of photoperiod are at the origin of numerous biological and behavioral changes in animals. so, it is admitted that the photoperiodism is the main factor controlling the seasonal variation of the sexual activity of ewes. however, the external manifestations of heat in ewes are difficult to observe, as they are very discreet. so, the use of indirect methods of heat detection or the control of cycles is therefore justified in ovine species. the purpose of this study was to check if the ouled djellal ewes followed a seasonal or an unseasonal profile in the region of chlef, and to study the hormonal profiles: pituitary hormones (fsh and lh) and gonadal hormones (progesterone and œstradiol). the study involved 60 ewes of ouled djellal breed, adults, healthy and non-pregnant. the blood samples were carried out from the jugular vein, 2 times a week in spring (april to june) and in autumn (october to december). the hormones have been assayed using the elisa technique. the results did not reveal any significant difference (p < 0.05) between hormone concentrations during both seasons. it is so showed that the ovarian follicular activity of the ouled djellal ewes in the region of chlef is also present during the seasonal light anoestrus; this leads us to confirm the unseasonal profile of this breed. keywords: autumn, hormonal assay, fsh, lh, œstradiol, progesterone, spring. concurrent session iii: bioactive compounds & genetic diversity co-chair: el hassan achbani, inra, morocco co-chair: karen midden, southern illinois university, usa o1. environmentally and genotypic induced shifts in reproductive phenology of some almond [prunus dulcis (mill.) d.a. webb] cultivars grown in north-eastern morocco el hassan sakar*, mohamed el yamani, abdeali boussakouran, and yahia rharrabti laboratory of natural resources and environment, polydisciplinary faculty of taza, unviersity sidi mohamed ben abdellah, b.p 1223, taza, morocco. *corresponding and presenting author: hassan.sce@ gmail.com/elhassan.sakar@usmba.ac.ma. abstract in morocco, almond [prunus dulcis (mill.) d.a. webb] is the most important nut crop among the rosaceous family both in terms of cultivated acreage and production. the modern moroccan orchards are characterized by the dominance of some commercial cultivars such as marcona, fournat de brézenaud, ferragnès, ferraduel, and tuono which were released by european almond breeding programs. to optimize agronomic practices in almond orchards (pollination, pruning, harvesting …), key phenological events must be known. unfortunately, an accurate knowledge of their phenology is far to be investigated before, hence the novelty of our work. we investigated some key reproductive phenological events of almond from the five widely almond cultivars: marcona (mar), fournat de brézenaud (fbz), ferragnès (frg), ferraduel (frd), and tuono (tuo). this study was undertaken over two crop seasons (2016 and 2017) across five different sites from north-eastern morocco namely: aknoul a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 433 (ak), bni hadifa (bh), rislane (r), sidi bouhria (sb), and tahar souk (ts). these cultivars were marked in triplicate. since 2015 season, these were visited periodically and their reproductive phenology was assessed according to the general bbch scale and reported as julian days. phenological events investigated consisted were as follow: initial blooming date (bbch61), full blooming date (bbch65), final blooming date (bbch69), blooming duration, and ripening date (bbch89). anova analyses demonstrated significant effects of cultivar, site, crop season, and their interactions on the whole studied phenological events. lsd’s test demonstrated wide variabilities between cultivars as regards to their reproductive phenology. besides, important shifts were detected among the five localities, but also between the two crop seasons for flowering and ripening times. the north’s sites (ak, bh, and ts) showed an extended blooming duration as compared to the eastern’s sites (sb and r). blooming time in our cultivars ranged from middle (fbz and mar) to later (frd, frg, and tuo). our results revealed also, a strong negative correlation was found between initial blooming date and blooming duration, while ripening time was independent from blooming time. shifts in ripening time between cultivars are highly desirable trait when several cultivars are grown simultaneously in the same orchard. keywords: reproductive phenology, commercial almond, north-eastern morocco. o2. gas chromatography-mass spectrometry profile of essential oil from asteriscus graveolens (forssk) and its antifungal activity against fusarium oxysporum f. sp. albedinis, the causal agent of “bayoud” disease on date palm. el mustapha chibane1*, adil essarioui2, mohamed ouknin1, amal boumezzourh1, and lhou majidi1. 1 laboratory of natural substances & molecular synthesis and dynamics. fst, errachidia, morocco; 2 national institute for agricultural research regional center, errachidia, morocco. . *corresponding and presenting author: elmustaphachibane@gmail.com. abstract date palm (phoenix dactylifera) is an important food source and commercial perennial crop in the sahara and north africa. “bayoud”, a vascular wilt disease, incited by the soilborne fungal pathogen fusarium oxyxporum f. sp. albedinis (foa), has become a serious threat to date production in date palm-growing regions in morocco. substantial body of research reported antifungal properties of a wide range of plant extracts. the present work aims to determine chemical composition and anti-foa activity of essential oil extracted from the aromatic and medicinal plant asteriscus graveolens (forssk). gas chromatography-mass spectrometry analysis revealed the presence of twenty-five distinct chemical components in the essential oil samples. mycelial growth and spore germination of foa were found to be strongly inhibited by the oil when tested using agar dilution assay. the minimum inhibitory concentration of the oil was 0.6% (v/v), and its fungicidal effect was reached at a concentration of 13.33% (v/v). these findings contribute significant insight into the potential of using plant extracts for controlling “bayoud” disease. keywords: “bayoud” disease, asteriscus graveolens (forssk), fusarium oxysporum f. sp. albedenis, antifungal activity o3. antioxidant activity of camel milk: effect of incorporation of argania spinosa’s sub products. ikram merch1,2*, nazha lakram2, med rachid kabbour2, aouatef benali2, fouzia zkhiri1, and el haj el maadoudi2. 1 laboratory of virology, microbiology & quality / eco-toxicology & biodiversity university hassan ii, faculty of science and techniques of mohammedia, pb 146 mohammedia 20650 morocco; 2 the national institute of agronomic research, rcar-rabat, pb 6570, institutes rabat, 10101, rabat morocco. *corresponding and presenting author: ikram.mercha@ gmail.com. abstract phenolic compounds are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food. many studies reported that beneficial bio molecules compounds have been identified from various parts of the argania spinosa’s (l.) skeels and can play an important role in fighting diseases and could be used in pharmaceutical and a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 434 personal care products. in this present study we investigate the effect of the incorporation of the argan tree’s sub product in animal feeding, on the antioxidant activity of camel milk collected from essaouira region and compare it to the milk of a control population argan free. the content of total phenolics in the extracts was determined spectrometrically according to the folin-ciocalteu procedure and calculated as gallic acid equivalents (gae). the antioxidant properties were evaluated by the scavenging test of the diphenyl picryl hydrazyl (dpph) radical. the results obtained show that the polyphenol content of camel milk extracts following a diet based on the argan sub products is higher compared to that of the control group, also a greater capacity to trap the dpph radical with an average of 82.85%. keywords: camel milk, argania spinosa, polyphenolic compounds, antioxidant activity. o4. preservation of aromatic and medicinal plant resources: case of thymus spp. zineb nejjar el ansari* and ahmed lamarti laboratory of plant biotechnology, department of biology, faculty of sciences, abdelmalek essaadi university, m’hannech ii, b.p.2121, tetouan, morocco. *corresponding and presenting author: zinebnejjarelansari@hotmail.com. abstract the extension of thymus spp. culture, as an endemic species in morocco and as producers of essential oil rich in bioactive molecules (thymol, p-cymene, γ-terpinene, β-caryophyllene, carvacrol...), requires to make a very large number of plants available to farmers in a very short time. these plants must be able to give a high yield of essential oil and be morphologically and phonologically homogeneous, to facilitate cultural operations. if the high oil content can be obtained by varietal selection, the obtaining of homogeneous material passes through in vitro propagation. this latter comprises a number of techniques divided into three categories, namely micropropagation by apex culture, micropropagation by adventitious budding and somatic embryogenesis. to find a large-scale application, propagation methods must be reliable, simple and reproducible. for now, only micropropagation from axillary buds seems to be the technique to develop, if we can reduce culture cycle and significantly increase the rate of multiplication to make production profitable. thus, we established an in vitro vegetative multiplication protocol from stem nodes of three thymus species (thymus broussonetii bois., thymus pallidus coss., thymus vulgaris l.). as a result, we found that mineral and hormonal compositions, as well as the succession of different culture media influenced the multiplication and development of explants. indeed, ms (murashige and skoog) and n30k (margara) macronutrients give the best results. in addition, the type of cytokinin present in the medium plays a determining role in the proliferation of apex. also, the presence of polyamines seems to be favorable for in vitro propagation. on the other hand, the acclimatization of rooted explants of 1216 weeks has been successfully established. thereby, this study suggests that the production of thymus vitro-plants still needs to be improved, by modernizing protocols that provide producers in the sector with plant material that is homogeneous, adapted to the growing conditions and performing well in terms of yields and costs. keywords: preservation, aromatic and medicinal plants, thymus spp., apex culture, in vitro propagation. o5. chemical composition, antioxidant, and antibacterial acitivity of argania spinosa pulp. fatima zahrae zouhair1,2*, loubna rachidi2, younes ennahli2, aouatif benali2, mohamed rachid kabbour2, mohamed bouksaim2, and azzouz essamri1. 1 laboratory of agroresources and process engineering, faculty of sciences, university ibn tofail, b, p14000 kenitra, morocco; 2 laboratory of food technology urpaf, national institute for agricultural research, rabat, morocco. *corresponding and presenting author: fatidoc.89@gmail.com. abstract argan (argania spinosa (l) skeels), endemic and emblematic tree to southwestern morocco, where it represente the lonely species of the tropical of sapotaceae, occupies an area of around 820,000 ha (charrouf et al., 2002). it is the most remarkable in north africa, due to its economic, botanical and bioa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 435 ecological interest as well as its social value. the yield of fresh fruit varies according to tree, environment and climate season. it is 500 kg / ha / year per hectare on average (m’hirit, 1989) and about 15 kg / tree (rahali, 1989). recently, many beneficial bio-molecules compounds have been identified from various parts of the a. spinosa, which can play a beneficial role in fighting disease and could be used in pharmaceutical and personal care product industries. the puprose of this work is to optimize the parameters extraction of bioactive molecules from the pulp (pericarp) and oil cake part of argania spinosa (l.) skeels (a. spinosa) of tiznit region. the first step was devoted to study the effect of solvent polarity and extraction technique on the phytochemical screening and quantitative caracterisation of the extracted compounds. the second step was designed to the investigation of the antioxidant capacity and the antibacterial activity of the extracts containing more phenolic compounds. keywords: argania spinosa pulp, optimization, extraction, bioactive molecules, antioxidant, antibacterial activity, phenolic compounds. o6. validation of antifungal activity of essential oils from competitive-ruderal plants khadija basaid1*, b. chebli1, j.n. furze2, e.h. mayad2, and r. bouharroud3 1 biotechnology and environmental engineering team, laboratory for process environmental and energy engineering, national school of applied sciences, ibn zohr university, po box: 1136/s, agadir, morocco; 2 laboratory of biotechnologies and valorization of natural resources, faculty of sciences agadir, ibn zohr university, agadir, morocco; 3 integrated crop production unit, institut national de la recherche agronomique, agadir, morocco. *corresponding and presenting author: khadija.basaid@gmail.com. abstract senecio glaucus ssp. coronopifolius (maire) and ridolfia segetum (l.) are competitive-ruderal plants growing in morocco. their essential oils were obtained by hydro-distillation, using a clevenger-type apparatus, and tested for antifungal activity in vitro against botrytis cinerea. s. glaucus and r. segetum produced 68% and 80% inhibition of mycelial growth at 640 µl in the volatile phase, and 83% and 98% at 16 µl/ ml by the poisoned food method respectively. essential oils evaluated in this study are antifungal agents against b. cinerea, and can therefore be considered as a natural substitute for chemical pesticides. this study constitutes the first step in calculation of inhibition of the fungus by the oils. qualification and quantification of essential oils will specify measurable units, creating prediction of inhibition. keywords: senecio glaucus, ridolfia segetum, competitive-ruderal, essential oils, antifungal, botrytis cinerea. o7. evaluation of antioxidant and phytotoxic activity of extracts of flower buds of capparis spinosa fatima-zahra ennacerie1*, fouzia rhazi filali1, najia moukrad1, amar bentayeb2, and mohammed bouidra2 1 department of biology, team microbiology and health, laboratory chemistry biology applied to the environment, faculty of science, university moulay ismail, meknes, morocco; 2 department of chemistry, team physical chemistry condensed matter, faculty of science, university moulay ismail, meknes, morocco. *corresponding and presenting author: amsam.ennacerie@gmail.com. abstract the purpose of this study is to valorize the spontaneous capparis spinosa plant harvested from western morocco and to rationalize its use by evaluating its antioxidant activity and to test the degree of its toxicity on plant eukaryotic cells. the antioxidant activity of aqueous and ethanolic extracts of flower buds was evaluated by two methods frap and dpph. the cytotoxicity of the extracts was evaluated by the seed germination inhibition test of lepidium sativum. the different extracts have an antioxidant activity that is similar to that of vitamin c for the aqueous extract of flower buds. the test of toxicity of the aqueous extract of this organ is revealed by anti-germinating effect of moderate intensity, which is reversible after rehydration of lepedium sativum seeds. his antimitotic activity is expressed by inhibition of elongation and growth seedlings. capparis spinosa is a plant of nutritional qualities particular for its high antioxidant effect, and its tolerance by eukaryotic cells vegetaria tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 436 an. keywords: capparis spinosa, flower buds, ethanol extract, 10% decoction, antioxidant activity, antimitotic effect, germination, lipidium sativum seeds. o8. multivariate screening of locals and foreign figs (ficus carica l.) in northern morocco lahcen hssaini1,2*, hafida haninie2, rachid razouk1, ibtissame guirrou1,3, assia ejjilani1,2, abderrahman mekaoui1, and jamal charafi1§ 1 national institut of agricultural research (inra), regional center of meknes, morocco; 2 laboratory of bioprocess and bio-interfaces, faculty of science and technology, beni mellal, morocco ; 3 laboratory of food technologies, qualipole, meknes, morocco. *presenting author: hssaiini@gmail.com. §corresponding author: jcharafi@gmail.com. abstract this work aims to assess pomological and biochemical characteristics of fig fruit of 140 known local and foreign genotypes belonging to inra meknes collection in ain taoujdate. multivariate analysis was made on the basis of 43 pomological and biochemical characters on samples of 15 fruits for each genotype. results showed a very high significant variability in pomological traits between genotypes except for ostiole width. fruit weight varied form 12.39±0,9 g to 87.03±2,8 g with an average 36,04±1,84g. also,a very highly significant variability was revealed among samples based on their colorimetric parameters. more than 65% of genotypes have a very high lightness index (l*) values that varied between 46 and 83.6. the variables chart has shown some strong correlations varied from r²=0.4 (p<0.05)to r²=0.86 (p<0.01) distinguishing neck and ostiole dimensions from the fruit form descriptors. however, the total soluble solids was less discriminant. the agglomerative hierarchical clustering based on pearson correlation has revealed tree distinct and homogeneous groups. particularly, the genotype “grosse dama blanche 2259” has been largely distinguished from the others. the qualitative characterization has highlighted an important variability among genotypes regarding their morphological quality. the results showed a dominance of globose (67%) and ovoid (70%) shapes of figs. about 82% of genotype present ribs and over than 72% present skin cracks. the multiple correspondence analysis (cma) has revealed tree groups especially differentiated by shape, color, juiciness and pulp characteristics. biochemical analysis has also revealed a very high significant variability among samples in total soluble sugars, total phenols and total flavonoids contents. the values recorded, attest of a high nutritional potential of the evaluated genotypes. keywords: ficus carica l., pomology, biochemical analysis, characterization, multivariate analysis. o9. characterization and phylogenetic diversity of allorhizobium vitis isolated from grapevine in morocco khaoula habbadi1,2,3*, quentin duplay3*, david chapulliot3, isabelle kerzaon3, rachid benkirane2, abdellatif benbouazza1, florence wisniewski-dyé3, céline lavire3, ludovic vial3, and el hassan achbani1 1 laboratoire de recherche et de protection des plantes urpp-inra-meknès, morocco; 2 laboratoire de botanique, biotechnologie, et protection des plantes, faculté des sciences, kenitra, morocco; 3 université de lyon, université claude bernard lyon 1, cnrs, umr 5557, ecologie microbienne, inra, umr1418, 10 villeurbanne, f-69622, france. *corresponding and presenting author: khaoula405@gmail.com. abstract crown gall was observed in recently planted vineyards of the meknes region (morocco). 82 isolates from 10 affected vineyards were characterized by reca sequencing and were found to belong to a. tumefaciens genomospecies g1, g4 or g7, rhizobium rhizogenes, and to allorhizobium vitis. only the 39 a. vitis isolates showed to be pathogenic in vitro on tomato and mlsa phylogenetic analyses revealed a poor genetic diversity, with the definition of only four genomic groups. definition of the a. vitis genomic groups correlates with specific pathogenic traits: indeed, genomic groups differ with respect to the severity of hypersensitive response symptoms on tobacco leaves, the intensity of necrotic response on grapevine explants, and opine profiles. both vitopine and octopine were detected by uhplc in tumors induced by isolates of three genomic groups, an opine signature scarcely ever reported. keywords: crown a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 437 gall, grapevine, allorhizobium vitis, agrobacterium tumefaciens, phylogenetic diversity. concurrent session iv: water & soil management co-chair: clarice coyne, usda-ars, pullman, wa, usa co-chair: ahmed elamrani, university mohamed i, morocco o1. enhancing faba bean (vicia faba l.) germplasm for increased resilience to temperature extremes and improved nutrition quality. jinguo hu1*, fouad maalouf2, yu ma3, erik landry1, haixiao dong3, charles hawkins3, zhiwu zhang3, clarice coyne1, long-xi yu1, aladdin hamwieh4, and shiv kumar5. 1 usda, agricultural research service, western regional plant introduction station, washington state university, pullman, wa, usa; 2 international centre for agricultural research in dry areas (icarda), terbol-lebanon; 3 department of crop and soil sciences, washington state university, pullman, wa, usa; 4 international center for agricultural research in dry areas (icarda), cairo egypt; 5international center for agricultural research in dry areas (icarda), rabat-morocco. *presenting and presenting author: jinguo.hu@ars.usda.gov. abstract our research on the enhancement of faba bean (vicia faba l.) has the overall objectives of increasing resilience to temperature extremes and improving nutritional quality. we conduct traditional field/greenhouse experiments and adopt contemporary genomics approaches in our independent and collaborative research projects. we released four winter-hardy germplasm lines for cover and pulse crop development and we quantified the low molecular weight carbohydrates in mature and immature seeds of 40 faba bean genotypes. more recently, in collaboration with icarda, terbol, lebanon and supported partially by a grant from the cgiar-u.s. university linkages program, we collected data for eight agronomic traits on an icarda faba bean reference set of 140 accessions grown under high temperature at different locations in lebanon and usa. we genotyped the reference set with the genotyping by sequencing (gbs) technique which produced approximately 4 million reads per genotype on an illumina hiseq 2500 sequencer. the single nucleotide polymorphism (snp) marker calling program uneak (universal network enabled analysis kit) found 10,950 variant loci from the sequence data. after fileting snps with missing rate over 60% and minor allele frequency less than 5%, 2,632 snps were obtained for final analyses. the final dataset contained 131 accessions with snp call rate above 70%. the missing snps were imputed by linkimpute. genome-wide association study (gwas) was performed using the program blink (bayesian-information and linkage-disequilibrium iteratively nested keyway). four snp markers were identified to be significantly associated with plant height (2 snps) and seed size (2snps). we look for new opportunities in collaborative research on faba bean germplasm, genetics and genomics. keywords: faba bean, low molecular weight carbohydrates, heat tolerance, cold. o2. agronomic performance of commonvetch (vicia sativa l.) and forage pea (pisum sativum l.) in pure culture and mixed withbarley in the souss-massa region and their effect on soil fertility. mimouni abdelaziz*, wifaya ahmed, bouharrou rachid, moukrini fouad, and karra youssef. national institute for agricultural research, crra, agadir, morocco. *corresponding and presenting author: mimouniabdelaziz@yahoo.fr. abstract in the souss-massa region, the rotations adopted are generally characterized by a dominance of cereals, fodder (maize for silage and alfalfa) and vegetables. also, in some area the rotations practiced are based on barley, corn and saffron with a lack of rotations based on legumes. the introduction of forage legumes in the region is an alternative for a diversification of livestock feed. trials in different loa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 438 calities have shown that vetch and pea fix significant amounts of atmospheric nitrogen under the edaphic and climatic conditions of the souss region. the vetch yield average was 132 kg /ha and 126 kg / ha in mixed crops and peas yields were 114 and 93 kg / ha, respectively. howeve, the contribution of symbiotic fixation in the accumulation of nitrogen by vetch was 61% and 82% in mixture. this contribution for peas was 76% and 94% in mixed crops. mixed crops also have the advantage of reducing disease attacks, pests and weeds. these two legumes have a symbiotic nitrogen binding capacity negatively affected by lack and excess of water, excess nitrogen and deficiency of phosphorus and potassium. trials results also showed that barley-corn and maize-maize rotations decreased soil organic matter by 22-31%. on the other hand, rotations based on both legumes significantly improved soil organic matter. the barley-corn and maize-maize rotations have the same behavior and induced an increase in the apparent density of 4 and 8%, respectively. however, legume cultivated alone or in mixed crops reduced apparent soil density by 10%. barley-corn and maize-maize rotations induced respectively a severe decrease in soil water retention between 29 and 36% compared to the initial value. conversely, the two legumes cultivated alone and in mixed crops could significantly improve soil water retention between 24 and 26%. rotations of vetch and pea cultivated alone and mixed crops significantly enriched soil with microorganisms relative to barley-corn and maize-corn rotations. keywords: intensification, vetch, pea, symbiotic fixation, soil fertility. o3. irrigation management improvement of greenhouse tomato by transpiration models in the soussmassa region. ahmed wifaya1*, mohamed boutfirass2, rachid bouharroud1, khalid azim1, fouad elame1, lahcen bouirden3, lahoucine gourdo3, khalid lekouch3, mohamed el jazouli3, youssef karra1, fouad mokrini1, and abdelaziz mimouni1. 1 regional center for agricultural research agadir, morocco; 2 regional centre for agricultural research settat, morocco; 3 thermodynamics and energetics lab., faculty of sciences agadir, morocco. *corresponding and presenting author: wifaya_ahmed@ yahoo.fr. abstract this study compares the performance of four models to estimate the daily evapotranspiration (et) under greenhouse. the first, de villele model (etdv), penman-monteith model (etpm), stanghellini model (etst) and finally the transpiration model based on the heat balance equation at the cover, takakura (ettk). the work was conducted during two tomato production seasons 2012 and 2013 under two greenhouses types; monospan and canarian, located at experimental station of melk zhar in balfae (inra agadir). st models and pm correctly simulate tomato transpiration for both measured values for daily gain of the tomato stem (mc/dg) monospan greenhouse (r ² = 0.55 and 0.62). similarly, we have reached a satisfactory relationship between the eff-tr (the efficiency of plant transpiration) and the report mc / dg using the st model under monospan and canarian greenhouses (r² = 0.67 ; 0.58) respectively. consequently, we identified critical thresholds or baselines water status of tomato; water comfort (0.40 <mc/ dg <0.50 and 70 <eff-tr <90%); overestimation of irrigation (mc / dg <0.42 and eff-tr <70% and water stress (mc/dg > eff-tr 0.46 and> 90%). in addition, the comparing means depending on the yield level has identified three groups of et models ; sta (318t / ha)> tkb (272t/ha)> ~ dvc pmc (236249t / ha). keywords: tomato, greenhouse, irrigation management, evapotranspiration models. o4. a comparative study of the effect of 2,4-dichlorophenoxyacetic acid hormonal pretreatment and cold pre-treatment on the gynogenetic response of five moroccan barley varieties (hordeum vulgare l.). rabouan oubaidou1*, s. hentour1, y. el goumi1, s. essayagh2, o. lamsaouri1, and m. fakiri1. 1 agri-food and health lab, hassan i university, faculty of science and technology, b.p. 577, settat, morocco; 2 biochemistry and neuroscience lab, hassan i university, faculty of science and technology, b.p. 577, settat, morocco. *corresponding and presenting author: rabouanoubaidou@gmail.com. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 439 abstract haplomethods are key during the process of varietal creation. we aimed to improve the protocols of the haplomethods used in our laboratory to enhance the induction rate in calluses and/or embryos and to increase the regeneration rate in chlorophyllous plants. using in vitro cultures of unfertilized ovaries, a technique called gynogenesis, we evaluated the effect of 2,4-dichlorophenoxyacetic acid hormonal pre-treatment on production yields and compared it to what we are used to obtain using a cold pretreatment at 4°c as previously described in our published works. we applied gynogenesis, on five moroccan varieties of barley namely tamelalt, aglou, asni, massine and arig and compared three variations of gynogenesis protocols. firstly, we investigated the effect of light and darkness on our cultures. secondly, we incubated unfertilized ovaries at 4°c for 14 days. thirdly, we injected donor spikes with a solution of 2,4-dichlorophenoxyacetic acid or incubated donor spikes in a solution of 2,4-d at 4°c for 14 days. the results obtained show the benefit of darkness over light. we also noted the benefit of 2.4-d pre-treatment in cold compared to a cold pre-treatment alone. statistical analysis revealed a significant correlation between the genotype and the photoperiod on the outcome of pre-treatments protocols. we hilighted better yields when ovaries were pretreated with 2.4-d and incubated in the light. we also showed that a cold pretreatment alone had no effect for all genotypes and that only ovaries that had undergone a 2,4-d hormonal pretreatment displayed improved induction rates in the aglou and asni varieties and improved regeneration rates of in the asni variety. the latter gave the highest regeneration rate (13.43%) when spikes were pretreated by 2.4-d combined with a cold pretreatment and ovaries were placed in light. moreover, the plants that stemed from this method were stronger. hence, a hormonal pretreatment using 2,4-d could be an alternative to overcome the hurdles faced when using other types of pre-treatments. keywords: in vitro culture, haplomethods, gynogenesis, hordeum vulgare l., 2,4-diclorophenoxyacetic acid (2,4-d), cold. o5. some biochemical responses of the use of arbuscular mycorrhizal fungi in date palm under salt stress mohamed ait el mokhtar*, said wahbi, raja ben laouane, mohamed anli and abdelilah meddich. plant biotechnology and physiology lab, faculty of sciences, cadi ayyad university, marrakech, morocco. *corresponding and presenting author: mohamed. aitelmokhtar@gmail.com. abstract mycorrhizal and nonmycorrhizal date palm seedlings were subjected to salt stress in order to study some biochemical responses of the effect of arbuscular mycorrhizal fungi (amf) in alleviation of salt stress. two months after germination, the plants were mycorrhized and were subjected to 0mm and 240mm nacl after five months. the activities of two antioxidant enzymes were assessed besides of protein, sugar and polyphenols contents. activities of peroxidase (pod), and polyphenol oxidase (ppo) and polyphenol content were increased significantly in both mycorrhizal (m) and nonmycorrhizal (nm) plants grown under nacl salinity while protein and sugar contents decreased. the results showed that both enzymes activities were very high in roots compared to leaves especially for the ppo. the application of amf was found to enhance significantly the activities of pod and ppo under salt stress. in the same conditions, mycorrhizal date palm plants showed a high content on polyphenol in shoots compared to control plant under salt stress. the sugar and protein contents were high in shoots than in roots and under salt stress, the application of amf improved the both contents. keywords: date palm, arbuscular mycorrhizal fungi, salt stress, biochemical responses. o6. agricultural practices in the souss-massa region and their consequences on soil fertility mimouni abdelaziz*, bouharrou rachid, wifaya ahmed, moukrini fouad, karra youssef. national institute for agricultural research, crra, agadir, morocco. *corresponding and presenting author: mimouniabdelaziz@yahoo.fr. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 440 abstract using a participatory study and monitoring of the history of soil fertility indices, organic matter, total nitrogen, salinity and others, indices of degradation of soil fertility have been identified. indeed, the cropping system through rotations and techniques practiced in the region have influenced in various ways the soil fertility and subsequently crop performance. indeed, in the plain of souss-massa, agriculture has undergone a great evolution over time, it has reached a level of intensification very high at present. the intensified areas have evolved very rapidly, reaching in 2015 about 56434 ha, mainly in monoculture citrus (39808 ha) and early crops (16626 ha). the significant expansion of these areas was largely the result of the substitution of crops for argan plantations with the dominance of vegetable monoculture, maize and no practice of rotations based on legumes or regenerative soil fertility crops. over approximately forty years, the clearing of the argan tree and the cultivation of the soil caused a degradation of the organic matter with respective losses of 68% in the massa and 57% for the souss. total nitrogen losses were 52% and 41%, respectively. also, analyzes of 300 soil samples in different subregions showed that soils in the souss-massa region have very low levels of organic matter and total nitrogen. in fact, in the four subregions, massa, taroudant, ait baha and taliouine, the samples analyzed showed organic matter contents of less than 0.5% and absence of samples with contents greater than 2%. 65% of the analyzed samples have total nitrogen contents of less than 0.01%. keywords: intensified crops, rotations, organic matter, total nitrogen. o7. effect of nitrogen and seed germination rate on yield, biomass, and grain quality of moroccan durum wheat. oussama hnizil1*, sedri naima1, nsarellah nasselehq2, baidani aziz1, idrissi omar2, sahri ali2, and amamou ali2. 1 university hassan ii, settat, morocco; 2 not provided. *corresponding and presenting author: o.hnizil@ uhp.ac.ma. abstract durum wheat is a typical mediterranean crop. its importance comes from the fact that durum wheat grain is consumed in different forms namely bread, pasta, couscous, boulghour... however, these different forms require grains of durum wheat of good quality (protein content, gluten strength, grain color and baking strength). nitrogen (n) element is the nutrient that most frequently limits yield and plays an important role in regulating productivity and quality of durum wheat grain. in the other hand, seed rate (s) is also a vital factor which influence yield and quality of wheat. the objective of this study is to evaluate the effect of different nitrogen treatments and seed rates on yield, total biomass and end use quality of five moroccan durum wheat varieties. the trial was carried out during the 2017 cropping season, at three experimental stations (sidi el aidi, marchouch and tassout). the five moroccan cultivars were sown following split-plot design with two replications, three seed rates (s: 300, 400 and 500 grains/m2) and three nitrogen treatments (n: 45, 90 and 135 kg/ha). data were recorded for quantitative characters: yield and components, biomass, ndvi, chlorophyll content and grain protein content. with regard to the results, both n and s had a significant effect on grain yield, biomass, physiological treats and protein content. in addition, highly significant genetic variation was observed between genotypes and the genetic response to n supply. the new released varieties showed high response to the variation of n application. the difference between environments was also observed and discussed. the experiment is carried out also on 2018 to validate the result found during the 2017 cropping season. keywords: nitrogen fertilization, planting density, durum wheat, yield and its components, grain quality. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 441 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) concurrent session v: plant health & production ii co-chair: alan walters, southern illinois university, usa co-chair: mohamed amine serghini, university ibn zohr, morocco o1. insight into the mediterranean olive tree domestication and diversification. ahmed el bakkali1*, adelmajid moukhli2, and bouchaib khadari3,4 1 inra, ur amélioration des plantes et conservation des ressources phytogénétiques, meknès, morocco; 2 inra, ur amélioration des plantes, marrakech, morocco; 3 agap, university of montpellier, cirad, inra, montpellier supagro, montpellier, france ; 4 conservatoire botanique national méditerranéen de porquerolles (cbnmed), umr agap, montpellier, france. *corresponding and presenting author: ahmed_elbakkali@yahoo.fr. abstract the olive tree (olea europaea l.) is one of the most important oil crops in the world and 95% of total olive oil production derived from the mediterranean basin (mb). since the ancient times, out-performed olive genotypes have been selected by local farmers and vegetatively propagated leading to the present varietal assortment with almost 1,200 cultivars around the mb. therefore, the elucidation of the olive origin, domestication and diversification process is important from a cultural and agricultural standpoint since it can inform on the origin and history of human civilizations as well as the management of genetic resources, while offering guidance for modern breeding. primary domestication from wild olive probably occurred around 6000 bp in the middle east. however, the question remains as to whether current cultivated olive derived from single domestication event in the levant followed by secondary diversification or whether it was the result of independent domestication events. here we analyzed a comprehensive sample from 35 wild populations (oleasters, 722 individuals) and 410 cultivars from across the mb using nuclear and plastid dna markers. our genetic findings from bayesian model clustering implemented in the structure program and maternal lineages argue in favor of a single primary domestication event in the eastern mb, followed by diffusion of the first domesticated olive and introgression with wild olive from the central and western mb. these results should be examined to gain further insight as to be able to accurately design sampling of mediterranean olive germplasm suitable for innovative breeding within the sustainable oliviculture and climate change context. keywords: olea europaea, oleasters, simple sequence repeat (ssr), plastid maternal lineage, genetic structure, admixture, single domestication center. o2. econometric analysis of market integration and price transmission in morocco’s apples markets: application of threshold cointegration and cointegration with structural break. ouahiba elalaoui1*, abdelouafi ibrahimy2, aziz fadlaoui3, and redouane arrach4. 1 agro-economist engineer, graduate of the national school of agriculture; morocco; 2 professor of applied statistics and economics, national school of agriculture; morocco; 3 agro-economist researcher, national institute for agricultural research; morocco; 4 director of strategy and statistics directorate, ministry of agriculture, fisheries, rural development, water and forests, rabat, morocco. *corresponding and presenting author: ouahiba.elalaoui@gmail.com. abstract the level of market integration and transmission of food prices is a key determinant of price stability and food security. thus, integration can be regarded as a way of assessing efficiency of agricultural markets. this study investigated the degree of market integration using the time series techniques of threshold cointegration and cointegration with structural break. the purpose of this study is twofold. firstly, it attempts to determine whether price transmission sufficiently improved throughout the period under examination. secondly, it purports to explore the nature of price transmission. in order to achieve these objectives, we sampled five wholesale markets, comprising two sura tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 442 plus markets (casablanca and meknes) and three deficit markets (agadir, marrakech and oujda). the dataset for the analysis includes monthly price series from september, 1993 to june, 2017. the results reveal that apple markets are integrated and hence price signals are transmitted from surplus to deficit regions. however, the extent of market integration is low and price transmission is slow and incomplete. furthermore, the findings indicate that the intensity of price transmission has not improved throughout this period, in spite of the development of road infrastructures and telecommunications. overall, the empirical evidence confirms the presence of asymmetric price transmission, sign of market inefficiency. nevertheless, the nature of asymmetry does not confirm the research hypothesis stipulating that increases prices are transmitted more quickly than decreases because of the market power of middlemen in the food marketing chain. keywords: market integration, cointegration, apples, price transmission. o3. culture method of cercospora beticola sacc, sensitivity test of isolates with methyl thiophanate and detection of resistance in morocco. zakariae el housni1,2*, said ezrari1,3, abdessalem tahiri1, rachid lahlali1, and abderrahman ouijja2. 1 phytopathology unit, departement of plant protection, ecole nationale d’agriculture de meknès, bps 40, meknès, morocco; 2 departement of biology, faculty of sciences, university moulay ismail, p.o. box 11201, meknes, morocco. *corresponding and presenting author: atahiri@enameknes.ac.ma. abstract the sugar beet crop (sb) is considered the first source of extraction of refined sugar for morocco. it is cultivated in four large areas: gharb, doukkala, tadla and moulouya. the bs has a large list of enemy that significantly reduces yields. cercospora leaf spot (cls) is the main fungal disease. in the last years, we have observed a decline in efficacy of methyl thiophanate (mt), active ingredient belongs of benzimidazole, which was been used extensively in the management of cercospora leaf spot (cls) in morocco. several samples of cercospora beticola sacc were collected from the 4 major production areas for laboratory tests. tests on cultivation techniques have been conducted to see if there is an effect of the following factors: the culture media (pda & bla), the light, the sampling area, the method of isolation, on the growth rate of the fungus, the percentage of germination and the percentage of sporulation. with pcr, all isolates were verified by specific primers (cbactin959 f and cbactin959 r) encoding 959 bp of the active gene to ensure that the species to study is cercospora beticola sacc. radial mycelia growth of each isolate in pda medium was compared to mycelia growth in pda medium modified with mt with levels of 1,5, 10 and 50 ppm. the percentage of inhibition obtained was used for the ec50 calculation. all the isolates showed resistance to the mt molecule with different degrees to stand out with 3 groups: low resistance with ec50 less than 100 ppm, moderate resistant with ec50 between 100 and 1000 ppm and very resistant with ec50 more than 1000 ppm. keywords: cultivation techniques, cercospora beticola, pcr, sensitivity, resistance, methyl thiophanate, ec50, morocco. o4. a dynamic multimarket analysis of fat food sector in morocco. sara essaten1* and abdelkader ait el mekki2 § 1 agronomic and veterinary institute, rabat, morocco; 2 national school of agriculture, meknes, morocco. *presenting author: sara.essaten@gmail.com. §corresponding author: amekki@enameknes.ac.ma. abstract due to a severe deficit of the domestic supply, the fat food demand in morocco is mainly covered by the imports which ensure as much as 80% of the households’ consumption. thus, moroccan fat food security is largely defined upon the government efforts to enhance domestic production on one hand, and the world market patterns, on the other hand. in this context, the fat food sector performances should largely be determined by the government price and trade policy knowing that morocco has signed preferential trade agreements with its main food suppliers (eu and usa) and is an active wto member. this research aims at analyzing the impact of trade openness and government farm and marketing price policy on the fatty a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 443 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) products sector in morocco. in this respect, a dynamic multimarket model, including olive oil, edible oilseed, butter and margarine, will be developed in order to evaluate the effects of price and trade policies related to exogenous variables on the whole fat food value chains from the farmer to the consumer. indeed, the multimarket model will be made up of six blocks where four of them represent the behavior of economic agents at agriculture, processing, international trade and consumption steps. the other two blocks will be devoted to the market equilibrium and cross price relationship along the value chains. all dynamic behavioral parameters will be econometrically estimated using appropriate microeconomic theory. keywords: fat food sector, dynamic multimarket model. o5. genetic variability revealed by ssr (simple sequence repeat) markers of apricot (prunus armeniaca l.) issued from various genetic and environmental resources in morocco. jamal ayour1,6,7*, imane elateri2, mohammed alami3, abderrahim alahyane1, mohamed najib alfeddy4, jean-marc audergon5, catherine m.g.c. renard6,7, and mohamed benichou1 1 faculté des sciences, université cadi ayyad, laboratoire sciences des aliments, m-40090 marrakech, morocco; 2 laboratory of molecular biology and food, center of analysis and characterisation, faculty of sciences-semlalia, cadi ayyad university, p.o. box: 2390, marrakesh, morocco; 3 laboratory of microbiology and molecular biology, faculty of science, mohammed v university, rabat, morocco; 4 laboratoire de phyto-bactériologie, unité de recherche protection des plantes au centre régional de la recherche agronomique, inra, bp 533, marrakech, morocco; 5 inra, ur1052 génétique et amélioration des fruits et légumes, f-84143 montfavet, france; 6 université d’avignon et des pays du vaucluse, umr408 sécurité et qualité des produits d’origine végétale, f-84000 avignon, france; 7 umr408 sécurité et qualité des produits d’origine végétale, inra, f-84000 avignon, france. *corresponding and presenting author: jamal.ayour@ced.uca.ac.ma. abstract the knowledge of the available genetic resources is of first importance to optimize its preservation and/ or its potential use in breeding programs. the objective of this study was to assess the genetic diversity of moroccan apricot (prunus armeniaca l.) accessions available in collection using 21 microsatellite markers. genotypic diversity of 92 genotypes, issued from a prospection organized in all the country, was determined using diversity indices, molecular variance analysis, principal component and cluster analysis. polymorphic parameters indicated that the least informative prime was bppct001 with 3 genotypes while the most informative primer was udp98409 with 23 genotypes. the pic values for ssr loci ranged from 0.08 for ampa109 to 0.43 for bppct001, with the mean value of 0.19. whereas the shannon’s information index was ranged from 0.37 to 1.94 for ampa109 and udp98409 respectively, this shows an importance genetic variability of analyzed apricots regarding their geographic origin. indeed, the genetic variance was about 87% among apricot clones and 10% among geographic region. the results of neighbour-joining, tree analysis and pcoa indicated that the genetic structure of the 92 apricot clones could be grouped in different populations according to their agronomic performances. information obtained in this study may be useful for apricot breeding improvement programs and it could enable the enlargement of the apricot genotype in the mediterranean basin scale. keywords: prunus armeniaca, clone, genetic diversity, microsatellite markers, variability. o6. genetic diversity of moroccan sesame (sesamum indicum l.) populations using issr markers meriem el harfi1,2*, abdelghani nabloussi1, hafida hanine2, and jamal charafi1§ 1 plant breeding and genetic resources unit, national institute of agronomic research, regional agricultural research centre of meknes, morocco; 2 laboratory of bioprocess and biointerfaces, faculty of science and technology, beni mellal, morocco. *presenting author: elharfi.meriem@gmail.com. §corresponding author: jcharafi@gmail.com. abstract sesame (sesamum indicum l.) is an ancient oilseed crop known for its nutty seeds and high-quality edible oil. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 444 it is an unexplored crop with a great economic potential. in morocco, sesame has been grown for decades and it is one of the specific crops of the tadla-azilal area. the present study deals with assessment of genetic diversity among 33 sesame populations. seven inter simple sequence repeat (issrs) primers were used for molecular analysis of these populations. a total of 57 markers were detected and the number per primer varied from 4 to 14, with an average of 8 bands. the primers generated 82% (47 bands) of polymorphic fragments and the pairwise comparison of the 33 genotypes showed that 74% of genotypes pairs were distinct by less than 11 markers. this low diversity between genotypes is confirmed by the genetic distances obtained. the simple matching coefficient of similarity ranged from 0.509 to 1, with an average of 0.79 and the polymorphic information content (pic) ranged from 0.002 to 0.349. the hierarchical analysis using the upgma method showed that, despite subdivision into groups, several genotypes had the same genetic profile and were lowly diversified. the results obtained show that sesame in morocco has a low genetic diversity and may be probably derived from a limited genetic pool. keywords: sesamum indicum l., issrs markers, genetic diversity, polymorphism. o7. effect of moroccan seaweed polysaccharides on germination of tomato seeds (solanum lycopersicum l.). abir mzibra1,2*, abderrahim aasfar1, issam meftah kadmiri1, and ahmed bamouh2 1 laboratory of green biotechnology of moroccan foundation for advanced science, innovation and research (mascir), rabat design centre, rue mohamed al jazouli, madinate al irfane, rabat, morocco; 2 institut agronomique et vétérinaire hassan ii, production, protection et biotechnologie végétales, bp: 6446, rabat, morocco. *corresponding and presenting author: abiir.mzi@gmail.com. abstract polysaccharides extracted from marine seaweeds can act as plant biostimulants by applying them to seeds, plants, or growing substrates, but always at low concentration, which is provided by specific formulations. the aim of this study was to evaluate the effect of polysaccharides enriched extracts (pees) obtained from 17 moroccan seaweeds belonging to chlorophyceae (5 species), phaeophyceae (5 species) and rhodophyceae (7 species) on 3 germination parameters: germination percentage (gp), germination speed (gs) and mean time of germination (mgt) of tomato seeds (solanum lycopersicum) under laboratory conditions. tomato seeds were cultivated in the half-strength murashige-skoog medium and supplemented with different pees at 3 concentrations of (0.1; 0.05 and 0.02 mg ml-1 (w/v) or with sterile distilled water for control seeds. our results showed that seeds treated with pees extracted from u. rigida, c. tomentosum, c. decorticatum, gigartina sp., b. bifurcata, f. spiralis, s. polyschides in the 3 concentrations enhanced germination (significant increase in germination percentage associated with lower significant mean germination time and high significant germination speed) and seeds treated with pees of g. pistillata, c. acicularis, g. crinale, c. foeniculacea and c. tamariscifolia enhanced significantly germination parameters just at lower concentrations (0.02 mg.ml-1). g. pistillata at higher concentration (0.1 mg.ml-1) showed an inhibition of germination. this study emphasizes the use of moroccan pees as biostimulants inducing an early tomato seed germination and establishment with the corresponding concentration. keywords: biostimulant; polysaccharides enriched extracts; seaweed; seed germination, germination parameters. o8. ethnobotanical uses of arbutus (arbutus unedo l.) in morocco. faida rahima1,2*, j aabdousse1, a boulli1, s bouda2, and n wahid1§ 1 laboratory of environment and valorization of agro-resources (evar), department of life sciences, faculty of science and technology, sultan moulay slimane university, beni-mellal, morocco; 2 laboratory of management and valorization of natural resources, department of life sciences, faculty of science and technology, sultan moulay slimane university, beni-mellal, morocco. *presenting author: faida.rahima@gmail.com. §corresponding author: wahid2na@ hotmail.com. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 445 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) abstract arbutus is a fruit tree with high ornamental, environmental, economic and medical value, because of the properties attributed to different biological parts. given this interest, the present study is interested in describing and compiling available data on the ethnobotanical uses of arbutus in morocco in comparison with other studies at the international level. it is very present in the everyday uses of foresters, farmers and rural populations. the international bibliographic analysis shows that the different biological parts of arbutus, especially fruits, have been widely used by herbalists. the related literature is generally in line with the medicinal use of the plant by moroccan inhabitants. on the other hand, the consumption and the industrial valorization of the fruits of arbutus are very limited in morocco in comparison with those on the international scale. it is imperative to sensitize the local population and landlocked douars on the knowledge of the valorization of the fruit of this species. keyswords: arbutus unedo l., ethnobotanical uses, prospection, ecology, arbutus fruit. o9. valorization challenges to almonds and their coproducts: characterization of oils extracted from broken almonds springing from shelling and sorting operations. ahmed elamrani1*, r. melhaoui, n. houmy, m. addi; m. abid, a. mihamou, m-l. fauconier2, m. sindic3, and serghini-caid1 1 laboratoire lbpm, faculté des sciences, université mohamed ier, oujda; morocco; 2 laboratoire de chimie générale et organique, gembloux agro bio-tech, université de liège; belgium; 3 laboratoire, qspa, gembloux agro bio-tech, université de liège; belgium. *corresponding and presenting author: ahmed.elamrani@gmail.com. abstract the almond tree (prunus dulcis) is one of the most popular nut trees grown worldwide under arid conditions. in north eastern region of morocco, after the olive tree the almond tree is the second most important tree crop in this region, due to its resistance and capacity against water shortage and irrigation deficit. during this decade, new orchards of almond trees have emerged, thanks to the program green morocco, supported by the btc (*btc-profao project). thus 6.000 hectares of new orchards of almond trees were planted using the association ferragnes / ferraduel, which is a couple of french cultivars known for their late blooming. in the aim to improve the income of this rural population, cooperatives and economic interest groups were created; farmers have been trained for good almond cultivation practices as well as almond harvesting and processing. in eastern morocco, traditionally almonds are hulled and shelled, manually by rural women, but currently, almonds processing machines are under installation. manually or mechanically, almonds’ hulling and shelling operations generate many byproducts which are hulls (thin mesocarp, green shell cover), shells (hardened endocarp) but also broken kernels from almonds sorting and brown skin as byproduct of almonds’ blanching. taking in consideration the importance of valorization of almond byproduct, this work deals with the agro economical relevance and added value that could be generated by using broken almonds (as a co product) for almond oils extraction. thus this oral communication focuses on (i) the characterization of almond oils produced by cooperatives in this region as an adding value to this almond’s co-products (ii) paths of research for the valorization of the other by-products mainly almonds’ shells and hulls. briefly, almond oils were extracted from broken almond (as a co-product) by screw press, oil yield rage between 48 and 56%. fatty acids (fa) profile, determined by gc-fid, shows dominance of unsaturated fatty acids fraction [usfa > 85% ] which is mainly represented by oleic acid (61-69%) and linoleic acid (22-27% ). saturated fatty acids fraction (sfa <11%) is represented mainly by palmitic acid (7-8%). and stearic acid (23%). total phenol content range between 40-45 mg/ kg-oil, however total tocopherols content is important and range between 510-530 mg/kg-oil with a large dominance of α tocopherol. the presence of these compounds is important in relation to oil stability and as nutritional quality label. thus, consumption of almond oil is recommended thanks to their beneficial effects on health, but it’s also recommended for cosmetic uses. keywords: almond tree, almonds, almond co-products, almond oils. *btcprofao: projet filière amandes de l’oriental, “the almond value chain in eastern morocco, pillar ii a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 446 of the moroccan green plan, supported by belgian development agency (btc 2011-2017). session vi: africa sustainable agriculture (plenary session) chair: khalid meksem, southern illinois university, usa o1. a strategy for incorporating sustainability into tertiary agriculture education. john w. groninger* department of plant, soil, and agricultural systems, southern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: groninge@siu.edu. abstract where agriculture production systems are rapidly evolving, such as across sub-saharan africa, shortterm gains in crop yields can occur at the expense of system resiliency needed to achieve sustainability. post-secondary (tertiary) education institutions have great potential to better facilitate large scale sustainability initiatives. agricultural education resources that do not reflect present industry conditions and the declining prestige of agriculture in urbanizing societies further hinders reform efforts. based on a 2014 assessment of samaru college of agriculture at ahmadu bello university in nigeria (funded by the united states agency for international development farmer to farmer program and implemented by winrock international), i identify strategies for using increasing interest in agricultural sustainability to revitalize agriculture teaching institutions by broadening their value to students and better demonstrating the relevance of agriculture institutions to society. my presentation will focus on education system reforms that can address watershed management issues. particularly critical are curriculum reforms that promote entrepreneurship, build creative problem-solving skills, and encourage meaningful industry engagement among students and staff alike. i conclude with suggestions for increased engagement among universities in morocco and the united states to work cooperatively with colleagues in sub-saharan africa to address ongoing and emerging agriculture sustainability issues among all parties. o2. assessing the impacts of agroforestry on community and household resilience in africa: an analysis of the modified taungya system in ghana kofi akamani* department of forestry, southern illinois university, carbondale, il 62901, usa. *corresponding and presenting author: k.akamani@siu.edu. abstract the promotion of sustainable development on the african continent continues to be challenged by several problems, including population pressure, widespread poverty, food insecurity, resource degradation, and climate change vulnerability. owing to the shortfalls of industrial agriculture and sustained-yield forest management approaches, agroforestry has been receiving attention as an integrative land use strategy for achieving the sustainable development goals in africa. agroforestry, a land use strategy that involves the integration of trees with annual crop cultivation and other farm activities, promises several benefits including food security, ecosystem restoration, as well as climate change mitigation and adaptation. however, the relationship between agroforestry and community resilience, i.e. the capacity of communities to adapt to change while maintaining their well-being, has not received adequate attention in the literature. importantly, the issue of scale has not received explicit recognition in the assessment of agroforestry outcomes. since the adoption of the modified taungya system (mts) in ghana in the early 2000s, a collaborative approach to agroforestry has been pursued as a means for forest restoration, as well as food and timber production. this presentation utilizes qualitative and quantitative data gathered from two forest-dependent communities in the ashanti region of ghana to assess the impacts of the mts program on social-ecological resilience at the level of the community and the household. the purpose is to determine whether the impacts of the mts program on socialecological resilience at the community and household a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 447 which is dominated essentially by an informal system constitute a potential leverage to enhance productivity and encounter climatic changes. for this purpose, actions should be defined for the promotion of both the formal and the informal sector within their respective contexts. keywords: africa, climatic change, agricultural model, seed sector. o4. status of vegetable variety improvement for morocco alan walters* department of plant, soil, and agricultural systems, southern illinois university, carbondale, il usa. *corresponding and presenting author: awalters@siu.edu. abstract the choice and utilization of vegetable varieties by growers depends upon many different factors, and the dynamics leading to eventual variety choice and use is highly varied, even in morocco. the globalization of agriculture, along with the privatization of the plant breeding industry in the last few decades, has directly influenced which vegetable varieties are available to farmers in developing countries. however, new vegetable variety development is normally non-existent in developing countries, with seed generally sourced from developed countries. this results in developing countries having a dependence on those developed countries that have bred the varieties to supply vegetable seed for their needs. morocco’s goal for future vegetable market development is to produce high-quality products to be sold in international markets that allow the highest revenue returns, and this generally requires the use of hybrid varieties that were not necessarily developed for the specific environments in which they are being recommended. so, this situation often times results in vegetable seeds being marketed into regions having environments where they are not always best suited to maximize their productivity. thus, the adoption of modern hybrid varieties has lagged in some regions, especially marginal environments that have overall low production potential, which are often most suitable for local traditional varieties (landraces). new variety development works best if breeding is conducted in the environment in which it will be grown to maximize a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) levels are equivalent to each other. analysis of qualitative data at the community level indicated that when comparing current community conditions to conditions prior to the implementation of the mts program, both communities have experienced marginal improvements in the various capital assets that shape community resilience. at the household level, a statistical comparison of current household capital assets with capital assets prior to the mts program showed varying levels of decline in household resilience across the two communities. these results suggest the impacts of agroforestry on social-ecological resilience may be sensitive to the scale at which assessments are made. keywords: agroforestry, community resilience, livelihoods, sustainable development, scale. o3. the future agriculture in africa in the context of climate change: seeds perspective lamiae ghaouti*. institut agronomique et vétérinaire hassan ii, rabat, morocco. *corresponding and presenting author: lamiaeghaouti@hotmail.com. abstract agriculture is highly exposed to climate change, as farming activities directly depend on climatic conditions. the modern agriculture through the maximization of natural resources use contributed in a considerable proportion to the climatic change through the release of greenhouse gases into the atmosphere. in comparison to the other continents, africa is the least contributing to the carbon dioxide emissions but is paradoxically the most impacted area in the world by climatic changes. therefore, rethinking the agricultural model in africa where agriculture is a crucial sector is a complex issue but compulsory within the view of the present situation. africa holds a relevant asset through genetic diversity as it is a center of diversity for a wide range of crops. it is an essential component in the equation of maintaining the food production level while decreasing the agro-ecosystems vulnerability. genetic diversity is a key factor for the management of heat, drought, salinity, pests and diseases that are more acute due to climatic changes. cropping systems should be adopted depending on their efficiency of use of natural resources within the targeted agro-ecosystems. rethink the agricultural production paradigm. seed sector in africa a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 448 future crop yield and productivity. therefore, the development of new varieties specific to environments in developing countries is a critical step for future food security in these regions. keywords: agricultural globalization; climate change; food security; landraces. concurrent session vii: plant health & production iii co-chair: john groninger, southern illinois university, usa co-chair: fouad mokrini, inra, morocco o1. resistance to pratylenchus penetrans and p. thornei in international wheat lines and its durability when inoculated together with the cereal cyst nematode heterodera avenae, using qpcr for nematode quantification fouad mokrini1*, nicole viaene2,4, lieven waeyenberge2, amer dababat5, and maurice moens2,3 1 national institute for agricultural research (inra), rue far, inzegane, agadir, morocco; 2 institute for agricultural and fisheries research, plant, crop protection, burg. van gansberghelaan 96, b-9820 merelbeke, belgium; 3 faculty of bio-science engineering, ghent university, coupure links 653, b-9000 ghent, belgium; 4 department of biology, ghent university, ghent, belgium; 5 international maize and wheat improvement center (cimmyt), p.k. 39 06511, emek, ankara, turkey. *corresponding and presenting author: fmokrini.inra@gmail.com. abstract the root lesion nematodes pratylenchus penetrans and p. thornei cause high yield losses in rainfed wheat fields in morocco, as well as worldwide. growing resistant varieties is one of the most effective methods for controlling nematodes. therefore, a collection of 14 lines of spring wheat and 11 lines of winter wheat (triticum aestivum and t. durum), developed at cimmyt, were screened for resistance to p. penetrans and p. thornei in tubes (15 x 20 x 120 mm3) under the greenhouse conditions. the resistance level was evaluated based on the numbers of nematodes extracted from roots and soil nine weeks after infestation. the number of p. penetrans or p. thornei were determined visually using a microscope and with a qpcr assay. three lines (l9, l12 and l13) were found moderately resistant to p. thornei and one of these (l9) was also moderately resistant to p. penetrans. to investigate the durability of this resistance, we co-inoclulated juveniles of heterodera avenae, a cereal cyst nematode widely present in moroccan wheat fields, and assessed the reproduction of both lesion nematodes p. penetrans and p. thornei on the three moderately resistant lines. our results showed that the lines l9 and l9, l12, l13 remained moderately resistant to p. penetrans and p. thornei, respectively, in the presence of h. avenae. these findings are promising; however, the field performance of these lines against root lesion nematode attacks should be evaluated. keywords: durability, resistance, root lesion nematode, screening, wheat. o2. transfer and molecular mapping of aegilops tauschii-derived hessian fly resistance genes (h22, h23, h24, and h26) from d genome of triticum aestivum onto a genome chromosomes of triticum turgidum by induced homoeologous recombination. moha ferrahi1*, b. friebe2, j.h. hatchett3, and b.s. gill2 1 national institute for agricultural research (inra), regional center of meknes, bp 578, meknes, morocco; 2 dept. of plant pathology, wheat genetics resource center, throckmorton plant sciences center, kansas state university, manhattan, ks 66506-5502, usa; 3 dept. of entomology and usda-ars, waters hall, kansas state university, manhattan, ks 665065502, usa. *corresponding and presenting author: mohaferrahi@yahoo.fr. abstract aegilops tauschii coss. (2n=14, dd) is a rich source of disease resistance genes for the improvement of cultivated wheat including several resistance genes against hessian fly. to date, five hessian fly resistance genes (h13, h22, h23, h24, and h26) have been transferred from ae. tauschii to common wheat (triticum aestivum l.). in this study, we attempted the transa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 449 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fer of four genes h22 (1d), h23 (6ds), h24 (3dl), and h26 (4d) from t. aestivum d genome onto a genome chromosomes of t. turgidum. the t. aestivum resistant parents wgrc01 (h22 on 1d), wgrc03 (h23 on 6ds), wgrc06 (h24 on 3dl), and wgrc26 (h26 on 4d) were crossed with t. turgidum cv. langdon disomic substitution lines ldn 1d(1a), ldn 6d(6a), ldn 3d(3a), and ldn 4d(4a). we targeted the transfer of hessian fly resistance genes into d-genome substitution chromosomes of t. turgidum by homologous recombination. in total 88 crosses were made. the resulting f1 plants (345 seeds) were backcrossed with the ldn 5d(5b) substitution line in which chromosome 5b is absent and replaced by a pair of 5d chromosomes with the objective of transferring d genome hessian fly resistance genes onto a or b genomes of t. turgidum by homoeologous recombination. a total of 2,053 segregating bc1f1 plants were tested for hessian fly resistance, and the resistant plants (1,132) were backcrossed again with lnd 5d(5b) to produce bc2f1 and selfed to produce bc1f2. in the bc1f1 populations, 24 families segregated for an excess of resistant plants than the expected 1:1 resistant to susceptible plants suggesting that they were putative a-d genome positive recombinants. mapping analysis using microsatellites was used in these families to identify recombinants between aand dgenome chromosomes. the data indicated that h22 recombinants were recovered consisting of the distal part of the short arm of 1a, the proximal of 1ds, and the complete long arm of 1d. the recombinant can be described as t1as-1ds.1dl. the recombinant involving h23 probably consisted of the whole short arm of 6d and the long arm of 6a, and is described as t6ds.6al. the centromeric marker indicated that this recombinant has the centromere from chromosome 6a. in addition, monosomic substitution lines were recovered for the remaining resistance genes h24 and h26. these monosomic substitution lines are useful germplasm for further manipulation aimed at transferring genes h24 and h26 to durum wheat. keywords: triticum turgidum, triticum aestivum, aegilops tauschii, hessian fly, induced homoeologous recombination, microsatellites mapping. o3. analysis of genetic diversity among safflower accessions of different origins using agro-morphological traits and issr markers karim houmanat1,2*, jamal charafi1, hamid mazouz2, mohamed el fechtali1, and abdelghani nabloussi1§ 1 inra, research unit of plant breeding and plant genetic resources conservation, crra of meknes, p.o. box 578, meknes, morocco; 2 university moulay ismail, laboratory of biotechnology and molecular biology, faculty of sciences, meknes, morocco. *presenting author: k.houmanat@gmail.com. §correspomding author: abdelghani.nabloussi@gmail.com. abstract safflower (carthamus tinctorius l.) is one of the oilseed crops that have important agronomic and environmental benefits, mainly adaptation to arid and semi-arid environmental conditions. this enables it to be a good alternative oilseed crop for morocco and other mediterranean countries which are exposed to an increased climate change trend. the present study aimed to evaluate 61 safflower accessions, from different geographical origins, for agronomic, morphological, phenological, pathological and technological attributes. in addition, molecular analysis, using issr markers, was carried out to characterize these accessions which were planted in two consecutive years, 2013 and 2014, in inra-experimental station of douyet. results obtained for agro-morphological study exhibited a large variability among the genetic material evaluated. hence, average seed yield was about 2 t/ha, with a variation from 1.14 to 4.3 t/ ha. seed oil content varied between 22% and 39%. significant differences were also observed among the accessions for their flowering time, their resistance to brown rust and broomrape, for the color of the petals and for their spinesness. results of molecular analysis showed that a high number of reproducible issr bands exhibited a high percentage of polymorphism, indicating a high genetic diversity among the material studied. polymorphic profiles were identified according to an index of genetic diversity, generally variable according to the genotypes, from 0.13 to 0.40, with an average of 0.23. dendrogram similarity relationships revealed some clustering according to different genetic pools. the ascending hierarchia tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 450 cal classification (ahc) of molecular data showed two large distinct groups and four subgroups, whilst ahc of agro-morphological data revealed two large distinct groups with a very high level of similarity (0.977). by analyzing and comparing both ahc, one could observe that classification of some accessions in one group is almost similar for both dendrograms. the findings of this study were very interesting and would be useful for safflower breeding program in morocco as well as in other countries of the world. keywords: genetic resources, safflower, variability, genetic pools. o4. assessment of codling moth sensitivity (cydia pomonella l.) to certain insecticides used in apple orchards in morocco, azrou region salma el iraqui1*, ahmed el bakkali1, and m’hamed hmimina2§ 1 national institute of agricultural research, meknès regional center, bp 578, meknès, morocco; 2 zoology department, institut agronomique et véterinaire hassan ii, b.p. 6202, rabat, morocco. *presenting author: iraquisalma@gmail.com. §corresponding author: hmimina@yahoo.fr. abstract the codling moth, cydia pomonella (l.), is the key pest of apple production worldwide. in morocco, there is a sustainable presence of codling moth causing considerable damage in apple orchards despite frequent applications of broad spectrum insecticides. at azrou region (morocco), the chemical control is widely and massively used (12 to 15 treatments per season) exclusively to control this pest. this technique seems to be in the advantage of the insect since the traps captures are usually exceeding the action threshold. the failure of codling moth control in the azrou orchards led us to examine the insect sensitivity to some insecticides and explore the mechanisms implied in their detoxification. the efficacy of azinphos-methyl, chlorpyriphos-ethyl, diflubenzuron, thiacloprid, methoxyfenozide, spinosad, and deltamethrin, was evaluated on neonate larvae and compared with a laboratory sensitive strain. toxicological tests showed that the populations tested are developing a resistance. in fact, biotests established a decrease of sensitivity for 5 products out of 7 and exhibited the presence of a cross-resistance between the organophosphates, the benzoylureas, the pyrethroids, the neonicotinoids and the diacilhydrazines. this resistance is mainly linked to the esterase activity unlike the mixedfunction oxidase activity which was the same between local and sensitive strains. finally, the kdr mutation, linked to deltamethrin resistance, was found with a low frequency in azrou population attesting that resistance is being selected there.keywords: cydia pomonella l, sensitivity, resistance, enzymes, esterase, mixed-function oxidase, kdr. o5. using amplicon sequencing analysis to decipher the microbiome for date palm “bayoud” disease suppression in suppressive soils adil essarioui1*, daniel cameron schlatter2, harold corby kistler3,4, and linda kinkel4 1 national institute for agricultural research, regional center of errachidia, morocco; 2 usda, washington dc, usa; 3 cereal lab disease, usda-ars, st paul, mn, usa; 4 department of plant pathology, university of minnesota, usa. *corresponding and presenting author: essar002@umn.edu. abstract “bayoud”, a vascular disease caused by the soilborne fungus fusarium oxysporum f. sp. albedinis, is the principal enemy of date palm trees that put at stake the future of date industry in morocco. previous work showed that some soils are naturally suppressive to the disease. however, our knowledge of how bayoud pathogen is suppressed in these soils is still limited. to improve our understanding of mechanisms by which “bayoud” is suppressed in suppressive soils, we characterized fungal and bacterial community composition and structure in suppressive and conducive soils using amplicon sequencing analysis. fungal and bacterial populations were analyzed by amplifying and illumina sequencing the internal subscribed spacer (its1) and the ribosomal (16s) genes, respectively. results showed that the suppressive soils have greater microbial density and diversity than conducive soils. additionally, suppressive soils are more enriched in microbial taxa known for their prolific production of antimicrobial compounds such as fusarium, aspergila tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 451 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) lus, and diverse actinomycetes. we hypothesize that “bayoud” suppression is the results of two complementary mechanisms: competition for nutrients between the pathogen and a highly dense saprophytic microbial community, and antagonism from antibiotic producing microbial taxa. keywords: bayoud disease, suppressive soils, amplicon sequencing. o6. genome wide association studies on yield components using a lentil genetic diversity panel jamin a. smitchger1, yu ma2, ping zheng2, clarice j. coyne3*, rebecca j. mcgee3, and doreen main2 1 department of crop and soil sciences, washington state university, pullman, wa 99194; 2 department of horticulture, washington state university, pullman, wa 99194, usa; 3 united states department of agriculture, pullman, wa 99194, usa. *corresponding and presenting author: clarice.coyne@ars.usda.gov. abstract the cool season food legume research community are now at the threshold of deploying the cutting-edge molecular genetics and genomics tools that have led to significant and rapid expansion of gene discovery, knowledge of gene function (including tolerance to biotic and abiotic stresses) and genetic improvement of many crop species. we employed two-enzyme genotyping-by-sequencing to genotype a 367 accession lentil diversity panel (usda lentil core collection) for use in genome wide association studies for yield components. the snp markers were filtered for a minimum site count of 75% and minimum allele frequency of 0.05 resulting in 4,084 total snps in the final data set. we will present preliminary snp marker discovery based on one year of field phenotyping for days to 50% flowering, plant height, height to lowest pod, average seed per pod, pod dehiscence, 90% maturity and seed yield per plant. concurrent session viii: micropropagation & tissue culture co-chair: abdelghani nabloussi, inra, morocco co-chair: kofi akamani, southern illinois university, usa o1. in vitro micropropagation of almond rootstock soufana safih1,2*, a. bakkali1, n. ben bassou1, k. makroum2, and m. belfaiza2 1 unité de recherche amélioration des plantes et conservation des ressources phytogénétiques; centre régional de recherche agronomique de meknès, morocco; 2 laboratoire de biotechnologie végétale, ecologie et valorisation des écosystèmes, université chouaib dokkali, el jadida, morocco. *corresponding and presenting author: soufana.safih@gmail.com. abstract almond tree can produce in difficult conditions such as drought, salinity… due to the rootstock resistance and tolerance. many micropropagation protocols were developed for almond rootstock; it’s a suitable and fast method for obtaining a large number of genetically identical plants. the objective of the present work was to evaluate different protocols of almond micropropagation rootstock under the same condition to see the more efficient method. in shoot multiplication, two mediums were evaluated ms (murashige & skoog 1962) and ql (quoirin & lepiovre, 1977) supplemented with variable combination of iba and bap. the best shoot proliferation was observed through the mean number of shoots and the mean shoot length; to see the efficient protocols to increase the shoot length ms was added with bap and with ga separately. rooting is considered a critical stage in prunus sp. micropropagation; the routing effect of various concentrations of iba and ana added to 1/2 ms medium was compared; the best method corresponds to the highest in vitro rooting rate as well as root number per shoot. keywords: prunus, micropropagation , rootstock, almond. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 452 o2. micropropagation of local “beldi” almond ecotypes mohamed addi*, s. kodad, r. melhaoui, h. serghini, a. elamrani, a. mihamou, and m. abid lbpm laboratoire, faculté des sciences, université mohammed premier, oujda, morocco. *corresponding and presenting author: mohamedaddi78@gmail. com. abstract the almond tree, after the olive tree, is the fruit-bearing species, which occupies the most surface in morocco. in the eastern region of morocco, the almond tree occupies a surface of 26,000 ha producing 14 to 15% of the national production out of almonds (1). many native almond trees (local ecotypes) are still grown in the north eastern region of the country and new species have been introduced particularly cultivars imported from france and spain. local genetic resources have not yet been systematically conserved. the conventional methods of maintaining fruit-tree crops, both of local ecotypes and introduced cultivars, for germplasm preservation for long periods require extensive space and labour. tissue culture has been exploited to maintain genetic variability of local ecotypes from which crop plants could be improved, to produce healthy planted materials and to increase the number of desirable germplasm useful for almond breeding program (2). the present study was performed to determine the best combination of some growth regulators for the in vitro germination of isolated zygotic embryos in newly harvested beldi almond seeds. preliminary results showed multiple shoot initiation on ms medium containing 30 gl-1 sucrose, 1 mgl-1 bap and 8 gl-1 agar. keywords : micropropagation, almond, beldi ecotypes, genetic variability, tissue culture. (1) veille économique-secteur amandier 2017, minisère de l’agriculture et de la pèche maritime. http://www.agriculture.gov.ma/pages/rapportsstatistiques/campagne-agricole-2015-2016 (2) conservation of biodiversity through tissue culture sujata mathur research and reviews: journal of microbiology and biotechnology volume 2 | issue 3 | july – september, 2013. o3. demonstration of the neutralizing effect of lumbricus terrestris on ph abdellatif el harti and mohammed raouane* reserch team : lumbricidae, improving soil productivity and environment (lapse), centre « eau, ressources naturelles, environnement et développement durable (cern2d) » mohammed v university in rabat, ecole normale supérieure (ens), avenue mohamed belhassan el ouazani, b.p. 5118, takaddoum, rabat, morocco. *corresponding and presenting author: mohamed_raouane@yahoo.fr. abstract this study attempts to clarify the role of lumbricus terrestris in the regulation of soil ph. the experimental approach adopted for this study involved using fasting worms following a totally in vitro protocol, which made it possible to rule out all possible interference with the soil components. immersion of the worms in the reaction solutions at different phs allowed the delimitation of a tolerance zone between ph = 2 and ph = 12. in this zone, the more the ph deviates from the neutrality (6≤ph≤8), the more intense the worms’ reaction, and the more the production of cutaneous excretions becomes abundant. there is a tendency to progressively neutralize the reaction solutions. the regular spraying of the worms with the same reaction solutions made it possible to observe that the more the ph deviates from neutral, the greater the variation in the weight of the live worms. this drop in the fresh weight of the worms is indicative of the release of cutaneous excretion under the effect of ph. the neutralizing power is all the more important as the fresh weight of the worms is substantial. the application of cutaneous excretions alone obtained by stimulation with petroleum ether causes the neutralization of the acidic and basic reaction solutions. given these results, it can be said that in nature, lumbricus terrestris would directly affect the ph of the soil through its skin excretion. the ph factor is a determining factor in soil productivity and the distribution of plant groups. conditions of high soil acidity or alkalinity significantly affect plant growth because of the effect of ph on the solubility of mineral elements and their uptake by the root system. in fact, the majority of cultivated plants grow in soils with a ph close to a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 453 neutrality. the neutralizing effect of cutaneous excretion of lumbricus terrestris would explain the positive role that earthworms play in plant production. keywords: lumbricus terrestris, cutaneous excretion, ph, neutralizing effect. o4. valorization of essential oils of oregano and eucalyptus in vitro and in vivo biological control of fire blight caused by erwinia amylovora afaf ameur1,2*, naima rhallabi2, houda benchnikh1,3, marie epiphane doussomo1,3, abdellatif benbouazza1, my mustapha ennaji2, and el hassan achbani1§ 1 laboratory of bacteriology and biocontrol, plant protection unitinra-meknes morocco; 2 laboratory of virology, microbiology, hygiene and bioactive molecules and quality/eco-toxicology and biodiversity, faculty of sciences and techniques, hassan ii, mohammedia, university mohammedia, morocco; 3 departement of biology, faculty of sciences, kenitra, morocco. *presenting author: afaf.ameur@gmail.com. §corresponding author: achbani105@gmail.com. abstract fire blight is a disease caused by erwinia amylovora, which affects several plant species, mainly belonging to the rosaceae family and ornamental maloids. in morocco, fire blight made its first appearance in 2006 in the region of meknes. since then, it has led to significant economic losses and in the absence of effective control of this disease, research has now been turning to biological control. the present work aims to evaluate the effectiveness of different essential oils in vitro and in vivo against e. amylovora. essential oils are extracted from aromatic and medicinal plants using the hydrodistillation technique. the minimum inhibitory concentrations were determined by the dilution method in the liquid medium. and also tests were performed also in vivo on immature fruit of pear and in the field against the bacterium e.amylovora. for the in vitro test, most essential oils show strong antibacterial activity at very low concentrations. the choice is focused on oregano and eucalyptus, in particular, exert an antibacterial effect against the pathogen; they inhibit the development of e. amylovora with inhibition percentages respectively of 78.37% and 63.27%. the mic and mbc of the oils vary between 32 and 10 µg/ml and the mbc/mic ratio shows that both oils are bacteriolytic. for the in vivo test, the oils have been shown to be remarkably effective on immature pear fruit and at the field level. keywords: erwinia amylovora, rosaceae, biological control, essential oils, oregano, eucalyptus. o5. influence of application of silicon on black cumin (nigella sativa l.) cultivation in salt stressed environments jamila fahimi1,2*, fouad achemchem1, zakia bouzoubaâ2, nabil saffaj1, rachid bouharroud2, and rachid mamouni1. 1 team of materials, catalysis and natural resources development, department of chemistry, university ibn zohr, fsa. agadir; 2 agrophysiology & post harvest laboratory natural resources and local product research unity, crra-agadir, inra, morocco. *corresponding and presenting author: jamila. fahimi@edu.uiz.ac.ma /f.achemchem@uiz.ac.ma. abstract salinity is a harmful abiotic factor to agricultural production particularly in arid and semi-arid regions. in salt conditions almost every physiological and biochemical pathway in the plants are affected. the application of silicon is considered as an alternative approach to mitigate salt stress in plants. for that, this research aims to study the influence of silicon on black cumin nigella sativa (l) cultivation grown under three concentrations of salinity (0, 50 and 100 mm). the results showed a significant decrease of photosynthetic pigments contents, the ratio k/na and biomass in salt conditions. while, the addition of silicon acted positively in nigella sativa (l). keywords: silicon; nigella sativa (l); salinity; tolerance. o6. breeding effect on grain yield and protein content of six moroccan durum wheat varieties released during the last three decades abdelali boussakouran1*, el hassan sakar1, mohamed el yamani1, mouna taghouti2, and yahia rharrabti1 1 laboratory of natural resources and environment, polydisciplinary faculty of taza, unviersity sidi mohamed ben abdellah, taza, morocco; 2 national agricultural research institute (inra), bp 6356, rabat, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 454 morocco. *corresponding and presenting author: boussakouran@yahoo.fr. abstract durum wheat (triticum turgidum l. var. durum) is an important crop in mediterranean environments where yield potential is usually constrained by drought stress and requires efforts towards the release of new varieties not only more productive, but also offering improved yield stability across a range of environmental conditions. the present work aims to explore breeding effects during the last three decades on grain yield and protein content of six moroccan durum wheat varieties grouped in old, intermediate and modern and subjected to two contrasting environments (irrigated and rainfed) during 2016-17 crop season. measurements consisted in the following parameters: grain yield (gy) and its components spikes per square meter (sm2), grain per spike (gs), and thousand grain weight(tgw); protein content (pc); yield components were determined as: plant height (hp) and harvest index (hi). analyses of variance demonstrated the predominance of water regime effect on the majority of investigated traits since it explained more than 77% of their total variance. breeding effects on gy was marked by a genetic gain of 179 kg.ha-1.yr-1 under favorable conditions but a significant loss of 66 kg.ha-1.yr-1 under stressed conditions. old varieties displayed a relative yield stability across environments compared to modern ones which had a bad performance under water shortage conditions. on the contrary, pc showed a significant genetic gain under both irrigated and rainfed condition (0.084 %.yr-1 and 0.096 %.yr-1, respectively). under rainfed conditions, correlation analyses revealed that gy was positively correlated to sm2 (r = 0.952**), ph (r = 0.829*) and tgw, (r = 0.934**). however, a negative association was shown between gy and pc (r = -0.897*). regarding irrigated treatment, gy was highly dependent on its main components sm2 (r = 0.935**) and positively correlated to pc (r = 0.901*), however gy was negatively associated to ph (r = -0.871*) and tgw (r = -0.960**). relationship between gy and pc was of a curious pattern. in fact, while under favorable conditions increases in gy were accompanied by higher levels of pc, the opposite was observed under stressed conditions where higher gy was obtained in detriment of good grain quality (lower pc). keywords: durum wheat, grain yield, protein content, breeding effects, morocco. session ix: us-morocco research & higher education collaboration co-chair: khalid meksem, southern illinois university, usa co-chair: ahmed bouaziz, iav, morocco o1. the moroccan american commission for educational & cultural exchange mission. the fulbright mission in morocco. james miller. the moroccan american commission for educational & cultural exchange (macece), rabat, morocco. abstract not provided. o2. application process to the graduate school at southern illinois university, carbondale, il, usa oumaima chetto iav hassan ii, rabat, morocco. propsective graduate student: department of plant, soil, and agricultural sciences, college of agricultural sciences, southern illinois university, usa. abstract not provided. o3. meeting challenges and demands of agriculture through research, education and outreach and a need for more young people karen stoelzle midden*. college of agricultural sciences, southern illinois university, carbondale, illinois, usa. *presenting and corresponding author: kmidden@siu.edu. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 455 abstract there is a new paradigm for professional and scholarly demands in agriculture yet much of the public, especially students, fail to be aware of the critical need and exciting opportunities in these expanding areas. today’s global agriculture encompasses traditional farming and now also includes much broader practices and challenges. the need to address growing global population, urbanization, climate change and other world developments has inspired scientist, humanitarians, planners and leaders to seek agriculture practices to serve our changing society. these practices include alternative food systems and securities, urban agriculture, new seed development, food engineering, conservation of natural resources, recreation, and other areas connected to economics, politics, social, cultural, environmental, mental and physical human health; each that demand attention in education and research. to meet the demands and challenges, it is critical to engage talented students to pursue careers in research, education and industry for the future of agriculture. data from the united states department of agriculture states that, in the us alone, an average of nearly 58,000 high-skilled agriculture and related job openings are expected annually with only 35,000 graduates qualified to fill those positions. (https://www.purdue.edu/usda/employment/). there is a need for more young people to pursue their education, research and careers in agriculture. this presentation will discuss today’s agriculture as it relates to educational opportunities to prepare students for meaningful careers in agriculture, agriculture programs at southern illinois university and potential collaboration between southern illinois university and moroccan universities to serve the global agriculture community. keywords: agriculture research, careers in agriculture, graduate education, employability, global agriculture. o4. advice to graduate students khalid meksem* southern illinois university, usa. *presenting and corresponding author: meksem@siu.edu. abstract not provided. concurrent session x: plant pathology, nematology, and disease resistance co-chair: lamia ghaouti, iav, morocco co-chair: mustapha el bouhssini, icarda, rabat, morocco o1. current status of the root-knot nematodes (meloidogyne spp.) attacking vegetable crops in the souss region of morocco fouad mokrini1*, soukaina janati2, abdellah houari2, aicha el aissami3, ahmeh wifaya1, abdelaziz mimouni1, and mohamed sbaghi4 1 integrated crop protection research unit, nematology laboratory, inra, agadir, morocco; 2 faculté polydisciplinaire de taroudant, laboratoire des biotechnologies, valorisation et environnement, university ibn zohr, taroudant, morocco ; 3 faculté des sciences, université mohammed v, rabat, morocco; 4 national institute of agronomic research, scientific division, rabat, morocco. *corresponding and presenting author: fmokrini.inra@gmail.com. abstract root-knot nematodes (meloidogyne spp. ; rkn) are the most common nematodes attacking vegetable crops and causing serious damage worldwide. therefore, we aimed to review the current status and impacts of this nematode on vegetable crops in the saiss region of morocco. the earliest report of nematodes attacking vegetable crops in morocco was in 1982, by janati, who recorded the presence of four species of meloidogyne, m. javanica, m. incognita, m. arenaria and m. hapla. since then and up to now several surveys of nematodes associated with vegetable crops were conducted and revealed the presence of four species. however these surveys were limited and primitive since not all vegetable crops production areas were sampled to detect rkn. an intensive survey of rkn was conducted on vegetable crops growing a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 456 regions of sous-massa and showed that the root-kont nematodes (rkn) were detected in seventy-five out of 87 localities sampled (= 86%). four species were detected, viz. m. javanica, m. incognita, m. arenaria and m. hapla. both m. javanica, m. incognita were the most dominant species. the highest density of j2 of meloidogyne spp. was recorded in khmit ait amira with 349 j2 (meloidogyne spp.) (100 g soil)1. in view of the estimates of nematode densities obtained in this survey, once can assume that this genus damage vegetable crops in many cases. field studies on the population dynamics and the damage function are necessary to estimate the economic impact of this nematode on vegetable crops in the souss region. the current status of rkn on vegetable crops in the souss region is advanced and much work is needed on the distribution, damage threshold and on the management issues. keywords : root-kont nematodes, vegetables crops, survey, souss-massa. o2. a new source of resistance to hessian fly (mayetiola destructor (say)) in durum wheat (triticum durum desf.) hajar brahmi1,2*, m. el bouhssini2, f. bassi2, s. el haloui3, a. sabraoui2, k. el fakhouri2, and a. lazraq1 1 laboratory of functional ecology and environment, department of biology, faculty of science and technology, fez, morocco; 2 international center for agricultural research in the dry areas (icarda), rabat, morocco; 3 national institute of agronomic research, regional center of settat, inra, crra, settat, morocco. *presenting and corresponding author: hajar. brahmi01@gmail.com. abstract durum wheat (triticum durum desf.) is a major component of human diet worldwide, and is grown over 1 million ha in morocco. however, this crop suffers from numerous pests, in particular hessian fly (mayetiola destructor (say)) causing serious economic damage. the use of resistance cultivars has been the economic and environmentally friendly approach to protect wheat from pest damage. several resistant varieties to hessian fly have been developed and registered in the moroccan catalogue. however, because of the development of virulent biotypes, there is a need for continuous search of new sources of resistance. the present study was carried out under controlled conditions at inra in settat for two years (spring 2015 -2016) to screen a collection of 380 genotypes of durum wheat. promising genotypes from the initial screening were re-evaluated for confirmation in replicated trials. the results of phenotypic identification revealed one source of resistance to this pest. genetic studies of this new source will be undertaken to determine if this genotype carries a new gene for resistance to hessian fly in morocco. keywords: durum wheat, hessian fly, resistance, screening. o3. morphological, pathogenic and molecular characterization of rhizoctonia solani strains isolated from potato tubers imad kotba1,2,3*, mohammed achouri1, amina ouazzani touhami2, allal douira2, and el hassan achbani3 1 iav, complexe horticole, agadir, morocco; 2 laboratoire de botanique, biotechnologie et de protection des plantes, faculté des sciences, université ibn tofail, kenitra, morocco; 3 laboratoire de phytobacteriologie et de lutte biologique du crra de l’inra, meknès, morocco. *corresponding and presenting author: imad.kotba@gmail.com. abstract rhizoctonia solani kühn [teleomorph: thanatephorus cucumeris (frank) donk.] is an important fungal pathogen widespread in all potato growing areas of the world that causes stem canker and black scurf of potato (solanum tuberosum l.). the aim of this study was to find a simple and reliable technique for determining the pathogenicity of rhizoctonia solani isolates. for this purpose, sixty isolates of r. solani obtained from sclerotia on potato tubers, collected from different markets of agadir and casablanca regions, were studied for their morphology, pathogenicity and molecular characteristics. they were morphologically characterized by the production of sclerotia and moniloïd cells, and by the mycelium growth capacity at 15, 20, 25, 30 and 35°c. this morphological characterization leads to three groups of isolates. the first group contained p01 and p03 isolates, which were able to develop under 35°c. however, under 25°c, they didn’t develop sclerotia. the second group, only a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 457 formed by l17.1 isolate, did not form sclerotia under 25°c and was not able to develop under 35°c. the third group, formed by several isolates, developed sclerotia under 25°c conditions and were not able to grow under 35°c. also, a positive correlation was consistent between the production of sclerotia and moniloïd cells formation. the anastomosis reaction revealed that p01, p03, l17.1, and l4.1 isolates were identified as ag-4 and ag-3 for the other isolates. the pathogenic characterization has shown that p01, p03, l4.1, and l17.1 isolates caused important damping off of radish, tomato, beans, zucchini, and melon. however, the other isolates showed only a minor damping off disease rate. the molecular characterization confirmed the classical anastomosis grouping of the isolates into ag-3 and ag-4 anastomosis groups. the molecular characterization is the most rapid and reliable technique to determine the anastomosis group of unknown isolates. the three tests including the pathogenicity, the cultural anastomosis grouping, and the molecular method helped separating the studied isolates to two groups. keywords: rhizoctonia solani, potato, anastomosis, morphology, pathogenicity, molecular characteristics. o4. characterization and diversity of plant-parasitic nematode communities associated with saffron (crocus sativus l.) in taliouine-taznakht (morocco) and their relationships with some soil physicochemical characteristics hinde benjlil1,2*, k. elkassemi1, z. ferji1, e. mayad2, and k. cherifi2 1 iav hassan ii, campus d’agadir, département de protection des plantes, bp 18, agadir, morocco ; 2 université ibn zohr, faculté des sciences, département de biologie, laboratoire biotechnologie et valorisation des ressources naturelles, bp 8106, agadir, morocco. *corresponding and presenting author: hinde.benjlil@gmail.com. abstract plant-parasitic nematodes (ppn) are the most destructive group of plant pathogens in the world, and they are an economically important group of soil pathogens. moreover, they cause 12.3% losses to agricultural products worldwide. saffron (crocus sativus l.) considered as the most expensive spice in the globe; it has been grown in morocco for centuries in the region of taliouine-taznakht. this latter, has a great saffron reputation nationally and internationally. the saffron’s crop receives increased attention and cultivation according to morocco’s green plan. as this situation may increases saffron crop sensitivity to many pathogens as well as plant parasitic nematodes (ppn). in order to manage this bioagressors a study of their biodiversity is necessary. in this context, nematode communities were characterized in 163 soil samples collected from 11 rural communes in different altitude. fifteen genera were identified belonging to 12 families; four genera of ppn considered to be harmful for saffron were detected: ditylenchus, helicotylenchus, pratylenchus, and aphelenchoides. their frequencies are respectively 92 %; 31%; 63%, and 43% in taliouine; while in taznakht, 92%; 21%; 64%, and 56%. regarding assessing the diversity of (ppn) associated with saffron in all sites, the genus richness (g) ranged from 3 to 9 whereas shannon diversity index (h’) varies from 0, 9 to 1, 47 and evenness (e) tends to 1. the relationship between plant-parasitic nematodes and the soil physicochemical properties were investigated with principal component analysis and co-inertia analysis. keywords: frequency; diversity; plant parasitic nematodes; saffron; taliouine taznakht. o5. evaluation of diverse sets of barley germplasm for resistance to scald disease (rhynchosporium secalis) houda hiddar1,2*, sajid rehman2, zakaria kehel2, athanasios tsivelikas2, ramesh p.s. verma2, abdelkarim filali-maltouf1, and ahmed amri2 1 microbiology and molecular biology laboratory, department of biology, faculty of sciences, mohammed v university, rabat, morocco; 2 international center for agricultural research in the dry areas (icarda), rabat, morocco. *corresponding and presenting author: hiddar.houda@gmail.com. abstract leaf blotch, also known as scald, is one of the most destructive diseases of barley (hordeum vulgare l.); caused by the haploid imperfect fungus rhynchosporium secalis. scald is widely distributed throughout the a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 458 world and can cause 40% reduction in grain yield. the use of resistant cultivars remains to be the most effective, economical and environmental friendly way to control the disease but the genetic resistance to moroccan isolates is poorly understood and only few sources of resistance have been identified. this study aims at the identification of new and effective sources of resistance to scald within various subsets: best bet subset selected using the focused identification of germplasm strategy (figs; 80 accessions), reference set identified within the generation challenge program (gcp; 204 accessions), and an association mapping panel (284 accessions) developed by icarda breeders. all the accessions were sown and evaluated at seedling stage under controlled conditions. we have found that of the 568 barley accessions tested, 78 barley accessions (14%) were found to be resistant, 86 accessions (15%) to be moderately resistant, 104 accessions (18%) to be susceptible, and103 accessions (18%) to be highly susceptible, respectively. most of the resistant accessions (125; 22%) showed immune reaction. these new sources of resistance will be a useful resource for resistance barley breeding programs around the world. keywords: barley, scald, rhynchosporium secalis, resistance. o6. diversity and distribution of entomopathogenic nematodes in morocco youssef ben seddik1,2*, abdelali blenzar1, abdelmalek boutaleb joutei2, and said amiri2 1 college of sciences, university of moulay ismaïl, meknes, morocco; 2 national school of agriculture, meknes, morocco. *corresponding and presenting author: benseddikyoussef@gmail.com. abstract a survey was carried out in three moroccan regions: saïs, middle atlas and tafilalet. the aim of the survey was the selection of moroccan isolates of entomopathogenic nematodes (epn) for their use in biological control against soil-dwelling pests. 169 soil samples were tested for the presence of epn by galleria baiting technique. 14 epn isolates were found in 13 soil samples (2 isolates were found in the same sample): 8 isolates are heterorhabditidae nematodes and 6 isolates are steinernematidae nematodes. soils where epn were found differ as regards some characters (texture, ph and organic matter content). keywords: entomopathogenic nematodes, galleria baiting technique, heterorhabditidae, steinernematidae. concurrent session xi: walter management & uses resistance co-chair: abdelaziz mimouni, inra, agadir, morocco co-chair: meryem el mrini, iav, rabat, morocco o1. the public-private partnership in irrigation: what is the impact on the technical efficiency of citrus farms in the souss-massa region? nassreddine maatala1*, aziz fadlaoui2, philippe lebailly3, and majid benabdellah4 1 hassan ii agronomy and veterinary institute, rabat, morocco; 2 inra, regional agricultural research center of meknes, p.o. box 578, meknes, morocco; 3 department of economics and rural sociology, gembloux agro-bio-tech, university of liege, belgium; 4 department of social sciences, hassan ii agronomy and veterinary institute, rabat, morocco. *corresponding and presenting author: m.nassreddine@gmail.com. abstract the public-private partnership (ppp) is a cooperative arrangement that allows the government to entrust to a private company some public facilities or incorporeal investments from conception to management for a specific period. in the irrigation sector, this type of partnership appeared in 2005 and concerned the el guerdane project in the souss-massa area. since then, the government has continued to implement other projects all over the country. however, the existing literature consulted allowed us to identify a significant deficit in the evaluation and the impact of such projects on farm performance as well as the use of water resources. the aim of this research is to evaluate the impact of two irrigation modes related to water service management on the technical perfora tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 459 mance of citrus farms, which are in our case the ppp mode adopted in the el guerdane project and the conventional mode whose management is entrusted to a public institution. the selected sample includes 120 farms located in the two perimeters of el guerdane and issen. apart from the fact that issen perimeter is managed by the regional office for agricultural development of souss-mass, the two perimeters have similar pedo-climatic conditions of production mainly because of their proximity. in addition, the citrus growers in both areas adopt the same production technology. the farms in our sample were selected using the propensity score matching method, and the estimation of efficiency scores was performed using the parametric stochastic frontier approach to the production by the frontier 4.1 software. the results revealed that technical efficiency scores range from 16% to 92% for all farms selected in our sample. the average technical efficiency is 68%. basically, this result demonstrates that there is still room for increased production without additional input from factors of production including irrigation water. furthermore, the determinants analysis shows that el guerdane’s farms are relatively more technically efficient than issen’s farms. also, it has been found that membership of professional organizations contributes to the improvement of technical efficiency. in view of these results, it is recommended that the agricultural council structures intensify their support by targeting the least efficient farms. keywords : public-private partnership, irrigation, technical efficiency, el guerdane project. o2. plant water status changes induced by water deficit during phenological growth stages of young olive trees (olea europaea l.) grown in northern morocco mohamed el yamani*, el hassan sakar, abdelali boussakouran, and yahia rharrabti. laboratory of natural resources and environment, polydisciplinary faculty of taza, b.p 1223, taza-gare, university sidi mohamed ben abdellah, taza, morocco. *corresponding and presenting author: elyamani.med90@ gmail.com and mohammed.elyamani@usmba.ac.ma. abstract the present work investigates plant water status changes of young olive trees (olea europaea l.) belonging to three moroccan varieties (moroccan picholine, menara and haouzia) grown under three water regimes namely t100 (unstressed), t50 (medium stress) and t0 (high stress) in northern morocco (taza province). leaf relative water content (lrwc), water potential (ψw), and stomatal conductance (gs), were measured during the principal growth stages; dormancy (bbch code: 00), leaf development (code: 15), flowering (code: 65), fruit development (code: 71), and fruit maturity (code: 81) for the growing season 2015-2016. anova analyses showed that the three traits examined were mainly influenced by the water regime and the phenological growth stage; lrwc and ψw were under the effect of both factors in equal proportion, while gs was mostly affected by the water regime. impacts of variety and interactions were of a very minor magnitude. water deficit reduced lrwc by an amount of 12% in high stressed olive plants, while ψw and gs were more affected with a reduction of 34%. the maximum reduction was shown during fruit development. between phenological growth stages, the highest values of the three traits were recorded during dormant stage, whilst the lowest values were observed at fruit development stage. among varieties, no significant differences were shown for gs, whereas menara and moroccan picholine displayed the lowest values for lrwc and ψw, respectively. relationships among water status traits highlighted a close positive correlations between them (r>0.90***). stage-to-stage fluctuations in rainfall and temperatures and water needs, and adaptive mechanism of olive trees under water deficit could explain the findings. keywords: olea europaea l., growth stage, water deficit, water status. o3. effects of water regime and organic amendment on flowering capacity, growth and development of replacement corms of saffron under different planting densities youssef karra1*, abdelghani tahiri1, ahmed wifaya1, mohammed amine serghini2, and abdelaziz mimouni1 1 research unit “natural resources and local products”, inra, crra, agadir, morocco; 2 biotechnology and natural resources valorization lab, department of biology, faculty of sciences, ibn zohr university, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 460 agadir, morocco. *corresponding and presenting author: youssef.karra@gmail.com. abstract in morocco, saffron cultivation is mainly located in taliouine region (95%), it’s a mountainous area (>1500m of altitude) characterized by a semi-arid climate. saffron is organically grown where local corms are multiplied to serve as a propagation material. the number and size of corms are main parameters for saffron yield expression which is affected by water supply, replacement corms intensity and organic amendment rates. the objective of this study is to study the effect of planting density, irrigation and compost amendment on growth and yield of saffron. corms were planted according to four levels of planting densities (25, 50,100 and 140 corms.m-2) in two trials plot. in the first plot, different water regimes were allocated to different percentages of evapotranspiration reference (100%, 75%, 50% and 30 % of eto). in the second plot, compost was amended twice at different rates. the results obtained showed that the irrigation regime at 75% eto allowed a significant efficiency on corm and stigma yields, the high planting densities revealed a positive effect on number of corms and shoots, although, low densities showed interesting results on corms multiplication rate.on the other hand, the compost amendment had a positive effect on corm development by promoting their multiplication, to the extent that the second input was benefic for the development phase of replacement corms. finally, the choice of the optimum planting density and the appropriate irrigation and amendment rate is extremely crucial to ensure good growth and a significant yield of saffron as a perennial crop. keywords: saffron of taliouine, planting density, corms multiplication, water regime, organic amendment. o4. adaptive strategies in date palm revealed by confocal imaging technologies ting ting xiao* and ikram blilou§ plant cell and developmental biology, biological and environmental science and engineering, king abdullah university of science and technology, thuwal 23955-6900, saudi arabia. *presenting author: tingting.xiao@kaust.edu.sa. §corresponding author: ikram.blilou@kaust.edu.sa. abstract date palm are confronted by harsh environmental conditions and have therefore adapted various strategies to survive the hostile environment. to unravel the underlying mechanisms of adaptation to desert conditions we conducted a detailed analysis of date palm tissue anatomy at different developmental stages. using confocal imaging we reveal new anatomical features and complex structures in roots, shoot and leaves explaining strategies of adaptation of date palm to desert conditions. o5. evaluation of spatio-temporal variability of runoff and sediment yield for a nakhla watershed using swat model roukia boukhari taleb1*, mustapha naimi1, mohamed chikhaoui1, damien raclot1,3, mohammed sabir2. 1 iav hassan ii, rabat, morocco; 2 ecole nationale forestière d’ingénieurs, tabriquet, salé, morocco; 3 institut de recherche pour le développement, umr lisah, montpellier, france. *corresponding and presenting author: rokia.boukhari.taleb@gmail.com. abstract deterioration and degradation are two main factors caused by the increase and intense use of natural resources, soil, water as well as the aggressive natural context that affects both quality and quantity. to solve this problem, evaluating the states and evolution over time and space are essential for a planned and a sustainable future to soil and water management. swat 2012 (soil and water assessment tool) model was thus implemented on a daily time frame for the nakhla watershed, which is located at 20 kilometers in the south of the city of tetouan. treatment of environmental data (mnt, pedology, satellite imagery and climatic data) has allowed us to describe the natural characteristic of the field at a resolution adapted to the objectives of the study. the adopted methodology was composed of the combined use of the swat agro hydrological model implemented in the qgis geographical information system open source for opa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 461 erations of analysis and modeling of hydrological processes at the level of the watershed area and of swat-cup tool to optimize the operations of sensitivity and uncertainty analysis. the calibration process was performed on two temporal scales; monthly and daily; and on the two stations timezouk and nakhla; and over three periods: warm-up period from 1980 to 1983, calibration period from 1984 to 1990, and the validation period from 1991 to 1994. the results acquired after calibration (ns = 0.8, r² = 0.8) and validation (ns = 0.7, r² = 0.8) at monthly timescale were adequate and presented good level of realism in the representation of the interactions between the different compartments of the model; as well as the simulation of the flow rates; and also in assessing the spatial distribution of the importance of erosion across the basin and quantifying sediment yield. keywords: qgis, swat, hydrology, sediment, nakhla, morocco. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) poster presentations abstracts wednesday & thursday may 9 & 10, 2018 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 462 p001. in vitro germination of orange tree’s (citrus sinensis) immature embryos karim mahmoudi1,2*, najat handaji1, mohammed ibriz2, najat arsalane1, tarik aderdouri1,2, kawtar label1,2, and hamid benyahya1 1 laboratoire d’amélioration et conservation des ressources phytogénétiques, institut national de la recherche agronomique, kenitra, bp 257, morocco; 2 laboratoire de génétique et biométrie, université ibn tofail, faculté des sciences kenitra. morocco. *corresponding and presenting author: karim1.mahmoudi1@gmail.com. abstract oranges constitute the major part of the production of citrus fruit, which is the most important fruit group in international trade. the creation of new triploid hybrids via the rescue of immature embryos allows diversification of the varietal profile of orange trees. the objective of this study is to optimize the in vitro germination of immature embryos according to the chemical composition of four in vitro culture media in two variétise of orange trees (pinéale and parsons brown). at the maturity stage, the fruit was harvested and the extracted seeds were classified according to their size. only small or flat seeds were cultured in a base medium of murashig and tuker (mt) under sterile conditions. the different growth regulator concentrations were tested to obtain the best medium for seedling development: m1 (mt + 1 mg / l ga3), m2 (mt + 1mg / l kenitin + 0.5 mg / l bap + 0.1 mg / l ana), m3 (mt + 25 mg / l adenine sulphate), m4 (mt + 0.5 mg / l kenitin + 0.5 mg / l bap + 1 mg / l ga3). for bot orange variétise pinéale and parsons brown, the germination rate is maximum in m3 medium respective ly at percentages of 100% and 90%. variétise be tween 6 and 7 dayas. with respect to growth rate (mm / week), bot variétise knew a variation in the four media. similarly, the maximum acclimation rate in the m1 medium is 80% and 90% respective ly for the pinéale and parsons brown variétise. in general, the smaller the embryos, the more sensitive they are to the composition of the culture medium. it is therefore essential to optimize the components of the medium to promote their growth and their in vitro developments. therefore, the medium m1 (mt + 1 mg / l ga3) remains the best to promote good germination in short time and a better acclimatizationrate. keywords: citrus, orange tree, triploidy, in vitro germination, immature embryos. p002. the potential of cactus pear as food and forage crop for sustainable agriculture in the arid and semiarid regions mohamed arba* plant ecophysiology and cultures of arid zones laboratory, department of horticulture, hassan ii institute of agronomy and veterinary medicine in agadir, b.p. 121, ait melloul 5615, morocco. *presenting author: arbamohamed@yahoo.fr. abstract this paper describes the importance of cactus pear and cladodes as food and forage crop in the arid and semi-arid regions and its worldwide distribution. the importance of cactus pear in the sustainable agricultural systems of the arid and semi-arid regions has been discussed. the physiological features of cactus pear and the importance of this plant in morocco are presented. the potential of cactus pear as food crop and the contribution of cactus pear fruits and cladodes in the human nutrition are highlighted. the possibilities of valorization of cactus pear to byproducts, which enable high income for rural populations and the nutritional value of the fruits and young cladodes as food for human are explained. the forage resources of the arid and semi-arid regions and the area of cactus pear used for animal feed in the world are presented. the potential of cactus pear as fodder and the nutrients contents in the pads useful for animals are investigated. the possibilities of mixing pads with other fodders and food intakes developed for ruminants are suggested. the crude proteins contents and minerals in the cactus pear pads have been studied in southern morocco regarding their requirements by cows and sheep. keywords: sustaina tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 463 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) able agriculture; food crop; animal feed; fodder; human nutrition; animal nutrition. p003. olive growing for mitigation of climatic changes effects oumkeltoum krimi bencheqroun*, a. hadiddou, m. alghoum, a. mekkaoui, and l. hssaini inra meknes, morocco. *corresponding and presenting author: oumkaltoumkrimi@yahoo.fr. abstract the olive tree is one of the emblematic species of the mediterranean where it has been cultivated for more than 6000 years. in fact, 95% of the world’s olive-growing heritage is in the mediterranean basin. this tree is known for its great hardiness and its high power of adaptation to the severity of natural conditions. therefore, it is one of the plant species capable of contributing significantly to the mitigation of the effects of climate change and this, thanks to its high tolerance to drought and the high adaptability of its root system to different types soil which gives it its important role in erosion control. indeed, the olive tree is known among the populations of the mediterranean as being “the rich vegetable of poor soils”. indeed, the robustness of its root system, its important development and its capacity of adaptation to the physicochemical characteristics of the ground, its structure and its texture increase its capacity of fixation of the soil. no need to mention that the olive tree is characterized by a broadly spreading foliage that allows a clear mitigation of the effects of torrential rains on the ground and facilitates, therefore, the infiltration of runoff water. similarly, the evergreen leaves (which are usually renewed every three years) which characterize the olive tree and which have fine hairs on their underside, simultaneously allow the preservation of moisture and increase the absorption capacity. carbon in the olive tree. hence its great role as protector of the environment. genetically, it is one of the plant species that has great richness and remarkable variability. at present, more than 2000 olive varieties have been identified and are cultivated worldwide. in spain, 262 varieties have been recorded, 476 varieties in italy and more than 150 varieties in algeria. in this plant species, tolerance to drought is a varietal character. indeed, the level of resistance varies according to the different varieties and according to the pedoclimatic conditions. this is due to the importance of the genotype x environment interaction. hence the interest of comparative trials for the identification of the most successful varieties and best adapted to different climatic conditions. in morocco, in addition to its ecological role, olive growing is of great socio-economic importance. the latter has always occupied more than half of the national arboreal surface. recently, the area reserved for olive trees has exceeded 1,000,000 ha. given the importance of the added value of the olive sector on the national economy, the benefits of products, good quality, this tree (table olives and olive oil) on human health, which explain the rise demand at the national and international levels, morocco is consciously committed to the extension of the area reserved for olive growing. this relevant choice will give it a good preservation of the environment and a better mitigation of the effects of climate change on the earth and this thanks to the many characteristics and natural characteristics of the blessed tree including the olive tree. keywords: olive tree, climactic changes, natural specificities, characteristics, mitigation, preservation, environment. p004. economic water management under climate change impact elame fouad1*, hayat liounboui1, doukkali rachid2 1 institut national de la recherche agronomique, inra, morocco; 2 institut agronomique et vétérinaire hassan ii, rabat, morocco. *corresponding author : abstract the recent statistics show a clear downward trend in global and per capita availabilities of water due in particular to climate change. in addition to the recurring droughts that morocco experienced during the last two decades, this tendency is accentuated by an unceasingly increasing demand, in particular under the effect of the demographic pressure and the economic development. in a context of scarcity and a limitation of potential resources not yet mobilized, morocco is confronted with the need of changing its supply policy to a demand management one. the a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 464 implementation of such policy requires the adoption of new management instruments and new institutional forms of organization. accordingly, the present study proposes a tool of modeling and decision-making support which integrates the economic, institutional, hydrological and agronomic aspects. the proposed approach is based on optimization techniques and positive mathematical programming to calibrate an empirical model. using climate change impact scenarios, this integrated economic model is tested for the basin of souss-massa. these simulations include changes in water availability and economic conditions, as well as demand management policy. the study results show that the demand management policy at the river basin level should take into account the regional specificities. the basin’s water resources are substitutable and water management policy cannot ignore this aspect and should integrate surface and groundwater resources at the same time. in drought conditions, the water marginal value ‘shadow price’ increases considerably such that water pricing policy alone cannot result in a rational and a sustainable use of the resource. keywords: water resources, climate change, drought, river basin, integrated economic model, scenario. p005. monitoring milk urea concentrations as strategies to improve the efficiency of nitrogen use in haouz dairy herds in central morocco napoléon munyaneza* and youssouf el hachimi department of biology, laboratory of food, environment and health of cadi ayyad university of marrakech. *corresponding author: munyaneza305@ gmail.com. abstract the aim of this study was to determine the milk urea concentrations variation and their potential relationships with feeding systems and milk production. a total of 251 bulk milk samples was collected in 83 dairy herds and measured in urea by calorimetric methods at the laboratory of food, environment and health at cadi ayyad university in marrakech, morocco. feeding strategies and milk production data were collected as part of a formal survey and analyzed using statistical package for social science (spss). the average of urea concentrations obtained was 11, 4 ± 1, 4 mg n/dl, with a range of 8, 1 to 15, 9 mgn/dl. out of all samples, 21, 8% had values outside the reference range (12 ± 2 mgn/dl), 3, 1% had values >14mgn/dl and 18, 7% had values < 10 mgn/dl. the milk production ranged of 4 to 28 liters/day/cow. data analysis on feeding systems used by farmers revealed that about 12% used alfalfa alone, 72,2% alfalfa plus cereals straw, 7, 3 % alfalfa and cereals straw plus residues of beet and 8, 5 % used alfalfa plus cereals straw supplemented by cereals bran. the correlation between urea concentrations and milk production was observed (r = 0,731; p < 0, 01). when yield milk was > 13, 5 liters /cow (51, 3%), milk urea concentrations was > 12 mgn/dl. the milk production was lower and urea in milk less to 10 mgn/dl (20%) when the cows received alfalfa alone or associated to cereals straw. the results showed a large variations of urea milk concentrations in relation with yield milk and feeding systems between farmers. thus, monitoring urea concentrations in milk would provide information that could help farmers to use nitrogen inputs and increase the profitability under the specific conditions of each farm. keywords. urea milk, dairy herds, nitrogen efficiency, milk production. p006. pseudomonas savastanoi pv. savastanoi, causal agent of olive knot disease in morocco: molecular identification and biological control abdelaaziz bouaichi1.2*, rachid benkirane2, khaoula habbadi2, said el kinany3, hanane lougraimzi4, mohamed ou-zine3, soumia sadik5, abdellatif benbouazza1, and el hassan achbani1 1 laboratory of phytobacteriology and biocontrol, national institute of agronomic research, meknes, morocco; 2 laboratory of botany, biotechnology and plant protection, faculty of science, ibn tofial university, kenitra, morocco; 3 laboratory of microbial biotechnology, faculty of sciences and techniques, sidi mohamed ben abdellah university, fez, morocco; 4 laboratory of health, nutrition and environment, faculty of sciences, ibn tofail university, kenitra, morocco; 5 laboratory of plant biotechnology and molecular biology, faculty of sciences, moulay ismail university, meknes, morocco. *corresponding and presenting author: bouaichi.inra@gmail.com. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 465 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) abstract olive knot disease caused by pseudomonas savastanoi pv. savastanoi (pss) is considered one of the most serious diseases affecting olive trees (olea europaea l.) in most olive growing regions worldwide and mainly in mediterranean countries, which can lead to severe damage in olive groves, causing serious losses in terms of production. this study aimed to isolate and identify the organisms causing olive knots on stems and branches in different regions in morocco as well as biological control with bio-agents like essential oil and antagonistic bacteria. bacteria were isolated from young knots on selective media. biochemical tests, pathogenicity tests and pcr were performed to identify the strains causing disease. fifty-nine isolates were identified as pss using biochemical, pathogenicity tests and pcr technique using specific primer. based on the inoculation of olive plants (cv. arbequina) with isolated strains, twenty-five caused typical knots at the site of inoculation. to control pss, essential oils (eo) thymus ciliatus and the origanum compactum exhibited in vitro inhibition of pss strains. their inhibition rates were respectively 56.25 % and 50%. also, some biocontrol agents, rahnella aquatilis, enterobacter ludwiqii, paenibacillus brasiliensis, and bacillus subtilis selected from collection of regional centre of agronomic research revealed in vitro antagonism toward pathogen strains. the most efficient strain, b. subtilis 2515-1 was selected for pots experiment using olive trees (cv. arbequina). this bacterium significantly reduced number and weight of knots on trees inoculated with two pathogens strains (pss 2064-8 and pss 2102-4). keywords: pseudomonas savastanoi pv. savastanoi, olive knot disease, molecular identification, biological control, essential oils, antagonistic bacteria, bacillus subtilis. p007. improving the growth of date palm in morocco through the use of biofertilizers including the plant growth promising rhizobacteria (pgpr) oufli m1,2*, elkinany s1, oouz-ine m1, hilali h2, and el hassan achbani1 1 laboratoire de microbiologie et protection des plantes crra, inra, meknès, morocco; 2 laboratoire de microbiologie, faculte des sciences et techniques, university mohamed i, settat, morocco. *corresponding and presenting author: m.oufli@uhp.ac.ma. abstract excessive application of agrochemicals to improve agricultural production can cause several problems related to their toxicity to humans, soil and groundwater pollution, and biodiversity loss. the current trend is the reduction in the use of pesticides and mineral fertilizers. to achieve this, the use of rhizobacteria has proven to be a promising approach. rhizobacteria known as plant growth promising rhizobacteria (pgpr) are now considered an important means for sustainable agriculture because of their ability to improve yield and fight against biotic and / or abiotic stresses. which will significantly affect the mitigation of the use of chemicals and thereby reduce their harmful effects. rhizobacteria are endowed with the potential of biological fixation of nitrogen, which reduces the nitrogen fertilizer supply. thus, they can be considered as a renewable resource. recently, the use of plant growth promoting rhizobacteria (pgpr) in agriculture has become an alternative to reducing the harmful effects of chemicals on the environment and providing sustainable agriculture. keywords : pgpr, date palm (phoenix dactylifera l.). p008. the moisture sorption isotherms and the thermophysical characterization of durum wheat “triticum durum l.” haytem moussaoui*, idlimam ali, and abdelkader lamharrar laboratory of solar energy and medicinal plants, teacher’s training college, cadi ayyad university, bp 2400 marrakesh, morocco. *corresponding and presenting author: haytem.moussaoui@gmail.com abstract durum wheat “triticum durum l.” is a cereal, a wheat variety cultivated since prehistoric times, known for its hard and vitreous grain. it is rich in protein and gluten. durum wheat is mainly used for pasta, especially in countries of europe and north america, while in other regions (middle east and north africa), it is also used as couscous and different types of bread. the ‘karim’ verity is the most popular variety in morocco and the a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 466 most widespread. it is a cultivar with wide adaptation (different environments: semi-arid and irrigated); it is also early cultivar as well as tolerant to few diseases (septoria and rust). the objective of this work is to develop and validate a trial model of durum wheat by identifying the adsorption and desorption curves and optimum water activity. these data constitute an important source of information for establishing stability and the typical storage conditions for the product. when you want to keep a dry product, it is necessary to know the water content of the product needed for its stability. in the hygroscopic field, there is a specific relationship between the moisture content of the product and the relative humidity of the surrounding environment. therefore, when we want to determine the appropriate water content to keep the product at a certain temperature, it is necessary to know the relative humidity of the surrounding air, which is respected to store properly. keywords: durum wheat, hygroscopic equilibrium, moisture sorption, optimal water activity, sorption isotherms. p009. quality proprieties and the proceeding evolution of the phenols composition during the maturity of the diploid and triploid vitis vinifera l. grape in marrakesh region hicham jediyi*, khalid naamani, abderrehim ait elkoch, nawal zhar, naima lemjiber, and a elkeroumi laboratory of protection and valorization of vegetable resources. faculty of sciences semlalia marrakech, cadi ayyad university, my abdallah street, pb: 2390, marrakesh 40000, morocco. *corresponding and presenting author: jedhicham@gmail.com. abstract the objective of this study was to determine the quality proprieties according to the effect of phenolic compounds on grape during maturity of three introduced triploid varieties: superior, early sweet, flame, and two others locale diploid varieties: abbou and adari in oudaya of marrakesh region. the varieties samples were evaluated by 12 quality parameters and phenols, flavonoids and tannins compounds. the descriptive quality panel found that introduced or locale grape attributes were significant difference (p≤0.05) among varieties samples. the statistical test established that the variation of quality proprieties generally and maturity index (°brix/acidity) especially was influenced by the phenolic compounds and the stage of maturity according to fruit maturity. were the analysis shown as that the phenols, flavonoids and tannins compounds decreased for the triploid and increased up for the diploids during maturity accompanied an increased up of quality proprieties except acidity, and that for of all varieties. the analysis revealed that the phenolic compounds, the rapport of sugar/acidity remain the principals’ attributes determinant the maturity of fruits. a classification of fruit maturity stage according to variety and quality proprieties according to phenols composition by pca analysis is provided. as regards the distribution of variables, we have noted that the juice content, sugars, maturity index and °brix for pc2, with phenols, flavonoids and tannins for pc1 are the most variables that have contributed to the discrimination of the grapes according to their stage of maturity and varieties. keywords: vine varieties. quality proprieties. phenols composition. maturity grape stages. p010. evaluation of antibacterial activity of mentha pulegium essential oil against strains of phytopathogenic bacteria hanane lougraimzi1*, meryem el jemli2, el hassan achbani3, abdellah aghraz4, abdelaaziz bouaichi5, and mohamed fadli1 1 laboratory of health, nutrition and environment, faculty of sciences, ibn tofail university, kenitra, morocco; 2 laboratory of pharmacology, faculty of medicine and pharmacy, mohammed v university, rabat, morocco; 3 laboratory of phytobacteriology and biocontrol, national institute of agronomic research, meknes, morocco; 4 laboratory of biotechnology, protection and valorisation of plant ressources, faculty of sciences, cadi ayyad university, semlalia, morocco; 5 laboratory of botany, biotechnology and plant protection, faculty of sciences, ibn tofail university, kenitra, morocco. *corresponding and presenting author: hananelou04@gmail.com. abstract aromatic and medicinal plants have interesting biological activities. the aim of the study is to examine a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 467 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) the in vitro antimicrobial activitie of essential oil of the leaves of menhta pulegium (l) against of phytopathogenic bacteria and to propose the alternative solutions based on the use of natural products instead of chemicals while avoiding the presence of toxic substances in the air and soil. the essential oil of this plant was obtained by the hydrodistillation method (clavenger). the inhibitory effects of this essential oil were tested against phytopathogenic bacteria by using agar disc diffusion and dilution broth methods. the results obtained showed a sensitivity of the different strains to the essential oil of mentha pulegium, which allowed presenting a therapeutic potential of this plant which could be exploited as a natural antibiotic for the treatment of several infectious diseases caused by these germs and could be helpful in understanding the relationship between traditional remedies and current antibiotics. keywords: antibacterial, essential oil, mentha pulegium, sensitivity. p011. synergistic effects of the inoculation with plant growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on the plant growth in the field anas raklami1,2*, noura bechtaoui1, abdel-ilah tahiri1,2, zakaria es-salmi2, abdelilah meddich2, and khalid oufdou1 1 laboratory of biology and biotechnology of microorganisms, faculty of sciences semlalia, cadi ayyad university, po box 2390, marrakech, morocco; 2 laboratory of biotechnology and plant physiology, faculty of sciences semlalia, cadi ayyad university, po box 2390, marrakech, morocco. *corresponding and presenting author: anas.raklami@gmail.com. abstract through many researches, arbuscular mycorrhizal fungi (amf) and plant growths-promoting rhizobacteria (pgpr) have been shown to promote plant growth, yield and nutrient uptake through a range of mechanisms that include improved nutrient acquisition especially phosphorus (p) nutrition, producing plant growth hormones, improving host physiological processes and inhibition of plant pathogens. the purpose of this study was to evaluate the effect of the inoculation with plant growth-promoting rhizobacteria and arbuscular mycorrhizal fungus and their potential to stimulate two of the most widely grown crops over the world. they are not only an important source of proteins in food and feed, but also a significant component of different agrosystems. these plants are the legume plant vicia faba l. (aguadulce variety) and the cereal plant triticum aestivum (karim variety). the effect of double inoculation, was studied in field experiment in agricultural soil with six treatments, the control without inoculation, pgpr alone, rhizobia alone, mixture of pgpr and rhizobia, amf alone and a mixture of pgpr, rhizobia and amf. the greatest effects were observed in the inoculation with a mixture of pgpr-rhizobia-amf. this inoculation improved the growth parameters (length, fresh and dry weight of shoot and root, number of leaves) of v. faba and t. aestivum. this indicates that the mixed inoculation significantly stimulated the growth of crops and could be used as potential bio-fertilizers to optimize plant growth and yield. keywords: arbuscular mycorrhizal fungi, rhizobia, growth, vicia faba l., triticum aestivum, fields, pgpr, biofertilizers. acknowledgments: this work is financially supported by the project pprcnrst. p012. characterization and identification of olive varieties using simple sequence repeats markers ahmed el bakkali* and abderahman mekkaoui inra, ur amélioration des plantes et conservation des ressources phytogénétiques, meknès, morocco. *corresponding and presenting author: ahmed_elbakkali@yahoo.fr. abstract olive (olea europaea l.) is an important fruit species cultivated for oil and canned fruit in the mediterranean basin. ex situ conservation is essential for the optimal management and use of genetic resources for both plant breeding and sustainable production. the collection of ain taoujdate (at), meknes, is one of the ancient ex situ olive germplasm bank in morocco with 78 varieties from 9 mediterranean countries. the characterization of all varieties provide useful information for managing olive germplasm to be preserved, exchanged and used in breeding program. in this work, we used 17 preselected microsatellites markers (ssr) to characterize 95 accessions in at cola tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 468 lection and results were compared to the world olive germplasm bank of marrakech (wogbm) database with 554 olive accessions. over all the analyzed accessions, low genetic diversity was observed in at collection compared to the wogbm with 48 and 402 genotypes, respectively, in which 37 genotypes are common between both collections and 11 genotypes exclusively present in at collection. additionally, fifty five errors related to mislabeling plantation and high proportion of redundant genotypes for “picholine marocaine” and “frantoio” varieties were revealed. we were able to identify six genotypes considered as molecular variants and 18 cases of synonyms in at collection such as “loretana” and “dritta di moscufo” varieties. even with the low genetic diversity identified in the at collection compared to the wogbm, 135 and 261 alleles, respectively, the principal coordinates analysis (pcoa) revealed that genotypes of at collection span the range of all the 405 genotypes identified in the wogbm. this study highlighted the powerful of ssr markers used to discriminate between olive varieties and represents a preliminary step towards managing the ex situ olive collection, authenticating varieties and developing further association mapping studies. keywords : olea europaea, ain taoujdate olive collection, world olive germplasm bank of marrakech, microsatellites (ssr), genetic diversity management. p013. analysis of phenotypic diversity of moroccan barley accessions maintained ex situ at the national genebank of inra, morocco ali sahri1*, hassan ouabbou1, naima qariouch1, ali amamou1, and abderrazek jilal2 1 institut national de la recherche agronomique (inra), centre régional de la recherche agronomique de settat, b.p : 589, settat, morocco; 2 institut national de la recherche agronomique(inra), centre régional de la recherche agronomique de rabat, b.p : 6356 rabat, instituts, morocco. *corresponding and presenting author: sahriali@gmail.com. abstract morocco has long been considered one of the major centers of diversity for barley (hordeum vulgare l.). it is one of the most important traditional crop and major source for proteins and minerals, in addition to its multiple uses as food and feed. the national genebank of inra-morocco based in settat conserves and manages more than 3000 barley accessions (representing old cultivars and local landraces) collected from the different regions of morocco. the characterization of diversity in barley germplasm is very important for utilizing the collection efficiently and effectively. in this study, a representative collection of 200 accessions of barley landraces, belonging to eight different agro ecological regions of morocco, were analyzed for its diversity of ten agro-morphological traits. based on shannon-weaver diversity indices (h’), moroccan barley landraces display an indice of 0.66, indicating the presence of significant degree of variability among the studied populations conserved at the national genebank. for the agroecological regions, the highest mean diversity index (h’ = 0.70) was observed for accessions from the atlas and the rif mountains. a multiple correspondence analysis (mca) was carried out on the basis of the ten agro-morphological traits to define the structure of the diversity. the three first axes of the mca explained 38% of the total variance in the collection and separated the accessions into two main groups. the first group includes the accessions from the rif and atlas mountains and the second group includes the accessions from the other agro ecological regions. the results of the mca indicated that the geographical proximity and altitude play a main function in the discrimination and the structure of the studied barley accessions. the information generated from this study (high variability) could be used in conventional breeding programs and in situ conservation. keywords: barley accessions, national genebank, agro-morphological traits, shannon-weaver diversity index, variability, multiple correspondence analysis. p014. effects of five biostimulants on peach production and quality in sais plain (morocco) meriame laita1,2, zerhoune messaoudi1*, rachid benkirane2, and lhoussain ait haddou1 1 department of arboriculture-viticulture, national school of agriculture, b.p. s40, meknes, morocco; 2 botanical, biotechnology and plant protection laboratory, faculty of sciences, university ibn toufail, bp 133, kénitra, morocco. *corresponding and presenta tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 469 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ing author: messaoudiz@yahoo.fr. abstract improvement of production and quality is among the biggest challenges of peach growing under calcareous soil and dry and hot summer conditions of sais (morocco). high soil ph induces low micronutrients uptake which favors leaf chlorosis and extreme temperature and dry air during end of spring and summer time altogether with other conditions induce high incidence of fruit drop and uncertain fruit quality. the present experiment was carried out during 2017 season in a conventional orchard of the zee lady variety of peach (the rootstock is the almond) in sais, morocco with an aim to evaluate biostimulant effects of five products on peach yield and quality. six variants namely vitazyme, protifert lmw 10, protifert lmw 6,3, protifert zinc and naturfer biostimulants and control (unsprayed trees) were compared according to a completely randomized design with 3 repetitions. data collected indicated a remarkable effect of biostimulant on fruit number per tree and harvest size and some pomological fruit parameters namely fruit weight, dimensions, core weight and core/flesh ratio. keywords: peach, fruit drop, fruit quality, biostimulant, pomological. p015. new insights of climate change effects on codling moth (cydia pomonella l.) voltinism in apple orchards and perspectives of efficient control strategy salma el iraqui1* and m’hamed hmimina2§ 1 plant protection department, national institute of agricultural research, meknès regional center, bp 578, meknès, morocco; 2 zoology department, institut agronomique et véterinaire hassan ii, b.p 6202, madinat al irfane, rabat-institutes, rabat, morocco. *presenting author: iraquisalma@gmail.com. §corresponding author: hmimina@yahoo.fr. abstract codling moth (cydia pomonella l.) is the worldwide k&ey pest of apples causing heavy economic losses in fruit production. in morocco, during the last decades, a massive flight of moths leads to more than 12 chemical treatments per year. to improve the control of cydia pomonella, this study aims to accurate its voltinism in relation with temperature variations. the progress of codling moth generations was investigated during 11 years in a moroccan apple orchard based on catches of sexual traps. the degree days (dd) method was also investigated to verify its adaptability to phenological forecasting models. the impact of temperatures of two periods was investigated: temperatures matching with the beginning of the life cycle of cydia pomonella (april and may) and temperatures prevailing in the period conducive to diapause (july 10 to early august 5). results revealed that the voltinism of codling moth had evolved in 2, 3 to 4 generations, depending on the season’s temperature. three complete generations and a fourth partial one characterized mostly its life cycle. the accomplishment of the fourth generation is highly expected in seasons recording high temperatures in april and may (360dd: case of 2014 and 2015). these high temperatures allow an earlier and faster development of the first generation which impacts the progress of the following ones, extends the presence of the insect and consequently increases its voltinism. moreover, temperatures during, july to early august, are always high in morocco and thus inhibit insect diapause and extends its duration of presence until the harvest. finally, the estimated generations, based on dd method, were in concordance with codling moth flight dynamics, supporting the use of phenological models. climate scenarios predict a rise in temperatures in the future, favoring the accomplishment of the fourth generation and consequently more damages in productions and more insecticides used. keywords: cydia pomonella l., voltinism, temperatures, degree days. p016. isolation and identification of aerobic bacterial community in earthworm (lumbricus terrestris) chloragogen tissue lamia yakkou*, maryam chelkha, mohammed raouane, souad amghar, and abdellatif el harti reserch team : lumbricidae, improving soil productivity and environment (lapse), centre “eau, ressources naturelles, environnement et développement durable (cern2d)” mohammed v university in rabat, ecole normale supérieure (ens). avenue med belhassan el ouazani. bp 5118, takaddoum-rabat, morocco. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 470 *corresponding and presenting author: yakkou.lam@ gmail.com. abstract microorganismes living in association with earthworms have a fundamental ecological and agronomic interest. several studies have demonstrated the involvement of these invertebrates’ intestinal flora in improving soil productivity, particularly in the processes of humification, decomposition and recycling of organic matter. in this work, we investigated the isolation and identification of other microbial species living in association with earthworms (lumbricus terrestris) in organs other than the gastrointestinal tract. the microscopic observation of chloragogen tissue smears, taken by dissection of the worm, revealed the presence of microbial entities of various shapes and sizes. isolation and cultivation made it possible to inventory seven bacterial strains. phenotypic and genotypic studies (pcr amplification of 16s rdna), allowed identification: pseudomonas fluorescens, pseudomonas montelii, citrobacter freundii, enterobacter aerogenes, staphylococcus epidermis, teribacillus aidengensis and pseudomonas rhizosphaerae. among the identified bacterial strains, the three species of pseudomonas (fluorescens, montelii and rhizosphaerae), along with enterobacter aerogenes are all of ecological and agronomic interest, particularly in improving plant productivity and soil bioremediation. keywords: lumbricus terrestris, chloragogen tissue, microorganisms, pcr-16s rdna, soil productivity. p017. study of chemical composition and antibacterial activity of thymbra capitata essential oil and its use in carrot juice preservation saoulajan charfi*, nadia boujida, jamal abrini, and nadia skali senhaji laboratory of biotechnology and applied microbiology, team biotechnology and applied microbiology, department of biology, faculty of science, abdelmalek essaadi university, tetouan, morocco. *corresponding and presenting author: sawlajan@gmail. com. abstract the aim of this work was to investigate the composition and antibacterial activity of moroccan thymbra capitata essential oil (tceo) and to evaluate its application as food preservative in carrot juice conserved at 20 and 5°c. chemical composition was analyzed by gc/ms. tceo antibacterial activity was evaluated by diffusion method and microtitration assays against escherichia coli o157:h7 cect 4267, listeria monocytogenes cect 4032 and staphylococcus aureus cect 976. in carrot juice, tceo activity was evaluated against these pathogens during 30 days storage at 20 and 5°c. analysis of tceo chemical composition showed that the main constituent of tceo was carvacrol (75.52 %), followed by p-cymene (9.75%) and β-caryophyllene (6.45%). tceo exhibited an antibacterial activity in vitro against the tested bacteria. in carrot juice, tceo was able to inhibit the growth of all pathogenic bacteria at both storage conditions. l. monocytogenes was the most sensitive strain to tceo when tested with diffusion method and in carrot juice, followed by s. aureus and e. coli o157:h7. tceo was more efficient against e. coli o157:h7 in carrot juice conserved at 5°c than at 20°c. on the other hand, no significant difference between storage temperatures was noted for l. monocytogenes and s. aureus. these results show the ability of tceo to inhibit pathogenic bacteria and to maintain the safety of carrot juice. keywords: thymbra capitata essential oil, chemical composition, antibacterial activity, carrot juice. p018. effect of six aromatic plants essential oils on storage keeping quality of fresh strawberries and raspberries asmaa mziouid1*, bouchra chebli1, and el hassan mayad2 1 laboratory of mechanic, process, energy and environmental, biotechnology and environmental engineering team, national school of applied sciences, ibn zohr university, po box: 1136/s, agadir, morocco; 2 laboratory of biotechnologies and valorization of natural resources, faculty of sciences agadir, ibn zohr university, agadir, morocco. *corresponding and presenting author: mmesadek@hotmail.fr. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 471 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) abstract six essential oils (eos) were hydrodistillated in a clevenger-type apparatus from: artemisia absinthium, artemisia herba-alba, cladanthus arabicus, lavendula dentata, thymus broussonnetii and thymus leptobotrys plants growing in morocco. the eos were tested for their antifungal activity first in vitro against botrytis cinerea using agar dilution and disk diffusion methods. and then the eos showing the lowest minimum inhibitory concentration were tested for their ability to extend the shelf life of fresh strawberries and raspberries, conserved at 4°c. in vitro, cladanthus arabicus had the lower antifungal property. the essential oils of thymus leptobotrys and thymus broussonnetii inhibited the growth of fungus at 250 µl/l and 500 µl/l respectively and were found to be more efficient preservatives for strawberries and raspberries during 11 days of cold storage. the results suggest that essential oils vapour may improve fruit quality during fresh strawberries and raspberries storage and transit. keywords: artemisia absinthium; artemisia herba-alba;cladanthus arabicus; lavendula dentate; thymus broussonnetii ; thymus leptobotrys; botrytis cinerea; essential oils; shelf life; strawberries; raspberries p019. fertilization practiced by olive growers of sais and its impact on olive orchards. karima bouhafa1*, lhoussaine moughli2, khadija bouchoufi1, and abdelkader sdouq1 1 regional agricultural research center, p.o.box 578, meknes, morocco; 2 department of soil science, hassan ii institute of agronomy and veterinary medicine, p.o.box 6202, rabats-instituts, rabat, morocco. *corresponding and presenting author: bouhafakarima@ gmail.com. abstract a study on fertilization practices adopted by farmers was carried out, through surveys, soil and leaf analysis, at 58 olive orchards randomly selected in sais region. soil samples were collected from the 0-30 and 30-60 cm layers during december-january. leaf samples were also collected during the same period. the results of the surveys showed that about 48% of the olive growers do not use any mineral fertilizer for their olive trees. this has affected the olive yield, which averaged 3 t / ha in fertilized orchards and 1.9 t / ha in unfertilized orchards. nitrogen is the most provided by the farmers with an average dose of 37 kg n / ha, followed by phosphorus with 20 kg p2o5 / ha and potassium with 9 kg k2o / ha. these doses are low for the olive tree. in addition, phosphorus is provided by some farmers at very high doses that have reached 138 kg p2o5 / ha. times of fertilizer intakes were generally concentrated over the period from january to april, indicating a lack of inputs during other periods where the need for mineral elements being important for the olive tree such as the fruit enlargement phase. these data indicate the existence of a failure in the fertilization practices adopted by olive growers concerning both the fertilizer doses brought and the timing of fertilizer inputs. soil tests showed that 50% and 84.5% of the soils studied are low in phosphorus, respectively, for the 0-30 cm and 30-60 cm layers. soil potassium levels are low to medium in 15.5% and 55% of olive orchards, respectively, for 0-30 cm and 30-60 cm soil layers. these soil analysis results from these olive groves confirmed the existence of a failure in the fertilization practices adopted by farmers. leaf analyzes showed that all the studied orchards require nitrogen inputs and nearly 91% of orchards require potassic fertilization. for phosphorus, only a third of the orchards sampled need an application. this study showed that fertilizer is not well controlled by olive growers and olive orchards are not well fed with essential elements for their production. keywords: fertilization, survey, sais, soil fertility, nitrogen, phosphorus, potassium. p020. impact of pgpr solubilizing phosphate bacteria on growth and phosphorus uptake of legumineuse plante vicia faba l. bechtaoui noura1*, raklami anas1, el alaoui abdelkhalek1, benidire loubna1, göttfert michael2, and oufdou khalid1 1 laboratory of biology and biotechnology of microorganisms, faculty of sciences semlalia, cadi ayyad university, po box 2390, marrakech, morocco; 2 technische universität dresden, institut für genetik, helmholtzstr. 10, d-01069 dresden, germay. *corresponding and presenting author: noura.bechtaoui@ a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 472 gmail.com. abstract phosphorus (p) is a critical element for plant growth; its deficit severely limits crop yields. a considerable amount of phosphate is concentrated in the upper part of soil in mineral or organic composition. however, these forms are no longer available, because they rapidly react with soil components, therefore only a small quantity of soluble phosphate can actually be absorbed by the roots. a low amount of phosphate limits plant growth, especially in periods of high use, flowering and fruiting stage. some of the rhizospheric bacteria have the ability to solubilize the complex forms of p in the soil and make it available to plants. these bacteria, in addition to improve phosphate nutrition, my also promote plant growth by producing phytohormones and some other beneficial substances such as siderophores and antibiotic compounds. the aim of the present study is to screen the phosphate solubilizing bacteria from agricultural soils and to test the synergistic or antagonistic effects of a mixed inoculation on the growth of the legume vicia faba l. the in vitro test of phosphate solubilizing capacity of strains used during this study was carried out on nbriy medium (nautiyal, 1999). the capacity of bacteria to produce siderophores, auxins and exopolysaccharides was also analysed. all of the tested strains produce a significant amount of siderophores and auxins. inoculation experiments with faba bean revealed that some symbiotic combinations result in an increase in biomass and p content of the inoculated plants. during this study, we prove through our laboratory tests and under greenhouse experimentation, that phosphate solubilizing bacteria could be used as potential bio-fertilizers to optimize the phosphorus supply of plants in deficient soils. keywords: solubilizing pgpr bacteria, rhizosphere, siderophores, vicia faba. p021. selection and characterization of rhizobacteria isolated from the rhizosphere of peanut (arachis hypogaea l.) ouissale chaghouaoui*, imane achkouk, amin laglaoui, mohammed bakkali, abdelhay arakrak equipe de recherche de biotechnologies et génie des biomolécules (erbgb), faculté des sciences et techniques de tanger b.p. 416, tanger, morocco. *corresponding and presenting author: chaghouaouiouissale@gmail.com. abstract the rhizosphere supports the development and the activity of a microbial community including the plant growth-promoting bacteria or the pgpr, they play an important role in the biofertilisation of where the interest of the use as alternatives of chemical fertilizers in order to respond to the need of food for the world population, they may intervene either directly by providing the nutrients or by producing growth regulators or indirectly by removing the phytopathogenic agents. in this context of sustainable agriculture and the protection of the environment, this work led to the isolation and characterization of the solubilising bacteria the phosphate (bsp) associated with three varieties of peanuts (arachis hypogaea) (jumbo, valencia and beldi) grown in pots under greenhouse conditions for 28 days. various tests have been carried out on the isolates studied to evaluate the different phytobenificial properties in vitro. a total of 79 isolates on 300 isolates tested (26.33%) have been capable of dissolving the tricalcium phosphate (tpc) on mid pikovskaya solid, and whose diameters of halo have been between 1 and 10 mm. the isolates with diameters of solubilization ≥ 4 mm (49 isolates) were selected and evaluated for the production of indole acetic acid, siderophores, and ammonia which the percentages are respectively 69.38%, 91.83% and 100%. 6 bsp (jp19, jr33, jr41, jr48, bp49 and br44) have been tested for the solubilization of the ptc in mid pvk liquid with the highest concentration (49.96± 0.85 mg/l) has been registered by jr19. keywords: arachis hypogaea, biofertilisation, bsp, pgpr, rhizosphere. p022. pesticide wastes management in some horticultural farms in the souss massa abderrahim hormatallah* laboratoire des pesticides. institut agronomique et vétérinaire hassan ii, complexe horticole d’agadir, bp 18/s. agadir, morocco. *corresponding and presenting author: phytopharmacie@yahoo.fr. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 473 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) abstract horticulture is one of the most relevant segments of the moroccan agriculture sector. morocco country reaches the self-sufficiency in vegetable production. this sector provides also an employment for the working rural population and important source of hand currency towards exportation to the european union, canada, russia and usa. the intensive conditions of the horticulture crops (citrus, tomato, pepper, green bean…) seem to be favorable to many insects (leaf miners, thrips, whiteflies ..) and the development of some diseases (powdery mildew, downy mildew, gray mold…) that cause more damage and affecting the yield quality. growers are often applying pesticides to protect their production and the yield quality. pesticide waste disposal is a concern for moroccan horticultural producers. in order to meet the objectives of “green morocco plan”, to complain to national legislation (law 42-95 regarding pesticides regulations, law 28-00 related to waste management and their disposal) and international regulations (fao’s code of conduct for pesticide use and disposal) and the requirements of quality control standards of agricultural products and the environmental management (globalgap, nature’choice, iso 14000, brc, it will be very important to know the practices used by farmers to manage pesticide wastes. 60 horticultural producers of the souss massa region were interviewed personally with a questionnaire focused on pesticides waste management. the souss massa, located in the south atlantic coastal, is a major horticultural producing and exporting area of morocco. the survey indicated that pesticide wastes management depends mainly on the certification of the farms. the majority of pesticide wastes were related to empty pesticide containers and pesticide effluents. all farmers are aware of human health risk and the environment pollution potential of pesticide wastes. the producers are face problems in terms of eliminating the empty containers due to the absence of laws and facilities for pesticides containers recycling. certified farms make triple rinsed pesticide containers and stored them under lock and key, or they reuse them for cultural operations after cleaning. in the case of the non-certified farms, the empty containers are burned or are sold via informal ways. burning plastic bottles and pesticide box can lead to the formation of toxic gas in the air. the component 2 of gcp /mor/041/ gff project in morocco will be developed a management system including outreach campaign to users for triple-rinsing and safe storage, collection and recycling of all types of containers, firstly through a regional pilot program in the souss massa. concerning leftover spray solutions, most farmers (73%) was reported that they apply them to the border lines of the field area or to weed grasses until the spraying tank is empty. 16% of farmers release the leftover spray solutions into an evaporation area and 11% were used a bioremediation system in phytobac or heliosec. keywords: pesticide wastes, empty pesticide containers, pesticide effluents, phytobac, heliosec, souss-massa, gcp /mor/041/gff project, fao. p023. effect of mineral fertilization on olive quality karima bouhafa1*, lhoussaine moughli2, khadija bouchoufi1, ahmed douaik3, and khalid daoui1 1 regional agricultural research center, p.o. box 578, meknes, morocco; 2 department of soil science, hassan ii institute of agronomy and veterinary medicine, p.o. box 6202, rabats-institutes, rabat, morocco; 3 regional agricultural research center, po box 6356, avenue mohamed belarbi alaoui, rabat, morocco. *corresponding and presenting author: bouhafakarima@gmail.com. abstract two fertilization trials were conducted in two olive orchards under rainfed conditions in oued amlil (s1) and ain taoujdat (s2). the objective was to study the effect of nitrogen (n), phosphate (p) and potassium (k) fertilizer inputs on the olive quality. the experimental design adopted for these trials is a factorial in incomplete random blocks. four doses of each of the elements n, p and k were tested: 0, 0.25, 0.5 and 1 kg n / tree / year; 0, 0.12, 0.25 and 0.5 kg p2o5 / tree / year and 0, 0.5, 1 and 2 kg k2o / tree / year. the average levels of olive oil, expressed as% dry matter, were 28.3 and 37.3%, respectively, in s1 and s2. the effect of nitrogen on olive oil content was insignificant at s1 and highly significant at s2. at the s2, the nitrogen supply induced a decrease in the oil content and the control recorded the maximum level of olive oil (38.5%). phosphorus had no significant effect on olive oil content in s1. on the other hand, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 474 the input of p caused a significant increase in the oil content in s2. potassium did not affect the olive oil content at both sites. regarding the effects of mineral elements on the olive oil quality, it is noted that nitrogen is the only element that significantly affected the two measured parameters (peroxide index and acidity). the intake of n induced an increase of the peroxide index to reach the maximum value by the dose 0.5 kg n / tree / year in the site 1, and by the dose 1 kg n / tree / year in the site 2. on the other hand, a decrease in the acidity level of the olive oil was observed with the addition of nitrogen at s2 where the best quality of the olive oil (minimum acidity) was obtained by the dose 0.5 kg n / ha / yr. the effect of nitrogen on olive oil acidity was not significant at s1. keywords: olive, nitrogen, phosphorus, potassium, acidity, peroxide index. p024. assessment of beta-glucan and micronutrients in a collection of barley’s wild relatives from icadra’s gene bank fadwa elouadi1*, a. amri2, a. jilal3, g. salih3, and m. ibriz1 1 laboratoire de génétique et biométrie, faculté des sciences. université ibn tofaïl, b.p. 133, kénitra. morocco; 2 international center for agricultural research in the dry areas, rue hafiane cherkaoui, rabat, morocco. po box 6299, rabat institutes, rabat, morocco; 3 ru plant breeding, conservation and valorization of plant genetic resources, inra, rcar-rabat, p.o. box 6570, rabat institutes, rabat, morocco. *corresponding and presenting author: elouadi.fadwa@ gmail.com. abstract barley is one of the oldest domesticated crop in the word, although barley food uses remains important only in some cultures particularly in asia and northern africa. whereas recently the consumer has become aware of the interest of barley as a food grain because of his impressive health benefits, and as a major source of soluble and insoluble fibers, also his antioxidant activity. the objective of this study is to investigate the genetic variation in a population of barley’s wild relatives. beta-glucan, and micronutrient concentration were determined, as well the identification of quantitative trait loci (qtl) associated with these traits in a population of wild barley from icard’s gene bank using a genome-wide association study (gwas). accessions of wild relatives were grown under greenhouse for genotyping, and field conditions for multiplication (2015-2016/2016-2017), characterized for beta-glucan, and micronutrient concentration, and then genotyped with 50k single nucleotide polymorphism (snp) markers. the population exhibited a wide range of variation for these traits, further some accessions had shown high levels of beta-glucan and micronutrient. this study demonstrates the variation of beta-glucan and micronutrient concentration in wild barley and the interest of genotyping to underline genes controlling these traits. key words: barley, beta-glucan, micronutrient, genome-wide association, quantitative trait loci. p025. time evolution of transpiration and heat transfer inside arch greenhouse crop using computational fluid dynamics allal senhaji1*, mhamed mouqallid1, and hassan majdoubi1,2 1 equipe de recherche en energétique et mécanique des fluides, ensam, meknès, morocco; 2 crmef fesmeknès, meknès, morocco. *corresponding and presenting author: senhaji145@gmail.com. abstract the most important factor affecting plant growth and development in greenhouses is solar radiation because, in addition to its indirect effect on the distribution of the greenhouse’s internal climate, it governs two important physiological mechanisms of plants, namely the transpiration and photosynthesis. the amount of solar radiation entering the greenhouse depends on the design of the greenhouse, the thermophysical and optical properties of the material, and the weather conditions, there are important variations in the amount of solar radiation reaching the crop, which strongly impacts climate and therefore crop activity. the aim of this paper is to develop a specific numerical submodel to simulate the distributed transpiration and microclimate inside greenhouses under day-time conditions. the numerical simulation was adopted in three dimensions (cfd), using the computer a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 475 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) code fluent v6.3.26 which is based on solving the navier-stokes equations. these equations are solved in the presence of turbulence model (k-ε) and a biband radiative submodel distinguishing short and long wavelength radiations was employed through a discrete ordinates (do) model to solve the equation of heat transfer by radiation (etr). simulations were carried out for a greenhouse of 810 m2 equipped with continuous roof vents. the analysis focuses on the heterogeneity of the distributions of climatic parameters (speed, temperature and humidity). but the main point concerns the heterogeneity of the radiation reaching the canopy and the latent and sensitive heat flux inside the culture. keywords: solar radiation, transpiration, cfd, crop, greenhouse, microclimate. p026. micropropagation and plantlet regeneration of saffrom (crocus sativus l.) meryem maiski*, o. lamsaouri, m. fakiri, and y. el goumi laboratoire de recherche agroalimentaire et santé, faculté des sciences et techniques, université hassan 1er, settat, morocco. *corresponding and presenting author: meryem.maiskii@gmail.com. abstract this study consists in evaluating various methods of micropropagation of saffron (crocus sativus l.).different protocols of surface sterilization have been tested with the aim to decrease the contamination porcentage. the effect of growth regulators combinations has been also tested on the induction of caulogenesis. the stirilization procedures tested did not show their efficiency on corms. however, in the last protocol, the explants showed a significant lower contamination rate (2,44%). the growth regulators used in the induction phase had allowed obtaining the best results of caulogenesis (96%). moreaver, it was observed that the use of an ather combination of cytokinin and auxin was favorable for buds’ growth. the yield was 1,06 buds per explant newly formed. p028. variability analysis of the seeds and oil yields of several accessions of citrullus colocynthis collected in morocco abdelhamid ben moumou* and saïd el madidi§ biometrics and bio resources, laboratory biotechnologies and natural resources development (lbvrn), faculty of sciences, university ibn zohr, agadir, morocco. *presenting author: benmoumou36@yahoo.fr. §corresponding author: s.elmadidi@uiz.ac.ma. abstract citrullus colocynthis has been garnering interest in recent times as a potential biodiesel feedstock crop due to its high seed oil content. this plant is a drought tolerant species, as established from the fact that it grows as a weed in arid and semi-arid lands. widely distributed in the sahara-arabian desert in africa and the mediterranean region. it is one of the few spontaneous species of plants that grow in arid conditions in southern mediterranean countries and middle eastern countries and could be used as raw material for biodiesel. in addition to its potential as a biodiesel feedstock the root and callus extracts of c. colocynthis have been reported to have anti-microbial, anti-inflammatory,anti-diabetic and anti-oxidant effects. c. colocynthis is a rich source of functionally important bioactive compounds and therapeutics such as polyphenols, glycosides, triterpenes and cucurbitacins and its fruit has been widely used for the treatment of many diseases including diabetes, rheumatism, paronychia, ulcer and cancer. the ripe fruits of c. colocynthis were harvested in 12 different localities in morocco. geographic coordinates, morphological characteristics were recorded. the measured traits are: fruit weight (fw), seed weight (sw), seed number (sn), weight of 100 seeds, and oil yield. the results of anova (glm) show significant and very highly significant effects according to the characters measured. significant variability was observed for seed and oil yields. this variability could be exploited for the selection of accessions with high oil yields. keywords: citrullus colocynthis, variability, seeds, oil yield, morocco. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 476 p029. climate change and land suitability in central morocco: impact and adaptation a labaioui1*, k bouchoufi2, and c kassimi2 1 regional center of agricultural research, meknes, morocco; 2 soil analysis laboratory, regional center of agricultural research, meknes, morocco. *corresponding and presenting author: amlabaioui@gmail. com. abstract the climate change will bring about changes in the areas suited to the growing of several species. the effects in terms of production potentiality will be very variable according to the region and the vegetal covers. they can come out by a moderate stimulation in moderate climate and for a warming limited to 2° 3°c. beyond, as for the low latitudes, excessive temperatures and an increased frequency of the droughts will lead to negative effects. in our study, we used current and future climatic data up to the year 2050, the ecocrop model integrated in the diva-gis software and soil map to predict the impact of the climate change on the land suitability of the meknes area in central morocco, for wheat, chickpea, the olive and the almond trees, so that these changes can be taken into account in the planning of the strategies of adaptation to the climate change. the results show that for wheat, the olive and the almond trees, the aptitude of the soil will increase in spite of the increase of the temperature and the reduction in rainfall. consequently, the strategies of development and of improvement of these three crops will not resist the important constraints related to the climatic conditions and should concentrate on the improvement of agronomic management and on the reinforcement of the capacities of farmers by including the techniques of control of the pests and diseases. for chickpea, however, land suitability will decrease, with very marginal land increasing from 5% to 75% of the total land area. so, the areas that will lose planting capacity need to be maintained, using targeted strategies such as, irrigation and switching to more drought-resistant varieties and or changing planting date. while areas that are likely to suffer from a significant decrease in aptitude should begin to diversify, moving to other crops as soon as chickpea is no longer appropriate. keywords: climate change, land suitability, morocco, adaptation. p030. evaluation of five mutants of rapeseed (brassica napus l.) for earliness, agronomic traits and response to changing environments in m2 population souhail channaoui1,2, mostapha labhilili3, mohamed mouhib4, hamid mazouz2, mohamed el fechtali1, and abdelghani nabloussi1* 1 research unit of plant breeding and plant genetic resources conservation, national institute of agricultural research, regional agricultural research center of meknes, morocco; 2 laboratory of plant biotechnology and molecular biology, department of biology, faculty of science, university moulay ismail, morocco; 3 research unit of plant biotechnology, national institute of agricultural research, regional agricultural research center of rabat, morocco; 4 research unit of nuclear techniques, environment and quality, regional agricultural research center of tangier, morocco. *corresponding and presenting author: abdelghani.nabloussi@gmail.com. abstract mutagenesis breeding is an efficient strategy to improve rapeseed for several productivity and quality parameters. five new m2 mutant populations (h2m1, h2m-2, h2m-3, h2m-4 and h2m-5) were selected from gamma irradiated and ethyl methane sulphonate (ems) treated material. a field experiment was conducted in two inra experimental stations, at douyet and sidi allal tazi, to evaluate rapeseed mutants for earliness, agronomic traits and response to changing environments. the variety ‘inra-czh2’ (wild type) was used as a check. data on days to flowering, days to maturity, plant height, number of branches/ plant, number of pods/plant, number of seeds/pod, pod length, pod diameter and 1000-seed weight were recorded. all the parameters showed significant differences among the mutants studied for both environments. mutant h2m-1 was earlier to flowering and maturity and was characterized by a higher number of pods/plant than the check. mutants h2m-2, h2m4 and h2m-5 produced higher number of branches per plant and 1000-seed weight, compared to the check variety, whilst mutant h2m-3 exhibited a rea tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 477 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) duced plant height and stature. among the five mutant lines, h2m-1 seemed to be the most interesting and further investigations and trials are needed in order to confirm its performance and stability as a promising and useful germplasm for releasing a new rapeseed variety. keywords: rapeseed, mutants, environments, earliness, seed yield. p031. effect of organic fertilizer and commercial arbuscular mycorrhizal fungi on the growth of micropropagated date palm cv. feggouss el kinany said1,2,5*, achbani el hassan2, faggroud mohamed3, ouahmane lahcen4, el hilali rania1,4, haggoud abdellatif, and bouamri rachid1§ 1 department of plant and environment protection, national school of agriculture, meknes, morocco; 2 laboratory of plant protection urpp-national institute for agricultural research, meknes, morocco; 3 department of agronomy, national school of agriculture, meknes, morocco; 4 laboratory of ecology & environment, faculty of sciences semlalia, cadi ayyad university, marrakesh, morocco; 5 laboratory of microbial biotechnology, sidi mohamed ben abdellah university, faculty of sciences and techniques, fez, morocco. *presenting author: said.elkinany@ usmba.ac.ma. §corresponding author: rbouamri@ enameknes.ac.ma. abstract date palm is an important crop in morocco, tunisia and many other drylands of the world, but its growth is often limited due to the low soil fertility and harsh environmental conditions of oases ecosystems, which can hardly be compensated by the sole application of high dosages of chemical fertilizers. for the first time, we investigated the effects of compost application and inoculation with a commercial strain of the arbuscular mycorrhizal fungus (amf), glomus iranicum, on the growth of micropropagated date palm plantlets (cv. feggous). after twelve months of growth, plantlets transplanted into compost amended substrate inoculated with amf showed increased biomass production (root and shoot biomass), chlorophyll and mineral nutrient contents than plantlets transplanted into compost amended substrate or without compost addition. thus, this inoculum reinforced the promoting effect of compost and was successful in colonizing the root system. according to our results, sandy substrate enriched with compost and inoculated with g. iranicum can be recommended for improving the growth and nutrition of micropropagated date palm plantlets. keywords: phoenix dactylifera l., compost, mycorrhizal fungi, organic fertilizer. p032. valorization of natural resources from burundi: case of myrianthus holstii engl niyukuri jonathan*, jihane raiti, and hafidi abdellatif laboratory of food sciences of cadi ayyad university of marrakech, morocco. *corresponding and presenting author: jonaniyu@gmail.com. abstract the aim of this work is to investigate the chemical composition of the oleaginous seeds of myrianthus holstii engl by determining the physicochemical properties of its lipid fraction and the chemical composition of its oilcake. the fruit which is fleshy is edible and a sclerified pericarp surround the seeds which are the object of our study. samples were harvested in burundi in three forests following: vyanda nature reserve, kibira national park and bururi natural reserve. analysis were conducted at the laboratory of food science at cadi ayyad university in marrakech, morocco. the oil extraction is performed with hexane using a soxhhlet apparatus. the obtained yield of oil is 46.58 ± 6.82% of dry matter (dm), unsaponifiable yield is 1.51 ± 0.87%, the oil acidity is 1.38 ± 0.23%, the iodine value is 25.37 ± 6.63 g iodine / 100 g of oils and the peroxide value is 5.53 ± 0.61 meq / kg of oil. the ethanol (80%) extracts was used to analyze the composition of the cake. a total polyphenols 0.34 ± 0.03 g gallic acid equivalent / 100 g dm was obtained by the folin-ciocalteu method., sugars content were formed for reach 6.05 ± 0.37 g glucose equivalent / 100 g d m and for total proteins 0.08 ± 0.01g bovine alumina serum equivalent / 100g dm. the results show the potential of use of this species as an oleaginous seed. nevertheless, this research requires others complementary work for being qualified as safe and edible for human consumption. keywords: lipid fraction, unsaponifiable. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 478 p033. the complementarity of irrigation technology adoption and groundwater development; evidence from the trifa plain of morocco aaron r. hrozencik1*, emily burchfield2, dale t. manning1, and aymen frija3 1 department of agricultural and resource economics, colorado state university, fort collins, co 80523, usa; 2 department of environment and society, utah state university, logan, ut 84322, usa; 3 international center for agricultural research in dry areas, tunis, tunisia. *corresponding and presenting author: aaronhroz@gmail.com. abstract a growing world population and changing climate place increasing pressure on agricultural production and already scarce water resources. promoting irrigation technology adoption and investing in additional sources of water supply are favored policy options to address water scarcity concerns and increase agricultural productivity. while many studies have investigated the effects of irrigation technology adoption on resource use, the literature has largely ignored how technology adoption decisions influence investments in additional water supply and vice-versa. this research addresses that gap in the literature by exploring the interaction between irrigation technology adoption and investments in groundwater development. we utilize remote sensing and survey data from the trifa plain of northeastern morocco to measure the complementarity between sets of irrigation technologies and investments in groundwater pumping capacity. the trifa traditionally relied on imported water to support the region’s robust agricultural economy. however, growing irrigation demand and climatic variability precipitated governmental efforts to promote water conservation and irrigation efficiency through technology adoption, particularly the adoption of on-farm water storage and drip irrigation systems. imported water deficits also catalyzed increased investment in groundwater pumping infrastructure with the number of irrigation wells in the trifa increasing by over 46% between 2000 and 2012. we leverage the simultaneity of groundwater development and irrigation technology adoption, and the exogenous nature of groundwater availability in the trifa to empirically test for complementarity between pumping capacity development and irrigation technology adoption. this work contributes to the literature exploring technology adoption and resource use decisions while also advancing knowledge regarding the impact of investment in groundwater development. keywords: groundwater, production economics, agricultural development, technology adoption. p034. chemical composition and antibacterial activity of essential oil of artemisia absinthium against erwinia amylovora fire blight agent laaziza doukkali1*, farthat guenoun1, bouchra tazi3, and abdessalem tahiri2§ 1 laboratory of bioactive molecules and environment, moulay ismail university of sciences b.p 11201, meknes, morocco; 2 laboratory of phytopathology, department of the protection of the plants & environment ; national school of agriculture, bp s 40 meknès, morocco; 3 laboratory of chemistry, department of the basic sciences; national school of agriculture, bp s 40 meknès, morocco. *presenting author: elaazizadoukkali@yahoo.fr. §corresponding author: atahiri@enameknes.ac.ma. abstract dealing with resistance and toxicity problems caused by the massive use of plant protection products (pesticides), and with the fact that regulation organizations are questioning the use of antibiotics in agriculture, it is particularly urgent to find alternatives to provide efficient protection of crops against plant diseases. the main objective of this paper is to study the antibacterial activity of essential oil (eo) extracted of artemisia absinthium against erwinia amylovora. the extraction was carried out by water distillation clevenger’s type apparatus. the chemical compositions of eo were investigated by using a gc–ms/fid and showed that eo of artemisia absinthium was characterized by the presence of β-thuyone, camphor and terpinen-4-ol as the main chemical components. the in vitro antibacterial activity against erwinia amylovora was evaluated using the method of aromatogram and the method of microdilution. the results revealed that eo of artemisia absinthium showed an antibacterial activity against erwinia amylovora. keywords: a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 479 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) fire blight, erwinia amylovora, chemical composition, antibacterial, activity, chemical, artemisia absinthium. p035. evaluation of five mutants of rapeseed (brassica napus l.) for earliness, agronomic traits and response to changing environments in m2 population. souhail channaoui1,2, mostapha labhilili3, mohamed mouhib4, hamid mazouz2, mohamed el fechtali1, and abdelghani nabloussi1* 1 research unit of plant breeding and plant genetic resources conservation, national institute of agricultural research, regional agricultural research center of meknes, morocco; 2 laboratory of plant biotechnology and molecular biology, department of biology, faculty of science, university moulay ismail, morocco; 3 research unit of plant biotechnology, national institute of agricultural research, regional agricultural research center of rabat, morocco; 4 research unit of nuclear techniques, environment and quality, regional agricultural research center of tangier, morocco. *corresponding and presenting author: abdelghani.nabloussi@gmail.com. abstract mutagenesis breeding is an efficient strategy to improve rapeseed for several productivity and quality parameters. five new m2 mutant populations (h2m1, h2m-2, h2m-3, h2m-4 and h2m-5) were selected from gamma irradiated and ethyl methane sulphonate (ems) treated material. a field experiment was conducted in two inra experimental stations, at douyet and sidi allal tazi, to evaluate rapeseed mutants for earliness, agronomic traits and response to changing environments. the variety ‘inra-czh2’ (wild type) was used as a check. data on days to flowering, days to maturity, plant height, number of branches/ plant, number of pods/plant, number of seeds/pod, pod length, pod diameter and 1000-seed weight were recorded. all the parameters showed significant differences among the mutants studied for both environments. mutant h2m-1 was earlier to flowering and maturity and was characterized by a higher number of pods/plant than the check. mutants h2m-2, h2m4 and h2m-5 produced higher number of branches per plant and 1000-seed weight, compared to the check variety, whilst mutant h2m-3 exhibited a reduced plant height and stature. among the five mutant lines, h2m-1 seemed to be the most interesting and further investigations and trials are needed in order to confirm its performance and stability as a promising and useful germplasm for releasing a new rapeseed variety. keywords: rapeseed, mutants, environments, earliness, seed yield. p036. genetic diversity and variability of the biochemical characteristics of moroccan loquat (eryobotria japonica) y. faiq1,2*, h. hanine1, s. ennahli2, h. latrache1, and o. kodad2 1 laboratory of bioprocess and bio-interfaces, fst, béni-mellal, morocco; 2 department of pomologie, national school of agriculture meknes, morocco. *corresponding and presenting author: youssef.faiq. cqpaa@gmail.com. abstract the diversity of climatic conditions gives morocco a vocation of fruit production in general and fruit rosacea in particular. this sector, which has expanded considerably since the beginning of the 80s (thanks in particular to private sector initiatives), has a great socio-economic potential and thanks to this important role the new moroccan agricultural strategy ‘the green morocco plan’ particular importance to the development of small-scale farming and the promotion of local products. in this aspect, the green morocco plan through projects for the intensification of the loquat in the zegzel valley and in the surrounding areas has made it possible to improve the quality of zegzel loquat and to enhance the value of its production through the protected geographical indication. “igp loquat of zegzel”. however, the high genetic diversity observed in the loquat orchards of the berkane area prevents the adoption of an adequate technical itinerary for each cultivar, and consequently a stable quality production from one year to the next. nowadays, the characterization and evaluation of the diversity and the evaluation of the interannual variability of the physicochemical and biochemical characteristics of the fruit which determine the quality and the specificity of zegzel loquat within the loquat populations remain unknown. in the perspective of asa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 480 sessing the diversity of loquat cultivated in northeast morocco that the present study was undertaken. the main objective of this study is to study the genotypic variability and to evaluate the biochemical quality of the fruit of the main genotypes selected from the traditional orchards of the zegzel loquat. a set of 35 individuals were selected and studied. the results of the present study confirmed that the biochemical characteristics of the fruit of the different genotypes of the loquat from the region of berkane present a wide variability for all the characters measured, they show that some genotypes have presented interesting performances from a quality point of view. fruit. however, the t9 and t11 genotypes have fruits with a high content of carotenoids, flavonoids, and total polyphenols. keywords: japanese loquat (eriobotrya japonica), biochemical marker, genotype, environment, flavonoids, carotenoids, total polyphenols. p037. hepatoprotective effect of methanolic extract of zygophyllum album against ccl4 –induced hepatotoxicity in rats wassila saiah1*, halima saiah2, hanane halzoune3, el-hadj ahmed koceir1, and naima omari1 1 bioenergetics and intermediary metabolism, laboratory, biology and organisms, physiology department, university of sciences and technology houari boumediene, bab ezzouar, algeria; 2 laboratory of natural bioresources, faculty of snv, department of biology, university hassiba ben bouali, algeria. *corresponding and presenting author: saiahbouchra@ yahoo.fr. abstract liver diseases pose a major challenge to international public health. hepatic damage is associated with distortion of metabolic functions of the liver. the present study was carried out to evaluate the hepatoprotective effect of the methanolic extract of zygophyllum album in ccl4induced hepatotoxicity in albino rats. hepatotoxicity was induced in rats by a single intrapéritonéal injection of ccl4 suspended in olive oil after every 72h for 10 days (group ii). hepatotoxicityinduced rats were treated with 100 mg/kg b.w. of methanolic extract of z. album for 10 days, and also, ccl4 was given as in group ii of rats. the effect of the drug was compared with the standard drug silymarin. the effect of the studied extract on antioxidant enzymes, serum glucose, urea, createnine serum bilirubin, total protein and lipid profile was determined. treatment with z. album extract showed a significant reduction in blood glucose levels, marker enzymes and lipid profile. the drug significantly increased the activity of antioxidant enzymes in the liver of hepatotoxic rats. the results of the present study suggest that treatment with z. album extract enhanced the recovery from ccl4 –induced hepatic damage due to its antioxidant and hepatoprotective property. key words: hepatotoxicity, ccl4, zygophyllum album, methanol extract, antioxidant. p038. pomological evaluation of four mains introduced almond cultivars in north eastern region of morocco reda melhaoui1*, m addi1; m abid1, a mihamou1, h serghini-caid1, m sindic2, and a elamrani1 1 laboratoire lbpm, faculté des sciences, université mohamed ier, oujda; morocco; 2 laboratoire de chimie générale et organique, gembloux agro biotech, université de liège; belgium. *corresponding and presenting author: reda_melhaoui@hotmail.fr. abstract almond (prunus dulcis l.) is one of the most important cultivated fruit trees in north eastern region of morocco. recently, as part of the green morocco program, north eastern region of morocco was supported by the belgian development agency through the “profao*” project for planting 6000 ha of almond trees. ferragnes and ferraduel, two french cultivars known for their late flowering in march to escape the late frosts of spring, were chosen for the planting of these new almond orchards , instead of “marcona” and “fournat” which also have been previously introduced in this region but appears to be sensitive to spring frost due to their flowering in february. this work focuses on the pomological characterization of these four almond cultivars (ferragnes, ferraduel, marcona and fournat) in five studied areas in north eastern region of morocco. for each almond cultivar, recorded pomological measurements for the fruit and kernel are: weight, width, length, thickness, volume and percentage of the double kernels. nuts weight ranges bea tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 481 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) tween a minimum value of 2.65g for ferragnes and a maximum value of 4.41g for fournat, while the kernels’ weight ranges between 0.73g for marcona and 1.38g for fournat. calculated mean values of nuts and kernels volumes are respectively 4000.98mm3 and 846.09mm3 for marcona, considered as small fruits, and 9667.20mm3, 1553.01mm3 for fournat considered as big fruits in this study, however intermediate values have been recorded for nuts and kernels of ferragne and ferraduel. finally, yield after shelling and the percentage of doubles kernels have been determined, they range from 20.27% to 33.54% and from 0% to 15% respectively. *profao: projet filière amandes de l’oriental, “the almond value chain in eastern morocco, pillar ii of the moroccan green plan, supported by belgian development agency (btc 2011-2017). keywords: almond, nut, kernel, ferragnes, ferraduel, marcona, fournat. p039. distribution of a plant of agronomic interest in morocco: case of common myrtle aabdousse jamal*, r. faida, a. boulli, and n. wahid§ ecology and sustainable development team (esdt), department of life sciences, faculty of science and technology, sultan moulay slimane university, benimellal, morocco. *presenting author: abdojamal.fstbm@gmail.com. §corresponding author: wahid2na@ hotmail.com. abstract myrtle is an aromatic and medicinal plant well known for its therapeutic, cosmetic and food properties. in traditional medicine, fruits and leaves are used as antiseptics, antibacterial, antihyperglycemic, analgesic and as anti-inflammatory agent, so in morocco it is widely used in the control of hair loss and skin toning. these diverse properties make myrtle a plant of interest not only therapeutically but also economically and agronomic. in morocco, it is exploited from the spontaneous natural state for the extraction of essential oils from leaves. its uncontrolled exploitation, coupled with the restriction of its natural environment, the degradation of its ecosystem and the reduction of its density within often isolated stations, increase the risk of a rapid genetic erosion of the species. for the sustainable management of this species, it is necessary to define a precise vision of the potential uses of myrtle and the prospects of increasing their supply through domestication. thus, a broad knowledge of the distribution of this species would facilitate the establishment of a seed bank for the development of agricultural domestication and genetic improvement programs. in order to determine the biogeographical range of the myrtle in morocco, we have carried out prospecting missions in different regions of the kingdom ranging from the central plateau to the western rif. these missions allowed us to determine the bioclimatic stage as well as the vegetation stage of the myrtle, the optimal climatic conditions for the installation, also the domestication and genetic improvement of this species. keywords: myrtus communis l., biogeographical distribution, bioclimatic stage, ecology. p040. phosphate solubilizing bacteria: potential biofertilizers for sustainable agriculture qessaoui redouan1,2*, r. bouharroud2, a. amarraque2, a. ajerrar1,2, h. lahmyed1,2, e h. mayad3,4, and b. chebli1 1 laboratoire génie de l’environnement et de biotechnologie, ecole nationale des sciences appliquée, agadir, morocco ; ² research unit of integrated crop production, centre regional de la recherche agronomique d’agadir (inra), morocco; 3 laboratory of biotechnologies and valorization of natural resources faculty of sciences agadir, ibn zohr university, agadir, morocco; 4 biotechnology and environmental engineering team, laboratory for process environmental and energy engineering, national school of applied sciences, ibn zohr university, po box: 1136/s, agadir, morocco. *corresponding and presenting author: qessaoui_bio@hotmail.fr. abstract the concept of sustainable agriculture is a response to the decline in the quality of the natural resource base associated with modern agriculture. the use of chemicals for agricultural needs will result loss in soil physical condition and water pollution. the exploitation of beneficial microbes as a biofertilizers has become paramount importance in agriculture sector for their potential role in food safety and sustainable crop production. phosphorus is the second maca tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 482 ronutrient essential for growth and development of plants. however, the solubility of this macronutrient is very low and 75 to 90% of phosphate fertilizers are precipitated. two isolates of pseudomonas (p. putida qef11b and p. luteola qef13b) isolated from the rhizosphere were tested in the phosphate solubilization in both solid and liquid media. the ph evolution was followed. p. putida qef11b shows a significant solubilization of phosphate which varies from 0.53 to 2.92 µg/ml for 24h and 96h respectively. in the solid media each isolate shows a halo around colony. this will be explained by the production of organic acids. the ability of this strain to acidify the media confirmed by the decreased of ph for both isolates compared to control. to circumvent phosphorus deficiency, this phosphate-solubilizing microorganism could play an important role in supplying phosphate to plants in a more environmentally-friendly and sustainable manner, and reducing problems associated with the use of chemical fertilizers. keywords: pseudomonas, phosphate solubilization, sustainable agriculture p041. responses of differential barley genotypes to moroccan isolates of pyrenophora teres f. teres karima taibi1*, f. bentata2, s. rehman3, m. labhilili2, a. el aissami1, r.p.s. verma3, and s. gyawali3 1 laboratory of botany, mycology and environment, department of biology, faculty of sciences, university mohammed v-agdal, rabat, morocco; 2 l’institut national de la recherche agronomique-maroc (inra-m), inra avenue ennasr, rabat, maroc bp 415 rp rabat, morocco ; 3 biodiversity integrated gene management program (bigm), international center for agricultural research in dry areas (icarda), p.o. box: 6299 rabat-instituts/rabat, morocco. *corresponding and presenting author: ktaibi1@gmail.com. abstract barley net blotch, caused by pyrenophora teres f. teres (ptt), is one of the most important foliar diseases in morocco. the disease causes appreciable yield losses under favorable environmental conditions. to identify effective sources of resistance to net blotch, the understanding of virulence spectrum of ptt is essential. sixteen barley genotypes were inoculated at seedling stage with 15 ptt isolates that were collected from different agro-ecological zones of morocco during the disease survey of 2009-2010. the experiment was conducted in factorial arrangement of treatments in randomized complete block design with three replicates. anova and gge biplot were employed to understand the barley-ptt interactions. the anova revealed highly significant effects of genotype (g), isolate (i) and g×i interactions. furthermore, the results indicated that race nonspecific interaction and aggressiveness of ptt isolates dominated over gene-for-gene interaction (race specific/ virulence). the study of virulence of ptt isolates revealed highly diverse virulence pattern of moroccan isolates. in addition, gge biplot revealed that moroccan cultivars, taffa and aglou, showed increased level of stable resistance to ptt comparatively while coast and rabat071 were the most susceptible genotypes. the emergence of new ptt pathotypes which are highly virulent to durable resistance in rabat071 poses a greater risk of breaking down currently deployed resistance to net blotch in morocco. gge biplot also discriminated virulence of ptt isolates of which pt2, pt7, pt8, and pt4 being the most virulent isolates, while pt10 and pt11 were found the least virulent isolates. a careful evaluation and selection of ptt isolates based on virulence pattern to barley genotypes is essential for successful barley breeding for resistance to net blotch in morocco. keywords: virulence, barley, gge biplot, net blotch, pyrenophora teres p042. civil development organizations and ict: two vectors of social change in rural mountain areas of the middle atlas (morocco) noureddine bahri1*, a. bentaïbi1 and t. desrues2 1 regional center of agricultural research of meknes, bp. 578 meknes vn, morocco; 2 institute of advanced social studies (iesa) cordoba, c/ campo santo de los mártires, 7 14004 cordoba, spain. *corresponding and presenting author: bahrinour@yahoo.fr. abstract the positioning of civil organizations (ngos) as an important stakeholder within the institutional landscape for local development is now recognized as a real characteristic of the new development dynamics a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 483 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) that morocco is experiencing during the last two decades. local development associations and cooperatives are a tool for mobilizing the population around concrete projects and a key partner for their participatory realization. they are also a local anchor of sustainability and scalability capable of assimilating the social demand and transforming it into innovative initiatives for the production of products and services. such organizations finally provide the advantage of having a broad credit to the various actors and are therefore able to play an integrative role for the various sectoral development interventions. access to information and communication technologies (icts) is a phenomenon illustrating the dynamics of change recorded in the kingdom and is considered as another vector of social change. as a result, ngo’s access to icts would allow: 1) to break their isolation, fostering internal and external communications; 2) to provide information on local development strategies and needs; 3) to obtain resources and establish partnerships or raise awareness among policy and administrative leaders; and 4) to promote the participation of their members and local people. these hypotheses are supported by the results of our study, icts are, however, resources or instruments that are part of a pre-existing social, economic and political order, so it is appropriate to check in accordance with each local situation the current reconfigurations and whether the latter promote the reproduction of the old positions of power and inequality or are, on the contrary, sources of change. we analyse also the icts and development actions carried out by the three selected ngos. secondly, we deepen the question of the place of women in the activities and decision-making processes of these ngos and, in particular, we return to the empowerment opportunities that icts offer to women. in conclusion, we present some of the development opportunities offered by icts and certain identified limits, especially in terms of capacity building and gender. keyword: icts, ngos, local development, agricultural development, collective action, social change. p043. characterization and phylogenetic diversity of allorhizobium vitis isolated from grapevine in morocco khaoula habbadi1,2,3*, quentin duplay3*, david chapulliot3, isabelle kerzon3, rachid benkirane2, abdellatif benbouazza1, florence wisniewski-dye3, céline lavire3, ludovic vial3, and el hassan achbani1 1 laboratoire de recherche et de protection des plantes urpp-inra-meknès, morocco; 2 laboratoire de botanique, biotechnologie, et protection des plantes, faculté des sciences, kenitra, morocco; 3 université de lyon, université claude bernard lyon 1, cnrs, umr 5557, ecologie microbienne, inra, umr1418, 10 villeurbanne, f-69622, france. *corresponding and presenting author: khaoula405@gmail.com. abstract crown gall was observed in recently planted vineyards of the meknes region (morocco). 82 isolates from 10 affected vineyards were characterized by reca sequencing and were found to belong to a. tumefaciens genomospecies g1, g4 or g7, rhizobium rhizogenes, and to allorhizobium vitis. only the 39 a. vitis isolates showed to be pathogenic in vitro on tomato and mlsa phylogenetic analyses revealed a poor genetic diversity, with the definition of only four genomic groups. definition of the a. vitis genomic groups correlates with specific pathogenic traits: indeed, genomic groups differ with respect to the severity of hypersensitive response symptoms on tobacco leaves, the intensity of necrotic response on grapevine explants, and opine profiles. both vitopine and octopine were detected by uhplc in tumors induced by isolates of three genomic groups, an opine signature scarcely ever reported. keywords: crown gall, grapevine, allorhizobium vitis, agrobacterium tumefaciens, phylogenetic diversity. p044. biological control of bacterial onion diseases using a bacterium, pantoea agglomerans 2066-7 soumia sadik1,2*, hamid mazouz2, abdellatif benbouazza1, and el hassan achbani1 1 laboratory of phytobacteriology and biocontrol, national institute of agronomic research, bp. 578meknès vn 50000, km 13 route haj kaddour, meknes morocco; 2 laboratory of plant biotechnology and molecular biology, faculty of sciences, b p 11201, avenue zitoune, meknes, morocco. *corresponding and presenting author: sadik.soumia@hotmail.com a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 484 abstract epiphytic microorganisms, isolated from the olive knots, apple fruits and trees, quince, compost and water from different areas were screened for antagonistic activity against pseudomonas marginalis, pseudomonas viridiflava, xanthomonas retroflexus and pantoea ananatis on onion bulbs. from 77 microorganisms tested for antagonistic property against bacterial onion diseases, the strain 2066-7 of pantoea agglomerans was selected. complete control against pseudomonas marginalis and pseudomonas viridiflava at 107cfu. ml-1 concentration of 2066-7 and an inhibition percent higher than 90% against xanthomonas retroflexus and pantoea ananatis were obtained on wounded onion bulbs inoculated with 106cfu.ml-1 of pathogens under cold conditions. the inhibitions percent were decreased under 25°c and 30°c. keywords: onion, bacterial onion diseases, storage, biological control, morocco p045. effect of parturition and lactation on ponderal performance in bedouin goat under harsh climatic environment henna kamilia, kouri fatima, kouri amina, amirat zaina, khammar farida, and charallah salima* research laboratory of arid lands, faculty of biology, usthb, bp 32 el-alia, algiers, algeria. *corresponding and presenting author: charalla@yahoo.fr. abstract in various animal species, many studies have shown weight fluctuations during parturition and lactation, however, no ponderal study was carried out during these physiological stages in the native bedouin goat in the algerian sahara. given its perfect adaptation to the hostile conditions of the arid zones, it seemed interesting to describe the evolution of body weight (bw) during these critical phases. this study is conducted on 14 females kept at the research station of beni-abbes (southwest algeria: 30° 07’ n, 2° 10’ w). these females are weighed in the morning, on the day of parturition (d0) and weekly for six weeks post-partum (w1 to w6). the weight data obtained are analyzed by statistical tests kruskal wallis and dunn (xl stat). the mean bw on day 0 is 23.2± 5.4 kg but becomes significant from w2 to w5 and drops very significantly in w6, respectively (20.5±5.1, 20.2±5.1, 19.9±3.9 et 19.2±4.00 kg, p= 0.01; 18.9±3.1 kg, p= 0.006). the statistical difference between mean weekly bw from w1 to w6 is not significant. the weight losses recorded during lactation would indicate the low level of ingestion capacity leading to an energy deficit that the lactating female must compensate by using her body reserves accumulated during the second half of the gestation period. at the same time, a progressive average daily gain is noted in newborns. the average bw in d0 would indicate the accumulation of lipid reserves during pregnancy and nutritional quality ingested. these weight variations reflect the interaction between the food supply and the mobilization of body reserves according to the physiological stage. keywords: parturition, lactation, ponderal performance, goat, arid zone. p046. in vitro evaluation of the effectiveness of peel pomegranate aqueous extract in controlling brown rot caused by monilinia spp. assia elkhetabi1,2*, rachid lahlali1, said ezrai1,2, abdessalem tahiri1, latifa askarne3, jovana hrustic4, lahsen el ghadaroui2, and said amiri1 1 department of plant protection, phytopathology unit, ecole nationale d’agriculture de meknès, bps 40, meknès, morocco; 2 laboratory of functional ecology and environment, sidi mohamed ben abdellah university, po. box 2202, route d’imouzzer, fez, morocco; 3 laboratory of microbial biotechnology and plant protection, université ibn zhor, faculté des sciences, agadir, morocco; 4 institute of pesticides and environmental protection, laboratory of applied phytopathology, banatska 31b, 11080 belgrade-zemun, serbia. *corresponding and presenting author: elkhetabiassia@gmail.com. abstract brown rot is considered as one of major pre-and postharvest pathogen of tree fruit and it is commonly caused by both fungal pathogens monilinia laxa and m. fructigena. this disease was usually controlled based on integrated strategy, which includes cultural practices and fungicide spray. however, due a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 485 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) to environmental and consumers health concerns, alternatives strategies need to be developed in order to substitute the frequent use of chemical substances. therefore, this study aimed at evaluating the in vitro antifungal activity of peel pomegranate aqueous extract against both fungal pathogens causing brown rot. the preliminary results showed a promising effect of the peel pomegranate aqueous extract on the in vitro mycelial growth of both pathogens when tested at 2.5 mg/ml of pda medium. in addition, obtained results highlighted a positive correlation between the concentration rate of peel pomegranate aqueous extract and the mycelial growth inhibition. interestingly, the inhibition rate was increased with increasing the concentration rate of peel pomegranate aqueous extract from 0.5 mg to 5 mg/ml. it was concluded that the aqueous extract of peel pomegranate might be a sustainable alternative control strategy of brown rot. as a result, studies on fruit and mechanisms behind its effectiveness are further considered in more details. keywords: brown rot, tree fruit, alternative control strategy, aqueous extract, peel pomegranate. p047. survey and prevalence of viruses infecting cucurbit crops in morocco nabil radouane1,2*, soukaina ermadi1, abdessalem tahiri1*, meryem benjelloun2, and rachid lahlali1 1 department of plant protection, phytopathology unit, ecole nationale d’agriculture de meknès, bps 40, meknès, morocco; 2 laboratory of functional ecology and environment, sidi mohamed ben abdellah university, po box 2202, route d’imouzzer, fez, morocco. *corresponding and presenting author: atahiri@enameknes.ac.ma. abstract cucurbit species are important vegetable crops in morocco and many mediterranean countries. a survey carried out within 2016 and 2017 in open field has emphasized severe yellowing, chlorosis, and symptoms of viral infections in major cucurbits growing areas of morocco; in particular azmour, el jadida, kenitra, taroudant, zagoura, and agadir. accordingly, samples having virus symptoms in two cucurbits corps zucchini (cucurbita pepo) and watermelon (citrulus lanatus) were collected, dried with calcium chloride, and analyzed by das-elisa technique for detection of the most common cucurbit viruses such as cucumber mosaic virus (cmv), zucchini yellow mosaic virus (zymv), water melon mosaic virus (wmv), papaya ringspot virus (prsv) and cucurbit aphid born yellow virus (cabyv). in addition, both pcr and rtpcr were used for specific detection of tomato leaf curl new delhi virus (tolcndv), cucumber mosaic virus (cmv), cucumber vein yellow virus (cvyv), and melon necrotic spot virus (mnsv). among 136 cucurbit samples (86 samples of zucchini and 50 samples of watermelon), the das-elisa revealed that 79.07% of the zucchini samples and 60% of the watermelon samples were positive for zymv, wmv, prsv and cabyv. surprisingly, the pcr bioassay revealed the presence of tolcndv for the first time in the country. regardless of the locality, the cmv was detected only by rt-pcr in most samples, while the cvyv and the mnsv were absents. keywords: cucurbita pepo, citrulus lanatus, cmv, zymv, prsv, cabyv, cmv, cvyv, mnsv, tolcndv, das-elisa, pcr and rt-pcr. p048. effect of silicon application on seed germination of coriandrum sativum (l.) grown under salt conditions jamila fahimi1,2*, fouad achemchem1, zakia bouzoubaâ2, nabil saffaj1, rachid bouharroud2, and rachid mamouni1 1 team of materials, catalysis and natural resources development, department of chemistry, ibn-zohr university, fsa. agadir, morocco; 2 agrophysiology & post harvest laboratory natural resources and local product research unity (ur rn &pdt) inra-crraagadir, morocco. *corresponding and presenting author: jamila.fahimi@edu.uiz.ac.ma /f.achemchem@ uiz.ac.ma. abstract salinity is one of the main factors limiting the growth of plants. agricultural losses caused by salinity are alarming and in continuous increase. silicon (si) is the second abundant element in the earth’s crust, performing a great role in healing plants in response to abiotic stresses. seed germination is critical step of the plant development. for that, this work aims to evaluate the germinative behaviour of coriander coa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 486 riandrum sativum (l.) in salt stress conditions in presence and absence of si. seeds were germinated in petri dishes in three levels of salinity (0, 25 mm and 50 mm of nacl) with and without si supply, the number of seeds germinated was recorded daily for 10 days. our results exhibit that salt treatments acted negatively on germination percentage (%), coefficient of velocity of germination (cvg), germination rate index (gri) and mean germination time (mgt). whereas, the application of si enhanced these germination indexes. keywords: silicon; salinity; coriandrum sativum (l.); germination. p049. improvement of tomato rhizobial inoculant by incorporation of plant growth promoting rhizobacteria (pgpr) as biocontrol against the bacterial canker caused by clavibacter michiganensis subsp. michiganensis meryam moustaine1,2*, r. elkahkahi3, a. benbouazza1, r. benkirane2, and el hassan achbani1 1 laboratory of bacteriology and biological control, the national institute of agronomic research of meknes. morocco; 2 laboratory of botany and plant protection, faculty of sciences, university ibn tufail, kenitra, morocco; 3 centre for innovation and technology transfer, university moulay ismail, marjane 2, meknes. morocco. *corresponding and presenting author: meryamenam@gmail.com. abstract three bacterial strains from the compost were tested for their antagonistic properties against the pathogenic bacterium clavibacter michiganensis on tomato. their efficacy was compared with that of the pantoea agglomerans bacterium (2066-7) whose ability to control clavibacter michiganensis is recognized. the results show that pantoea agglomerans reduces the onset of 70% disease symptoms, serratia proteamaculans to 45% and bacillus cereus to 75%. subsequent trials will verify the antagonistic efficacy of the combination of these three rhizobacteria on clavibacter michiganensis. keywords: clavibacter michiganensis, rhizobacteria, antagonistic. p050. study of several physicochemical properties of four sweet cherry cultivars (prunus avium l.) in the middle atlas mountains of morocco mina el baji1,2*, h. hanine2, h. latrache2, and o. kodad1 1 department of pomologie, national school of agriculturemeknes, morocco; 2 laboratory of bioprocess and bio-interfaces, fst béni-mellal, beni mellal, morocco. *corresponding and presenting author: m.elbaji92@gmail.com. abstract fruit samples analyzed in this study were harvested in 2015 and 2016 from four sweet cherry cultivars, namely “burlat”, “van”, “napoleon” and “pigeon heart” grown in the middle atlas of morocco (toufselt and imouzzer). some physicochemical properties of fruit were investigated. fruit weight ranged from 6.39 g and 6.90 g, but statistical differences between “burlat”, “van” and “pigeon heart” were non-significant. fruit volume in different cultivars was between 4.89 and 5.26 cm3. the highest values were recorded in “van” and “pigeon heart”. the lowest values of flesh width were in cultivars “burlat” (7.26 mm) and “napoleon” (7.12 mm). the epidermis of “napoleon” cherries had low firmness, whereas high firmness values were measured in “van” and “pigeon heart”. titratable acidity was between 8.36 and 11.05 g malic acid/l, “napoleon” having the highest value. soluble solids content in different cultivars was between 14.5 °brix and 16.07 °brix. the highest value was recorded in “van”. fruit weight and firmness were higher in 2016 than in 2015 while fruit length and thickness were lower. “toufselt” location shows high values of fruit weight and size. the fruits produced in “imouzzer” locations are characterized by a high firmness. keywords: sweet cherry, cultivar, physicochemical properties. p051. humics substances produced during composting process of the olive mill wastes and municipal organic wastes barje farid* and hafidi mohamed laboratory of ecology and environment, faculty of a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 487 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) science semlalia, cadi ayyad university, marrakesh, morocco. *corresponding and presenting author: fbarje@gmail.com. abstract the humics substances form an important fraction of soils’ organic compounds; their chemical nature has put forward various hypotheses on the mechanisms of their formation. in this context, to follow their evolution,the humic acids(has) and fulvicacids (fas) extracted at different stages of co-composting of olive mill waste and organic household refuse with added phosphate, were analyzedwith 13c-nmr and ft-ir spectroscopy. the findings highlight the contrasting variations in theproportions of c-aromatic and c-carbonyl of has as the h/c atomic ratio changes. the comparative 13c-nmr study of the fas with the same tendancy,that showlow aromatic (c=c) content in the initial stage of composting, which contained a greater proportion of carbonyl (c=o), o,n-alkyl and alkyl carbon. the ft-ir spectroscopy of has showed a decreasingabsorption in bands of aliphatic bonds -ch-, -ch2-, carboxylic acids, esters, amides c=o,the c-o-bonds of polysaccharides and alcohols with respect to ether bonds c-o-c;they was linearlycorrelated with the h/c ratio. for the fas, the main absorption bands were in the region of 3393 cm-1 (o-h), near 2931 cm-1 (aliphatic c-h), a distinct shoulder between 1717 cm-1 (c=o of carbonyl) and 1640 cm-1 (c=o of amide and ketone), 1547 cm-1 (aromatic c=c with conjugated c=o or amide n-h), near 1400-1384 cm-1 ( coostretch or oh deformation of cooh). the ratio of has to fas carbon content (cha/cfa), which is representative of the polymerization degree, showed a positive linear correlation (r =+0.56, p < 0.05) with the degree of aromaticity of has. the increase in the cha/cfa ratio with increasing humic acidsaromaticity,is representative ofa remarkable aspect of the restructuring brought, with respect to the mechanisms ofpolymerization during composting. keywords: humic acids, fulvic acids, composts, organic wastes. p052. physicochemical characterization of the pomegranate (punica granatum) in inra collection: a first step of selection of performants cultivars assia ejjilani1,2*, hafida haninie2, rachid razouk1, lahcen hssaini1, abderrahman mekaoui1, hakim outghouliast1, and jamal charafi1§ 1 national institute of agricultural research (inra), regional center of meknes, morocco; 2 laboratory of bioprocess and bio-interfaces, faculty of science and technology, beni mellal, morocco. *corresponding and presenting author: assiaejjila@gmail.com. §corresponding author: jcharafi@gmail.com. abstract in order to support the development of the pomegranate (punica granatum) in morocco, a collection of several important varieties has been installed in experimental station of the national institute of agricultural research (inra) located in aïn taoujdate. this study was carried to assess the appropriate physicochemical compounds of fruit for a future selection of performing varieties for agriculture and industry. eighteen cultivars were characterized including 10 local genotypes and 8 foreigners. measurements concerned parameters related to the external, internal and qualitative characteristics of fruit (weight, dimensions, weight of arils, pips, brix degree of juice, …). our results show a large diversity based on the used descriptors. for fruit weight, it varies between 198.14g for local cultivar “chelfi” and 589.28g for foreign cultivar “red pomegranate 2”. the fruit length differs between 58.93 (chelfi) to 96.3 mm (red grenade2) and its diameter between 72.29 (chelfi) and 109.99 mm (red pomegranate 2). total arils weight varies between 127 and 463g with an average of 237g. thus, the weight of one aril varies from 2.6 to 8.9 g with an average of 4.3. fruit juice content reached from 25 to 52 %. the high values of juice content were observed for cultivars zheri of autumn’,’ zheri precoce’, and ‘red pomegranate’, thus presenting an interest in industry. the flavor and the hardness of arils have a great influence on fruit taste quality. the cultivars red pomegranate, sefri and yellow pomegranate have a good quality marked by a sweet taste (17-20 °brix), a high richness in anthocyanins and a softness of their pips. these primary results show that this ex-situ collection of pomegranate contains a large physicochemical diversity and can be a basis for selecting the performing varieties for different regions. keywords: pomegranate, punica granatum, characterization, physicochemical, quality, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 488 ex-situ collection p053. valorization of the therapeutic virtues of salvia mouretii extracts from atlas mountains of morocco sanae jennan1*, abdellah farah2, fatima mahjoubi1, and dalila bousta3 1 materials engineering and environnement lab, department of chemistry sciences, sidi mohamed ben abdellah university, faculty of sciences dhar el mahraz, bp 1796 fez, morocco; 2 applied organic chemistry lab, department of chemistry , faculty of sciences and techniques, fez, morocco; 3 régulations neuroendocriniennes liées à l’environnement lab, faculty of sciences dhar el mahraz, bp 1796 fez, morocco. *corresponding and presenting author: jennansanae@gmail.com. abstract this study aims to investigate in vitro antioxidant activities of salvia mouretii and to examine the in vivo antiinflammatory and antinociceptive effects of the aqueous extract of aerial parts of s.mouretii. the aqueous and ethanol extracts were screened for their possible antioxidant activities by three complementary test systems, namely dpph free radical-scavenging, reducing power and molybdenum systems. anti-inflammatory activity of the aqueous extract was evaluated by carrageenaninduced rat paw oedema method. the antinociceptive effect was tested by using the plantar test. the ethanol extract showed better activity (ic50 = 0.4mg/ ml) in the dpph system. the aqueous extract showed greater activity in the phosphomolybdenum method (389.75 equivalent to ascorbic acid mg/g of extract and 188.73 equivalent to bht mg/g of extract) and exhibited the highest reducing power. the aqueous extract of s. mouretii at 10% inhibited the inflammation induced by carrageenan in rats at 75% compared to 67.5% for diclofenac at 1% after 4h of inflammation induction. however, at dose of 20%, s. mouretii showed a pro-inflammatory effect. in the plantar test, the tolerance time of the rats treated with 10% of the aqueous extract was significantly higher compared to the control group. these initial results tend to support the traditional use of s. mouretti used in morocco for the treatment of burns. keywords: salvia mouretii, antioxidant activity, anti-inflammatory activity, antinociceptive activity. p054. characterization of rhizobacteria isolated from compost and their potential effect on promoting tomato (solanum lycopersicum l.) growth m. moustaine1,2*, r. elkahkahi3, a. benbouazza1, r. benkirane2, and el hassan achbani1 1 laboratory of bacteriology and biological control, the national institute of agronomic research, meknes, morocco; 2 laboratory of botany and plant protection, faculty of kenitra ibn tufail sciences, kenitra, morocco; 3 centre for innovation and technology transfer, university moulay ismail, marjane 2, meknes. morocco. *corresponding and presenting author: meryamenam@gmail.com. abstract rhizobacteria promote plant growth (pgpr) are soil bacteria which colonize roots of the plant, improve growth and live symbiotically with it. these microorganisms can promote plant growth by nutritional and hormonal balance control, production of plant growth regulators, the solubilization of nutrients (such as phosphate) and including resistance against plant pathogens. the objective of this work was to study the pgp activities and production of antimicrobial enzymes of three bacterial strains belonging to the genus bacillus cereus (2027-2), pantoea agglomerans (2066-7) and serratia proteamaculans (2025-1), which are promoter’s rhizobacteria plant growth and having been tested on the culture of the tomato field. only the strains 2066-7 solubilize the phosphate in the culture medium pvk. although all isolates produce iaa 2066-7 product the high concentrations of this strain phytohormone in addition to its ability to synthesize all antimicrobial enzymes. all isolates showed an acetic acid indole activity in large amounts when cultured in the presence of tryptophan precursor to 1 g / l. this characterization has allowed to know the attributes benefits, which account for the benefits of competitiveness of these bacteria in the rhizosphere of crops, which, with their ability to perform biological nitrogen fixation, could promote the growth of tomato plants. keywords: pgpr, aia, solubilization phosphate, nitrogen fixation, production of ammonia (nh3) and production antimicrobial enzymes. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 489 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) p055. conservation of moroccan saffron with molecular identification by microsatellite markers (crocus sativus l.) mohamed lachheb*, m. ben el caid, l. salaka, k. lagram, mohamed amine serghini, and abdelhamid el mousadik laboratory of biotechnology and valorization of the natural resources (lbvrn), department of biology, faculty of science, university ibn zohr, agadir, morocco. *corresponding and presenting author: lachheb.mohamed91@gmail.com. abstract crocus sativus l. is a male sterile vegetatively propagated plant. its flower produces stigmas that when dried, constitute the most expensive spice in the world commonly known as saffron‘ red gold’, the name saffron is usually used to refer toboth the spice and the plant itself. this plant is cultivatedin anumber ofcountries in the world (iran, turkey, india, greece, morocco, etc.).in morocco, saffron is primarily cultivated in the area of taliouine/taznakht. the moroccan saffron is very popular at both national and international level. the conservation and enhancement of the moroccan saffron impose their morphological, biochemical and molecular characterization. the present study has for objectives, the development of a molecular identity of moroccan saffron, the investigation and evaluation of the genetic variability by molecular markers and the construction of a database allowing fora better management, selection, exploitation and conservation of the moroccan saffron as anational heritage. the samples used in this study, were collected from four different selected areas (provenances) in the region of taliouine/taznakht (sidi hssaine, agadir melloul, taznakht and askaouen) with an average of four samples of leaves from each provenance. concerning the genomic dna, it was extracted from 40mg of leaves, using ctab procedure and it has served as the matrix for the amplification by pcr. the microsatellite markers were selected for the identification and analysis of the genetic variability of cultivars of the moroccan saffron, because of their large polymorphism, their specificity, their codominance, their reproducibility and stability. 359 est sequences of genomic dna of the saffron were collected from the national center for biotechnology information (cnib). the ssrit software has allowed the identification of 14 est-ssr (ssre) and 12 ssr from the genomic dna (ssrg), the primers specific for each region ssr were determined by the software primer 3 version 4.0.0. in addition, 12 pairs of primers developed by nemati et al. (2012) among the iranian saffron and three pairs of primers determined at iris ensata, were selected for the molecular characterization of the moroccan saffron. the first results show that moroccan saffron is genetically different compared to the iranian saffron, this data is promoter for developing of a conservation strategy for the moroccan saffron. the provenance of sidi hssaine is a clone genetically and agro-morphologically different from other provenances, this data is in favor of a high potential for the improvement of the productivity of the saffron of taliouine with a selection assisted with ssr markers. keywords: microsatellite, saffron (crocus sativus), molecular markers, genetic diversity, molecular identity. p056. phytochemical and antibacterial study of thymus zygisin the azrou region fatima zahrae radi1,2*, sanae amine1,2, mohamed regragui1,2, najia hamzaoui1,2, hamid oulhaj1,2, and touriya zair1,2 1 research team in the chemistry of bioactive molecules and environment. department of chemistry. moulay ismail university, faculty of sciences, po box 11201, 50000 zitoune meknès, morocco; 2 laboratories of materials chemistry and biotechnology of natural products (chima-bio).department of chemistry. moulay ismail university, faculty of sciences, po box 11201, 50000 zitoune meknès, morocco. *corresponding and presenting author: fati_radi2007@ hotmail.com. abstract as a part of the valuation of the moroccan cultural heritage in aromatic and medicinal plants, a phytochemical and antibacterial study of a species widely used in traditional medicine was carried out, it is the thymus zygis of the azrou region. this plant’s yield of essential oil extracted by hydrodistillation is of the order of 5.25± 0,01. the essential oil (eo) was analyzed using gas chromatography coupled with a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 490 mass pectrometry (gc-ms). it contains as majority compounds: the thymol (32.46%), and the carvacrole (18.29%). this plant has undergone a phytochemical screening which consists of detecting the different chemical compounds existing in the plant using precipitation and coloring reactions. thus, it is rich in tannins, flavonoids, sterols and triterpenes, and free from the alkaloids of coumarins, and mucilage. for the antibacterial activity two methods were used: the first one is the method of diffusion on the disk and the second one is the method of the minimal inhibitory concentration (mic) and the minimum bactericidal concentration (cmb). the essential oil was tested against six bacterial species: escherichia coli, staphylococcus aureus, salmonella typhi, acinetobacter baumanii, shigella dysenteria, and enterobacter cloacae. the results obtained showed that the plant’s essential oil inhibited the activity of all the species at low volumes (2µl / ml). the cmb was identical to the mic(2µl / ml). according to the results of this study, we conclude that this medicinal plant possesses very important biological properties which can be widely used in various fields such as medicine, pharmacy, cosmetology and agriculture. keywords: aromatic and medicinal plants, thymus zygis, phytochemical study, bacterial species, essential oil and antimicrobial power. p057. numerical simulation of the airflow, temperature and humidity fields distribution inside multi-span greenhouse equipped with insect proof under different outside climate conditions reda errais1*, ahmed bekkaoui1, hassan majdoubi2,3, younes el fellah1, and allal senhaji3 1 département energie et agroéquipements. institut agronomique et vétérinaire hassan ii, rabat, morocco; 2 laboratoire de recherche scientifique et développement pédagogique. crmef, fès-meknès, morocco; equipe de recherche en energétique et mécanique des fluides. ecole nationale supérieure d’arts et métiers, meknès, morocco. *corresponding and presenting author: redaerrais@gmail.com. abstract the agricultural greenhouses are structures that create a favorable and optimal microclimate adapted for the growth and the development of each crop by controlling the climate and by reducing the introduction of the various insects harmful to plants. air circulation, temperature and humidity are the most important parameters of greenhouse climate that needs to be controlled in order to achieve optimal plants growth and maximize the yield. the aim of the current study is to predict the distributed climate inside multi-span tomato-greenhouses equipped with fine insect proof in sidewalls ventilation openings. also, we try to analyze the greenhouse ventilation efficiency under two different external climate conditions in order to determine the effect of the outside weather conditions on the airflow , temperature and humidity fields distribution inside and at the greenhouses openings. for this purpose, a 3d numerical simulation using a computational fluid dynamics (cfd) was carried out to estimate the dynamic and thermal and humidity behavior by using a commercial software package fluent v6.3.26 based on the finite volumes method to solve the mass, momentum and energy conservation equations. the turbulent transfers were described by model. likewise, the dynamic influences of insect screens and tomatoes crop on air flow movement were modeled by means of the concept of porous medium. atmospheric radiations contribution was also included in the model. keywords: modeling, simulation, greenhouse, microclimate, balance energetic, tomato crop, insect proof, cfd, fluent. p058. meat quality traits of béni guil lamb raised in eastern morocco kamal belhaj1,2*, f. mansouri1, caid serghini1, m. sindic2, m-l. fauconnier3, m. boukharta4, and ahmed elamrani1 1 lbpm laboratory , faculte of sciences, university mohamed ist, university mohamed premier, oujda, morocco; 2 qspa laboratory, gembloux agro-bio tech, gembloux, university of liège, belgium; 3 laboratory of general and organic chemistry, gembloux agro bio-tech, university of liège, belgium; 4 haute ecole charlemagne, isia-huy, belgium. *corresponding and presenting author: belhaj.kamal90@ gmail.com. abstract béni guil bred is the main ovine breed that dominates livestock farming in the semi-arid region of eastern a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 491 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) morocco. no previous data is available on the quality of béni guil lamb meat raised on the natural pasture of this area. therefore, this study aims to provide chemical composition and nutritional traits of this lamb meat. the animals were slaughtered at 33-37 kg live weight, with a strong fattening condition, corresponding to notation 4, according to the community scale (scale 1-5) of grading sheep carcasses europ (1) and r for conformation, according to the seurop grid (2). chemical analysis was conducted on trimmed lyophilised samples of the longissimus lumborum muscle (llm). chemical composition traits of llm, namely dry matter (25.72%) protein (19.43%) intramuscular fat (5.14% and ash percentage (0.94%) were similar to those of other indigenous breeds. total saturated fatty acids (sfa), monounsaturated fatty acids (mufa) and polyunsaturated fatty acids (pufa) content in the intramuscular fat was on average of 49.45 %, 38.48% and 12.4% of total fatty acids, respectively. the ufa/sfa and n-6/n-3 pufa ratios are 1.04 and 3.78, respectively, and are comparable to those recommended for a balanced diet. amino acid (aa) composition analysis conducted by a high-performance liquid chromatography allowed the identification of 17 aa, including eight essential amino acids (his, met, ile, leu, lys, thr, val, phe). the chemical index and the protein digestibility-corrected amino acid score values were 132 and 127, respectively. keywords: béni guil bred, lamb meat, fatty acids, amino acids, nutritional quality. 1la grille europ de classement pour l’état d’engraissement [internet]. centre interrégional d’information et de recherche en production ovine, france. 2011. available from: www.reconqueteovine.fr. 2la grille europ de classement pour la conformation [internet]. centre interrégional d’information et de recherche en production ovine, france. 2011. available from: www.reconquete-ovine.fr. p059. effect of silicon on growth and grain yield of wheat bendidi abderrazzak1*, rachid dahan1, el housin bouichou1, chams dha khalfi1, mohamed ibriz2, wafae sellami3, and mohamed el yaalaoui3 1 national agricultural research institute, morocco; 2 faculty of sciences, university ibn tofail, kenita, morocco; 3 faculty of science and technology of fès, morocco. *corresponding and presenting author: abendidi1@yahoo.fr. abstract the objective of this study was to determine the effects of silicon on the growth and grain yield of bread wheat and durum wheat. the trial was conducted at the greenhouse at inra meknes morocco using a randomized complete block design with three replications in 2016/2017. two factors (1dose of silicon; si-= 0 and si+= 400 kg si/ha), and 2genotype; g1= bread wheat; arrehane and g2= durum wheat; vitron) were tested. statistical analysis revealed no significant effect of silicon on grain yield and its components for bread wheat and durum wheat. however, we noticed that silicon improved length, volume and root biomass as well as leaf area, plant height and biomass produced for bread wheat, unlike durum wheat. probably some benefits of silicon (improved resistance to drought, fungal diseases and salinity) were masked because of the conduct the test in the greenhouse. the question that arises is the interest of bringing silicon for wheat in the saus region of morocco. keywords: silicon, bread wheat, durum wheat, growth, yield. p060. the chemical and biochemical characterization of moroccan carob fruits fatima ezzahra eddabbeh1,2*, abdelilah rahou2, and laila nassiri1 1 soil & environment microbiology unit, faculty of sciences, moulay ismail university, 50050, pb : 298, marjane, meknes, morocco; 2 biotechnology and valuation of plant resources, faculty of sciences, moulay ismail university, p.b. 298, marjane meknes, morocco. *corresponding and presenting author: eddabbeh_ fati_e@yahoo.fr. abstract the carob tree (ceratonia siliqua l.) is an agro-sylvopastoral tree specific to the mediterranean region. in morocco, it covers an area of about 130.000 hectares. there is a great agro-economic interest in a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 492 the carob treebecause of its low agronomic requirement and its high benefits and revenues. the results of the chemical and biochemical analyzes made on the moroccan carob from diverse regions showed a great interest for the industrialists as well as for the valorization of the carob fruit. this explains the induction of new plantations in different regions and the creation of cooperatives for the valorization of its fruits. the present paper synthesizes the chemical and biochemical diversity of carob seed and pulp in morocco. keywords: carob tree, chemical diversity, biochemical diversity. p061. evaluation of soil fertility of benslimane region habiba sehlaoui1,2*, h. dakak2, a. zouahri2, a. douaik2, a ghanimi3, and r. hassikou1 1 university mohammed v, faculty of sciences, department of biology, mycology and environment, rabat, morocco; 2 national research institute of agricultural research, crrar, research unit on the environment and conservation of natural resources b.p. 6356 institutes, rabat, morocco; 3 university mohammed v, faculty of sciences, department of chemistry, rabat, morocco. *corresponding and presenting author: sehlaoui.habiba@gmail.com. abstract morocco has always made the development of the agricultural sector a priority and a strategic choice. but being an essentially arid and semi-arid country, intensive agricultural development has led to an improvement in agricultural production, and it has, on the other hand, generated; degradation of soil quality. this deterioration constitutes a serious danger since it causes a decrease in its fertility. in order to better understand this problem, an assessment of the physico-chemical quality of soils becomes necessary. it is in this context that this study was conducted to examine the chemical fertility of the irrigated perimeter cherrat, which belongs to the benslimane region. this region has undergone some economic and particularly agricultural expansion in recent years. a survey of about thirty points was carried out. at first, measurements of ph, electrical conductivity, organic matter content, available phosphorus and exchangeable potassium were made. in a second step, a geographical information system (gis) was established in order to study the spatial variations of its parameters. keywords : soil, fertility, benslimane, cherrat. p062. agro-morphological analysis of moroccan sesame (sesamum indicum l.) populations meriem el harfi1,2*, abdelghani nabloussi2, marouane jbilou3, hafida hanine1, hajar rizki1, and jamal charafi2 1 laboratory of bioprocess and biointerfaces, faculty of science and technology, béni mellal, morocco; 2 research unit of plant breeding and plant genetic resources conservation, national institute of agronomic research, regional agricultural research center of meknès, morocco; 3 regional agricultural research center of afourer, morocco. *corresponding and presenting author: elharfi.meriem@gmail.com. abstract characterization of a given plant material contributes to the optimization of its conservation and its use as a germplasm in breeding programs. the aim of this study was to characterize 33 populations of moroccan sesame (sesamum indicum l.) on the basis of agro– morphological traits and to estimate genetic diversity among and within populations. the material studied had been collected in 2012 from different zones in the tadla area and, then, was evaluated in field experiments at the inra experimental station of afourer, using randomized block design during 2013 and 2014. the study was done on 13 agromorphological traits, namely growth rate, height of the plant, number of branches, height of the first branch, height of the first pod, number of pods per plant, width of pod, length of pod, number of seeds per pod, thousand seeds weight, seed yield per plant, seed yield per ha and oil content. significant differences between sesame populations in two different environments, afourer 2013 and afourer 2014, were observed only for three measured traits: height at the first pod (75.27 99.46 cm), number of seeds per pod (55.30 – 70.35) and thousand seeds weight (3.07 – 3.50 g). however, the environment has a significant effect on the majority of the traits studied. also, effect of year was more pronounced for afourer environment, being 2014 more favorable than year 2013. the a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 493 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) limited genetic diversity among the populations studied might indicate these were very close genetically and likely were derived from a same original variety. keyswords: sesame, populations, agromorphological traits, diversity, environment, morocco. p063. evaluation and selection of lactic acid bacteria as starter cultures from natural biotopes moussaid siham1,2*, k. ounine2, a. benali1, m. r. kabour1, and e. h. maadoudi1 1 laboratory of food technology and quality, food safety/environment, crra, inra, avenue ennasr, bp 415 rp, rabat, morocco; 2 nutrition, health and environment laboratory, faculty of science, university ibn tofail, b.p. 133 kenitra. morocco. *corresponding and presenting author: sihamfstg@gmail.com. abstract for thousands of years, humans have used microorganisms in the pharmaceutical, food, agronomic and industrial fields. these ubiquitous microorganisms of our environment and our food occupy an increasingly an important place in our life and are currently at the origin of the rise of biotechnology field. lactic acid bacteria (lab) are among the microorganisms used in the processing and preservation of food by producing organic acids, proteases, lipases, exopolysaccharides, antibacterial agents, etc. the objective of our study is to isolate and screen lab that have important technological skills. a total of forty-eight bacteria were isolated from camel milk, goat milk and argan residues. the evaluation of the acidifying ability by ph monitoring every two hours for twenty-four hours showed that for some bacteria the ph decreased from 6.7 to 3.8 after 24 hours of fermentation. the opa method revealed that 14% of the isolated have a significant proteolytic activity releasing up to 7 mm glycine. the dosage of fatty acids released by the isolates using the titrimetry method has shown that its amount varies between 0.6 µmol / ml and 2.6 µmol / ml. whereas the anti-free radical activity measured by the dpph method is 5% for a sterile skimmed milk while it is maximum for the same milk inoculated by our isolates to reach 34% compared to vitamin c for certain isolates. 30% of the isolates have an antibacterial activity against either staphylococcus aureus, e. coli or salmonella enterica or on the three strains at the same time whose diameter of the zone of inhibition of the pathogenic strains is between 2 mm and 12 mm. on the other hand, 49% of isolates are able to produce exopolysaccharides responsible for the texture of processed products. isolated bal have shown an important property to allow them to be considered as good starter cultures. keywords: lactic acid bacteria; acidifying activity; protease; lipase; antioxidant activity. p064. genotypic and environmental effects on doublekernelled fruits of some almond [prunus dulcis (mill.) d.a. webb] cultivars grown in northern morocco el hassan sakar* and yahia rharrabti laboratory of natural resources and environment, polydisciplinary faculty of taza, b.p 1223, tazagare (taza), unviersity sidi mohamed ben abdellah, taza, morocco. *corresponding and presenting author: hassan.sce@gmail.com / elhassan.sakar@ usmba.ac.ma. abstract almond tree [prunus dulcis (mill.) d.a. webb] is an important nut crop commercially grown throughout the world. it is cultivated for its kernel of high commercial and nutritional values. along with nutritional richness, fruit physical aspect is considered as an important fruit quality trait especially for almond processing and commercialization (consumer acceptance). in this regard, production of double-kernelled fruit is undesirable trait resulting in small and deformed kernels. in this work, we aimed to investigate fruit double kernels from the five widely almond cultivars: marcona (mar), fournat de brézenaud (fbz), ferragnès (frg), ferraduel (frd), and tuono (tuo). to assess environmental effects on this trait, fruits were sampled from three different environments in northern morocco: aknoul (ak), bni hadifa (bh), and tahar souk (ts) over two harvest seasons 2015 and 2016. at maturity stage, which fits 89 on the general bbch phenological scale, samples of 1.5 kgs were harvested in triplicate. once at laboratory, for each sample, sub-samples of 100 fruits were used for double-kernelled fruit estimation. nuts were cracked, and double kernels were counted and expressed in percentage (%). anova analyses a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 494 outcomes demonstrated significant effects of cultivar, site, harvest season, and their interactions on doublekernelled fruit production. this trait was genetically controlled since genotypic effects were more important and accounted for more than half of the total variance. moreover, environmental effects were also significant, but climatic factors (harvest season effect) being the most important. among sites, bh presented the lowest values of double kernels, while for cultivars frd and frg exhibited lower scores of this trait during the two harvest seasons and tuo showed the greatest values. for harvest seasons, 2016 was relatively dry as compared to 2015. 2016 presented the lowest values of double kernels. it could be concluded, that double-kernelled fruit trait was a genotypic trait, but climatic factor could enhance or reduce its expression. keywords: commercial almond cultivars, double kernels, genotypic and environmental effects, market and consumer acceptance. p065. development of the tree strawberry varieties (fortuna, fragaria and san andreas) houda tahiri1*, ahlam hamim2, and amal el amrani3 1 in vitro culture laboratory/inra tangier, morocco; 2 vegetable biotechnology laboratory /faculty of science tetouan, morocco. *corresponding and presenting author: houditahhiri@gmail.com. abstract the in vitro culture of strawberry plants allows to obtain from a small fragment of a plant placed on a synthetic nutrient medium an infinity of identical healthy plants. in this context, the present work revolves around two main axes. the first axis aims at studying the in vitro micropropagation of strawberry. the response of the explants to various factors, the basic culture medium, growth regulators. in order to have the answer, different concentrations were studied. in vitro germination of strawberry seeds is a mandatory step for the production of the necessary seedlings for micropropagation. the seeds of three cultivars “fortuna”,”fragaria” and “san andreas” scarified and sterilized by sulfuric acid h2so2 (36n), (5min); showed a maximum germination capacity after a short passage on the mineral medium of murashig and skoog (1962) agar. for the apex multiplication phase, murashig and skoog (1962) were selected with hormones with different concentrations of growth regulators (1 mg / l aib, 0,5 mg / l ba, 0,1 mg / l ag3) for mass production of strawberry plants. for the rooting phase, the results showed that rooting gives better results on the ms medium with different concentrations of aib (0.5, 1, 2 or 3 mg l-1). ‘aib are different depending on the cultivar and after 5 weeks the seedlings are grown at variable frequencies. the regenerated seedlings are easily acclimatized on a pre-sterilized turmeric-vermiculite substrate. concerning the second axis, a test of the resistance of the vitroplants to the mites tetranychus urticae was carried out. the results showed that phytohormone aib especially at 3 mg / l can increase the resistance of the vitroplants in the three varieties studied. keywords:in vitro culture, micropropagation, strawberry, fortuna, fragaria, san andreas, apex, hormones, tetranychus urticae. p066. antibacterial activity of cinnamon essential oil and its major constituent cinnamaldehyde on escherichia coli strains of avian origin khribch jamila1*, zrira saadia2, nassik saâdia3, el houadfi mohammed3, and oukessou mohamed1§ 1 department of biological and pharmaceutical sciences, hassan ii agronomic and veterinary institute, bp 6202, 1010, rabat, morocco; 2 department of food and nutrition sciences, agronomic and veterinary institute hassan ii, bp 6202, rabat, morocco; 3 avian pathology unit, department of pathology and veterinary public health, agronomic and veterinary institute hassan ii, bp 6202, rabatinstituts, rabat, morocco. *presenting author: j.khribch.inrh@gmail. com. §corresponding author: m.oukessou@gmail.com. abstract the development of bacterial resistance to common antimicrobial agents constitutes a great challenge for actors in the field of human as well as animal health. this situation prompted research of new antimicrobial agents from various sources including mainly plants. the present study consists in evaluating the antibacterial activity of essential oil of cinnamon and its major active constituent the cinnamaldehyde. this activity was evaluated using the usual techniques of antibioa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 495 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) gram, aromatogram and determining the minimal inhibitory concentrations (mic) and the minimal bactericidal concentrations (mbc) in a broth medium against two reference strains (escherichia coliatcc25922 and staphylococcus aureus atcc25923) and strains of e. coli isolated from broiler chickens admitted to the avian pathology unit at iav hassan ii resistant to the main antibiotics (enrofloxacin and florfenicol). in total, cinnamaldehyde has been tested on about twenty bacterial strains, and the essential oil of cinnamon has been evaluated on about 11 escherichia coli strains. in the second part, the association of cinnamaldehyde with certain antibiotics was tested for possible synergies. furthermore, cinnamaldehyde kinetic-killing profile was studied using the same germ reference strains. according to the results obtained in this work, essential oil of cinnamon was more active than cinnamaldehyde. the essential oil inhibited the growth of e. coli strains by expressing inhibition halts of 29,3 and 25,1 mm for the reference strain and strains of avian origin respectively, while cinnamaldehyde gave inhibition zones of 26,5 and 23,23 mm for the reference strains and the wild-type strains respectively. the average values of the mics obtained with cinnamaldehyde against the reference strains were 0,031%, 0,041% for escherichia coli and staphylococcus aureus respectively. the same product expressed an average mic of 0,039% for strains of avian origin, while the cmb values were on average 0,031% for e.coli and 0,026% for s. aureus. the comparison of mic and cmb values for bacterial strains indicates that this natural product is bactericidal. finally, the bactericidal activity of cinnamaldehyde is close to the time-dependent kind antimicrobial and cinnamaldehyde-antibiotics combinations were most likely indifferent agents against both s. aureus and e. coli bacteria. keywords: essential oil, cinnamon, cinnamaldehyde, resistance, escherichia coli, diffusion method on agar. p067. contract farming within the green morocco plan: case of cereal sector bouichou el houssain1,2*, a fadlaoui1, k allali2,3, and r arrach4 1 national institute for agricultural research (inra), meknes, morocco; 2 agronomic and veterinary institute hassan ii (iav), rabat, morocco; 3 national school of agriculture (ena), meknes, morocco; 4 ministry of agriculture and fisheries: directorate of strategy and statistics, rabat, morocco. *corresponding and presenting author: bouichouelhoussain@yahoo.fr. abstract contract farming (cf) has long been practiced but is since the launch of green plan of morocco (pmv) it has regained an important interest with noticeable diversification in structures and involved actors. under cf, aggregators (ag) and aggregated (ag) make advance agreements and clauses on volume, quality, time of delivery, use of inputs, and price or pricing formula. the main purpose of the study is to characterize and analyze principles governing the contracts of cereal aggregation project, implemented on a national scale. emphasizing the objectives, details the undertakings of the contracting parties, as well as, state’s undertakings. these investigations sensed the implementation of 120 agricultural aggregation project (aap) in the main cereal crops. the number of the aap for cereals having received the certificate of aggregation amounts to 7. the project aggregation contract of cereals crops analysis launched at national level, allowed revealing that they are structured in five clauses categories: 1) inputs supply, technical support and productions methods; 2) quantities, quality and terms of payment; 3) elements of the fixing of purchase prices and trading conditions; 4) management and sharing of risks; and 5) mediation, agree dispute resolution. this preliminary analysis stressed, inter alia, that great efforts were made of the regulation, and formalization of the contractual relationship. however, the contracts are faced with many shortcomings and insufficiency. indeed, the principal ones concern the heterogeneous contents which do not include quality advantages and the lack of the ag’s implication in the negotiation process. the latters are the ones who support all the risks inherent to the production. knowing that he use of specialized bodies for the drafting of contracts is rarely carried out. significant efforts remain of their content and the standardization of their structure which would take into account the guidelines of the law on aggregation. as well as other specific interventions, based on information, training and dissemination of the expected benefits from coordination should increase farmers’ willingness to join contract farming. keywords: a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 496 agricultural aggregation project, cereal, contractual clauses, contract farming, supply chains, vertical coordination. p068. effect of feeding argan by-products on the quality of fattening lamb’s meat sanaa moutik1,2*, n. lakram2,3, m. bendaou2, e. h. el maadoudi2, r kabbour2, n. essafi1, and n el housni2 1 physico-chemistry laboratory for inorganic and organic materials, ecole normale supérieure, mohammed v university, mohamed bel hassan el ouazzani avenue, rp bp 5118, rabat, morocco; 2 inra, rcarrabat, p.o. box 6570, rabat institutes, rabat morocco ; 3 laboratory of zoology and general biology, faculty of sciences, mohammed v university, ibn battuta 4 avenue rp bp 1014, rabat, morocco. *corresponding and presenting author: moutik.sanaa@ gmail.com. abstract conventional method of fattening of lambs consists of using rations based on concentrate, to ensure a faster growth. indeed, the studies accomplished have shown that the daily food intake of lambs is composed of about 80% of cereals with 50% of manufactured concentrate, 50% between barley and corn. however, the increase of grain’s price, claim to review ways of lamb’s feeding. an interesting approach is to use the by-products of agriculture. compared to other byproducts, the pulp and the meal of the argan offers nutritional advantages, they are rich respectively in sugar (12%) and protein (42%). the purpose of this work is to test the effect of incorporating these byproducts into the lamb’s diet. the feeding trials were carried out in real medium on two lots of lambs of the sardi breed. one fed with argan tree by-products and the other fed a traditional diet. the results obtained so far are very encouraging.keywords: meal, pulp, argan, lambs, fattening. p069. characterization of flavonoid by hplc-ms analysis, antioxidant and antibacterial potential of capparis spinosa l. imane tagnaout1,2*, hannou zerkani1,2, zakariya khiya1, aziz drioiche1,2, and touriya zair1,2 1 research team of chemistry bioactive molecules and the environment, university moulay ismail, faculty of sciences, bp 11201, zitoune, meknès, morocco; 2 laboratory of materials chemistry and biotechnology of natural products, university moulay ismail, faculty of sciences, zitoune, meknès, morocco. *corresponding and presenting author: i.tagnaout@edu. umi.ac.ma. abstract capparis spinosa l. is a species which has a great importance in the field of traditional medicine in arid and semi-arid regions whose nutritional and medicinal value remains ambiguous in morocco. it is in this context that our work is aiming at the recovery of this species through a phytochemical study and an evaluation of antibacterial and antioxidant activities of flower buds of capparis spinosa l. spontaneous collected and sold by nour cooperative within the region of zerhoun, morocco. we achieved the solid-liquid extraction by two different methods in order to optimize the extraction yields of polyphenols; maceration and soxhlet by using two mixtures of solvents: methanol / water and acetone / water at 70%. the best yields are obtained with methanol / water mixture; 46% for soxhlet against 31% for maceration. the quantitative analysis of phenolic is being performed by colorimetry, extracts showed that the soxhlet method is more effective than the maceration. the qualitative analysis performed by hplc / uv-esi-ms also reported the existence of the same flavonoid glycosides in both hydromethanolic extracts in which the most important are the derivatives of kaempferol, quercetin and isorhamnetin. the antioxidant activity is determined by the dpph test, showed that the aqueous-methanolic extracts obtained by soxhlet and maceration expressed some approximate antioxidant powers with ic50 of respectively 0.73± 0.01 and 0.84± 0.01 mg / ml. however, the antibacterial activity evaluated against pathogenic strains such as staphylococcus aureus, escherichia coli, klebsilla pneumoniae, revealed the ineffectiveness of various extracts against the most tested strains. keywords: capparis spinosa l., zerhoune, polyphenols, flavonoids, hplc, antibacterial, antioxidant activity. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 497 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) p070. molecular and biochemical profiling of thymus satureioides genotypes cultured under in vitro and in vitro environments aicha nordine* and abdelmalek el meskaoui phytobiotechnology unit, national agency of medicinal and aromatic plants, pb 159, taounate, morocco. *corresponding and presenting author: aicha.nordine@usmba.ac.ma. abstract in this study, the in vitro and in vivo essential oil (eo) composition and genetic variability of six micropropagated genotypes of thymus satureioides coss., a mediterranean medicinal and aromatic plant, were analyzed by gas chromatograph coupled to a mass selective detector (gc/ms) and randomly amplified polymorphic dna (rapd). yield and composition of the essential oil varied between genotypes. cluster analysis based on rapd data and eo grouped the six genotypes in three groups in both culture conditions, thus showing considerable intraspecific genetic and chemical variations. applying the mantel test, the result showed a significant correlation between the two proximity matrices; rapd and eo obtained from in vitro genotypes whereas this correlation was not observed when using the eo obtained from the in vivo genotypes. keywords: carvacrol, essential oil, in vitro environment, thymol, thymus satureioides. p071. response of two varieties of banana (musa acuminata) small dwarf and large dwarf in vitro to salt stress meriem belfakih1*, mohammed ibriz1, and abdelmjid zouhair2 laboratory of genetics biometry, faculty of sciences, university ibn tofail, bp 133. 14 000. kenitra, morocco. *corresponding and presenting author: m_belfakih2000@yahoo.fr. abstract the objective of this study is to evaluate the effect of salt stress (nacl) on the in vitro regeneration of banana and the possibility of using this technique to improve the tolerance of this species to salinity. previously, the rejects were disinfected with 8% bleach and then grown in murashige and skoog medium (1962) containing growth hormones (appendix 1). the medium was fed with different salt concentrations (0, 2, 4 and 6 g / l of nacl). the test was conducted at a temperature of 28 to 30 ° c with artificial illumination and a photoperiod of 16 h. the results obtained show that both varieties have regeneration of shoots despite saline treatment. however, salt affects leaf length of the explants and the regeneration rate. so, the increase in saline concentration induce to a decrease in the number of shootings per bud. the small dwarf variety appears to be sensitive to the salinityeffect of the medium at all naclconcentrations at the regeneration stage and the seedling stage. keywords: salt stress, regeneration, growth, in vitro, banana. p072. impact of the combination of salt stress and mineral nutrition on the growth and biomass of alfalfa (medicago sativa l). meriem belfakih1* and mohammed ibriz1, and abdelmajid zouhair2 laboratory of genetics biometry, faculty of sciences, university ibn tofail, bp 133. 14 000. kenitra, morocco. *corresponding and presenting author: m_belfakih2000@yahoo.fr. abstract this work aims to study the combined salt stress effects (0.6 and 12g/l nacl) and the conditions of normal (hn) and limiting (hd) mineral nutrition, on growth and mineral nutrition of three varieties of alfalfa (sirever, demnate 203, and riche). the study was done under controlled conditions in a growth chamber for one month. the results obtained in this test showed that the salt treatment leads to a net reduction of the aboveground and root biomass upon addition of 6g/l. this detrimental effect of salinity becomes less under normal supply conditions, with the exception of the ‘demnate 203’ variety which showed the highest yield under limiting supply conditions. also, saline treatment leads to an accumulation of na+ cation and a reduction in k+ absorption. the ‘siriver’ variety was more sodium-inclusive under normal mineral supa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 498 ply (hn) conditions. in addition, the ‘demnate 203’ ecotype produced more dry matter under salt conditions and limiting mineral supply (hd). keywords: alfalfa, medicago sativa, salinity, mineral nutrition. p073. survey and multivariate characterization of some figs genotypes (ficus carica l.) prospected in northern morocco lahcen hssaini1,2*, hafida haninie2, rachid razouk1, ibtissame guirrou1,3, assia ejjilani1,2, abderrahman mekaoui1, and jamal charafi1§ 1 national institut of agricultural research (inra), regional center of meknes, morocco; 2 laboratory of bioprocess and bio-interfaces, faculty of science and technology, beni mellal, morocco ; 3 laboratory of food technologies, qualipole, meknes, morocco. *presenting author: hssaiini@gmail.com. §corresponding author: jcharafi@gmail.com. abstract in order to collect some of the most known and cultivated fig genotypes in the northern regions of morocco, the survey of family orchards in the area was performed. tree main fig growing regions were included into the field inventory (taounate, ouazzane and meknes). all collected material was morphologically and pomologically characterized according to internationally accepted descriptors for ficus carica l. the colorimetric analysis was carried out according to cie. all data were standardized (µ= 0 and σ = 1) to have a comparable scale. hence, analysis of variance has shown a very high significant level of variability (p<0.05) among evaluated genotypes for all variables. the principal component analysis (pca) has revealed two mean groups with a total inertia of 78.7% based on the quantitative traits. otherwise, the genotypes “ghani” and “ounq hmam_ps14” have been largely distinguished from the two groups. the colorimetric principal component analysis, has shown tree distinctive and homogeneous groups with a total inertia of 90.92%. thus, the first group contains the brightest and clearest fruit skin color (very high values of l*and c* coordinates), the second is related to fruits with skin color less bright and tending to be purple. while the last group characterize genotypes with a dark skin color (low values of l* and negative ones for a* and/or b* coordinates). qualitative characterization has shown a dominance of globose (74%) and rounded (61%) shape of figs. most of genotypes have skin ribes (82%) and 78% did not present the drop at the ostiole. hierarchical ascendant classification (hac) was been performed using the euclidian distance based on all 38 variables (color, morphological and pomological descriptors) to decide the ultimate number of clusters by which the accessions could be assessed. the result has shown two main clusters, each one is subdivided distinctively into two homogeneous subgroups with a main distance of 10. every subgroup is discriminated following a specific group of characters. with the exception of “ghoudan” and “ghani”, all genotypes with the same denominations were clustered into the same group. keywords: northen morocco, ficus carica, pomology, morphology, colorimetric. p074. assessment of genetic diversity in two moroccan populations of walnut (juglans regia l.) using issr markers jamal charafi*, abdellah kajji, and rachid razouk national institute of agricultural research (inra), regional center of meknes, morocco. *corresponding and presenting author: jcharafi@gmail.com. abstract walnut (juglans regia) covers an area of 7600 ha in morocco and performs an important socio-economic role. indeed, it constitutes a significant source of income for farmers despite the many constraints related to its production and valorization. the fact that seeds are the only mean of propagation used by farmers has largely contributed to its diversification. prospection was made in two main cultivation regions: haouz in the high atlas and midelt in the middle atlas. thus, assessing the genetic diversity of the walnut’s genetic resources is a first step towards conservation and the elaboration of a breeding program. a characterization of 33 moroccan walnut genotypes from these regions 25 genotypes from the haouz region and eight genotypes from the midelt region was carried out. in addition to eight bulgarian varieties by 13 issr, primers can detect a large genetic diversity. a total of 120 markers were detected and the number a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 499 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) per primer varied between 7 and 13 with an average of 9 bands. this number reflects the high level of polymorphism within the studied genotypes. the primers generated 75% of polymorphic fragments and the pairwise comparison of the 41 genotypes shows that all genotypes pairs are distinct by 10 to 50 markers. these large differences are due mainly to the multiplication mode dominated by seeding which favors the diversity explosion. this high diversity between genotypes is confirmed by the genetic distances obtained and hierarchical analysis using the upgma method. results show a minimum distance of 58%, a maximum of 91% and an average of 76%; the geographic structure is evident. the genotypes subgroups of the midelt region are distinct from those of the high atlas and the subdivisions into subgroups by village are remarkable. the presence of some genotypes in subgroups of other areas is explained by exchanges of plant material (nuts) between the agricultures. bulgarian varieties are distinct from local genetic material which confirms the specificity of the local walnut’s genetic patrimony. keywords: walnut, juglans regia., genetic diversity, molecular markers, issr. p075. earthworm feeding activity and mucus secretion can decrease entomopathogenic nematodes activity as biological control agents maryam chelkha1,2*, rubén blanco-pérez2,3, francisco ángel bueno-pallero2, abdellatif el harti1, souad amghar1, and raquel campos-herrera2§ 1 research team “lombricidae, improving soil productivity and environment” (lapse), ecole normale supérieure (e.n.s.), centre eau, ressources naturelles, environnement et développement durable (cern2d), mohammed v university in rabat, avenue mohamed bel hassan el ouazzani, bp : 5118. takaddoum rabat morocco; 2 meditbio, centre for mediterranean bioresources and food, universidade do algarve, campus de gambelas, fct ed. 8, 8005139 faro, portugal; 3 department of agriculture and food, universidad de la rioja, calle madre de dios, 51, 26006, logroño, spain. *corresponding author: marychalkha@hotmail.com. §corresponding author: rcherrera@ualg.pt. abstract earthworms are well-known beneficial organisms associated to plant production by indirect action (i.e. changes in soil aeration, structure and fertility) and by direct action (i.e. enhancing decomposition of organic matter). in addition, earthworms might promote the dissemination of beneficial soil organisms, including biological control agents, such as entomopathogenic nematodes (epns). despite the fact that epns result innocuous against earthworms, and their displacement can be favoured by the earthworm activity, the impact of other earthworm activities such as feeding behaviour or mucus production is poorly understood. we hypothesized that feeding activity might decrease the viable epns in the soil, reducing their ability to kill insects, whereas the presence of mucus will not affect the biocontrol skills. we evaluated the feeding activity and mucus secretion of the earthworm eisenia fetida (haplotaxida: lumbricidae) in combination with three epn species: steinernema feltiae, s. glaseri and s. carpocapsae (rhabditida: steinernematidae). first, we tested whether the presence of mucus can alter the infectivity of the three epns by using galleria mellonella (lepidoptera: pyralidae) as model insect. treatments included controls (water and mucus) and two concentrations (1.5 and 10 ijs/cm2) per epn species in the presence and absence of mucus. second, we evaluated the killing ability of epns against g. melllonella larvae when applied in autoclaved soil alone or in combination with earthworms or mucus. our results showed that the presence of mucus significantly reduced the larval mortality caused by s. feltiae and s. glaseri, while s. carpocapsae was not affected. similarly, the presence of earthworm and mucus reduced the larval mortality caused by s. feltiae and s. glaseri, resulting the mucus the most restrictive treatment. our results illustrate how the presence of earthworms or their secretions (mucus) can alter the long-term persistence in the soil of certain epn species as well as its action as biological control agents in a dense-dependent and species-specific manner. further studies will investigate whether this trend is general to other epn species as well as other entomopathogenic agents to establish to which extend the combined application of these beneficial organisms is compatible. keywords: earthworms, entomopathogenic nematodes, biological control, multitrophic interactions a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 500 p076. analysis of the oil content and fatty acid composition of 94 cultivars of olive tfarah boutzankad1*, a. mhais1, s. mousavi2, c. el modafar1, a. el antari3, a. moukhli3, h. zaher3, and m. a. tadlaoui1 1 ucam, fstg, bd. a. khattabi, marrakech, morocco ; 2 cnrisafom 128, via della madonna alta 06128 perugia, italy ; 3 inra, crra, marrakech, b.p.533 menara, marrakech, morocco. *corresponding and presenting author: tfarah.boutzankad@ gmail.com. abstract olive oil contains a high percentage of unsaturated fatty acids, that is why it is considered to have a big nutitional interest. in litterature, the selection of olive varieties is taken on the basis of their argonomic characters. in the present work, our objective is to characterize the nutritional quality of 94 cultivars (core collection) in term of the oil content and the fatty acids composition in the fruits and the correlation with the genetic profile of core collection cultivars, divided into their genetic structre groups (east, center and west. the statistical analysis showed significant effect of cultivar and interaction cultivar x replicates for the traits ; oil content, unsatfa/sfa ratio, palmitic, oleic and linoleic acids percentage. then there is only genetic effect for the palmitic and stearic acids percentage. indeed, regarding to the admixed cultivars from core collection, the oil content is high in eastern group followed by western and centeral groups. whereas, the unsatfa/sfa ratio is high in western group followed by eastern and then central. therefore, the cultivars which have high linoleic acid content are from east, followed by the centeral and the western groups. the principal components analysis (pca) carried out on data summarizes 69,30% of the general information on the first two dimensions. as a result, three groups were distinguished : the first is characterized by high values of oleic acid percentage and unsatfa/sfa ratio as well as low palmitic acid percentage. the second contains individuals with intermediate values and last group gave high palmitic acid, low oleic acid and unsatfa/sfa ratio. this investigation will be in process for two more years in order to estimate the year effect and to quantify the genetic effect via the heritability estimation. keywords: oil content , fatty acids composition, core collection, olive oil. p077. livestock farming system and reproductive performance of local goat in the argan tree park (arganeraie) of the agadir region in morocco houda el kheyyat* and saïd el madidi§ biometrics and bio resources, laboratory biotechnologies and natural resources development (lbvrn), faculty of sciences, university ibn zohr, agadir, morocco. *presenting author: elkheyyathouda@yahoo.fr. §corresponding author: s.elmadidi@uiz.ac.ma. abstract a study was conducted using a structured survey among 35 goat farmers randomly selected and distributed in 9 villages in the arganeraie of the agadir region. the livestock system is of an traditional extensive type whose diet is based on sylvo-pastoral resources. herd management is permanent wandering with confinement during rainy days. only 37% of the respondent goats’ farmers can provide their goats with drinking water, only 5.71% who have drinking troughs. the living area per goat is less than (1.5 m² / goat) for 70% of goats’ farms and less than (1 m² / goat) for 57% of the goats’ farms surveyed. the farmers surveyed were mostly illiterate (74.29%) with an age between 27 and 77 years old. the average herd size is 82 animals per farmer and 80% of the farms have between 10 and 100 animals. animal vaccination is only provided for 25.71% of goat farms and 74.29% of them do not have access to health care. all goat herds are composed of 72.54% females and 27.46% males. out of a total of 3190 goats observed, 459 are male goats, 1493 female goats, 417 male kids and 821 female kids. the goat herd is heterogeneous and composed mainly (86.4%) of 3 local breeds (alas, barcha and ghazalia) which represent respectively 57.4%, 22.3% and 6.7%. the number of kiddings is 1 for 71.4% of farmers, 1.5 for 28.6% of them. 8.6% of farmers do not know the age at first birth and 82.8% of them, this age is less than or equal to 12 months. the prolificacy rate is 114.3% and the abortion rate is 28.4%.the mortality rate for adults was 17.92% and that for the kids a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 501 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) was 12.76%.the main barriers to goat production in the region are food, housing, health and lack of subsidies. keywords: livestock farming system, argan tree park (arganeraie), reproductive performance. p078. preliminary standards for apple fertilization in the middle atlas mountains of morocco rachid razouk*, abdellah kajji, jamal charafi, mohamed alghoum, and el houssain bouichou regional agricultural research center of meknes, b.p 578, meknes, morocco. *corresponding and presenting author: razouk01@yahoo.fr. abstract until now, interpretation of soil and leaf analyses for the moroccan apple orchards is based on reference norms established on other countries such usa, france, netherlands, united kingdom and south africa. establishment of norms under local conditions is therefore necessary for an efficient management of fertilization in moroccan orchards taking into account local practices and production performances. for this objective, a work was carried out to evaluate use efficiency of nitrogen, phosphorus, potassium, calcium and magnesium in adult apple orchards in the middle atlas of morocco (imouzzer kandar) and to establish preliminary reference norms for fertilizing this crop under local conditions. the study was based on soil and leaf analysis and data regarding farming practices and yield level on forty apple orchards (cv. golden delecious/mm106) where nineteen are growing on silty-clay soil and twenty-one on sandyloam soil. the results showed significant correlations between leaf content for each nutrient and yield level following polynomial equations, thereby indicating local reference norms for apple leaf analysis: 2.803.10% for nitrogen, 0.20-0.22% for phosphorus, 1.90-2.10% for potassium, 1.30-1.45% for calcium and 0.25-0.28% for magnesium. the correlations between leaf and soil contents were significant, thereby indicating apple needs in nitrogen fertilizer: 88 kg/ ha on silty-clay soil and 130 kg/ha on sandy-loam soil. moreover, were determined references norms for soil richness in phosphorus (92-126 ppm on silty-clay soil and 82-106 on sandy-loam soil), potassium (350415 ppm on silty-clay soil and 320-410 ppm on sandy-loam soil), calcium and magnesium for an apple yield objective of 26 t/ha corresponding to the average yield obtained in the study area. the found norms for leaf analysis are less than international standards because of feebleness of yield level in the study region, largely related to deficient cultural practices adopted by farmers. in addition, investigation of leaf nutrients ratios n/k, n/ca, k/ca, k/mg and ca/ mg showed that there was a disharmony in uptake of these nutrients originating particularly from high soil richness in ca and mg. taking into account these considerations, the found references norms can be applied only under the adopted farming practices. nevertheless, by improving local practices, reference values may change. keywords: morocco, apple tree, soil analysis, leaf analysis, fertilization standards. p079. effect of potassium source and rate on productivity and quality of strawberry fruit amal nakro* and ahmed bamouh§ department of plant production, protection and biotechnology, hassan ii institute of agronomy and veterinary sciences, bp 6446 madinate al irfane, rabat, morocco. *presenting author: a.nakro@iav.ac.ma. §corresponding author: a.bamouh@iav.ac.ma. absract the purpose of this research is to investigate the effect of potassium fertilization source and rate on productivity and quality parameters of strawberry. the experiment was conducted in 2016-2017 at the experimental greenhouse of hassan ii institute of agronomy and veterinary sciences in rabat. strawberry plants of the cultivar ‘san andreas’ were planted on november 6th in 12-liter pots equipped with a gravity fertigation system. tested treatments were two potassium sources: (1) sulphate of potash for all the growing cycle (sop/sop) (2) nitrate of potash during the vegetative period and sulphate of potash during the fruit production period (nop/sop) combined with four potassium rates of 100, 200, 300 and 400 kg/ha of k2o. the experimental design is a split-plot of eight treatments with three replications. the potassium sulfate source was superior to potassium nitrate on the improvement of productivity and quality parameters of strawberry fruit. the potassium a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 502 sulphate improved the number of flowers by 16%, the fruit production by 21 %, the titrable acidity content by 0.5 g/l and the sugar content by 3%. the highest yield was recorded in plants treated with 300 kg/ ha of k2o of potassium sulphate. the rate of 300 kg/ha of k2o gave also superior results regarding quality parameters of strawberry fruit. this optimum potassium rate enhanced the number of flowers by 35%, the fruits produced by 44%, the sugar content and the titrable acidity content by 0.9 g/l. keywords: strawberry, fertilization, potassium, source, rate, potassium sulphate, potassium nitrate, productivity, quality, sugar content, titrabale acidity. p080. intensive urbanization at the expense of farmland el hirch kaoutar1,2,3*, ait yacine z.2, idrissi n.3, and wahid n.1§ 1 équipe d’écologie et développement durable (eedd), département de sciences de la vie, faculté des sciences et techniques, université sultan moulay slimane, béni-mellal, morocco; 2 équipe de génie de l’environnement, département de sciences de la vie, faculté des sciences et techniques, université sultan moulay slimane, béni-mellal, morocco; 3 équipe de traitement de l‘information et aide à la décision, département de l’informatique, faculté des sciences et techniques, université sultan moulay slimane, béni-mellal, morocco. *presenting author: kaoutarelhirch.cha2@gmail.com. §correspondig author: wahid2na@hotmail.com. abstract currently, the modern city has lost these properties and these landscape reflexes related to the phenomenon of urbanization. in fact, urban sprawl alters land use and the appearance of the countryside, which contributes to the accelerated evolution of habitats with regard to areas devoted to green spaces. despite the laws for the preservation of agricultural land, urban sprawl is intensifying to the detriment of land with high agronomic potential, increasingly in the face of economic growth. this is worsening more and more in the absence of a sustainable urbanization strategy. several studies have confirmed that unsustainable urban intensification has environmental consequences; risks on agricultural production, the disruption of agricultural ecosystems, the landscape quality of the city, and the quality of the climate. the current challenge is to define indices of the degree of regression of agricultural land or agricultural biodiversity, and the cost of loss generated by urban intensification. in this context, our work is to trace the evolution of urbanization in the city of beni-mellal, in order to evaluate its impact on the structuring of agricultural biodiversity and on the phenomenon of global warming. the evaluation and anticipation of its impacts are of interest to both scientists and territorial managers. keywords: urban intensification, agricultural biodiversity, global warming, béni mellal. p081. ability of non-pathogenic fusarium oxysporum strain fo47 to suppress rhizomania disease of sugar beets in morocco fatima nouayti1,2*, ilham madani2, abdessalem tahiri1, abdelali blenzar2, and rachid lahlali1 1 phytopathology unit, department of plant protection, ecole nationale d’agriculture de meknès (enameknès), km10, route haj kaddour, bp s/40, meknès 50001, morocco; 2 moulay ismail university, faculty of sciences. department of biology po box 11201 zitoune meknes, morocco. *corresponding and presenting author: fatimanouayti@gmail.com. abstract rhizomania is one of the most devastating diseases of sugar beet worldwide. the disease poses a serious threat to moroccan production and it is capable of significantly decreasing quality and yield of sugar beet plantations. the long-term survival of its fungal vector (polymyxa betae) in soil makes it a very difficult disease to manage. therefore, this study investigated the potential of a non-pathogenic fungal fusarium oxysporum strain fo47 to control polymyxa betae. this biocontrol agent was applied as soil treatment, seed treatment, or a combination of the both treatments. a bio-test was performed on treated soil. after four weeks of culture, the roots of sugar beet seedlings were retrieved and analyzed by the das-elisa test. results indicated that f. oxysporium fo47 reduced the activity and survival of p. betae when compared to a reference biocontrol agent trichoderma harzianum, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 503 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) which only revealed significant in reducing the viral load of beet necrotic yellow vein virus (bnyvv) as seed treatment. the non-pathogenic fusarium oxysporum fo47 was more effective as soil treatment and allowed almost the same reduction of bnyvv virus concentration as t. harzianum 908. therefore, our findings emphasizes that the performance of the biocontrol agent depends on the method of application. keywords: beet necrotic yellow vein virus; biocontrol, fusarium oxysporum strain fo47; trichoderma harzianum strain 908. p082. preliminary study of the effect of humic substances on saffron growth and corms production abdelghani tahiri*, youssef karra, meriyem koufan, naima ait aabd, and abdelaziz mimouni national institute of agronomic research, crra, agadir, av. des far, bp. 124, inezgane, morocco. *corresponding and presenting author: aghanitahiri@ yahoo.fr. abstract among the 85-species belonging to the genus crocus, saffron (crocus sativus l.) is a monocotyledonous plant that belongs to the iridaceae family. it has been cultivated for its stigmas that provide the most expensive spice which has been valuable since ancient times for its odoriferous, coloring, and medicinal properties. saffron, is an autumn-flowering, triploid male -sterile plant propagated vegetatively by means of corms as the flowers are sterile and fail to produce viable seeds. low multiplication rates and fungal infestation of corms reduce the productivity and quality, thereby restraining the availability of planting material. to overcome these problems, in this study an experiment was conducted to evaluate the potential effect of commercial humic substances (as biostimulant) on saffron growth, quality and corms production. keywords: saffron, corm production, crocus sativus, humic substances. p083. study of the antioxidant compounds of artemisia mesatlantica maire sanae amine1,2* , fatima zahra radi1,2, zakaria khiya1,2, smail amalich1,2, malika mahjoubi1,2, mohamed bourakhouadar1,2, and touriya zair1,2 1 research team of chemistry bioactive molecules and the environment, university moulay ismail, faculty of sciences, bp 11201, zitoune, meknes, morocco; 2 laboratory of materials chemistry and biotechnology of natural products, university moulay ismail faculty of sciences, b.p. 11201 zitoune, meknès, morocco. *corresponding and presenting author: aminasanae@hotmail.fr. abstract artemisia mesatlantica maire is an endemic species of morocco, belonging to the asteraceae family. located in the moroccan atlas region, it is very requested by the local population for its therapeutic properties. however, because of the limited studies conducted on this species, and in order to contribute inits valorization, we selected it in full bloom in the middle atlasregion for a phytochemical study and an evaluation of its antioxidant properties. the phytochemical screening of a. mesatlantica maire revealed its richness in polyphenols, sterols, triterpenes and mucilage. the extraction yield of the total polyphenols by macerationin a methanol-water mixture (80/20) was 10.10%. the determination of the total phenols by the folin-ciocalteu method, after fractionation of the polyphenolic extract ofa. mesatlantica maire, showed the abundance of these compounds in the butanol fraction. for the total flavonoids essay, using aluminum trichloride, the ethyl acetate and butanol fractions of a. mesatlantica maire were the richest. in addition, the raw extract was particularly rich in condensed tannins. the valuation of the antioxidant activity by reduction of iron (frap) and trapping of the free radical dpph* showed that all the extracts have a moderate antioxidant power. these first studies on a. mesatlantica maire of the middle atlas revealedits abundance in important chemical compounds, and due to the ability of this plant to trap radicals, it can be considered as a source of natural antioxidants. keywords: artemisia mesatlantica maire, antioxidant activity, polyphenols, flavonoids, tannins. p084. use of antibiotics for managing bacterial contamination and resistance in date palm tissue-culture media houria ben-amar*, reda meziani, and adil essarioui a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 504 national institute for agricultural research, regional center of errachidia, morocco. *corresponding and presenting author: houriabenamar@hotmail.fr. abstract date palm industry in morocco relies on tissueculture techniques for large scale production of plant material. unfortunately, bacterial contaminations of juvenile tissues constrain the improvement of palm micropropagation rate. antibiotics have been classically used to manage undesired bacterial growth in plant culture media. this can lead to resistance accumulation within contaminant populations if antibiotics are not properly used. in order to develop methods for managing bacterial contaminations and antibiotic resistance during date palm in vitro multiplication, we studied 9 clinical antibiotics for their inhibitory effect against 2 bacterial strains frequently associated with date palm juvenile tissues. antibiotics differed in their effect on the two bacterial isolates. one strain was sensitive to all antibiotics with an inhibition intensity ranging from 6% to 59%. however, the second isolate was completely resistant to 3 antibiotics and weakly sensitive to the others, with an intensity of inhibition smaller than 12%. this study highlights the importance of alternate use of multiple antibiotics for controlling bacterial growth and managing antibiotic resistance in date palm tissue-culture media. keywords: date palm, tissue-culture, bacterial contamination, antibiotics. p085. physiological analyses of limited transpiration rate under high evaporative demand during durum wheat domestication rachdad fatima ezzahra1,2*, danguy des deserts alice1,3, el bouhmadi keltoum2, ghanem michel edmond1§ 1 crop physiology lab, international center for agricultural research in the dry areas (icarda), rabat, morocco; 2 departement of biology, faculty of sciences ben m’sik, university hassan ii, casablanca, morocco; 3 montpellier supagro, france. *presenting author: rachdad.fz@gmail.com. §corresponding author: m.ghanem@cgiar.org. abstract to satisfy increasing wheat demands, scaling up wheat production will require boosting yield in suboptimal, drought-prone areas. this increase will be particularly challenging since in several major agricultural regions of the world, yield stagnation has been observed during last decades. in addition to soil drought, drought-prone environments tend to exhibit high levels of atmospheric vapor pressure deficit (vpd), which is an additional driver of crop water loss via transpiration (tr). recently, it was hypothesized that “water-saving” cultivars displaying tr decreases in response to high vpd, or in response to the soil drying, should lead to enhanced drought tolerance under terminal drought conditions by increasing the availability of stored soil moisture during the critical grain filling phase (sinclair, 2017). it is likely that during breeding, breeders favored high-yielding but more water-consuming genotypes, and disfavored / excluded low-yielding but more conservative species. for this purpose, we have examined the evolution of tr traits in a domestication series of durum wheat. tr response to an artificial increase in vapor pressure deficit (vpd) of 3 species was evaluated: triticum turgidum l. subsp. dicoccoides, triticum turgidum subsp. dicoccon, and modern elite lines of durum wheat (triticum durum desf.). our findings reveal a large diversity of responses especially in wild and primitive wheat. such considerations will be particularly useful to breeding and introgression of useful traits from crop wild relatives. keywords: domestication, drought, vpd, transpiration, water conservation traits. p086. diagnosis of clinical cases of infectious bursal disease using a developed rapid taq man-mgb real-time rt-pcr assay maryame cheggag1*, khalil zro2, ghizlane sebbar2, mohamed mouahid3, mohammed el houadfi1, and faouzi kichou1 1 institute of agronomy and veterinary medicine hassan ii, b.p. 6202, madinat al irfane, rabat, morocco; 2 biopharma, rabat, morocco; 3 veterinary clinic temara, morocco. *corresponding and presenting author: cheggagmaryame@gmail.com. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 505 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) abstract infectious bursal disease (ibd) is an important contagious viral infection of immune system of poultry. despite various types of commercially available vaccines against ibdv, this infection poses a permanent threat to the profitability of poultry industry worldwide. the aim of this work was to modify and to develop taqman-mgb rrt-pcr in one-step involving two fluorogenic taqman labeled probe for the detection of infectious bursal disease virus (ibdv) collected from suspected cases distributed in different regions of the country during the period 2013-2016. the method developed was validated for specificity, linearity, repeatability, sensitivity, reproducibility. it allowed reducing the test running time by 6 folds. this method was applied on 102 pools of bursa of fabricius (bf) samples collected from affected broiler farms suspected to be infected by ibdv. birds showing macroscopic lesions including muscle petechial hemorrhages, hypertrophy and hemorrhage of bf, were subjected to molecular analysis using modified protocol “taqman-mgb real-time rt-pcr”. the validation satisfied all criteria and the assay developed could be a useful tool fora very rapid diagnosis of ibdv and permit to detect and to discriminate in one-step very virulent (vv) from non-vv (classic and variant) ibdv strains in poultry. out of 84 ibdv positive samples, a prevalence of 33 % for hyper-virulent strains and 50 % for classical strains was noted. these results indicate that despite the vaccination hyper-virulent form of ibdv continues to cause serious problems for moroccan broiler chickens. keywords: ibdv, one-step rrt-pcr, taqman-mgb, macroscopic lesions. p087. synergistic effects between exogenous salicylic acid and arbuscular mycorrhizal fungi in date palm (phoenix dactylifera l.) seedlings under drought stress benhiba laila*, abdellatif essahibi, and ahmed qaddoury unit of biotechnology and symbiosis agro-physiology, faculty of sciences and techniques, cadi ayyad university, marrakesh, morocco. *corresponding and presenting author: lbenhiba.doc@gmail.com. abstract in morocco, country traditionally producer and consumer of date, the phoenicicole sector is suffering from serious biotic and abiotic constraints such as drought, salinity and bayoud (vascular fusariosis). however, the arbuscular mycorrhizal (am) symbiosis has been shown to improve plant growth, mineral uptake ability and tolerance to drought stress. it also induces resistance against soil pathogens. in addition, salicylic acid (sa) or orthohydroxy benzomethylic acid is a phenolic compound which influences various physiological processes and biochemical interactions in plants. indeed, this molecule has a regulatory role in a range of diverse physiological processes in plants, including photosynthesis, ion uptake and transport, chlorophyll synthesis, seed germination, membrane permeability and plant growth (aftab et al., 2010). salicylic acid influences also plant tolerance to drought (azooz and youssef, 2010), salinity (gunes et al., 2007) and heavy metal stress (moussa and el-gamal, 2010). the aim of this study was to determine the combined effects of mycorrhizal symbiosis and salicylic acid application (1mm) on the growth and development of date palm (phoenix dactylifera l.) seedlings in greenhouse conditions under different drought stress (ds) levels. results showed that both amf-inoculation and salicylic-acid can increase date palm tolerance to drought. the combination of the two factors further promoted drought tolerance, indicated by improving plants morphological parameters (leaf and root length, surface area, shoot and root fresh and dry weights) compared to control plants and increasing proline and total soluble sugar accumulation in the leaves of stressed plants. keywords: date palm (phoenix dactylifera l.), arbuscular mycorrhizal fungi, symbiosis, drought stress, exogenous salicylic acid (sa). p088. morphological and molecular characterization of fusarium spp. associated with dry root rot disease of citrus in morocco said ezrari1,2*, rachid lahlali1*, abdessalem tahiri1, adil asfeers3, abdelhadi ait houssa3, and abderrahim lazraq2 1 department of plant protection, phytopathology unit, ecole nationale d’agriculture de meknès, bps a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 506 40, meknès, morocco; 2 laboratory of functional ecology and environment, sidi mohamed ben abdellah university, po. box 2202, route d’imouzzer, fez, morocco; 3 louata field -providence verte group: po box 4 sefrou region, morocco.*corresponding and presenting author: rlahlali@enameknes.ac.ma. abstract the dry root rot (drr) is one of a devastating disease of citrus plantations with higher incidence under biotic or abiotic stresses. it might cause significant damage to younger citrus plantations depending on the rootstock. recently, this disease emerged as serious threat to moroccan citrus plantations; the wilting of young citrus trees was observed in different orchards with typical symptoms of vascular necrosis and root rot. furthermore, the diseased rootstocks displayed also a brown coloration in the stem. this disease is reported to be caused by a complex of different species of fusarium, but the fusarium solani was emphasized to be predominant. despite the occurrence of this disease in the past, there was a lack of informations about the identity of the fungal pathogens involved, the favorable conditions for their development as well as appropriate strategies for their control. therefore, this study aimed at identifying and phenotyping the fungal pathogens associated with this disease from different diseased plants and soil collected from citrus orchards and nurseries. the preliminary results underlined that the fungal pathogen responsible of dry root rot of citrus belongs to the genus of fusarium spp. based on morphological traits and molecular characterization. as a result, a depth study of morphological and molecular characterization of fusarium isolates is further considered in more details. keywords: citrus, dry root rot, fusarium spp., morphological characters, molecular tests. p089. evaluation of some new introduced hybrids of sunflower (helianthus annuus l.) under drought conditions in morocco rajae kettani1*, abdelghani nabloussi2, lahcen hssaini2, and chems doha khalfi1 1 research unit of agronomy and plant physiology, national institute of agricultural research (inra), regional center of meknes, po. box 578, meknes, morocco; 2 research unit of plant breeding and plant genetic resources conservation, national institute of agricultural research (inra), regional center of meknes, po. box 578, meknes, morocco. *corresponding and presenting author: r_kettani@yahoo.fr. abstract drought was always present in morocco’s history. its importance as a structural element of the country’s climate increased during the last decades, with a net reduction in the precipitation and an increasing temperature trend, as a result of climate change. the present study was carried out in meknes area (morocco) in order to characterize and evaluate new sunflower hybrids for their agronomic performance and tolerance to water stress often occurring at bud and flowering stage. seven introduced hybrids were conducted in ain jemaa under rainfed conditions during 2016-2017. agronomic parameters, namely plant height, head diameter, number of seeds/head, 1000-seed weight, seeds weight/head, and seed yield/ha, as well as physiological traits, like as chlorophyll level, stomatal conductance and relative water content, were measured. seed yield was shown to be significantly correlated with number of seeds per head, head diameter and plant height. the highest seed yield, 2.84 t/ha, was observed for the variety ‘laila’, while the lowest yield is recorded for the variety ‘merida’. in ‘laila’ hybrid, during flowering stage, there was a significant and strong positive correlation between stomatal conductance and seed yield. as flowering process was happening under drought conditions, it could be suggested that that hybrid was able to maintain open stomata and thus to yield better than the other studied hybrids. keywords: sunflower, hybrid, drought, seed yield, stomatal conductance. p090. effect of commercial organic fertilizers on soil fertility, nitrogen use efficiency and yield of broccoli grown under irrigated arid conditions in south of morocco kaoutar aouass1*, lahcen kenny1, and mariam bouchfar2 1 department of horticulture, complex of horticulture, agronomic and veterinary medicine hassan ii institute, rabat, morcco; 2 department of biology, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 507 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) university ibn zohr, agadir, morcco. *corresponding and presenting author: aouass.kaoutar@gmail.com. abstract a field research was conducted in order to determine the effect of three different types of commercial organic fertilizers (cof) as compared to compost on soil fertility, nitrogen use efficiency, growth and yield of broccoli in the souss massa area in south of morocco. broccoli seedling were raised under greenhouse and transplanted to sandy-loamy soils in open field prior to adding 200 kg/ha of n, 80 kg/ha of p and 180 kg/ha of k either as compost (2.5, 3, 2.5), a ternary cof ( 7-4-7), a mixture of one binary cof (8-12-0) and one simple cof (0-0-30) or a mixture of three simple cof (10-0-0, 0-18-0 and 0-0-30). the highest yields (30 t/ha) were obtained with compost and the ternary cof (28 t/ha). as for soil fertility parameters, a clear effect of organic inputs was registered on soil organic matter, electric conductivity and ph. the highest coefficient of nitrogen use efficiency (nue) was obtained with the ternary cof (64%) and the lowest (25%) with the simple cof. based on these findings, recommendations on how to build an organic fertilization program for leafy vegetables are discussed. keywords: organic fertilizer, compost, broccoli, nitrogen use efficiency, arid conditions. p091. variations in the proline, glucose and protein contents in origanum majorana l. grown saline conditions hamza elhaddaji1*, moustapha akodad1, abdelmajid moumen1, ali skalli1, and mourad baghour1 1 observatoire de la lagune de marchica de nador et région limitrophes (labo. olman-rl), faculté pluridisciplinaire de nador, université mohamed premier, bp 300, 62700, selouane, nador, morocco. *corresponding and presenting author: hamza.elhaddaji@gmail.com. abstract in recent years, low rainfall in north-eastern morocco has led to low water levels of water stored in dams. this leads the farmers to look for other alternatives to develop their agriculture, either by looking for other sources of water supply such as pumping groundwater, which is characterized in most cases by poor quality due to high salinity levels, or the introduction of other crops more resistant to salinity and with less water requirements. the objective of this study is to determine the effect of salt stress on the protein, proline and glucose contents in marjoram (origanum majorana l). we started our study by applying different concentrations of nacl (0, 50, 100 and 150 mm) for 44 days on plants grown in greenhouse at the multidisciplinary faculty of nador – fpn. different physiological and biochemical parameters were analyzed to evaluate the salinity tolerance of marjoram plants. our results showed that proteins, glucose and proline contents were affected positively by different concentrations of nacl, reaching high value of these parameters with high nacl level (150 mm). keywords: salt stress, protein, proline, glucose, marjoram. p092. improving tomato fruit production and quality by co-expressing lenhx2 and slsos2 mostapha maach1*, maría pilar rodriguez rosales2, kees venema2, fancisco javier gálvez2, maría elena sánchez romero2, mustapha akodad1, adelmajid moumen1, and mourad baghour1 1 observatoire de la lagune de marchica de nador et région limitrophes (olman-rl), faculté pluridisciplinaire de nador, université mohamed premier, bp 300, séloune 62700, nador, morocco ; 2 departamento de bioquímica, biología celular y molecular de plantas. estación experimental del zaidín, csic, calle profesor albareda, 1, 18008 granada, spain. *corresponding and presenting author: mostaphamaach12@gmail.com. abstract salinity is a major environmental factor limiting crop productivity and quality especially in arid and semiarid regions. the presence of salt in the medium impose impose both an ionic and an osmotic stress to most plants and negatively affect plant growth by reducing the uptake of essential ions, such as potassium. the transporters responsible for k+/na+ homeostasis as well as the proteins involved in their regulation are the main mechanisms of salt tolerance. the salt tolerance locus sos1 from arabidopsis has been shown to encode a plasma membrane na+/h+ antiporter. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 508 sos1 activity is regulated by a complex composed of the sos2 kinase and the sos3 ca2+ binding protein. on the other hand, nhx genes in plants encode intracellular cation/h+ antiporters regulating na+, k+ and h+ levels in cytoplasm and intracellular compartments, providing osmotic and ionic protection. in this study, transgenic tomato plants (cv. microtom) expressing both the endosomal antiporter lenhx2 and the protein kinase slsos2 were produced. our results suggest that co-expression of lenhx and slsos2 genes significantly improved growth, yield, fruit quality and enhanced salt tolerance of tomato plants growing under saline conditions. keywords: lenhx2, slsos2, co-expression, salt stress, tomato, yield p093. micropropagation of date palm (phoenix dactylifera l.) cv. mejhoul: morphological and physiological characteristics of regenerant plantlets respond to culture medium texture and composition reda meziani1*, mouaad amine mazri2, essarioui adil1, and fatima jaiti3 1 national institute for agricultural research, regional center of errachidia, morocco; 2 national institute for agricultural research, regional center of marrakechmorocco; 3 university moulay ismail, faculté des sciences et techniques d’ errachidia-morocco. *corresponding and presenting author: redameziani@ yahoo.fr. abstract date palm (phoenix dactylifera l.) has long been a major fruit crop in southern morocco. in these areas, date production is an important source of food and income for local populations and plays a key economic role by creating suitable microclimate for growing other crops. unfortunately, over the last century, morocco has lost about 12 million palm trees mainly because of bayoud, a severe wilt disease caused by the fungal pathogen fusarium oxysporum f. sp. albedinis. a few years ago, a strategic program was launched to restore disease-devastated areas and create new modern plantations outside traditional oases. the expected total number of palms to plant by 2020 is 3 million trees. the use of in vitro multiplication techniques is the best way to achieve this goal. among the techniques used for date palm micropropagation, organogenesis allows for the production of true-totype plantlets. however, the efficiency of this technique depends widely on the genotype and medium composition. the effects of various culture conditions on shoot elongation, rooting, plantletphysiological characteristics, and acclimatization were studied. adventitious shoots obtained through direct organogenesis of date palm cv. mejhoul were used as explants. the effects of medium texture (liquid or semi-solid), polyvinylpyrrolidone (pvp), adenine, myo-inositol, and l-glutamine on plantlet quality and acclimatization were evaluated. the most effective culture medium was semi-solid half-strength murashige and skoog medium (ms/2). in this medium, average shoot length reached 13.15 cm, the mean number of roots per shoot was 3.3, average root length extended to 3.15 cm, and acclimatization success rate attained 85 %. liquid medium, pvp, adenine, myo-inositol and lglutamine did not improve survival rate during acclimatization. some physiological characteristics of the plantlets were also determined. chlorophyll content and fluorescence, foliar surface and stomatal conductance were measured after 3 months of culturing in each medium. the ranges were as follows: chlorophyll content, 12.3-31.8 cci; chlorophyll fluorescence, 0.633-0.795; foliar surface, 7.35-13.03 cm2; and stomatal conductance, 10.5-34.6 mmol/m²/s. there was a positive correlation between physiological characteristics and acclimatization success rate. these findings provide valuable information for costeffective large-scale production of date palm cv. mejhoul plants. keywords: date palm, micropropagation, organogenesis, morphological characteristics, physiological characteristics. p094. efficiency of hedysarum flexuosum l. under stress environments anass el yemlahi*, abdelhay arakrak, amin laglaoui1, and mohammed bakkali equipe de recherche en biotechnologies et génie des biomolécules (erbgb), faculté des sciences et techniques de tanger, morocco. *corresponding and presenting author: elyemlahi@hotmail.fr abstract the success of such symbiosis between legume and a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 509 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) rhizobium may require the selection of both partners that have the ability to survive at various environmental conditions. in order to study the rhizobium-sulla efficiency under environmental constraints, we investigated the diversity of 36 isolates nodulating four hedysarum flexuosum l. known as sulla harvested from different environmental habitats. the isolates were tested for their tolerances to extremes of temperature, ph, salt, water deficit low, resistance to heavy metals, antibiotics, in addition to their ability to use different source of carbon and nitrogen. correspondingly, edaphic and climatic conditions at each harvested site were analyzed. a different pattern was observed between sampled sulla, estimated on the basis of plant growth, osmolytes accumulation, nodulation and subsequent nitrogen fixation depending on the most prevailing factors of their natural habitat and the degree of tolerance of their associated symbionts to stress-testing conditions. results suggest that such rhizobiums could operate interdependently and/ or synergistically to detoxify the stress molecules and alleviate therefore the effects of environments stress factors. key words: sulla, rhizobium, biological nitrogen fixation and environments stress factors. p095. optimization of the extraction technique for the determination of bioactive compounds in two aromatic and medicinal wild plants: matricaria chamomilla l. (wild chamomile) and cladanthus mixtus l. oberpr. and vogt. (moroccan chamomile) amina el mihyaoui1,2*, maria-emilia candela castillo2, maria-emilia requena candela2, and ahmed lamarti1 1 laboratory of plant biotechnology, biology department, faculty of sciences, abdelmalek essaadi university, tetouan, morocco; 2 phytopathology laboratory, department of plant biology, faculty of biology, university of murcia, campus de espinardo, 30100 espinardo, murcia, spain. *corresponding and presenting author: elmihyaoui.amina@gmail.com. abstract medicinal plant extracts are widely used in pharmaceutical and cosmetic products. matricaria chamomilla l, (german chamomile, matricaria recutita l., matricaria suaveolens l. chamomile) is a widely recognized medicinal plant from the asteraceae family, it is native to southern and eastern europe and cultivated also in countries of america and asia, also, it is one of the most popular medicinal plants commonly used in herbal tea, for food purposes and in folk medicine. the dried flowers of chamomile contain many terpenoids and flavonoids contributing to its medicinal properties. wild chamomile. cladanthus mixtus is an endemic plant of morocco, also known commercially chamomile of morocco, is a biennial chamenyphous asteraceae with many upright stems. in morocco, it is encountered in two different zones, the first between tangier, ouezzane, souk larbaa, moulay bousselham and azilah, and the second between kenitra, sidi slimane, khemisset and rabat. the extracts produced with the different solvents were determined using hplc/dad and hplc/ms. in the present study, we have determined the extraction solvent medium to obtain a well-illustrated chromatogram for the purpose of identifying bioactive compounds in the flowers of two medicinal plants: matricaria chamomilla and cladanthus mixtus. keywords: matricaria chamomilla, cladanthus mixtus, hplc/dad, hplc/ms. p096. potential of biocontrol in vivo and in vitro of bacillus amyloliquefaciens and trichoderma harzianum against strawberry anthracnose caused by colletotrichum acutatum rababe es-soufi*, mohammed l’bachir el kbiach, and ahmed lamarti plant biotechnology team, department of biology, faculty of sciences, abdelmalek essaâdi university, morocco. *corresponding and presenting author: rababeessoufi@gmail.com. abstract anthracnose is one of the most widely known strawberry diseases; it can be destructive to strawberry plants, it is caused by colletotrichum species and mainly by colletotrichum acutatum. the pathogen produced lesions on leaves, fruit and other parts of plant. the control of fungal diseases attacking strawberry plants is mainly done by treatment of the soil and the plants using chemical pesticides. but still a lot of work needs to be done before any environmentally friendly and consistent control strategies come a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 510 into existence. the use of the antagonists was done with a lot of procedures starting by with isolation and finishing by selecting strains highly effective for the biocontrol. relatively little is known about the exact mechanisms used by bacillus amyloliquefaciens in its behavior as a biocontrol agent on plants. here, the suspensions of bacillus amyloliquefaciens and trichoderma harzianum at concentration of 3.105ufc/ml and 104spores/ml respectively (previously selected as the minimal inhibitory concentrations of colletotrichum acutatum development) were tested in vitro and in vivo against colletotrichum acutatum. in vitro study: the wounded strawberry’s leaves were sprayed with the antagonist suspension and inoculated one hour later by colletotrichum acutatum, then held in moist chambers at 25°c±2. the leaves did not become infected with c. acutatum. in vivo study: the strawberry plants were sprayed with the mixture of the antagonist and pathogen suspensions, then held in moist chambers at 25°c±2. the plants didn’t get anthracnose symptoms. the concentrations studied were effective to control the strawberry anthracnose in vivo and in vitro, next we will start the study in field for this application. keywords: biocontrol, antagonism, anthracnose, bacillus amyloliquefaciens, trichoderma harzianum. p097. germination of somatic embryos derived from moroccan quercus suber l. trees safaâ rahmouni*, mohamed l’bachir el kbiach, and ahmed lamarti laboratory of plant biotechnology, department of biology, faculty of sciences, abdelmalek essaadi university, m’hannech ii, b.p. 2121, tetouan, morocco. *corresponding and presenting author: safaarahmouni@hotmail.fr. abstract cork oak (quercus suber l., fagaceae) is one of the most important species of the mediterranean basin due to its ecological and socio-economical interests. nevertheless, the increasing demand for cork and the low natural regeneration provides lost of cork oak forest. vegetative propagation by in vitro methods (techniques of micropropagation) presents many advantages compared to traditional methods. somatic embryogenesis is considered as the best method of micropropagation that can solve problems of yield and intraclonal variability. it had several successes in forest species allowing the production of trees from somatic embryos. the goal of our study is the regeneration of seedlings from moroccan cork oak somatic embryos of three lines (ain rami 1, ain rami 2 and bouhachem). to this end, we were interested in studying the effect of growth regulators, embryos size and the period of cold storage on the germination of cork oak somatic embryos. we noticed the presence of different types of shoots (shoots of normal size with or without roots, shoots of small size with or without roots and abnormal shoots). the frequency of germination, the mean number of shoots per embryos, the mean length of shoots, the mean number of leaves per shoot and the mean number of roots per shoot depend on the genotype and the plant growth regulator used. germination of large somatic embryos (>12 mm and with large cotyledons) from the three embryogenic lines was successful. stratification at 4°c for 2 months was the most efficient for stimulating somatic embryos germination of quercus suber l. keywords: cork oak, quercus suber l., fagaceae, secondary somatic embryogenesis, germination. p098. effect of 6-benzylaminopurine combined with different auxins on vegetative micropagation of origanum compactum l. rajae benkaddour* and ahmed lamarti laboratory of plant biotechnology, department of biology, faculty of sciences, abdelmalek essaadi university, m’hannech ii, b.p.2121, tetouan, morocco. *corresponding and presenting author: benkaddourrajae27@gmail.com. abstract morocco is well known by botanical diversity of medicinal and aromatic plants that show a socioeconomical interest. indeed, the medicinal and aromatic plants have constituted recently an important choice in various utilizations such as cosmetic, food and therapeutic fields. however, the anarchic collect of some endemic plants could constitute a main reason for their disparition; otherwise the research of alternatives ways by in vitro culture of these species is a promising strata tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 511 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) egy in biotechnology. in this context, our work rely on (i) improvement, valorization, conservation and characterization of medicinal and aromatic plants, already propagated in the north of morocco, (ii) production development by in vitro culture of four selected species belong to origanum genus (origanum majorana l., origanum vulgare l. origanum elongatum l.origanum compactum l.) in order to answer the farmers needs and the introduction of an alternative culture of oregano because of a high value added. a part of my research work aimed to obtain in vitro plants of origanum compactum l. with a good quality and to achieve its acclimatization by realizing different tests. first of all, we determined the most suitable mineral medium, afterward, realized the test of cytokinins to know the one that gives the best results in terms of budding, growth, hyperhydricity, regeneration and rooting. the best one was the 6-benzylaminopurine but the length of the root wasn’t good enough to succeed the acclimatization. to obtain the explants of origanum compactum l. having an important length of roots and then to success its acclimatization; we have tested four auxins (indole-3-acetic acid, naphtalene acetic acid, indole butyric acid, 2,4-dichlorophenoxy acetic acid.) each of them have been added to the medium at four concentrations (0,2mg/l-0,5mg/l-0,8mg/l and 1mg/l) in the presence of 0,5mg/l of 6-benzylaminopurine. our results show that the rooting and the acclimatization were the best when the combination between 1mg/l of indole3-acetic acid and 0,5mg/l of 6-benzylaminopurine was applied. our current work is about the study of other growth factors on the budding, growth, hyperhydricity, regeneration, rooting and acclimatization of origanum compactum l. keywords: aromatic and medicinal plants, origanum,phytohormones, auxins. p099. identification of the cœlomic microbial community of lumbricus terrestris (annelida: lumbricidae) lamia yakkou*, maryam chelkha, mohammed raouane, souad amghar, and abdellatif el harti. lumbricidae, improving soil productivity and environment (lapse), centre « eau, ressources naturelles, environnement et développement durable (cern2d) » mohammed v university in rabat, ecole normale supérieure (ens). avenue med belhassan el ouazani. bp 5118, takaddoum-rabat, morocco. *corresponding and presenting author: yakkou.lam@gmail.com abstract earthworms play an important role in soil fertilization. these annelids interact with soil microorganisms to promote decomposition, mineralization and recycling of the organic material. several studies have focused on the study of microorganisms associated with the earthworm’s digestive tract. in the present work, however, we were interested in the microorganisms possibly associated with the worm at the level of the coelomic liquid. in fact, microscopic examination of coelomic fluid smears taken by puncture of the coelomic cavity of lumbricus terrestris revealed the presence of microorganisms of various shapes and sizes. isolation and culturing revealed the presence of three bacterial strains. phenotypic and genotypic studies (pcr amplification of 16s rdna) allowed the identification of two of the bacterial strains: pseudomonas fluorescens and buttiauxella ferragutiae. the third strain, which is not listed in the ncbi database, may be a new bacterial species. the two bacterial strains that were identified have a proven ecological and agronomic interest: pseudomonas fluorescens, a rhizosphere-secreting bacterium that secretes indole-3-acetic acid (phytohormone), plays an important role in protecting and promoting plant growth. buttiauxella ferragutiae, on the other hand, is very little studied with regard to its activities at ground level. however, this strain has already been isolated from the intestine of the earthworm. keywords : lumbricus terrestris, coelomic fluid, microorganisms, pcr16s dna, aia, soil productivity, pgpr. p100. screening of phosphate solubilizing rhizobia isolated from hedysarum nodules in north of morocco samia hamane1*, saida aarab1,3, mounir hassani zerrouk2, anas el yemlahi1, amin laglaoui1, mohammed bakkali1, and abdelhay arakrak1 1 equipe de recherche de biotechnologies et génie des biomolécules (erbgb), faculté des sciences et techniques de tanger b.p.: 416 – tanger, morocco; 2 faculté polydisciplinaire de larache, b.p.: 745 – larache, morocco; 3 faculté des sciences et techniques d’al hoceima, b.p.34, ajdir al hoceima, morocco. *corresponding and presenting author: samiaa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 512 hamane@gmail.com. abstract plant-microorganisms’ interactions in the rhizosphere are the major determinants of plant productivity and soil fertility. the use of beneficial bacteria as biological alternative for chemical inputs needs the selection of competent rhizobacteria exhibiting plant growth promoting (pgp) attributes. this study was aimed to select phosphate solubilizing rhizobia strains isolated from root nodules of the legume hydesarum flexuosum grown in north morocco, and to evaluate their antagonistic ability against fusarium oxysporum and aspergillus ochraceus. out of 11 isolates, 7 bacteria were able to solubilize tricalcium phosphate (tcp) that were evaluated for more pgp activities. as results, none of these selected bacteria proved to be able to synthesize hydrogen cyanide (hcn), while the amounts of indole acetic acid (iaa) produced by these bacteria ranged between 0.27mg l-1 and 1.51 mg l-1. besides, only 3 strains presented a positive profile for siderophores production. as for extra cellular enzymes production (urease, cellulase, amylase, protease and chitinase), the percentage of isolates that produced these enzymes was respectively: 0%, 100%, 28%, 100% and 85%. a quantitative assay of tcp solubilization was conducted too, and solubilized p concentrations were between 52 mg/l and 81 mg/l, accompanied by a drop-in media ph. since the bacterial enzyme acc-deaminase plays a significant role in sustaining plant growth under abiotic stress conditions, 5 rhizobia were capable to use acc as the sole nitrogen source. this study reveals the potential of sometest rhizobia to be used as efficient bio-inoculants. keywords: hydesarum flexuosum, rhizobia, fusarium oxysporum, aspergillus ochraceus, pgp, tcp, biofertilizer. p101. assessment of soil erosion by usle model using remote sensing and gis a case study of moulay bouchta watershed, western rif, morocco a. zouagui1*, m. sabir2, m. naimi1, m. chikhaoui1, and m. benmansour3 1 unité de recherche: gestion conservatoire des eaux et des sols iav-hassan ii, b.p. 6202 instituts, 10101, rabat, maroc; 2 école nationale forestière d’ingénieurs (enfi), bp 511, tabriquet salé, maroc; 3 centre national de l’énergie, des sciences et des techniques nucléaires (cnesten), bp 1382, r.p.10001 rabat, morocco. *corresponding and presenting author: zouagui.anis@gmail.com. abstract soil erosion causes many environmental and socioeconomic problems: loss of biodiversity, decrease in the productivity of agricultural land, siltation of dams and increased risk of flooding. it is therefore essential to establish a detailed evaluation of this process before any spatial planning. to evaluate the effects of soil erosion spatially and quantitatively in order to face this phenomenon, and propose the best conservation and land development strategies, the universal soil loss equation (usle) coupled with remote sensing and geographic information system (gis) is applied. this model is a multiplication of the five erosion factors: the erosivity of the rain, the erodibility of the soil, the inclination and the slope length, the vegetation cover and the anti-erosion practices. the study area is the moulay bouchta watershed (7 889 ha), which is located in the western part of the rif mountains, is characterized by a complex and contrasting landscape. the resulting soil loss map shows an average erosion rate of 39.5 (t/ha/yr), 87% of the basin has an erosion rate above the tolerance threshold for soil loss (7 (t/ha/yr)). soil losses per subbasin range from 16.2 to 81.4 (t/ha/yr). the amount of eroded soil is estimated at 311,591 (t/yr), corresponding to a specific degradation of 12.1 (t/ha/ yr). in the absence of any erosion control, 25% of the soil losses would reach the new dam located a little upstream of the basin outlet, reducing its water mobilization capacity to 59,625 (m3/yr). the application of principal component analysis (pca) to soil erosion factors shows a significant influence of topographic factor (ls) on soil erosion process, followed by the effect of support practices (p), then by soil erodibility (k). keywords: erosion, siltation, usle, rif mountains, watershed. p102. differential evolution and the impact of pests on the biochemical defensive profile during ripening of five vitis vinifera l. grapes varieties in the marrakesh region hicham jediyi1*, khalid naamani1, and abderrehim a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 513 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ait elkoch1 1 laboratory of protection and valorization of vegetable resources. faculty of sciences semlalia marrakech, cadi ayyad university, my abdallah street, pb: 2390, marrakesh 40000, morocco. *corresponding and presenting author: jedhicham@gmail.com abstract vitis vinifera (l) is a plant sensitive to various ravages and diseases such as pests. in this study we want to develop the evolution of the phytochemicals of the five grape varieties involving three introduced (superior, early sweet and flame) and two other local varieties (abbou and adari) during the three stages of maturity (green, ripening and mature) in oudaya of marrakesh région. we wish to show the dynamic changes that have taken place in the clusters during the physiological changes complex issues related to the development and maturation, also the action of pests (diptera) on the evolution of the biochemical profile of these varieties during ripening grape, especially our unknown local varieties. the parameters concerned the amount of total sugars, proteins, the activity of phenylalanine-ammonia lyase (pal), tyrosine-ammonia lyase (tal) and the lipid peroxydation. the analysis of total sugars and proteins showed their gradual increase during the period of ripening fruit of all varieties. we recorded in the tal activity, a decrease in the course of the maturation of the fruit, and then we noticed an increase in the pal activity in the ripe fruit for all varieties. this evolution of the parameters analyzed by the ¨anova¨ test, showed a significant difference for all phytochemicals (p<0.05) between the different varieties of vines according to the stages of maturity studied and a significant effect on the case of fruit, attacked or healthy one for all varieties (p<0.01) translating a significant difference between the healthy and attacked mature vines fruit for all varieties. as regards the distribution of variables, on the basis of pca analyze of the development stages according to variety and phytochimicals, we have observed that the sugars, proteins and tyrosine ammonia-lyase for pc2 are the most variables that have contributed to the discrimination of the grapes according to their stage of maturity and/ or varieties. keywords: vine varieties, maturity grape stages, phytochemical compounds. p103. selected biofertilizers effects on plant growth and yield of vegetable crops under field conditions abdel-ilah tahiri1,2*, zakariya es-salmi2, anas raklami1,2, noura bechtaoui1, abdelilah meddich2, and khalid oufdou1 1 laboratory of biology and biotechnology of microorganisms, faculty of sciences semlalia, cadi ayyad university, po box 2390, marrakech, morocco; 2 laboratory of biotechnology and plant physiology, faculty of sciences semlalia, cadi ayyad university, po box 2390, marrakech, morocco. *corresponding and presenting author: abdelilah.tahiri.1993@ gmail.com. abstract at the present time, food security is considered as one of the most serious challenges facing society which will further enhance demand on the production of global food. the current pressure on agriculture has caused land degradation, expansion into more marginal areas and soil types, and higher expectations of agricultural productivity per unit area. therefore, reliable, environmentally friendly techniques are needed to sustainably meet growing global food demands. some plant-microbe interactions like plant growth promoting rhizobacteria (pgpr), arbuscular mycorrhizal fungi (amf) and composts has been known that they are widely used and enhance plant growth by different action mechanisms. in this context, the main objective of this study is to evaluate the impacts of selected biofertilizers on the plant growth and the yield of vegetable crops in field. the leek (allium porrum l.) and lettuce (lactuca sativa l.) plants were grown on soil with low levels of organic and mineral matter. we used bio-fertilizers based on amf, pgpr and compost obtained from plant and animal wastes, alone or in different combinations. the biological treatments applied showed noticeable results compared to the control without bio-fertilizers. indeed, the amendment by the compost m3 based on green waste and livestock unit manure has considerably improved biomass parameters and yields of the two cultures. these increases were clearly significant compared to the other treatments, followed by amf. nevertheless, some other combinations of pgpr-amf and compost are also shown to be beneficial for the improvement of a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 514 the yield of the tested cultures. thereby, the use of such bio-fertilizers can be an interesting strategy for the sustainable management and development. keywords: arbuscular mycorrhizal fungi, pgpr, compost, bio-fertilizers, yield, allium porrum l., lactuca sativa l., field. acknowledgments: this work is financially supported by the project ppr-cnrst. p104. caracterisation of the moroccan varieties for agronomique and quality traits for breadmaking kenza eddakir1,2*, a. rochdi1, and w. tadesse2 1 faculty of sciences, university ibn tofail, kenitra, morocco; 2 international center for agricultural research in dry areas (icarda), rabat, morocco. *corresponding and presenting author: kenza.eddakir@ hotmail.fr. abstract everywhere and because of the importance of wheat, farmers strive to maximize yield, minimize cost, reduce environmental impact and ensure a high baking quality. seventeen of the moroccan varieties from inra , sonacos and icarda are tested for agronomic and quality traits for two years 2016 and 2017. yield and its component were collected at harvest. proteins were determined using the kjeldahl method. glutenins were extracted from wholemeal flour and electrophoresis was carried out for one grain from each cultivar. for each of the cultivars dough strength, stability and water absorption were assessed using the chopin farinograph and the mixograph, and the samples were also assessed using sds sedimentation test. correlations were calculated between the data obtained in these tests of functionality and the quantity of protein in the grain. most of the values obtained in the functionality tests were correlated significantly with grain protein content. but in general, the results shows that the quality of the moroccan varieties still very poor and many efforts should be made to improve the local quality for bread making and insure a good yield to meet the population needs and insure the self-sufficiency. wheat breeders are strongly encouraged to overcome the challenge that face the culivation of wheat and create new varieties that would be tolerant to drought, resistant to dome diseases and with a good quality. keywords: triticum aestivum, gluten proteins, sds page, breadmaking, quality, yield. p105. the optimal condition of storage and enthalpyentropy compensation analysis of the powder of taraxacum officinale’s leaves in moisture sorption phenomena haytem moussaoui*, idlimam ali, and abdelkader lamharrar laboratory of solar energy and medicinal plants, teacher’s training college, cadi ayyad university, bp 2400 marrakesh, morocco. *corresponding and presenting author: haytem.moussaoui@gmail.com. abstract taraxacum officinale is a kind of flowering plant that belongs to a vast family of asteraceae. this family is represented by more than 2500 species. they are utilized for various purposes that include for dyspepsia, bile stimulation, bruises, rheumatism, eczema and muscle aches. moreover, the powder of dandelion are used as special ingredients in soups, salad green, teas and wine. the taraxacum officinale used in this study were collected locally in settat region, morocco. the aim of this study is to determine the experimental sorption isotherms, the optimal water activity, and the net isosteric heat of sorption in order to conserve the powder. these isotherms are a powerful tool to know the state of water inside the product and his functional availability in the biological and chemical substances. the sorption isotherm curves were determined experimentally for the dandelion seeds at three temperatures (30, 40, 50 °c) and relative humidity within the range of 5-90% commonly used in the drying and storage. the net isosteric heats of sorption of water were determined from the equilibrium data at different temperatures. the compensation theory was further used to good straight lines were observed for adsorption and desorption. the gibbs free energy values are positive (δgβ> 0), indicating that the sorption processes are not spontaneous. keywords: taraxacum officinale, moisture sorption, optimal water activity, enthalpy, entropy. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 515 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) p106. antioxidant activity of anysophyllea boehmii seeds from burundi jonathan niyukuri*, raiti j., and hafidi a. laboratory of food sciences of cadi ayyad university of marrakech, morocco. *corresponding and presenting author: jonaniyu@gmail.com. abstract the objective of this study is to valorize some indigenous trees. the samples of seeds were harvested in october at 3 sites namely: mumirwa (1000-1500 m of altitude), buragane (1200-1400 m of altitude) and buyogoma (1400-2000 m of altitude). the fruit have a mesocarp edible, a thin pericarp and a tenders’ seeds. the seeds were dried at room temperature in the laboratory. ethanolic (80%) extraction were performed from powder obtained by grinding the seeds. with ethanolic extract, total phenol content of samples were determined using folin-ciocalteu method and flavonoid contents were quantified using the colorimetric method of aluminum chloride. the antioxidant activity was evaluated by scavenging of dpph (2, 2-diphenyl-1-picrylhydrazyl) free radical and ferric reducing antioxidant power (frap) assay. the kinetics of dpph reduction by ethanol extract and ascorbic acid were performed by determining steady state. parameter, time to the equilibrium state of ic50 (tec50), allowed to calculate the effectiveness anti-radical (ea). the ethanolic extraction yield were 14.80±0.18% of dry weight (dw), total phenol contents obtained were 851.89±34.96 mg gallic acid equivalent/100 g of dw and flavonoids contents were 261.35±102.45 mg ec/100g ms. the ic50 of ethanolic extract, total phenol and ascorbic acid were respectively 0.080±0.012 mg of extract/ml, 0.0033±0.0012 mg gallic acid equivalent/ml and 0.095±0.003 mg of ascorbic acid/ ml. the tec50 of ethanolic extract and total phenol was 11±2.2 min and for ascorbic acid was 5±0.6. the effectiveness anti-radical of ethanolic extract, total phenol and ascorbic acid were respectively 166.67±27.78, 4040.40±673.40 and 52.63±3.7 ml/µg.mn the reducing power of plant extracts was 3217.80±415.36 mg aasc/100g de dw. these results show that of anysophyllea boehmii has a high antioxidant activity and even greater than that of synthetic antioxidants. thus, in light of this study, active ingredients can be isolated from anysophyllea boehmii polyphenols food industry, cosmetic, and pharmaceutical use. keywords: antioxidant activity, indigenous trees, phenol and flavonoids p107. traditional phytotherapy in the high atlas: importance and valorization salima boutahiri1,2*, ayoub asbabou1,2, najia el hamzaouiss1,3, malika mahjoubi1,2, amal bouzoubaa1,2, touriya zair1,2§ 1 research team of chemistry of bioactive molecules and the environment, faculty of science, moulay ismail university, meknes, b.p. 11201, meknes, morocco; 2 laboratory of materials chemistry and biotechnology of natural products, faculty of science, moulay ismail university, meknes, b.p. 11201 zitoune, meknes, morocco; 3 laboratory of microbiology, mohamed v prefectural hospital center, meknes, morocco. *presenting author: boutahirisalima@gmail.com. §corresponding author: touria.zair@yahoo.fr. abstract herbal medication is an ancient practice. human beings have always used plants to ensure their survival and to relieve their ills. in this sense, the objective of this work is to highlight the importance of the traditional phytotherapy and to enhance the supply of medicinal plants in the high atlas of morocco. for this purpose, an ethnobotanical study was carried out, and six remarkable spontaneous species were selected from the local pharmacopoeia of the region (thymus satureioides, mentha rotundifolia, lavandula mairei, pallenis spinosa, artemisia mesatlantica and teucrium polium) for a laboratory study (phytochemical screening, polyphenol and flavonoid assays and the valuation of antioxidant activity by dpph). the ethnobotanical study showed that 91.71% of respondents use plants (55.63% refer to their own experiences and those of their ancestors, and 36.09% resort to herbalists). 68.33% of these users are women, 50% are between 15 and 40 years old and 70% are illiterate. plants are used for a therapeutic purpose in 86.20% of cases. this medicinal flora is widely used for the treatment of the digestive system diseases (46.06%). the different diseases are mainly treated by decoction of the foliage, thus constituting a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 516 the preparation method and the vegetal organ the most used. 81.65% of cases show improvement after treatment with plants. the phytochemical screening revealed the presence of tannins and flavonoids in the six-studied species and a high content of saponins for thymus satureioides. the highest yields of total polyphenols were recorded in the species pallenis spinose (19.36%), artemisia mesatlantica (18.5%) and teucrium polium (16.23%). on this basis, the results for the total polyphenol assay were 2.733, 2.450 and 2.166 mg ga equivalent/g for pallenis spinosa, artemisia mesatlantica and teucrium polium respectively, and the results for the flavonoid assay were 2.083, 2.041 and 1.966 mg quercetin equivalent/g for the same respective species. extracts from the studied plants showed an interesting antioxidant activity. the best result was held by teucrium polium extract, which reduced 50% of the free radical dpph by a concentration of about 0.51 mg/ml. keywords: traditional phytotherapy, ethnobotanical study, thymus satureioides, mentha rotundifolia, lavandula mairei, pallenis spinosa, artemisia mesatlantica, teucrium polium, phytochemical screening, polyphenols, flavonoids, antioxidant activity. p108. phenotypic and symbiotic characterization of rhizobia nodulating lotus creticus in the north of morocco taoufik belechheb*, mohammed bakkali, amin laglaoui, and abdelhay arakrak equipe de recherche de biotechnologies et génie des biomolécules (erbgb), faculté des sciences et techniques de tanger, abdelmalek essadi university, morocco. *corresponding and presenting author: taoufik.belechheb@gmail.com. abstract plants belonging to the lotus genera are now considered to be promising legumes for sustainable development, because it was used as a pioneer plant, improving soil structure and providing symbiotic inocula to promote plant succession. moreover, by its creeping habit, this species reduces the effects of the severe mediterranean winds thus fulfilling a primordial function in the protection and the preservation of the ecosystems contributes to soil stabilization , provides high quality animal fodder and its productivity is important on a wide range of soils so they are increasingly utilized in pastures. lotus sp, may play a key role in the nitrogen cycle, as it comes into symbiosis with nodulating bacteria legumes (lnb) collectively known as rhizobium and make them excellent candidates to maintain soil cover, dune stability, restore or improve the fertility of degraded soils and use for sustainable agricultural systems. this work was conducted to evaluate and characterize the phenotypic diversity (biochemical and physiological tests) that exists in a rhizobial collection isolated from nodules of lotus creticus collected from different sites in tangier in the north of morocco. tolerance to salinity, high temperatures, acid and alkaline ph, heavy metals and carbon and nitrogen source assimilation as well as symbiotic and cultural characteristics allowed the description of a wide physiological diversity among tested isolates. the dendrogram prepared by the upgma method, allowed us to highlight the most resistant isolates to tested stress conditions which represent the best candidates for the production of good quality inoculum for plants in marginal areas. keywords: lotus creticus , phenotypic characterization, rhizobium, tangier, forage. p109. life strategy and social participation of moroccan youth in poor rural areas of morocco noureddine bahri1*, a. bentaïbi1, and t. desrues2 1 regional center of agricultural research of meknes, bp. 578 meknes vn, morocco; 2 institute of advanced social studies (iesa) cordoba, c/ campo santo de los mártires, 7 14004 cordoba, spain. *corresponding and presenting author: bahrinour@yahoo.fr. abstract this research is carried out about young people from the mountainous areas of the moroccan middle atlas to better understand their logics and strategies of life. the study was focused on three communes marked by a high rate of poverty and a pronounced distance from the main lines of communication and the political and economic decision-making centers. it has adopted a dual approach to data production, namely: the administration of a survey with more than 300 individuals (a quantitative approach) and open interviews (a a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 517 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) qualitative approach) with young people in addition to different key informers. the first objective of this research concerns the exploration of the various strategies of life of young people in situations of social, economic and ecological constraints (high poverty and unemployment rates, underemployment, agriculture subject to climatic risk, lack of non-agricultural employment opportunities and lack of infrastructure for culture or recreation). the second is focused on the perception of their situation with particular attention to the analysis of the level of citizen participation of rural young people and the relationships maintained with the main actors of local development in general and of agricultural development in particular. this research postulates that these populations of young people are currently experiencing mutations and profound ruptures that challenge current traditional systems. the identification of these mutations would give some visibility to the complexity of these areas and would affirm the need to incorporate this notion of diversity into the public policies in morocco in the field of social, economic and cultural integration of rural youth. this consideration would enable young people to be better mobilized in local development activities and to reduce their dissatisfaction with their situation. this research has established that the young people of the regions studied are carrying diversity at the ideological level or values, i.e. they confront the management of their daily life in a differentiated way according to a series of axiological variables highlighted. this diversity is itself dependent on the nature and extent of the capital (economic, social, educational-cultural) available among the young people concerned. keywords: social change, youth, local development, agricultural development, life strategy, social participation. p110. introduction of new binary forage associations for crop-livestock systems under semi-arid conditions rajae kettani1*, chems doha khalfi1, and driss chergui2 1 research unit of agronomy and plant physiology, national institute of agricultural research (inra), regional center of meknes, po. box 578, 50000 meknes, morocco; 2 experimental area of douyet, fes, morocco. *corresponding and presenting author: r_ kettani@yahoo.fr. abstract the improvement of forage protein autonomy of farms in semi-arid regions implies the increase of the share of adapted and productive associations fodder as legume and cereals. indeed, they have many agronomic advantages that must be emphasized: no need to irrigate, a balanced protein product in relation to pure cereals, better protein content, a good competitiveness against weeds and increased resistance of the mixture to diseases. the experimentation was carried out at inra experimental station in fes (morocco) for three years to test new binary associations with a seeding rate of 120 kg per ha and the same proportions of 50-50 mixed species combined with different doses of nitrogen. the associated species are vetch guich (inra variety) which is early, productive in green fodder and resistant to alternaria and botrytis and four early to moderately precocious tutors: a rust resistant and productive nasr oat (inra variety); two early and high barleys: amalo (inra variety) and kaws (spain variety) and a rustic triticale forricale, very tolerant to water deficit and pouring (french variety). the results obtained show that it is possible to improve significantly the production and quality of forage associations through a good choice of varieties in mixture and appropriate nitrogen fertilization adapted to the production objective. kaws-guich combined with 120 nitrogen units per ha produced 9 tons of dry matter per ha and allows a productivity increase of 45% compared to the region average obtained with local binary associations. it also triples the proportion of vetch at harvest and consequently improves forage quality, as well as an increase of dry matter content of 27% and therefore a better forage nutrient concentration. on another side, the forricale-guich combined with only 60 nitrogen units per ha produced 5.5 tons of dry matter per ha and offers a better stand composition with a proportion of vetch of 40% and an increase of forage dry matter content of 17% compared to the region data. these two associations harvested in the cheesy stage of grain before drought occurrence has allowed increasing the qualitative and quantitative forage. they represent an interesting response for self-sufficiency and less dependence on weather conditions and suit specifically for farms poorly suited to corn silage. keywords: binary forage associations, varieties, nitrogen, drought, yield, dry matter. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 518 p111. cytokinins influence the microtuberization and growth of potato (solanum tuberosum l) plantels in vitro aziz akdi1,2*, ahlam hamim1, imane saibari3, sliman el hani1, and nourdin mtili2 1 national institute of agricultural research, tangier, morocco; 2 faculty of sciences, abdelmalek essaidi university, tetouan, morocco; 3 faculty of science and techniques of tangier, abdelmalek essaadi university, morocco. *corresponding and presenting author: azizakdi99@gmail.com. abstract potato (solanum tuberosum l.) is a very important crop in agricultural production in morocco. however, certified seeds production still one of the major problems hindering the development of this crop. to overcome this problem, the tissue culture techniques including microtuberization are used worlwide to produce prebasic, virus-free seed potatoes know as microtubers. the microtubers are sown in a protected enviroment to produce minitubers. tuberization is the predominant type of reproductive development for tuberous plants; it is a complex process, whih is controlled by several factors. the main objectif of the present study was to standarize the media for potato plant growth and microtuber induction on ms medium (murashinge and skoog, 1962) with and without cytokinins. to do this, four culture media were tested: ms1 = ms + 80% sucrose; ms2 = ms / 2 + 80% sucrose; ms3 = ms / 2 + 80% sucrose + 4mg / l kinetin + 1mg / l bap (6-benzylaminopurine); ms4 = ms / 2 + 80% sucrose + 0.2mg / l kinetin + 5mg / l bap. the plant material consists of botanical seeds (true potato seeds-tps) of two varieties registered under serial numbers (cipn °) 997003 and 998010. induction of microtuberization was completed after 9 weeks of culture under dark conditions at 22°c. for both varieties, the best rates of tuberization were obtained on ms3 medium. the increase in the concentration of bap compared to kinetin causes a decrease in the rate of tuberization. however, the larger microtubers are produced on the ms1 medium for both varieties. the addition of cytokinins in the microtubercule production media promotes the germination of microtubers. keywords: solanum tuberosum l, microtubercules, bap, kinetin. p112. water resources vulnerability factors in a global environmental change context: a systematic review and meta-analysis in international cases soumaila kanga*, mustapha naimi, and mohamed chikhaoui soil and water management laboratory, department of natural resources and environment, hassan ii institute of agronomy & veterinary medicine, po. box 6202 madinat al irfane, rabat, morocco. *corresponding and presenting author: soumkanga@gmail. com. abstract assessing the vulnerability of water resources is increasingly becoming an essential practice in the integrated water resources management. accordingly, several studies have been carried out around the world during the last two decades. thus, the main objective of this paper was to review factors used internationally to assess the vulnerability of water resources at local scale in order to adapt them and use them in the assessment of the vulnerability of water resources in a context of climate change in morocco. to achieve this goal, we have pre-selected 187 papers on water vulnerability in the science direct database of elsevier publisher and the google scholar database. by applying the prisma protocol, only 13 articles out of the 187 selected met the inclusion or exclusion criteria previously defined for the analysis. the 113 water resources vulnerability factors identified were sorted out into five categories: physical or biophysical factors (35.4 %), socio-economic factors (31%), environment or eco-environment factors (22.1 %), infrastructure factors (8.8 %) and water governance and institutions factors (2.65%). based on the availability of data, the scale of application and the environmental problems of water resources in morocco, we were able to recognize 50 factors among all categories that could be used in the assessment of water resources vulnerability in morocco. keywords: water vulnerability factors, integrated water management, water vulnerability assessment, local scale. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 519 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) p113. evaluation of the irrigation water in the central bahira plains of el kalâa, morocco soukaina el hasini1*, mohammed el azzouzi1, houria dakak2, oumaima ibnhalima2, and abdelmajid zouahri2 1 laboratory of materials, nanotechnologies and environment, faculty of sciences, university mohammed v, av ibn battouta, bp1014, rabat, morocco; 2 research unit on environment and conservation of natural resources, national institute of agricultural research (inra) po box 6356 rabat-institutes, rabat, morocco. *corresponding and presenting author: elhasinisoukaina3@gmail.com. abstract the aridity of the climate due to the succession of drought periods in morocco leads to a situation of water paucity. these conditions make irrigation an unavoidable technical requirement for the development of agricultural lands. even so, agricultural intensification was accompanied by a lack of irrigation control which leads to several problems of degradation of water and soil quality in terms of salinity and alkalinity. this constitutes a serious danger to the sustainability of the land and water quality. in order to succeed in dealing with this situation as well as to ensure the sustainable preservation of these resources, the requirement of their monitoring and analysis becomes necessary. the present work falls within this framework suggests a synthesis of the hydrochemical situation by assessing the distribution of salinity and other major problems affecting the quality of groundwater in the bahira plain, particularly around sad al majnoun. an approach has been followed including a monitoring of the groundwater at the scale of the area according to a network of 33 water points. the measurements were carried out in situ (ce, ph, piezometric level) and in the laboratory for the major and secondary elements to specify (rsc, sar and n% ). moreover, the maps of the groundwater quality parameters have been established under gis allowing us to delineate the areas affected by the problems of degradation of this resource as well as constitute a source of information and management for a best future monitoring of the qualitative state of the water table. keywords: irrigation water, hydrochemical situation, cartography, gis, central bahira. p114. ability of non-pathogenic fusarium oxysporum strain fo47 to suppress rhizomania disease of sugar beets in morocco fatima nouayti1,2*, ilham madani2, abdessalem tahiri1, abdelali blenzar2, and rachid lahlali1 1 phytopathology unit, department of plant protection, ecole nationale d’agriculture de meknès, meknès, morocco; 2 moulay ismail university, faculty of sciences. department of biology, p.o. box 11201, meknes, morocco. *corresponding and presenting author: fatimanouayti@gmail.com. abstract rhizomania is one of the most devastating diseases of sugar beet worldwide. the disease poses a serious threat to moroccan production and it is capable of significantly decreasing quality and yield of sugar beet plantations. the long-term survival of its fungal vector (polymyxa betae) in soil makes it a very difficult disease to manage. therefore, this study investigated the potential of a non-pathogenic fungal fusarium oxysporum strain fo47 to control polymyxa betae. this biocontrol agent was applied as soil treatment, seed treatment, or a combination of the both treatments. a bio-test was performed on treated soil. after four weeks of culture, the roots of sugar beet seedlings were retrieved and analyzed by the das-elisa test. results indicated that f. oxysporium fo47 reduced the activity and survival of p. betae when compared to a reference biocontrol agent trichoderma harzianum, which only revealed significant in reducing the viral load of beet necrotic yellow vein virus (bnyvv) as seed treatment. the non-pathogenic fusarium oxysporum fo47 was more effective as soil treatment and allowed almost the same reduction of bnyvv virus concentration as t. harzianum 908. therefore, our findings emphasizes that the performance of the biocontrol agent depends on the method of application. keywords: beet necrotic yellow vein virus; biocontrol, fusarium oxysporum strain fo47; trichoderma harzianum strain 908. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 520 p115. optimization of the technical management of agroforestry (choice of cereal species and nitrogen fertilization) in olive groves bendidi abderrazzak1*, khalid daoui1, rachid dahani1, and mohamed ibriz2 1 inra, meknes, morocco; 2 faculty of sciences, university ibn tofail, kenitra, morocco. *corresponding and presenting author: abendidi1@yahoo.fr. abstract intercropping is practiced on a large scale in moroccan olive groves. according to the data from the feasibility study of the fruit growing project (paf), cereal cultivation is practiced intercropped in more than 50% of the olive groves. in order to determine the influence of intercropped cereal farming techniques on olives trees, we studied olive tree production in association with two different cereal species (bread wheat and durum wheat) with two nitrogen doses (40 and 120 kg n ha-1) in the khenifra region. the results of the first year of study, characterized by drought and cold during the winter months, showed that durum wheat (karim) allowed a grain yield (11.3 qx ha-1) higher than that obtained by bread wheat (amal) (8.8 qx ha-1). regarding the nitrogen dose, the highest grain yield for wheat was recorded with 120 kg n ha-1 with an average of 11.8 qx ha-1, while grain yield with 40 kg qx ha-1 was only 8.3 qx ha-1. while olive production of 45 kg / tree was not affected by either the cereal species or the amount of nitrogen added to the intercrop. these results show that the installation of cereals as an intercrop in moroccan olive groves (under 16 years old for the picholine marocaine variety) would allow the farmer to improve the productivity of his land and consequently the maximization of his income. keywords: intercropping, olive groves, bread wheat, durum wheat, nitrogen, production. p116. influence of photovoltaic panels on growth and productivity of tomato under canarien greenhouse kabira ezzaeri1*, hicham fatnassi2, lahoucine gourdo1, abderrahim bazgaou1, ahmed wifaya3, rachid bouharroud3, hassan demrati1, ahmed aharoune1, ahmed bekkaoui4, and lahcen bouirden1 1 thermodynamics and energetic laboratory, faculty of sciences, agadir, morocco; 2 inra, univ. nice sophia antipolis, cnrs, umr 1355-7254 institut sophia agrobiotech, 06900 sophia antipolis, france; 3 regional centre of agricultural research, agadir, morocco; 4 mechanization agricultural department, iav hassan ii, rabat, morocco. *corresponding and presenting author: k.ezzaeri@uiz.ac.ma. abstract solar energy is one of the cleanest sources of alternative energy. due to high energy demand in one hand, and environmental negative impact of fossil fuels on the other hand, many countries consider the alternative energy sources as a suitable and feasible option in industry and domestic usage. the use of renewable energy for greenhouse environment control to replace or reduce the consumption of fuel and powerline electricity is an important objective for sustainable greenhouse crop production. the photovoltaic greenhouse is a new technology for a “double use” of the resource land, because it generates energy in the form of biomass (crops, flowers, or algae) and, also, electric energy. the goal of our research is to study the development and yield of tomato inside a canarian greenhouse, with 10% of their roof was occupied by photovoltaic panels. the results shows that the shading provides by the photovoltaic panels have not a significant effect on the tomato development and yield. keywords: photovoltaic greenhouse, shading, tomato, yield. p117. epidemiology of multi-resistant bacteria in the hospital environment: service of major burns and plastic surgery, mohamed v hospital, meknes najia el hamzaoui1*, hassan ouallal2, abouddihaj barguigua3, and sbiti mohammed4 1 medical microbiology laboratory, mohamed v hospital, moulay ismail university, meknes, morocco; 2 membrane materials and separation processes laboratory, faculty of science and technics, irrachidia, morocco; 3 multidisciplinary research and development laboratory, department of biology-geology, polydisciplinary faculty, beni mellal, morocco; 4 medical biology laboratory of regional military hospital moulay ismail meknes morocco. *corresponding and a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 521 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) presenting author: najia.elhamzaoui@gmail.com. abstract following several cases of bacterial infections, in the service of major burns and plastic surgery of mohamed v hospital, meknes, samples were taken from this service to identify the bacteria responsible for these infections. environmental sampling was carried out by the swabbing method. the cultures were realized for 48 hours at 37 °c on lactose agar, cled, mannitol salt agar (chapman), blood agar and chocolate agar. the identification of the isolated bacterial species was carried out using the api gallery systems, the resistance level of the bacteria to the antibiotics was studied by the diffusion method in agar. the choice of antibiotics as well as the interpretation of susceptibilitytesting were performed according to ca-sfm recommendations. the surfaces and medical devices containing bacteria are of type: beds, surgical equipments,operating tables, bedside tables, door cuffs, stethoscopes, cupboards and washbasins. the identifiedbacteria are staphylococcus epidermidis and saprophiticus, klebsiella penumoniae, pseudomonas putida and acinetobacter baumanii, and the results of the antibiogram showed a predominance of multiresistant bacteria, especially acinetobacter baumanii and klebsiella penumoniae. many observations have suggested that the contamination often resulted from inappropriate application of cleaning procedures, and many factors that are related to microorganisms:lifetime on a support, surface adhesion, ability to produce a biofilm and its ability to withstand adverseconditions. considering the obtained results, the monitoring and the behavior study of bacteria, present in the service of the major burns, with regard to the more commonly used disinfectantsbecomes a necessity for the establishment of the appropriate disinfection procedures. p118. long term monoculture promotes selection of antagonistic phonotypes among streptomyces in soil. essarioui adil1* and kinkel linda l2 1 national institute of agronomic research, regional center of errachidia, morocco; 2 department of plant pathology, university of minnesota, usa. *corresponding and presenting author: essar002@umn. edu. abstract streptomyces possess strong antagonistic activities and inhibit diverse plant pathogens. however, the effects of plant community characteristics on streptomyces inhibitory activity have received little attention. we evaluated the impact of plant species and plant community richness on the frequency and intensity of inhibitory activity of rhizosphere streptomyces communities against 2 fungal plant pathogens, fusarium oxysporum f. sp. albedinis and fusarium oxyxporum f. sp. lycopersici, the agents of date palm tomato wilts, respectively. soil samples were taken from the rhizosphere of two plant species, andropogan geradii and lespedeza capitata, each growing in long term communities of 1 (monoculture) or 16 (polyculture) plant species. streptomyces inhibitory activity against the two pathogens was studied using an in vitro assay to reveal inhibition on each target pathogen. for each soil sample, the proportions of streptomyces that exhibit antagonistic activities against each pathogen and the mean size of the inhibition zone were determined. plant richness modified the impacts of both plant species on streptomyces antagonistic activity. regardless of plant species, monocultures supported higher frequencies and greater intensities of streptomyces inhibition than polycultures. streptomyces antagonistic capacity increased with decreasing plant community richness, suggesting that plant diversity modifies selection for antagonistic phenotypes among soil streptomyces. fostering monoculture conditions in agricultural settings may contribute significantly to reducing the impacts of soil-dwelling crop pathogens. keywords: streptomyces, inhibition, monoculture. p119. evaluation of phosphate solubilizing bacteria isolated from cytisus monspessulanus rhizosphere for their growth promoting and antagonistic activity against phytopathogenic fungus fusarium oxysporum imane achkouk1*, saidaaarab2, amin laglaoui1, mohammed bakkali1, abdelhay arakrak1 1 equipe de recherche de biotechnologies et génie des biomolécules (erbgb), faculté des sciences et techniques de tanger b. p. 416, tanger, morocco; 2 faculté des sciences et techniques d’al hoceima, b.p. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 522 34, ajdir al hoceima, morocco. *corresponding and presenting author: imaneachkouk@gmail.com. abstract plant growth promoting rhizobacteria (pgpr) are considered to be the most promising agents for agricultural crop via increasing crop yields and decreasing disease occurrence. the aim of this work is to evaluate phosphate solubilizing bacteria (psb) isolated from the rhizospheric soil of cytisus monspessulanus, for multiple plant growth promotion traits and to test their antagonistic effect against fusarium oxysporum. a total of 37 isolates (15.81%) among 234 isolated rhizobacteria were able to solubilize the tricalcium phosphate (tcp) on pvk solid medium. the 21 isolates with solubilization diameters ≥ 0.4 cm were selected and evaluated for the production of hydrogen cyanide, and the results showed that 52.38 % of these isolates (11 isolates) produced this compound, so theywere retained for further tests. the amount of iaa produced during the quantitative test fluctuated between 55.45±9.52 mg/l and 2.35±0.38 mg/l and all the strains were positive for the production of sidérophores except cmr67.the selected rhizobacteria were then evaluated for their ability to secrete enzymes such as cellulase, chitinase and protease. the percentages obtained were respectively 81.81%, 45.45% and 63.63%. in addition, the ability of these psb to produce ammonia was tested, and 9 strains (18.18%) were able to produce this compound. all test strains were assessed by tcp solubilizing quantitative assay in pvk liquid medium and the concentrations of solubilized p were between 147.49 ± 0.06 mg/l and 7.57± 0.04 mg/l. this solubilization was accompanied by a ph decrease of the medium from 7 to 4.18. the antagonism test against fusarium oxysporum showed that all the selected isolates were able to inhibit its growth except the isolates cmp55 and cmr22. the best performance on all qualitative and quantitative tests was recorded by the three isolates cmr64, cmr68 and cmr78 that can be used as bioinoculants to contribute to the rehabilitation of degraded marginal soils. keywords: psb, pgpr, cytisus monspessulanus, fusarium oxysporum. p120. ethnobotanical uses of arbutus (arbutus unedo l.) in morocco faida rahima1,2*, aabdousse j.1, boulli a.1, bouda s.2, and wahid n.1§ 1 ecology and sustainable development team (esdt), department of life sciences, faculty of science and technology, sultan moulay slimane university, beni mellal, morocco. 2 laboratory of management and valorization of natural resources, department of life sciences, faculty of science and technology, sultan moulay slimane university, beni-mellal, morocco. *presenting author: faida.rahima@gmail.com. §corresponding author: wahid2na@hotmail.com. abstract arbutus is a fruit tree with high ornamental, environmental, economic and medical value, because of the properties attributed to different biological parts. given this interest, the present study is interested in describing and compiling available data on the ethnobotanical uses of arbutus in morocco in comparison with other studies at the international level. it is very present in the everyday uses of foresters, farmers and rural populations. the international bibliographic analysis shows that the different biological parts of arbutus, especially fruits, have been widely used by herbalists. the related literature is generally in line with the medicinal use of the plant by moroccan inhabitants. on the other hand, the consumption and the industrial valorization of the fruits of arbutus are very limited in morocco in comparison with those on the international scale. it is imperative to sensitize the local population and landlocked douars on the knowledge of the valorization of the fruit of this species. keys words: arbutus unedo l., ethnobotanical uses, prospection, ecology, arbutus fruit. p121. livestock genetic resources support resilience to climate change in oasis production systems: a review mustapha ibnelbachyr* national institute for agricultural research, center of errachidia, po box 529, boutalamine, errachidia, morocco. *corresponding and presenting author: m_ a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 523 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) ibnelbachyr@yahoo.fr. abstract livestock, an essential component of production systems in oasis areas, is facing increasingly the effects of climate change. livestock genetic resources play a significant role in promoting biodiversity and improving resilience to this phenomenon in oasis production systems. this review addresses the current status of preservation of animal genetic resources in the oases of southeastern morocco, and presents the opportunities offered by these resources to meet future needs for oasis production systems. the focus is put on the typical oasis breeds; the tidili cow, the d’man sheep, and the drâa goat. the tidili cow is typical cattle of the valley located between the southern slopes of the high atlas and the siroua massif. the main assets of this local breed include its perfect adaptation to local environmental conditions and its interesting milk production. the d’man sheep is the most prolific moroccan sheep that has contributed to the improvement of meat production in the oases and in the crossbreeding areas. the drâa goat is the goat breed of the oases of the drâavalley which furrows the moroccan southeast. it is the only moroccan goat ableto get conceive at any period of the year and giving an important milk production. thus, it constitutes a resource of genetic and economic interest certain. overall, livestock breeds are genetically and economically important to improving resilience to climate change in oasis production systems. preservation and genetic improvement programs are needed to prevent extinction of these breeds in the future. keywords: climate change, resilience, local breeds, oasis. p122. protein, oil, and fatty acids contents of soybean mutant fm6-847 grown in fayetteville, north carolina safiah kassem1*, jiazheng yuan2, naoufal lakhssassi3, khalid meksem3, and my abdelmajid kassem2§ 1 pine forest middle school, 6901 ramsey st, fayetteville, nc, usa; 2 plant genomics and biotechnology lab, department of biological sciences, fayetteville state university, fayetteville, nc, usa; 3 department of plant, soil, and agricultural sciences, southern illinois university, carbondale, il 62901, usa. *presenting author: safiahkassem@gmail.com. §corresponding author: mkassem@uncfsu.edu. abstract proteins, oil, and fatty acids are important components of soybean [glycine max (l.) merr. ] seed composition. the objective of this study was to compare protein, oil, and fatty acids contents of the soybean mutant fm6-847 with three usda reference lines (ld00-2817, ld06-7620, and ld07-3395) in a field in fayetteville, nc in 2016. our results showed that the mutant fm6-847 had significantly higher levels of protein than ld07-3395 but not ld00-2817 and ld06-7620. the mutant fm6-847 had significantly higher oil and palmitic acid contents than all three usda reference lines. the mutant fm6-847 had a significantly higher stearic acid content than ld002817 and ld07-3395 but lower than the content in ld06-7620. for oleic and linoleic fatty acids, the mutant fm6-847 had significantly lower contents compared to all three usda reference lines. the mutant fm6-847 had significantly higher linolenic acid content than ld00-2817 but significantly lower levels than ld06-7620 and ld07-3395. the mutant fm6847 outperformed the usda reference lines in yield, oil and palmitic acid contents; therefore, it is well adapted to north carolina, is stable, and will benefit farmers in nc to increase their soybean yield, seed composition, and profit. key words: soybean, protein, oil, fatty acids, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid. p123. response of two varieties of banana (musa acuminata) small dwarf and large dwarf in vitro to salt stress meriem belfakih1*, mohammed ibriz1, and abdelmjid zouahri2 1 laboratory of genetics biometry, faculty of sciences, university ibn tofail, bp 133, kenitra, morocco; 2 unité de recherche « environnement et conservation des ressources naturelles», inra, crra de rabat, bp 6356 rabat instituts, morocco. *corresponding and presenting author: m_belfakih2000@yahoo.fr. abstract the objective of this study is to evaluate the effect a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 524 of salt stress (nacl) on the in vitro regeneration of banana and the possibility of using this technique to improve the tolerance of this species to salinity. previously, the rejects were disinfected with 8% bleach and then grown in murashige and skoog medium (1962) containing growth hormones (appendix 1). the medium was fed with different salt concentrations (0, 2, 4 and 6 g / l of nacl). the test was conducted at a temperature of 28 to 30 ° c with artificial illumination and a photoperiod of 16 h. the results obtained show that both varieties have regeneration of shoots despite saline treatment. however, salt affects leaf length of the explants and the regeneration rate. so, the increase in saline concentration induce to a decrease in the number of shootings per bud. the small dwarf variety appears to be sensitive to the salinity effect of the medium at all naclconcentrations at the regeneration stage and the seedling stage. keywords: salt stress, regeneration, growth, in vitro, banana. p124. impact of the combination of salt stress and mineral nutrition on the growth of alfalfa biomass (medicago sativa l). meriem belfakih1*, mohammed ibriz1, and abdelmjid zouahri2 1 laboratory of genetics biometry, faculty of sciences, university ibn tofail, bp 133, kenitra, morocco; 2 unité de recherche « environnement et conservation des ressources naturelles », inra, crra de rabat, bp 6356 rabat instituts, morocco. *corresponding and presenting author: m_belfakih2000@yahoo.fr. abstract this work aims to study the combined salt stress effects (0.6 and 12g/l nacl) and the conditions of normal (hn) and limiting (hd) mineral nutrition, on growth and mineral nutrition of three varieties of alfalfa (sirever, demnate 203 and riche) .the study was done under controlled conditions in a growth chamber for one month. the results obtained in this test showed that the salt treatment leads to a net reduction of the aboveground and root biomass upon addition of 6g/l. this detrimental effect of salinity becomes less under normal supply conditions, with the exception of the ‘demnate 203’ variety which showed the highest yield under limiting supply conditions. also, saline treatment leads to an accumulation of na+ cation and a reduction in k+ absorption. the ‘siriver’ variety was more sodium-inclusive under normal mineral supply (hn) conditions. in addition, the ‘demnate 203’ ecotype produced more dry matter under salt conditions and limiting mineral supply (hd). keywords: alfalfa, medicago sativa, salinity, mineral nutrition. p125. evaluation of diverse sets of barley germplasm for resistance to scald disease (rhynchosporium secalis) houda hiddar1,2*, sajid rehman2, zakaria kehel2, athanasios tsivelikas2, ramesh p.s. verma2, abdelkarim filali-maltouf1, and ahmed amri2 1 microbiology and molecular biology laboratory, faculty of sciences, mohammed v university, rabat, morocco; 2 biodiversity and integrated gene management program (bigm), icarda, rabat, morocco. *corresponding and presenting author: hiddar.houda@gmail.com. abstract leaf blotch, also known as scald, is one of the most destructive diseases of barley (hordeum vulgare l.); caused by the haploid imperfect fungus rhynchosporium secalis. scald is widely distributed throughout the world and can cause 40% reduction in grain yield. the use of resistant cultivars remains to be the most effective, economical and environmental friendly way to control the disease but the genetic resistance to moroccan isolates is poorly understood and only few sources of resistance have been identified. this study aims at the identification of new and effective sources of resistance to scald within various subsets: best bet subset selected using the focused identification of germplasm strategy (figs; 80 accessions), reference set identified within the generation challenge program (gcp; 204 accessions), and an association mapping panel (284 accessions) developed by icarda breeders. . all the accessions were sown and evaluated at seedling stage under controlled conditions. we have found that of the 568 barley accessions tested, 78 barley accessions (14%) were found to be resistant, 86 accessions (15%) to be moderately resistant, 104 accessions (18%) to be susceptible, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 525 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) and103 accessions (18%) to be highly susceptible, respectively. most of the resistant accessions (125; 22%) showed immune reaction. these new sources of resistance will be a useful resource for resistance barley breeding programs around the world. keywords: barley, scald, rhynchosporium secalis, resistance. p126. analysis of the hydro-sedimentary process of a watershed in western rif of morocco using the swat model: case of the tleta watershed fatiha choukri1*, mohamed chikhaoui1, mustapha naimi1, damien raclot2, and yannick pepin2 1 département des ressources naturelles et environnement, institut agronomique et vétérinaire hassan ii, rabat, morocco; 2 umr lisah, institut de recherche pour le développement, rabat, morocco. *corresponding and presenting author: choukri.fatiha@gmail.com. abstract swat model (soil and water assessment tool) is a tool for simulating flows in the landscape, useful for guiding management and groundworks within catchment areas. accordingly, this model was tested to reproduce, on a daily timespan, water and sediment flows arriving to ibn batouta reservoir over the period 1980-2010. various climatic (precipitation and temperature), morphologic (mnt) and thematic data (land use and pedology) were used for feeding the model. the measurements acquired at ibn batouta reservoir during the periods 1980-1982, 1983-1996 and 1997-2010 were used respectively for initialization, calibration and validation after running the sensitivity analysis to determine the most significant parameters. the performances indices of r², nse and pbias were adopted to assess and control the quality of predictions of the monthly flows of water and sediments. the performance of prediction of monthly water flows is very high with r² (0.95 ; 0.90), nse (0.94 ; 0.89) and pbias (-6 ; 2) respectively for both periods of calibration and validation. meanwhile the performance of predictions of the monthly solid transport is very satisfactory with r² (0.83 ; 0.64), nse (0.82 ; 0.61) and pbias (17 ; -6) respectively. finally, swat model allows to properly reproduce monthly water and sediment flows within the tleta catchment area and testing scenarios of management and groundworks to be considered. keywords: swat, hydrological modeling, water erosion, western rif, morocco. p127. performance analysis of a modified solar still equipped with solar tracker d. o. maliani*, a. bekkaoui, e. h baali, k. guissi, y. el fellah, and h. krim département energie et agroéquipements. institut agronomique et vétérinaire hassan ii, rabat, morocco. *corresponding and presenting author: drissi. oussama@gmail.com. abstract conventional solar still suffer from its low yield despite many attempts to optimize it. this is due to its high dependence of the low energy generated by the greenhouse effect. hence, the concentration seems to be an effective way to increase heat energy received by the still, and consequently enhance its productivity. however, many researches addressed in the field do not benefit from this powerful technique with an efficient way. they usually use an intermediate item to transport heat to the water inside the still, such pipes filled by a heat transfer fluid, which causes a loss of energy and decreases the thermal performances of the solar still. the new idea, presented in this work deals with the design of an innovative system of solar distillation that concentrate directly the sun’s rays on the water to be distilled while ensuring efficiency in a follow-up of the sun as well in the azimuth as elevation. the greenhouse effect will be used as an extra thermal energy. the aim was carried out to estimate the dynamic and thermal behavior of the concepts. an arduino electronic card will ensure the control of the system. the yield, economic aspects and payback period of different solar still are examined, and possible conclusions with integration of dissimilar aspects are reached. keywords: solar still, desalination, solar tracker, single slope. p128. effect of temperature on date-palm syrup (dibs) processing and quality noufia younés1*, outaleb zahra2, alem chakib3, and filali zegzouti younés4 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 526 1 national institute of agronomic research, errachidia, morocco; 2 université sidi mohammed ben abdellah faculté des sciences et techniques, fès, morocco; 3 research team in biochemistry & natural substances, fste-umi, errachidia, morocco; 4 research team in biology environment and health, fste-umi, errachidia, morocco. *corresponding and presenting author: noutfiaa@yahoo.fr. abstract in 2017, morocco produced about 112,000 tons of dates. this production is characterized by a high percentage of khalts (clones / varieties with low quality). these date palm clones are sold to noncompetitive prices or used for processing of others date-palm products. this investigation aims to contribute to the study and characterization of date-palm syrup as a by-product of this fruit. thus, five samples dates of about 1500g (about 1000g coreless) each were used to manufacture the syrup at two extraction temperature (70°c and 90°c). the main processing steps are: coring, juice extraction, filtration and concentration of the juice. the obtained results of date samples are: solids = 85.44 ± 3.53, ph = 5.08 ± 0.71, acidity = 0.15 ± 0.048 and ash = 1.86 ± 0.49. the physical and chemical composition of syrup was: brix = 74.03 ± 0.95, solids = 82.00 ± 1.42, ph = 4.74 ± 0.15 and ash = 1.62 ± 0.10. the yield production was about 64%. also, this study showed that the extraction temperature affects with a very highly significant manner (α=0.00018), the duration of juice concentration. this duration was being 9h and 6.4h for respective extraction temperatures of 90°c and 70°c. however, the extraction temperature does not seem to affect yield, ph and dry matter of the syrup. keywords: syrup, dibs, date-palm fruit, temperature, quality. p129. mycorhization effect on cork oak acorns and their eco physiological behavior (quercus suber l.) in drought conditions btissame bouther1*, younes abbas2, and moustapha arahou1 1 botanical, mycology and environment lab, department of biology, faculty of sciences, university med v rabat, morocco; 2 faculty polydisciplinary of beni mellal, university sultane moulay slimane, beni mellal, morocco. *corresponding and presenting author: bouther.btissame@gmail.com. abstract the cork oak is one of the most important species in morocco due to its socio-economic and environmental role. this species occupied a very large area whereas it decreases more and more these last decades, inspite of all the attempts of artificial regeneration which failed. in order to protect this species from extinction and subsequent strong deforestation, nursery cork seeds were planted in a substrate inoculated with three different ectomycorrhizal fungi under controlled conditions in order to evaluate the species: effect on the development and resistance to drought of the cork oak. the plants inoculated by the fungus 3 showed very strong resistance which is explained by very low values in the height of the stem, the number of leaves and the stem diameter. keywords: cork oak, mycorhization, artificial regeneration, drought. p130. efficacity of ruta graveolens powder to fight against callosobruchus maculatus (coleoptera, bruchinae) lalla fatima douiri1*, nada lafkih1, yasmine ghouati2, hayat bouhareb1, oulghazi said1, chakir meryem3, mgamat fouzia3, and mohieddine moumni1 1 department of biology sciences faculty, / moulay ismail university, p.b 11201, meknes, morocco; 2 food technology and biochemistry unit / national school of agriculture, p.b. s/40 50000 meknes, morocco; 3 department of biology sciences faculty, / ibn tofail university, pb : 14000, kenitra, morocco. *corresponding and presenting author: fatimadouiri@ yahoo.fr. abstract insects occur at every point along the chain of food production from the open field or greenhouse environment to larders and pantries in domestic premises. their presence causes concern not only because of their appearance and the direct spoilage they cause to food products, but also because of the microbiological contaminants and pathogens they may a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 427 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) carry, and because of possible allergenic reactions that their exuviae and frass may generate. this study focuses on the insecticidal activity of ruta geaveolens powders against c. maculatus elevated at the expense of cicer arietinum seeds (l). laboratory’s experiment was carried out under controlled conditions (27°c, 70 ±5% relative humidity and 12 hours of light). the results obtained for the studied parameters indicate that powders extrated from rosmary leaves and young twigs have an insecticidal activity against weevils, after (1 to 5) days exposure the lowest lc50 and lc99 range respectively from 87.58 to 13.19 and 157.34 to 26.52mg /50graines for females and 52.63 to 15.90mg/50graines and from 93.79 to 25.74 mg/ 50graines for males. in fact, longevity obtained with the control is 9.1 to 7.73 days, for males 5 to 3.7days and for females de 6 à 4.7days. the ruta powders exert negative effects on all the parameters of the weevil and can be used against c. maculatus. in addition, they are derived from botanical products, biodegradable, renewable source, economic and have low environmental impact and often undetectable. keywords: ruta geaveolens, cicer arietinum, powders, callosobruchus maculatus. p131. improve the effeciency of gibberellic acid to increase the fruit set and yield of seedless clementines mandarin of morocco ismail kaidi1,2*, l. messaoudi1, and z. messaoudi² 1 département de chimie, université moulay ismail, faculté des sciences, morocco; 2 département arboriculture fruitière oléiculture viticulture, ecole nationale d’agriculture de meknès, morocco. *corresponding and presenting author: ism.kaidi@gmail.com. abstract the sidi aissa clementine (citrus reticulata blanco) is a seedless mandarin of great fruit quality. it is the leading clementine cultivar produced in morocco. in recent years, moroccan growers have planted thousands hectares of sidi aissa clementine and more may be planted in the future. one of the major challenges to moroccan growers is the problem of low fruit set and small fruit size associated with the production of seedless sidi aissa and other seedless clementine mandarins. to overcome this problem, mandarin growers apply ga3 during bloom to enhance fruit set and yield. however, the current registration of gibberellic acid (ga3) for fruit set of clementine mandarins in morocco was based on experiments conducted in foreign countries and by private companies. in spain and south africa, ga3 is routinely used in the production of seedless mandarins to increase both fruit set and fruit size, but these strategies have not proven reliable in morocco. the label provides no detailed information telling growers how to use ga3 to increase fruit set and yield or yield of commercially valuable large size fruit annually and, thus, results for clementine mandarin production have been variable production have been variable. moreover, improper use of ga3 results in low fruit set, canopy damage, and potential reduction of flower formation in following years. the goal of this research is to identify the proper dose and best timing of ga3 application for increasing fruit set and quality of commercially valuable fruit of sidi aissa clementine mandarin and to determine the beneficts effects resulting from use of high rates of ga3 or from numerous applications of lower rates of ga3 the current and following years. on yield and fruit size in a comprehensive experiment testing the efficacy of combinations of ga3 concentrations and application times, the effect of which was unknown on mandarins, was undertaken to maximize the yield of quality valuable fruit of sidi aissa clementine mandarin. keywords: ga3, clementine, fruit set, citrus flowers. p132. status of wilt and root rot (wrr) disease of kabuli chickpea in several regions of morocco amine elbouazaoui1,2*, ilyass maafa2, ahmed s. kemal1, and allal douira1 1 ibn tofail university kenitra, kenitra morocco; 2 international center for agricultural research in the dry areas (icarda), rabat, morocco. *corresponding and presenting author: amine.elbou10@gmail.com. abstract kabuli chickpea is the most important temperate food legume in wheat-based system of morocco. the area covered by the crop is over 88,000 ha in 2015/16 with a production of 44,000t which is not enough to meet the demand of the population, a fact that pushed the country to imports chickpea to fill the gap. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 528 the low productivity is partly due to diseases and insect pests. major chickpea diseases are ascochyta blight and wilt/root rot (wrr) affecting spring and winter planted chickpea causing high yield losses. although wrr disease is known to impact chickpea production, knowledge on disease incidence, distribution and pathogen associated with wrr complex are not well studied. for this purpose, a survey was conducted in two regions of morocco (gharb and saiss) covering 19 fields, to determine the distribution and importance of wrr. the results showed that wrr incidence ranged from 1-30% where the majority of the field had 10 to 20% disease incidences. laboratory isolation revealed that the major pathogens were fusarium oxysporum f.sp. ciceris (59.8%), rhizoctonia bataticola (32.7%) and r. solani (7.3%) and very low incidence of pythium spp. these results showed the need to develop cultivars resistant to fusarium wilt and dry root rot. keywords: chickpea wilt root rot, fusarium oxysporum fs ciceri, morocco. p133. induction of two pathogenesis related pr proteins in pear (pyrus communis) by pathogenic and nonpathogenic bacteria naima lemjiber¹*, abdelhai dihazi¹, nawal zhar², hicham jediyi², younes rakibi², wassila arkiz², and khalid naamani¹ ¹ laboratory of biotechnology and valorisation of plant resources, faculty of sciences semlalia, departement of biology, cadi ayyad university, marrakech, morocco; ² laboratory of biotechnology for valorisation and protection of agro-resources, faculty of sciences and techniques marrakech, departement of biology, cadi ayyad university, marrakech, morocco. *corresponding and presenting author: naima. lemjiber2012@gmail.com. abstract plants represent a rich source of nutrients for many organisms including bacteria, fungi, protists, insects, and vertebrates. although lacking an immune system comparable to animals, plants have developed a stunning array of structural, chemical, and protein-based defenses designed to detect invading organisms and stop them before they are able to cause extensive damage.the accumulation of pathogenesis-related proteins such as proteases and peroxidase was studied in two pear varieties, var.williams and var.louis bonne inoculated by pathogen and non-pathogen bacteria. the bacillus pumilus strain increased also the activities of peroxidase and proteases after inoculation. the obtained results highlight the potential rol of bacillus pumilus in induction of defense-related enzymes. keywords: pathogenesis-related (pr) proteins, proteases, peroxidase, bacillus pumilus. p134. antioxidant activity of crataegus monogyna from morocco chaymae benkirane1,2*, hakima bahri1§, bouchra tazi2, and samira oulahboub1,2 1 laboratory of genetic resources and plant improvement, ecole nationale d’agriculture de meknès, , bps 40, meknès, morocco; 2 laboratory of chemistry, department of basic sciences, ecole nationale d’agriculture de meknès, bps 40, meknès, morocco. *presenting author: chaymae.1.benkirane@gmail. com. §corresponding author: hbahri@enameknes. ac.ma. abstract antioxidants are tremendously important substances which possess the ability to protect the body from damage caused by free radicals induced oxidative stress. the aim of this work was to investigate the antioxidant effect of hawthorn (crataegus monogyna) collected from ifrane region (morocco) as a potential source of new bioactive natural compounds. hawthorn is a medicinal plant widely used in phytotherapy for the treatment of many cardiovascular diseases. in this study, samples of flowers, leaves, ripened and unripe fruits were analyzed. the antioxidant activity was measured by dpph (2,2-diphenyl1-picrylhydrazyl) free radical scavenging method. then, folin–denis and aluminum chloride colorimetric assay were used to determine respectively the total polyphenol and the total flavonoid contents of crataegus monogyna extracts. the results showed that all the plant parts possess important antioxidant properties. unripe fruits and flowers revealed the highest antioxidant activity with ic50 values of 7.3 and 8.3µg/ml respectively. total polyphenol content in different plant parts ranged from 105.1 to 280.4 gae µg/100 mg extract and total flavonoid from 4.7 to 70.8 qe µg/100 mg extract. antioxidant activity shows a significant correlation with total polyphenol content. these results indicate that crataegus monogyna exhibits a good antioxidant activity and thus can present a great potential as a dietary supplement or in pharmaceutical industry. keywords: crataegus monogyna, antioxidant activity, total polyphenol content, total flavonoid content. p135. impact of partial root-zone drying and water deficit on glucose, protein, polyphenols and dry matter contents of potato grown under greenhouse conditions said zantar1*, sliman elhani1, m. haddadi1, and mounir hassani2 1 nuclear techniques, environment and quality research unit. national institute of agronomic research (inra), 78 bd. sidi med ben abdellah 90010 tangier, morocco; 2 department of biology, polydisciplinary faculty of larache, university abdelmalek essaâdi. b.p 745, larache, morocco. *corresponding and presenting author: zantar_said@hotmail.com. abstract water shortage is the most important factor constraining agricultural production all over the world. potato (solanum tuberosum l.) tuber yield and quality are impacted by irrigation management. the aim of this study was to evaluate the effects of two irrigation techniques on quality parameters of potato grown under glasshouse conditions. these techniques are deficit irrigation (di) and partial root-zone drying (prd). di consists in irrigating the whole root system of the plant with an amount of water less than the crop evapotranspiration (etc). prd, in some way, is an evolution of di in which the root system is divided between two containers to alternate wetting and drying of the root system. prd and di plants were irrigated according to transpiration demand for controls and the amount of water was reduced to 20%, 30% and 50% for both irrigation strategies. the following parameters were studied to evaluate the effect of prd and di on the quality: glucose, protein, polyphenol and dry matter content. tubers from water stressed plants presented less contents of dry matter, glucose and proteins than the well-watered plants. under prd strategy, total polyphenols were reduced significantly only under severe water stress (50% less than etc). at the same irrigation level, prd technique was more advantageous compared to di in particular under moderate water restriction for total polyphenols, glucose and protein content. keywords: potatoes, deficit irrigation, quality, polyphenols, glucose, protein. p136. estimation of biochemical propreties and antioxidant activity of several moroccan plum (prunus domestical.) cultivars youssef ait bella*, said bouda, and abdelmajid haddioui§ laboratory of biotechnologies and valorisation of plant genetic resources, faculty of sciences and technics, sultan moulay slimane university, p. b. 523, benimellal, morocco. *presenting author: youssef. gvrl@gmail.com. §corresponding author: ahaddioui@yahoo.fr. abstract in this work, we report on the quality of 15 plum cultivars grown in different regions of morocco. plum (prunus domestica l.) is among the most important stone fruit crop in the world. in morocco, prunus domestica l. is considered an important tree crops and is cultivated in different climatic regions. however, there is little information on the biochemistry of plums of the cultivars growing in morocco. fruits are analyzed by determining several parameters such as total polyphenols, flavonoids, antioxidant activity, total soluble solids, vitamin c, titratable acidity, ash, humidity, dry matter, juice ph, total sugars, fibers and crude proteins. the results showed that plum cultivars are characterized by a high nutritional value. in the other hand, the results showed significant differences between cultivars for the majority of the estimated parameters: total polyphenols, flavonoids, antioxidantactivity, total soluble solids, vitamin c, titratable acidity, ash, humidity , dry matter,juice ph, total sugars and fibers. besides, the fruit mineral composition analysis (zn, p, k, mg, ca…) revealed very high significant differences among the cultivars. the cultivars lmozari and tabarkakacht exhibited respectively the highest total polyphenols value (9.39 mg/g) and a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 529 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) antioxidant activity (70.65 %) comparing with others introduced varieties. therefore, some local cultivars with a particular chemotype could be recommended to cultivate at large scale. keywords: prunus domestica l., biochemical parameters, characterization, cultivar. p137. humic substances produced during composting process of the olive mill wastes and municipal organic wastes farid barje* and mohamed hafidi laboratory of ecology and environment, faculty of science semlalia, cadi ayyad university, marrakesh, morocco. *corresponding and presenting author: fbarje@gmail.com. abstract the humics substances form an important fraction of soils’ organic compounds; their chemical nature has put forward various hypotheses on the mechanisms of their formation. in this context, to follow their evolution, the humic acids(has) and fulvic acids (fas) extracted at different stages of co-composting of olive mill waste and organic household refuse with added phosphate, were analyzed with 13c-nmr and ft-ir spectroscopy. the findings highlight the contrasting variations in the proportions of c-aromatic and ccarbonyl of has as the h/c atomic ratio changes. the comparative 13c-nmr study of the fas with the same tendancy, that show low aromatic (c=c) content in the initial stage of composting, which contained a greater proportion of carbonyl (c=o), o,n-alkyl and alkyl carbon. the ft-ir spectroscopy of has showed a decreasing absorption in bands of aliphatic bonds -ch-, -ch2-, carboxylic acids, esters, amides c=o, the c-o-bonds of polysaccharides and alcohols with respect to ether bonds c-o-c; they was linearly correlated with the h/c ratio. for the fas, the main absorption bands were in the region of 3393 cm-1 (oh), near 2931 cm-1 (aliphatic c-h), a distinct shoulder between 1717 cm-1 (c=o of carbonyl) and 1640 cm-1 (c=o of amide and ketone), 1547 cm-1 (aromatic c=c with conjugated c=o or amide n-h), near 1400-1384 cm-1 ( coostretch or oh deformation of cooh). the ratio of has to fas carbon content (cha/cfa), which is representative of the polymerization degree, showed a positive linear correlation (r =+0.56, p < 0.05) with the degree of aromaticity of has. the increase in the cha/cfa ratio with increasing humic acids aromaticity, is representative of a remarkable aspect of the restructuring brought, with respect to the mechanisms of polymerization during composting. keywords: humic acids, fulvic acids, composts, organic wastes. p138. mapping of forest fire in morocco by modis products issameddine zidane1*, rachid lhissou2, abdelali bouli1, and mustapha mabrouki3 1 laboratoire d’environnement et valorization des agro-ressources, department science de la vie, university sultan moulay slimane, beni mellal, morocco; 2 équipe de recherche en télédétection environnementale et nordique, institut national de la recherche scientifique, québec, canada; 3 laboratoire de genie industrial, university sultan moulay slimane, beni mellal, morocco. *corresponding and presenting author: z.issam.13@gmail.com. abstract as in the mediterranean countries, the forests in morocco are exposed to a recurrent risk of fires due to the extreme flammability of forest species during the summer. faced with this situation, the identification of burned areas and their monitoring are essential information to ensure the proper management and conservation of these ecosystems. this research focuses on the use of modis-terra’s medium spatial resolution satellite imagery in a semi-arid environment, particularly in the rif region, which is in first place in terms of the number of fires and the area burned on a moroccan scale. our main objective is to map burned surfaces during the summer of 2016 using the two nasa forest fire mapping products derived from modis images: the mcd64a1 and mcd45a1. for this purpose, we carried out a field campaign to collect data on the presence of the fire and the measurement of the burnt surface combined with that of the hceflcd. the validation and comparison to show that mcd64a1 remains the most reliable in terms of fire presence, with a surface area greater than 25 ha and r² greater than 0.7. keywords : wildfire, remote a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 530 sensing, burned area, mcd64a1, mcd45a1, modis. p139. biochemical characterization of several moroccan walnut (juglans regia l.) accessions kabiri ghizlane*, bouda said, elhansali mohammed, and haddioui abdelmajid§ laboratory of biotechnology and valorization of plant genetic resources, faculty of sciences and technics, sultan moulay slimane university, b.p. 523, beni mellal, morocco. *presenting author: kghizlane84@ gmail.com. §corresponding author: ahaddioui@yahoo.fr. abstract juglans regia l. is a significant fruit in the nut species. walnut has a high calorie level and rich nutrient composition. in morocco the walnut is cultivated in various environments mainly on mountainous, as a traditional crop. the aim of this study is to investigate and to compare the biochemical parameters of 11 moroccan walnut accessions. walnut kernels are analyzed by determining several parameters such as the dry matter, the moisture, the total ash, the total oil, the crude protein, the crude fiber, the carbohydrates, the energy value, the mineral composition (p, k, mg, na, cu, zn, mn, fe, cr, ni, b), the phenolic compounds, the total flavonoid and the antioxidant activity. the obtained results show that moroccan walnut is characterized by high nutritional value. on the other hand, the result showed significant differences between accessions for the majority of the estimated parameters, meaning of a high intra-diversity. this result should be exploited to select the best walnut accession. keywords : juglans regia. l variability biochemical – accessions. p140. evaluation of some nematophagous fungi isolates for their biological control potential against rootknot nematodes (rkn), meloidogyne hafsa tazi1,2*, m. ait hamza1, h. boubaker2, and a. el mousadik1 1 laboratoire biotechnologie et valorisation des ressources naturelles, faculté des sciences, université ibn zohr, bp 8106, agadir, morocco; 2 laboratoire biotechnologie microbienne et protection des végétaux, faculté des sciences, université ibn zohr, bp 8106, bp 8106, agadir, morocco. *corresponding and presenting author: hafssatazi@gmail.com. abstract the root-knot nematodes (rkn), meloidogyne spp., are serious threat to many cultures around the world, particularly greenhouse crops. nematicides are the most used method against plant parasitic nematodes. however, the majority of them were banned in developed countries because of their negative effects on both the environment and the human health. hence looking for other ecological and effective management strategies has become a necessity. biological control is considered the most relevant and least damaging approach as it is ecofriendly, economically viable and offers a sustainable and cost-effective alternative to chemical nematicides. the nematophagous fungi (nf) have been studied extensively as agents of biological control. this work aims the evaluation of nematicide activity of some nf strains isolated in nurseries. strains were isolated, including a large diversity of orbiliaceae with trapping organs and of toxic hypocreaceae. egg-parasitic and endoparasitic species were also observed. in vitro predation tests revealed that 7 isolates have increased the immobility of juveniles (j2).the most effective isolate was ht21 which increased the percentage of immobility up to 76% followed by ht1 and ht2 with 66 %,ht30 with 48 %,ht13 and ht14;29 %, 20% respectively and finally ht22; 12%. the other isolates have shown a percentage of immobility under 10%. the active fungal isolates are candidates for studies on identification of potential nematicides. hence the strains of fungi that have shown their efficacy against plantparasitic nematodes will be used as bionematicides. keywords: root-knot nematodes, biological control, nematophagous fungi, bionematicides p141. analysis of quality parameters related to the maturity of peaches and nectarines in the sais region ibtissame guirrou1*, a. mamouni2, a. mekaoui2, and k. elfazazi3 1 unité de recherche gestion des ressources naturelles, socio économie et qualité, qualipôle-agropolis, a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 531 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) inra, meknès, morocco; 2 unité de recherche amélioration des plantes et conservation des ressources phyto-génétiques, centre régional de la recherche agronomique de meknès, inra, morocco; 3 laboratoire de recherche en agroalimentaire, centre régional de la recherche agronomique de tadla, inra, morocco. *corresponding and presenting author: guirrouibtissame@yahoo.fr. abstract as a perishable fruit, peach and nectarine can only be consumed fresh or transformed. so the choice of the date of harvest is very important because it directly influences the quality of the fruits, knowing that it is during the maturation that elaborates the organoleptic quality of the fruits having a strong impact on the market value: size, visual appearance, odor, and fruit preservation. before doing the experimental work, we have done a survey in order to collect the information, concerning the variety range used and the distribution of the periods of maturity as well as the spread of the availability of the fruits on the market in the region of sais. the diagnosis of the varietal range has shown the presence of a great diversity (more than 200 varieties). we have found a dominance of varieties: patty, gypsum, azurite for peaches and carolina, turquoise, emerald and zephyre for nectarines. the fruit maturity is spread over 5 months, from early may to late september. some experimental analysis has been done in order to focus on the identification of fruit quality parameters. the parameters studied are the measurement of size/ weight, firmness, color, moisture content, refractometric index (brix), acidity, ph and total phenolic content. in order to cover the maximum, for each variety, we have practiced between 5 and 6 passages more than what is practiced by the farmer (3 passages) for 3 exploitations at sais region. the results showed that early varieties have a smaller size, firmness and sugar content compared to seasonal and late varieties. while, late varieties have a relatively low acidity compared to early and seasonal varieties and therefore ph values > 4 classify them as non-acidic fruits. furthermore, it has been shown that the total phenolic compounds are not influenced by the season (early and late). these compounds are probably determined by the genetic potential of the variety. indeed, nectarines have a slight wealth compared to peaches. keywords: peaches/nectarines, maturity, quality parameters, early/seasonal/late varieties. p142. in-vitro synergic activity between essential oils and methanolic extracts of two medicinal plants from algeria elkolli hayet1* and elkolli meriem2 1 laboratoire des matériaux polymériques multiphasiques, département de génie des procédés, faculté de technologie, université ferhat abbas, setif 1, 19000, algeria; 2 laboratoire de valorisation des resources biologiques naturelles, faculté des sciences de la nature et de la vie, université ferhat abbas setif 1, 19000, algeria. *corresponding and presenting author: kolli_h@yahoo.fr. abstract due to the increased interest in alternative natural substances (essential oils and plant extracts) for their low toxicity nature, their combinations or mixings represented a potential new strategy for developing new anti-oxidant therapy. the use of combinations thus new approaches to increase their efficacy, taking advantage of their synergistic and additive effects. the purpose of this review is to provide an overview on the efficacy of these combinations. so we have investigated antioxidant activity of individual essential oils and methanolic extracts, then we have studied the antioxidant activity of their combinations. all the combinations gave a synergical effect. according to the ic50 parameter, the radical scavenging capacities are ranked in the following descending order: b. incrassatum eo> b. alpinum eo > eo mixing> quercetin> rutin> bht> b. alpinum ext > ext mixing > b. incrassatum ext. keywords: essential oil, methanolic extract, bunium alpinum, bunium incrassatum, combination, antioxidant activity, synergy. p143. effect of a sulla-based diet (hedysarum flexuosum) on the productivity and milk quality of local beni arouss goat mohamed ayadi1*, a. errassi2, m. chabbi2, and s. zantar1 1 laboratory of materials and valorization of rea tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 532 sources, faculty of science and technology, university abdelmalek essaadi, tangier, morocco; 2 national institute of agricultural research, 78 av. sidi mohamed ben abdellah, tangier, morocco. *corresponding and presenting author: ayadimohamed004@gmail.com. abstract productivity, chemical composition and fatty acid profile of milk were studied in order to highlight the effect of the incorporation of sulla (hedysarum flexuosum) into the diet of beni arousse goats in comparaison with alfalfa (medicago sativa). 14 goats were divided into two homogeneous groups and milk collection was carried out weekly. monitoring milk production during the first three months of lactation for each batch (n=7) has shown that the use of sulla as main food has affected significantly and positively the productivity of milk (+ 15%). no significant variation was recorded for ph and acidity. on the other hand, the analysis of the chemical composition using milkoscan revealed that the milk from the test batch is characterized by a slightly higher average content of lactose (5.23 vs 5.13%) and defatted dry extract (10.31 vs 10.12%), while the protein and fat content were not significantly affected (p> 0.05), knowing that the price of milk is estimated essentially from these two parameters. analysis by gas chromatography showed that the incorporation of sulla has beneficial effects on the fatty acid profile of the milk and which can be summarized essentially by a significant decrease of 10.51% for palmitic acid content (c16:0) and a significant increase of 28.99% in the family of omega-3 fatty acids which are considered as highly beneficial. in fact, these results show that the exploitation of hedysarum flexuosum appears as a promising strategy for improving the quality of the local goat’s milk. keywords : goats, lactation, sulla, chemical composition, fatty acids. p144. identification and pomological evaluation of apple (malus domestica borkh.) cultivars in morocco youssef khachtib*, said bouda, and abdelmajid haddioui§ laboratory of biotechnologies and valorization of plant genetic resources, faculty of sciences and techniques, sultan moulay slimane university, p. b. 523, beni mellal, morocco. *presenting author: yosf5achtib@gmail.com. §corresponding author: ahaddioui@yahoo.fr. abstract apple (malus domestica borkh) is the second most important tree fruit crop of the rosaceae family in terms of cultivated area in morocco, after almond tree; but global apple production involved only a few numbers of cultivars. besides, the role of local cultivars and landraces in large-scale production has become completely insignificant in front of the introduced ones. in order to identify and evaluate the genetic resources of apple in morocco, several prospecting were done and leading to collect 29 cultivars throughout its cropping area. 32 pomological traits were considered to analyze the phenotypic variability of the cultivars. the results of the analysis of variance showed very high significant differences between cultivars for almost all measured parameters, implying the existence of high phenotypic variability in moroccan apple cultivars. fruit shape, weight, length, and texture were revealed to be the most discriminating traits between analyzed cultivars. the autochthonous cultivars have shown the lowest values of considered characters compared with modern cultivars (introduced). this is clearly appeared with local lahlou cultivar that obtained the lowest values of fruit weight, fruit height and fruit size. nevertheless, the local and introduced apple cultivars were gathered in one group, according to principal components and hierarchical analyses, suggesting that they have had a common ancestor and a common genetic basis. furthermore; some mislabeling of apple cultivars could be detected. the results of this study are discussed in the light of how this variation within traditional cultivars can serve as an interesting potential of biodiversity for future apple breeding and germplasm conservation programs. keywords: apple, local cultivars, introduced cultivar, pomological, variation. p145. biocide effect of ammi visnaga (l) and asphodelus cerasiferus gay. powder on callosobruchus maculatus f. (coleoptera, bruchidae) fouzia mgamat1*, ahmed boughdad2, fatima douiri3, rachid zohair3, el ayadi rachida1, and hamid el ibaoui1 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 533 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 1 nutrition, health and environment laboratory, departement of biological sciences, faculty of sciences, ibn toufail university, kenitra, morocco; 2 departement of zoology, enam, meknes, morocco; 3 departement of biological sciences, faculty of sciences , my ismail university, meknes, morocco. *corresponding and presenting author: mg.fouzia@yahoo.fr. abstract in the perspective of looking for alternative methods of fighting a pest of stored products, it is proposed to evaluate the potentialities of ammi visnaga (l) and asphodelus cerasiferus gay. powder as botanical insecticides for the protection of chickpea seeds (cicer arietinum) against callosobruchus maculatus (fab). thus, 50 seeds of the chickpea were dusted separately and respectively with the powder of khella seeds and the roots of asphodel at the rate of 0; 0.16, 0.32, and 0.64 g for a. visnaga, and 0, 0.12, 0.23 and 0.46g for asphodel. the treated seeds were then presented to 10 pairs of newly emerged c. maculatus. three repetitions were done within each lot. the results showed that the powders of a. visnaga and a. cerasiferus did not affect neither the longevity, the fecundity nor the success rate of c. maculatus. keywords : cicer arietinum, ammi visnaga, asphodelus cerasiferus, botanical insecticides, stored products. p146. responses of stomatal conductance, leaf water potential and water-use efficiency in potato to partial root-zone drying and deficit irrigation under glasshouse conditions sliman elhani1*, said zantar1, and maroua haddadi1 1 nuclear techniques, environment and quality research unit. national institute of agronomic research (inra), 78, bd. sidi med. ben abdellah, tangier, morocco. *corresponding and presenting author: sliman_ elhani@yahoo.fr. abstract partial root-zone drying (prd) is a deficit irrigation (di) technique which consists in dehydrating half of the root system whereas the other half is well irrigated, so that abscisic acid produced in the drying roots reduces stomatal conductance and limits transpiration. aiming at comparing this technique to di under glasshouse conditions, an experiment was conducted on potato (solanum tuberosum l.) to evaluate stomatal conductance, leaf water potential (ψl) and water-use efficiency (wue). prd and di plants were irrigated on transpiration demand determined by the weight of the pots. treatments consisted of four levels of water supply for each irrigation strategy: 100%, 80%, 70% and 50% of crop evapotranspiration (etc). results showed no significant differences in tuber yield between irrigations strategies whereas statistically significant differences were found among treatments. prd strategy reduced stomatal conductance and increased leaf water potential. wue was higher under prd but did not show statistically significant differences with di. using the same amount of water, prd strategy allowed better yield and positive physiological responses to water restriction. keywords: potato, partial root-zone drying, stomatal conductance, leaf water potential, wue. p147. agronomic and molecular characterization of a collection of faba bean (vicia faba l.) chetto oumaima*, belqadi loubna, and arbaoui mustapha production, protection and plant biotechnology department, hassan ii institute of agronomy and veterinary medicine, rabat, morocco. *corresponding and presenting author: oumaima.chetto@gmail.com. abstract despite the importance of faba bean (vicia faba l.) in morocco, its acreage has decreased due to a number of biotic and abiotic constraints but also due to the small number of varieties available. in order to contribute to the genetic improvement and the creation of new varieties, a study of phenotypic and molecular diversity was carried out on a large number of moroccan and foreign accessions. this plant material was evaluated on the basis of nine agro-morphological traits in two locations; taounate and tiflet within the framework of seminov’s breeding program. in addition, the collection was analyzed using the molecular marker srap with six primers, in a laboratory of iav hassan ii. the study of diversity revealed a high polymorphism with an extremely significant environmental a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 534 effect as well as a genotype effect for the majority of agro-morphological traits. on the basis of these same features, distance analysis revealed that accessions of foreign origins are distant from moroccan accessions and constitute an isolated cluster. the mediterranean genotypes from spain and egypt, fit in among the moroccan accessions. furthermore, this study also contributes to develop the srap marker technique on agarose gel to evaluate genetic diversity. this characterization revealed an average polymorphism rate of 86.2%. the analysis of distance also revealed a grouping which fits partially with the phenotypic analysis results. the association between the srap markers and the quantitative traits revealed the presence of some significant associations to more than 95% (tstudent): m3-e1-5, m3-e2-1, m3-e2-2, m1-e2-2 and m1-e2-4 markers seemed to be associated with the orobanche tolerance; the yield is associated with m3e1-4, m3-e2-6 and m2-e6-3 markers; and the tgw to m2-e6-3 and m3-e1-6 markers. additional studies should be carried out on a larger number of genotypes to validate those molecular markers for their potential use in marker-assisted selection. keywords: vicia faba l., srap, genetic diversity, marker – trait association p148. optimization of controlled pollination in argan tree (argania spinosa l.) naima ait aabd*, mohamed boujghagh, rachid bouharroud, abdelghani tahiri, and abdelaziz mimouni national institute of agronomic research, crra agadir, bp 124, inezgane, agadir, morocco. *corresponding and presenting author: aitaabdnaima@gmail. com. abstract pollination is a critical stage in sexual plant reproduction and is a key step to assure fertilization and seed production for many plants. the argan tree is a monoecious species, with hermaphrodite flowers, alone or grouped in glomeruli. in fact, the pollination system of argan tree is similar to several angiosperms with more frequent insect pollination in natural conditions. however, few studies assess the quantitative field assessment of pollination modes and sexual breeding system of argan tree. in this work different steps have been optimized to assess self and cross-pollination experiments in argan tree in order to evaluate the reproductive biology and directed crosses in breeding programs. hence, we report the sexual and breeding system of 23 trees in inra orchard installed since 2010 in mzd, agadir. the breeding system was determined by hand, self and cross-pollination experiments. inflorescences were selected, bagged and the following treatments were assigned to each of the selected branch. open/natural pollination (control), autogamy (isolated pollination), hand self-pollination and hand cross-pollination. the obtained results show strength of incompatibility within trees and a. spinosa is largely self-incompatible. the trees showed high heterozygosity in floral and seed development. likewise, the obtained results are useful to optimize controlled pollinations in argan tree and can help to increase fruit production in breeding programs and avoid the incorrect interpretation from pollination experiments in studies of reproductive biology. keywords: arganiaspinosa, hand pollination, cross-pollination, self-incompatible, seed production, breeding system. p149. screening of actinobacteria from sugar beet soils in beni mellal region for their phosphate solubilizing capacity yassine aallam*, meriam bousselham, abdelmajid haddioui, and hanane hamdali§ laboratory of genetic and plant biotechnology, faculty of sciences and technology, university of sultan moulay slimane, b.p: 523 beni mellal, morocco. *presenting author: yassine.aallam@gmail.com. §corresponding author: hamdali_hanane@yahoo.fr. abstract agriculture will have to face the challenge of meeting the growing demand for food, which requires intensive application of chemical fertilizers like phosphate which is the second most important nutrient for plant growth, but its availability is often reduced. therefore, high quantities of expensive chemical soluble p-fertilizers are added to soil. therefore, it is becoming a necessity today to develop a modern and sustainable agriculture with alternative methods that are less expensive, less polluting but equally effeca tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 535 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) tive. like the direct use of phosphate rock (pr) as an alternative to chemical p-fertilizers associated to phosphate rock-solubilizing actinobacteria (prsa) in order to increase pr reactivity. therefore, the main objective of this work was to obtain competent prsa presenting other pgpr-associated traits to be used for the development of an inoculant to improve sugar beet growth and p nutrition. in this study, four different sugar beet soils of beni mellal region (morocco) were explored for the presence of phosphate solubilizing actinobacteria. 164 strains of actinobacteria were isolated and tested for their ability to solubilize a phosphate rock (pr). among tested isolates, more than 34% (57 strains) showed growth on synthetic minimum medium (smm) containing insoluble pr and 16% (27 strains) on smm containing tricalcium phosphate (tcp) as unique phosphate source. twenty isolates showed the most active growth and solubilization ability in the presence of pr and tcp in broth cultures. thus, this work could contribute to select some actinobacteria as potential prsa for use in low-input agriculture in low-p soils amended with pr. keywords: actinobacteria, sugar beet, soils, screening, phosphate rock solubilization, beni mellal. p150. modeling and simulation of convective solar drying of sewage sludge toky a. a. arisily1*, said hachimi2, abdellatif djerrari1, and ali hajji1 1 process engineering and environment research unit, institut agronomique et vétérinaire hassan ii, rabat, morocco; 2 ecole nationale supérieure des mines, university mohamed v, rabat, morocco. *corresponding and presenting author: tokyarisily@gmail.com. abstract in recent years, solar sludge drying has attracted more and more communities, including in morocco, due to its low operating costs and its respect for the environment. the objective of this work is to develop a lumped parameters dynamic model for convective solar drying of sludge from a waste water treatment plant (wwtp), then to simulate and validate the model with the experimental data and results found in the literature. a mathematical model of this process will make it possible to optimize the design and the exploitation of these driers, as well as the control of the characteristics of the sludge produced according to end-use or disposal chosen. first, based on the literature, a brief discussion is presented on some models, heat and mass transfer parameters, and sludge drying kinetics. secondly, a lumped parameters dynamic model representing the different exchanges of heat and mass within the solar dryer is developed. the finite explicit difference method is used for solving the equations. these equations have been programmed in excel vba to form a simulation tool. third, the experimental data and results of amadou (2007) were used for simulations and validate the established model. a good agreement was found between the experimental results presented by the author and those given by the model. keywords: sewage sludge, solar drying, modeling, simulation, wwtp. p151. genetic diversity of faba bean (vicia faba l.) landraces in the moroccan rif region using molecular aflp markers nora aqtbouz1*, lamiae ghaouti1, loubna belqadi1, and wolfgang link2 1 département de production, protection et biotechnologies végétales, institut agronomique et vétérinaire hassan ii, b.p. 6202, rabat-institut, rabat, morocco; 2 institute of agronomy and plant breeding, von siebold straße 8, 37075 göttingen, germany. *corresponding and presenting author: noraaqtbouz@ gmail.com. abstract faba bean (vicia faba l.) is the most important grain legumes grown in morocco. this crop is mainly produced based on locally maintained varieties and seeds (about 98 %), following an informal system of seed production and supply. these local varieties are characterized by a huge genetic diversity and are remarkably well adapted to the traditional agroecosystem and farming system. assessment of genetic diversity is the first step of every plant breeding program. in fact, use of new methods for studying genetic diversity seems important. the objective of the present study was to describe the genetic diversity of a representative collection of faba a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 536 bean local populations originating from four regions in the province of taounate, using the amplified fragment length polymorphism (aflp) molecular marker. four aflp primer combinations were used on 180 samples of faba bean local populations. a total of 139 amplified dna fragments, varying in size from 60 to 380 base pairs, were revealed. from those, 84 (60.43%) were polymorphic. the amova analysis showed significant levels of variation among and within the four regions analyzed. a large extent of variation occurs within regions (98.70%). the upgma dendrogram constructed based on the jaccard’s similarity coefficient indicated no clear specific pattern or grouping among these samples analyzed according to their geographical origin. all faba bean samples were displayed in one main cluster, divided in subgroups, which represent some degrees of similarity. keywords: faba bean, local population, aflp, genetic diversity, upgma, jaccard similarity coefficient. p152. occurrence of faba bean (vicia fabae l.) diseases in five regions in morocco krimi bencheqroun sanae1*, aouzal sarra1,2, lhaloui saadia1,3, and seid ahmed3 1 national institute of agricultural research (inra), settat, morocco; 2 mohamed v university, fstsettat, morocco. 3 international center for agricultural research in the dry areas (icarda), rabat, morocco. *corresponding and presenting author: krimisanae@ gmail.com. abstract survey were conducted during three successive cropping seasons 2013, 2014, 2015 in five major faba bean growing regions in morocco (abda, doukkala, saiss, zemour zair and chaouia). the aim of this study is to find out the occurrence and distribution of the principal cryptogamic diseases of faba bean affecting yield and quality of cropping. the results showed that chocolate spot (botrytis spp.) was the most widespread and destructive disease with high incidence in all surveyed regions. ascochyta blight (ascochyta fabae) and rust (uromyces fabae) were also important diseases mainly in humid regions (saiss and zemmour zaer). in some fields, the late infection of ascochyta blight in the season had affected yield and seed quality. other diseases of minor importance were recorded at low incidence levels in some regions were downy mildew (peronospora viciae) and root rots. therefore, screening for resistance to the major diseases observed in this study has to be intensified within faba bean breeding program in morocco. p153. solubilization of organic and inorganic phosphate by soil bacterial isolates nabil tirry1*, g. laghmari1, b. benbrik1, r. bouamri2, w. bahafid1, and n. el ghachtouli1 1 laboratoire de biotechnologie microbienne, faculty des sciences and techniques, université sidi mohammed ben abdellah, fès, morocco; 2 département de protection des plantes et de l’environnement, ecole nationale d’agriculture de meknès, morocco. *corresponding and presenting author: nabil.tirry@usmba. ac.ma. abstract phosphorus (p) is one of the major essential macronutrients for plant growth and development. however, p in soil is generally present in two main insoluble forms: mineral forms, such as apatite, hydroxyapatite and oxyapatite, and organic forms including inositol phosphate, phosphomonoesters, phosphodiesters and phosphotriesters. microorganisms are capable of converting insoluble phosphate into a bioavailable form through solubilization and mineralization processes. hence, in the present study fifty-seven phosphate solubilizing bacteria were isolated from tree different agricultural sites in morocco using nbrip-agar containing tricalcium phosphate (tcp) as the phosphate source. the isolates were able to solubilize tcp in nbrip liquid medium, with different capabilities (up to 190 µg/ml). p solubilization was accompanied by a decrease in the ph of the medium. crude alkaline and acid phosphatase activities of the strains were determined by p-nitrophenyl phosphate assay. 59 % of the isolates showed alkaline phosphatase activity, 72 % showed acid phosphatase activity, while 49 % displayed both alkaline and acid phosphatases activities. the isolates were tested for other pgp traits. results showed that 71.92 % of the bacterial isolates are able to produce siderophores, 45.61 % produce hcn and all isolates produce aia (up to 500 µg/ml). a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 537 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) the screening for drought tolerance revealed that 4 isolates were able to grow well on the luria–bertani medium containing up to 18 % nacl (w/v). based on the multifunctional properties, twelve isolates were selected to determine the effect on germination and growth on wheat seeds. the outcome of this research proves plausible practical applicability of these pgpb for crop production in soils of saline and arid environments. keywords: phosphate solubilization, plant growth-promoting rbacteria (pgpb), alkaline phosphatase, acid phosphatase, salt stress, seed germination. p154. characterization of soil degradation in northern morocco abderrahim el boukhari1*, mustapha naimi2, mohamed chikhaoui2, and damien raclot1,3 1 institut agronomique & vétérinaire hassan ii, po. box. 6608, madinat al irfane, rabat, morocco; 2 department of natural resources & environment, institut agronomique & vétérinaire hassan ii, po. box. 6608, madinat al irfane, rabat, morocco; 3 research institute for development (ird), umr lisah, visiting at institut agronomique & vétérinaire hassan ii, rabat, morocco. *corresponding and presenting author: abderrahimelboukhari@gmail.com. abstract water erosion is a major process of soil degradation. in morocco, it is considered the main cause of desertification. it leads to a reduction of the productive potential of soils and the siltation of dams reservoirs leading to a loss of the volume of mobilizable water and calling into question the durability of these hydraulic structures. negative effects are reflected in the areas of power generation, drinking water supply and irrigation. faced with the scale of this scourge, morocco urgently needs to multiply its interventions to assess the consequences of land degradation and also to develop appropriate conservation programs. in this context, a sample of 15 dams located in the northern region of morocco was selected to analyze siltation over periods of 8 to 47 years. the bathymetric data were provided by the drpe (directorate of research and water planning state secretariat for water), associated with this study. the present paper aims toi) characterize the specific degradation (ds) of the northern region of morocco through the study of 15 watersheds; ii) to deduce a range of variability of ds in this region; and iii) to evaluate the relevance of simple models based on the surface of the basin or the combination of the surface and the anual average water slide, to predict the ds. the approach adopted is based on the confrontation of an updated measure of the specific degradation derived from bathymetric surveys with assessments of the latter using established models to predict the siltation of dams in morocco. over periods ranging from 8 to 47 years, the review of the siltation of dam reservoirs by analyzing of bathymetric surveys shows an average total annual about 0,6*109 m3, representing a 13% of the initial total capacity that is of the order of de 4,6*109 m3 and a variability of the specific degradation ranging from 172 to 5365 t km-2 an-1, whether a ratio of 1 to 31. finally, this study shows the very strong variability of the prediction performance of the specific degradation based on simple models using the basin surface and the water slide. keywords: water erosion, dams, siltation, specific degradation. p155. interest of the use of wastewater in industrial plants ‘energy value’. case of jatropha curcas ouafae mokhtari1*, hassan el halouani1, kamal merghem1, imade hamdani2, abderrahim lahrach3, and belkhir hammouti4 1 laboratory of water science, environment and ecology, department of biology, faculty of sciences, university mohammed premier, oujda, morocco; 2 laboratory of applied analytical chemistry, materials and environment, faculty of sciences, university mohammed premier, oujda, morocco; 3 laboratory of geo-resources and environment, sciences and technologies faculty, university sidi mohammed ben abdallah, fez, morocco; 4 laboratory of applied analytical chemistry, materials and environment, faculty of sciences, university mohammed premier, oujda, morocco. *corresponding and presenting author: mokhtari.ouafae@gmail.com. abstract to assess the interest of the use of wastewater on the cultivation of jatropha curcas and the role of the lata tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 538 ter in the conservation of water and soil and in the production of renewable energy, tests were conducted under semi-arid climatic conditions of northeastern morocco (oujda). the aim is to monitor the growth and behavior of jatropha curcas irrigated with different types of conventional waters and unconventional (including the salinity may be elevated up to 2.82 mmhos / cm). the performances of growth of jatropha curcas l. were studied under different types of water of irrigation (wastewater, fertigation, freshwater) and others without irrigation (in rainfall regime). the results showed: (1) the significant effect of wastewater on the vegetative development and yield (85%, p <0.3%), (2) acceleration of the development cycle and formation up to 3 peaks of inflorescences per year, (3) the application of water stress significantly reduced the growth and yield of jatropha relative to the irrigated plants (a degree of 90%, p <0.2%). its development is slow, and the first flowering is largely delayed (8 months) only after the arrival of the first rains of november, (4) the fertigation has improved the performance of jatropha and accelerated the process of reproduction (3 peaks of inflorescences). these results are very similar to those obtained after irrigation with treated wastewater which is only another way of fertigation but much cheaper, and (5) jatropha curcas shows proper installation despite the high salinity of the soil (5,89mmhos/cm to 7 mmhos / cm). this shows a perfect adaptation of the plant to the different soil types: marginal, skeletal and fertile. the production of jatropha curcas is closely related to water intake, fertilizers, soil quality and climate. based on these results, the association of wastewater with jatropha curcas can be a means of: fight against desertification and erosion, of valorization of the marginal grounds by using jatropha curcas like a reforestation plant, what will contribute to the sustainable development of the country by the increase in the national heritage in terms of biodiversity, and in terms of renewable energy “a source of green energy”. keywords: waste waters, fertigation, jatropha curcas, reforestation, sustainable development. p156. development of an in vitro micropropagation system for carob (ceratonia siliqua l.) assia lozzi1*, rachid mentag2, rabha abdelwahd2, and abdelhadi abousalim1 1 department of crop production, protection and biotechnology, institute of agronomy and veterinary medicine hassan ii, rabat, instituts-6654, morocco; 2 biotechnology unit. regional center of agricultural research. national institute of agricultural research, rabat415, morocco. *corresponding and presenting author: assia.lozzi@gamail.com. abstract the carob tree (ceratonia siliqua l. fabaceae), is one of the most important component of mediterranean vegetation for economic and environmental reasons. the economic importance of this species arises from the industrial use of the locust bean gum obtained from its seeds, which is widely used as a natural additive (e-410) in the food industry to function as a flavor, stabilizer and thickener. the world production of carob pods is estimated at about 156,800 tons/year, the main producers are spain (22.8 %), italy (20.1 %), morocco (14 %), portugal (13.9 %) and turkey (fao, 2014). in morocco, a number of ambitious projects promoting the cultivation and exploitation of this genetic resource, have been launched under the green morocco plan. as the traditional carob propagation methods failed to meet the market request, the use of in vitro techniques seems appropriate to fulfil the increased demand for carob plant material. in vitro micropropagation can be applied for massscale production by a commercial outfit and may represent a good alternative methodology to the traditional propagation techniques. during our studies on carob micropropagation, a number of parameters have been investigated to optimize an appropriate protocol for mass propagation of this important plant species. the obtained results are promising and would set a basis for the further development of carob propagation. keywords: ceratonia siliqua, carob, micropropagation, in vitro. p157. soil fertility management in the irrigated perimeter of beni amir, morocco houria dakak1*, a. zouahri1, h. sehlaoui1,3, a. douaik1, a. ghanimi2, h. yachou1, and r. hassikou3 1 national research institute of agricultural research, crrar, research unit on the environment and conservation of natural resources b.p. 6356, rabat, moa tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 439 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) rocco; 2 university mohammed v, faculty of sciences, department of chemistry, rabat, morocco; 3 university mohammed v, faculty of sciences, department of biology, mycology and environment, rabat, morocco. *corresponding and presenting author: dakak_h@ yahoo.fr. abstract this work contributes to the rationalization of fertilizer use in order to sustainably manage soil fertility and limit water contamination. thus, the optimal nitrogen dose was determined on the basis of a calibration test that was installed under the cultivation of soft wheat at the farmer’s farm in the beni amir region, irrigated area of tadla. in addition, the statistical analysis showed that the relationship between grain yield and nitrogen dose was parabolic (quadratic), which made it possible to determine the optimal dose (150 kg / ha) which allowed the best grain yield, on the other hand the higher dose (200kg / ha) did not improve grain yield which constitutes a waste of unnecessary money and a risk of pollution by the excess of nitrates which have not been valued for enable better performance. keywords: nitrogen fertilization, rationalization, experimentation, optimal dose, wheat culture p158. psyttalia concolor (hymenoptera: braconidae) endoparasitoïde of ceratitis capitata (diptera: tephritidae): parasitism rate on argan ecosystem abdelhadi ajerrar1,2*, r. bouharroud2, m zaafrani1, b chebli1, a. amarraque2, r. qessaoui1,2, and el h. mayad3 1 equipe génie de l’environnement et de biotechnologie, ecole nationale des sciences appliquée, agadir, morocco; 2 centre régional de la recherche agronomique d’agadir, inra-morocco; 3 faculté des sciences ibn zohr, agadir, morocco. *corresponding and presenting author: ajirrar@gmail.com. abstract the mediterranean fruit fly, ceratitis capitata wiedmann is the most important pest of several fruits plants in the mediterranean area. argan trees (argania spinosa) known as a reservoir of this pest. psytallia concolor is the only ceratitis capitata’s endoparasitoïde known in morocco. the purpose of this study is to assess the parasitism rate of ceratitis capitata’s endoparasitoïde in souss region. fruits sampling were carried out monthly depending on fruit availability. fruits collected were immediately transported to the laboratory then incubated in ventilated boxes containing sand at 25 +/2 °c and 70 +/5% of relative humidity. the results obtained shows that psyttalia concolor activity’s begin early on december when the first ripe fruits appeared, but parasitism rate is very low during december, january and february (less than 1%). this rate were gradually increased to reach its maximum during june (6,07%). keywords: ceratitis capitata, endoparasitoid, argan, psyttalia concolor, souss. p159. genetic characterization and relationship analysis among a collection of barley cultivars solange nyiraguhirwa1,2,3,4*, zara grana1,2,3, fatima henkrar1, hassan ouabbou2, ibriz mohammed3, sripada m. udupa1§ 1 international center for agriculture research in dry areas (icarda), rabat, morocco; 2 institut national de recherche agronomique (inra) rabat, morocco; 3 ibn tofail university, faculty of sciences, kenitra, morocco; 4 institut national de recherche agronomique (inra), settat, morocco. *presenting author: hirwaso@ gmail.com. §corresponding author: s.udupa@cgiar. org. abstract barley is an important cereal crop in morocco and is used for animal feed, and human consumption (mainly as bead and malt). morocco is the 7th largest barley producer in the world. characterization and deployment of barley genetic diversity is the key for barley improvement. new sources of genetic diversity are being identified and deployed in the barley breeding programs to enhance grain yield, quality, and tolerance to abiotic and biotic stresses. here we characterize 113 barley accessions from 14 countries: morocco, algeria, tunisia, egypt, ethiopia, libya, nepal, italy, pakistan, yemen, syria, spain, afghanistan, mongolia using 14 ssr markers and show that this barley collection is genetically diverse. the a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 540 analysis estimated genetic diversity and pic values of 0.85 and 0.83, respectively, and detected 75 alleles indicating high degree of genetic diversity. both neighbor joining dendrogram and principal component analysis identified 2 major groups within the analyzed accessions, namely moroccan accessions vs. others. the results of this study have implications in genetic resources collection, conservation and utilization. keywords: genetic diversity, ssr markers, barley, horedum vulgare, differents origin. p160. assessment of transmission risk of arboviruses in morocco amal bennouna1*, t. balenghien2, h. elrhaffouli3, g. chlyeh4, and o. fassi fihri1 1 laboratoire de microbiologie, département de pathologie et santé publique vétérinaire, immunologie, maladies contagieuses, institut agronomique et vétérinaire hassan ii, rabat, morocco; 2 umr cirad / inra, contrôles des maladies animales, montpellier, france; 3 laboratoire de recherche et de biosécurité p3. hôpital militaire d’instruction mohamed v de rabat, morocco; 4 département de production, protection et biotechnologies végétales. unité de zoologie. institut agronomique et vétérinaire hassan ii, rabat, morocco. *corresponding and presenting author: amal397@yahoo.fr. abstract global changes, including international trade and travelling, have induced a recrudescence of vectorborne diseases, with an important change in their distributions. north africa is at risk of arbovirus emergence, because it links the mediterranean basin, where exotic vectors, such as aedes albopictus, are spreading leading to dengue or chikungunya outbreaks, and the sahel known as endemic area for some arboviruses such as rift valley fever (rvf), which is an acute viral zoonotic disease affecting both humans and domestic animals, and mostly transmitted by mosquitoes. morocco should be considered as risk of rvf emergence due to its proximity with mauritania where the disease is endemic. this work aimed to identify potential vectors of rift valley fever and to characterize the vector role of culex pipiens, a species known to carry this disease. we carried out transversal studies across the whole territory, by collecting adults and larvae with a standard protocol in the aim to have an estimation of the species abundance. we carried out adults and larvae collection in more than 140 different sites (urban, suburban and rural) during the springs 2015 and 2016, with a special interest for the south of morocco, considering the proximity of mauritania. it is the first recent and extensive prospection of culicidae in this part of the country. the main abundant collected species were: culex pipiens, culex theileri, culex simpsoni and culiseta longiareolata. aedes species could be underestimated due to the timing of collections. however, we identified for the first time in morocco the asian tiger mosquito aedes albopictus. specimens were identified by both morphology and molecular assay. the population located in rabat, capital of morocco, has overwintered during 2015/2016 demonstrating for the first time the establishment of this species in north africa. we after proceeded at a molecular analysis to characterize the vector role of culex pipiens by looking for all viral communities harbored by this species. this study is still in process to describe the population of pathogenic arboviruses hosted by culex pipiens. keywords: vector-borne diseases, emergence, arboviruses, culex, aedes. p161. assessment of flammability of moroccan forest fuels: new approach to estimate the flammability index salaheddine essaghi1,2,*, m’hamed hachmi², mohammed yessef1, mohammed dehhaoui1, and fahed el amarty2 1 institut agronomique et vétérinaire hassan ii, bp 6202, rabat-instituts, rabat, morocco; 2 ecole nationale forestière d’ingénieurs, bp 511, tabriquet, 11015 salé, morocco. *corresponding and presenting author: s.essaghi@gmail.com. abstract a new flammability index (fi) was developed, which integrated two parameters that are highly correlated to fuel moisture content (mc). these parameters are time-to-ignition and flame height. the newly obtained fi-values belong to the variation interval of {0; 20}. in addition to the six flammability classes defined in a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 541 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) the earlier work, a seventh class (fi > 16.5) was proposed to include fuel species with a high content of volatile flammable-compounds. flammability testing and mc measurement were performed at a range of mc obtained through a drying process of samples. as a result, fi was statistically highly correlated with mc for all 13 moroccan forest fuels tested in this study. following this, linear regression equations were established to predict the fi-value as a function of mc. therefore, the classification of flammability would depend on the species as well as the mc-value of the samples and the season in which they were collected. keywords: flammability testing method; flammability classification of fuels; correlation. p162. agro-morpho-phenological characterization of an interspecific f3 quinoa population (chenopodium quinoa wild) asmaa allaoui*, eric nathan jellen, and ouafae benlhabib iav hassan ii, rabat, morocco. *corresponding and presenting author: ipiapesa@iav.ac.ma. abstract multiple biotic and abiotic constraints impact the world food production. according to the fao, quinoa would be a good ally to fight against hunger and malnutrition for its high nutritional value and its adaptation to harsh agro-ecological zones. this investigation aims to characterize a population of 89 irl-3 quinoa interspecific lines developed from a natural cross between a chenopodium quinoa accessions co407and a wild accession (11-54) (chenopodium berlandieri) known for its adaptation to high temperatures and its resistance to pests. a completely randomized block design trial with three replications was conducted between february and july 2017 on an experimental plot at the agronomic and veterinary institute hassan ii of rabat. eleven quantitative and six qualitative traits related to the morphology, phenology and production potential were recorded. the analysis of the population descriptors illustrates clearly the large variability of the traits and the accessions. days to maturity fluctuated between 133 and 170 days, as was the variability of the seed and the inflorescencediameter and color, the plant height, the yield and its components. the analysis of variance (anova) showed highly significant differences among the irl lines for the studied traits. according to the pearson correlation coefficient, positive and significant relationships were found between the plant height and the length of the inflorescence (0.987) and between the yield and the aerialdry mater (0.812). the pca showed that the first principal component (pc1) accounted for 71.67% of the total variance; it is positively associated with the plant height, the inflorescence width and length and the yield. the pc2 accounted for 15.80% total variability and is the positively correlated with the days to maturity. the dendrogram displays five groups; the first includes 23 low production lines;the second includes early maturing accessions, tall and with long and broad inflorescences; the third includes the a little late to late lines. the fourth group is made of short and unproductive lines and the last brings together medium-sized and medium-yielding lines. keyswords: chenopodium quinoa wild, phenotypic diversity, characterization, genetic diversity. p163. effect of altitude on pomological and phytochemical traits of dallahia prickly pear fruits in northern morocco dramane y. koalaga1,2, zerhoune messaoudi1*, mohamed ibriz2, and lhoussain ait haddou1 1 laboratory of pomology, department of arboriculture-viticulture, national school of agriculture, b.p. s/40, meknes, 50001, morocco; 2 laboratory of genetic and biometrics, department of biology, faculty of sciences, ibn toufail university, university campus, bp 133, kenitra, morocco. *corresponding and presenting author: messaoudiz@yahoo.fr. abstract opuntia sp., commonly known as cactus pear, is originating from mexico and illustrates the impressive genetic diversity of cactaceae with 400 species and a great number of varieties for opuntia ficus indica. moroccan cactus presents a very high genetic variability and several cultivars exist. they are distinguished by the flowering period (early, late), the flower color (yellow, orange and pink), fruit and pulp colors (green, yellow, orange, red and purple), fruit a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 542 shape (oval, round or oblong), and organoleptic characteristics and antioxidant content of the fruit. cactus crop plays an important role on the ecological and economical scales but unfortunately, in morocco, huge amounts of the production are lost due to the lack of valorization prospects. dallahia prickly pear variety characterized by the green pulp color of the fruit and widely grown in northern morocco is among the lowest valorized cactus varieties due to the low oil content of its seeds. therefore, its fruits are mainly used for fresh consumption. the aim of this study is to discuss the effect of the altitude on the pomological (fruit dimensions, seediness and weight) and phytochemical (juiciness, sugariness…) traits of the fruits of this variety in northern morocco in order to reconsider the different possibilities to transform the excess of the production. keywords: cactus pear, opuntia ficus indica, dallahia, altitude, pomology, hplc, sugars, organic acids, fibers, gross energy. p164. phenotypic variability of moroccan prickly pear in southeast morocco abdelmonaim hormani bakali1*, chakib alem2, lalla laaziza ichir2, and el houssine el mzouzi3 1 national institute of agricultural research, errachidia, morocco; 2 faculty of science and technology of errachidia, univer-sity my ismail, morocco ; 3 national institute of agricultural research, settat, morocco. *corresponding and presenting author: homrani_bakali@yahoo.fr. abstract the genus opuntia (sensu stricto) has been the subject of various studies in morocco, but it lacks an assessment the saharan regions meanly in southeast of morocco. the overall objective of this work concerns the characterization of moroccan cactus accessions with a view to their extension to the pre-saharan ecosystem of morocco. the phenotypic study of 36 accessions from different regions of morocco was conducted in the experimental station of inra errachidia. statistical analysis of the data collected included analysis of variance, multivariate analysis and hierarchical classification. these statistical analyzes enabled us to: (i) classify the accessions into four groups and to choose six of them (77, 83, 134, 135, 181, t1) based on growth and production criteria. (ii) classify accessions into three categories based on cladode traits, then five accessions with large surface (36, 45, 70, 93). , t3) were chosen. (iii) make classifications according to the fruit criteria. thus, the best accessions from the caliber point of view that exceeded on average 120g were: 62, 149 and 182. on the other hand, the sweetest ones were: 33, 62, 70, 183 and t3. this study also includes a comparison between the growths, productions, cladode and fruit traits of the same accessions at agadir and errachidia stations. this comparison shows that the characteristics of each accession change according to the biotope. keywords: cactus, phenotypic variability, accessions. p165. control of chickpea leaf miner (liriomyza cicerina r.) using planting dates abdelhadi sabraoui1,2,3*, m. el bouhssini1, s. lhaloui2, a. bouchelta3, and k. el fakhouri1 1 international center for agricultural research in the dry areas (icarda), p.o. box: 6299rabat institutes, rabat, morocco; 2 entomology laboratory. plant protection research unit. regional center for agricultural research bp 589, settat, morocco; 3 plant protection and environment laboratory, moulay ismail university, b.p. 11201, zitoune, meknes, morocco. *corresponding and presenting author: a.sabraoui@cgiar. org. abstract in morocco, chickpea (cicer arietinum l.) is the first grain legume in the crop rotation system after cereals, occupying 45.12 % of food legumes area and producing around 441000 tonnes in 2016. however, the chickpea yield is still low and this is due to many biotic stresses. the chickpea leaf miner (liriomyza cicerina r.) is one of the main insect pests damaging chickpea in north africa and the mediterranean region. to determine which planting date will be optimum for the chickpea crop to reduce leaf miner infestation and losses caused by this pest, an experiment using four improved kabouli varieties planted in winter and spring, treated/untreated with insecticide, was carried out at two locations in morocco. the percent infestation by the leaf miner was about 25 %, 43 % and 3% for untreated winter planting, untreated a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 543 a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) spring planting and the treated plots, respectively. the grain yield losses caused by this pest were on average 42 % for the spring-sown crop and 20 % for winter planting. the use of winter planting date combined with biological insecticides could be included as options of integrated pest management for the control of chickpea leaf miner. keywords: planting date, leaf miner, chickpea, yield losses. p166. in vitro evaluation of compost extracts efficiency as biocontrol agent of date palm fusarium wilt el kinany said1,2,3*, achbani el hassan2, haggoud abdellatif3, ibijbijen jamal4, belmalha saadia1, rachidi fouad1, echchgadda ghizlane1, and bouamri rachid1 1 department of plant and environment protection, national school of agriculture, meknes, morocco; 2 laboratory of plant protection urpp, national institute for agricultural research, meknes, morocco; 3 laboratory of microbial biotechnology, faculty of sciences and technology, sidi mohamed ben abdellah university, fez, morocco; 4 department of biology, faculty of sciences, moulay ismail university, meknes, morocco. *corresponding and presenting author: rbouamri@ enameknes.ac.ma. or said.elkinany@usmba.ac.ma. abstract bayoud disease, vascular wilt of date palm caused by fusarium oxysporum f. sp. albedinis (foa), is widely distributed in all date palm growing regions of morocco. it is the most serious disease of the date palm. resistance to the pathogen was found in a few cultivars of date palm, which unfortunately produce poorquality fruit. compost is recognized for their ability to improve soil characteristics and to protect the crops against biotic and abiotic stress. in this experiment, in vitro effects of different concentrations of sterilized and unsterilized compost extract on the growth of f. oxysporum f. sp. albedinis were evaluated to control bayoud disease. all concentration of unsterilized compost extract significantly decreased radial growth of foa. in fact, fungal radial growth inhibition ranged from 20 to 97% depending concentrations. higher antifungal activities were noted in 30 and 40% concentration (more than 93%). nevertheless, sterile compost extract inhibited mycelia growth only for the 40% concentration with 18% fungal growth inhibition, while lower concentrations were not effective. this experiment constitutes a biological approach to protect date palm groves in oasis ecosystem in morocco. keywords: date palm, fusarium oxysporum f. sp. albedinis, compost extract, mycelium growth, inhibition rate. p167. study of olive fruit peroxidase activity against bactrocera oleae attack in attaouia region, haouz of morocco wassila arkize1*, m. a. el alaoui el fels2, k. naamani2, and a. elkeroumi3 1 laboratoire biotechnologie et valorisation des ressources végétales, faculté des sciences semlalia marrakech, morocco; 2 laboratoire biotechnologie et valorisation des ressources végétales, faculté des sciences semlalia marrakech, morocco; 3 laboratoire biotechnologie et valorisation des ressources végétales, faculté des sciences semlalia marrakech, morocco. *corresponding and presenting author: wassila. arkize@gmail.com. abstract bactrocera oleae (rossi) is a pest considered as a major biotic stress of olive fruit in morocco as well as other mediterranean countries. bactrocera oleae is causing considerable damages on the quality of table olives due to laying pits, but also on the olive oil production due to the olive fruit oxidation which results from the consumption of the olive fruit pulp by bactrocera oleae’s larvae. plants can defend against the pathogens like bactrocera oleae by synthesizing peptides and defense proteins, among these the pr proteins, among pr proteins are peroxidase which have several functions of plant protection against pathogens. peroxidases catalyzes the reduction of h2o2 from using different molecules such as phenolic compounds, precursors of the lignin and other auxins, indeed peroxidase can cope with biotic stress by strengthening the cell walls or producing active forms of oxygen. olive fruits were sampled from attaouia region, haouz of morocco, in twoo sites about 24 km apart, whitch differes in irrigation mode (site saguia: irrigated and site bour: not irrigated). the purpose of this study is to compare peroxidasic activity in infected and not infected olive fruits in both of study sites. keywords: bactrocera oleae, olive fruit, peroxidasic a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 544 activity, pr proteins. p168. interest of the use of wastewater in industrial plants ‘energy value’. case of jatropha curcas ouafae mokhtari1*, hassan el halouani1, kamal merghem1, imade hamdani2, abderrahim lahrach3, and belkhir hammouti4 1 laboratory of water science, environment and ecology, department of biology, faculty of sciences, university mohammed premier, oujda, morocco; 2 laboratory of applied analytical chemistry, materials and environment, faculty of sciences, university mohammed premier, oujda, morocco; 3 laboratory of geo-resources and environment, sciences and technologies faculty, university sidi mohammed ben abdallah, fez, morocco; 4 laboratory of applied analytical chemistry, materials and environment, faculty of sciences, university mohammed premier, oujda, morocco. *corresponding and presenting author: mokhtari.ouafae@gmail.com. abstract to assess the interest of the use of wastewater on the cultivation of jatropha curcas and the role of the latter in the conservation of water and soil and in the production of renewable energy, tests were conducted under semi-arid climatic conditions of northeastern morocco (oujda). the aim is to monitor the growth and behavior of jatropha curcas irrigated with different types of conventional waters and unconventional (including the salinity may be elevated up to 2.82 mmhos / cm). the performances of growth of jatropha curcas l. were studied under different types of water of irrigation (wastewater, fertigation, freshwater) and others without irrigation (in rainfall regime). the results showed: (1) the significant effect of wastewater on the vegetative development and yield (85%, p <0.3%), (2) acceleration of the development cycle and formation up to 3 peaks of inflorescences per year, (3) the application of water stress significantly reduced the growth and yield of jatropha relative to the irrigated plants (a degree of 90%, p <0.2%). its development is slow, and the first flowering is largely delayed (8 months) only after the arrival of the first rains of november, (4) the fertigation has improved the performance of jatropha and accelerated the process of reproduction (3 peaks of inflorescences). these results are very similar to those obtained after irrigation with treated wastewater which is only another way of fertigation but much cheaper, and (5) jatropha curcas shows proper installation despite the high salinity of the soil (5,89mmhos/cm to 7 mmhos / cm). this shows a perfect adaptation of the plant to the different soil types: marginal, skeletal and fertile. the production of jatropha curcas is closely related to water intake, fertilizers, soil quality and climate. based on these results, the association of wastewater with jatropha curcas can be a means of: fight against desertification and erosion, of valorization of the marginal grounds by using jatropha curcas like a reforestation plant, what will contribute to the sustainable development of the country by the increase in the national heritage in terms of biodiversity, and in terms of renewable energy “a source of green energy”. keywords: waste waters, fertigation, jatropha curcas, reforestation, sustainable development. a tla s jo ur na l o f bi ol og y is sn 2 15 891 51 . p ub lis he d by a tla s pu bl ish in g, l p (w w w .a tla spu bl ish in g. or g) 545 the international american moroccan agricultural sciences conference (amas conference; www.amas-conference.org) is an international conference organized by the high council of moroccan american scholars and academics (hc-masa; www.hc-masa.org).