British Miroiology Reserh Journl 16(2): 1-14, 2016, Artile no.bmrj.27883 ISSN: 2231-0886, NLM ID: 101608140 SCIENCEDOMAIN interntionl www.sieneomin.org Effet of Cynoteri on Reuing Dmping-off n Root Rot Iniene in Lupine Plnts, New Vlley Governorte, Egypt Montser Fwzy Ael-Monim 1*, Mohme Mhmou. Mzen 1 n Mrw Ael-All Mhmou Atw 1 1 Plnt Pthology Reserh Institute, Agriulture Reserh Center, Giz 12619, Egypt. Authors ontriutions This work ws rrie out in ollortion etween ll uthors. All uthors re n pprove the finl mnusript. Artile Informtion DOI: 10.9734/BMRJ/2016/27883 Eitor(s): (1) Joo Luio Azeveo, Deprtment of Genetis, University of So Pulo, Brzil. Reviewers: (1) Aul Hmee, Govt. College of Siene, Wht Ro, Lhore, Pkistn. (2) T. Pullih, Sri Krishnevry University, Ini. Complete Peer review History: http://www.sieneomin.org/review-history/15629 Originl Reserh Artile Reeive 23 r June 2016 Aepte 21 st July 2016 Pulishe 2 n August 2016 ABSTRACT This stuy ws onute to investigte the suppression effet of four ynoteri speies viz. Nosto musorum, Osilltori grhii, Spirulin pltensis n Anen spheri, ginst Rhizotoni solni, Fusrium solni n Mrophomin phseolin s the usl gent of lupine mping-off n root rot iseses uner greenhouse n fiel onitions. Cynoteril seonry metolites hve iverse ntgonisti tivity tht le to isintegrtion of miroil growth. All ynoteri were signifintly suppresse of the teste pthogeni fungi in vitro. The reution in the liner growth of the teste fungi ws iffere epening of type of solvent extrt. Aetone extrt of O. grhii ws reore the highest growth reution of ll teste pthogeni fungi. Contents of inole teti i (IAA), totl phenol n flvonoi ompouns n protese enzyme tivity showe n inrese in the extrts of N. musorum, O. grhii thn the extrts of S. pltensis n A. spheri. Also, etone extrt of ll teste ynoteri ws reore the highly seonry metolites thn methnol n wter extrts. Trete lupine sees (v. Giz 1) with ny of the ynoteri extrts efore sowing results signifintly reution of mping-off n root rot iniene ompre with untrete sees uner pot n fiel onitions. On the other *Corresponing uthor: E-mil: fowzy_2008@yhoo.om;
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 hn, treting lupine sees with ynoteri extrts inrese plnt growth n yiel prmeters uring oth growing sesons (2014-2015 n 2015-2016). In generl, O. grhii followe y N. musorum highly reue mping-off n root rot iniene oth in pot n fiel experiments s well s inrese of growth (pnt height n No. of rnhes plnt -1 ) n yiel prmeters (No. of pos plnt -1, No. of see plnt -1, see inex (gm) n totl yiel fe. -1 (Kg) in fiel. Also, etone extrt more effetive in this respet thn methnol or wter extrts. Keywors: Cynoteri; lupine; seonry metoli; soil orne pthogens; solvent extrts. 1. INTRODUCTION White lupine (Lupinus termis Forsk) is one of the olest griulturl rops wiely use in the worl not only s protein soure in foer proution ut lso for soil improvement [1]. Lupine elongs to the genus Lupinus in the Fee fmily. Lupine sees ontin onsierle nutrition ue to its high protein (35-45%) n oil ontent (10-15%). Unfortuntely in Egypt n severl ountries lupine legume plnts re infete y severl soil-orne fungi using mping-off n wilt isese whih effet oth quntity n qulity of the yiel. Soil-orne fungl iseses re mong the most importnt ftors limiting the yiel proution of lupine resulting in serious eonomi losses [2]. Mny soilorne fungi, inluing Rhizotoni solni Kühn, Fusrium solni (Mrt.) S, n Mrophomin phseolin (Tssi) Goi infet lupine plnts using mping-off n root rot iseses. Suh iseses use gret erese in see yiel [3]. The isese n e ontrolle y ertin fungiies [4]. However, isese mngement using fungiies is not eonomilly prtil or environmentlly sfe. Therefore, the inution of isese resistne in plnt my e n lterntive pproh to iminish the hzrous sie effets of hemil fungiies. Hene, strtegies iming t replement of hemil pestiies y hzrous free iologil gents n e resonly goo hoie. Potentil gents for ioontrol tivity re rhizosphere ompetent fungi n teri, whih in ition to their ntgonisti tivity re ple of inuing growth responses y either ontrolling minor pthogens or y prouing growth stimulting ftors. Interest in iologil ontrol hs inrese reently, fuelle y puli onerns over the use of hemils in the environment [5]. Biologil ontrol gents for plnt iseses re urrently eing exmine s lterntives to syntheti pestiies ue to their pereive inrese level of sfety n miniml environmentl impts [6] n whih reue the isese n re pereive s less hrmful thn onventionl fungiies [7]. It hs long een reognize s tht the iologil ontrol eme reently n effetive strtegy for fighting plnt pthogens [8]. Alge re one of the hief iologil gents tht hve een stuie for the ontrol of plnt pthogeni fungi, prtiulrly soil orne isese [9]. Vrious strins of ynoteri re known to proue intrellulr n extrellulr metolites with iverse iologil tivities suh s ntilgl, ntiteril, ntifungl n ntivirl tivity [10]. They hve reeive little ttention s potentil ioontrol gents of plnt iseses. Kulik [11] stte tht for numer of resons, ynoteri n lge re suitle nites for exploittion s io-ontrol gents of plnt pthogeni teri n fungi: Cynoteri proue lrge numer of ntiteril n ntifungl prouts, mny n grow in quntity in mss ulture n they re not thret to the environment [12]. The im of the present stuy is fouse on etetion the ility of some ynoteri in suppressing root rot iseses of lupine use y R. solni, F. solni n M. phseolin in vitro n in vivo n etermine their effets on some growth prmeters of Lupine plnts. 2. MATERIALS AND METHODS 2.1 Isoltion of Pthogeni Orgnisms Disese lupine plnts showing root rot were ollete from ifferent fiels lote t New Vlley Governorte for isoltion. The infete lupine roots were firstly wshe with tp wter n then the roots were surfe sterilize with 2% soium hypohlorite solution for 2 min. Isoltion proeures were rrie out oring to the metho esrie y Dhingr n Sinlir [13] using the Rose Bengl meium (Dextrose, 10 g; Peptone, 5 g; KH 2 PO 4, 1.0 g; MgSO 4.7H 2 O, 0.5 g; Rose Bengl 0.05 g; Agr, 15 g in 1 litre of istille wter n ph, 2
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 7.2±0.2). The resulte fungi were purifie using the hyphl tips n /or single spore tehnique on Rose Bengl meium n then suulture of eh isolte fungus on slnt meium for future stuies. Suultures of the otine isoltes were then kept on PDA slnts n store t 5 C for further stuies. The purifie ultures were then teste for their pthogeniity n those prove pthogeni were sumitte to ientifition, n to some pthologil stuies sheme uring the ourse of this work. 2.2 Pthogeniity Tests The purifie fungl isoltes seure from isese lupine plnt orgns were teste for their pthogeniity on helthy lupine plnts v. Giz 1 grown in pots (25 m in imeter) ontining sterilize soil n sown with isinfeste sees. Soil steriliztion ws rrie out using formlin solution 5%. The isinfeste soil efore sowing the see ws left to ertion for 3 weeks to get ri of the hemil remins. The otine isoltes (19 isoltes) were grown seprtely on rley grin meium in onil flsks for 7-10 ys to e use s soure of inoulum. Inoul of these teste fungi were pplie seprtely t the rte of 5% of the soil weight [4], mixe thoroughly with the soil then irrigte n left 7 ys for estlishment. Disinfeste lupine sees were sown in the infeste pots t the rte of 5 sees/pot. Four pots were use for eh isolte, (whih were onsiere s replites). Pots ontining sterile soil mixe with rley grins free of ny fungi were sown similrly with isinfeste lupine sees t the sme rte to e use s ontrol tretment. Pots were kept uner oservtion n irrigte s neee. 2.3 Disese Assessments Results were reore fter 30 ys of plnting for mping- off n fter 90 ys for rot root n wilt. The perentge of pre n post emergene mpe-off seelings ws estimte per eh replite. The root rot plnts of eh replite were remove from the soil fter the inoultion perio, wshe thoroughly to remove soil eris then isese severity ws estimte oring to the perentge of root isolortion s follows: 0 = roots without isolortion (no infetion), 1= 1-20%, 2= 21-40%, 3= 41-75%, 4= 75-100% n 5= ompletely e plnts. Disese severity inex (DSI) for eh replite ws lulte y the formul suggeste y Liu et l. [14] n lulte s follows: DSI = 100/ mx n Wheres: is the isese rting of eh plnt, mx is the mximum isese rting n n is the totl numer of plnts exmine in eh replite. Reisoltion ws rrie out from some of the experimentlly isese plnts to fulfill Koh's postultions n the eveloping fungi were ompre with the originl isoltes. Ientifition of the otine pthogeni fungl isoltes ws rrie out oring to Dhingr n Sinlir [13]. Representtive of these isoltes were sent to Assuit University Myologil Center (AUMC) for verifition. 2.4 Blue Green Alge (Cynoteri) Nosto musorum, Osilltori grhii, Spirulin pltensis n Anen spheri were otine from Miroiology Deprtment, Soil, Wter n Environment Reserh Institute, Agri. Res. Centre. 2.5 Cynoteri Growth n Different Extrts Preprtion Pure lgl ulture of the ynoteri, Nosto musorum, Osilltori grhii, Spirulin pltensis n Anen spheri were selete to evlute their ntifungl tivity ginst R. solni, F. solni, M. phseolin. BG11 meium ws use for mintenne of ynoteri [15]. The ulture mei were inute t 30±2 C without ertion n uner ontinuous illumintion of fluoresent lmps with intensity 2500 Lux. The ultures were shken every y to prevent lgl ell lumping n herene of lgl ells to the ontiners. 2.6 Extrts Preprtion The lgl pellets (37 gm) were extrte using seril Exhustive Extrtion Metho [16] with methnol, etone, n wter. All extrts were rie n weighe to estimte the onentrtion in 1 ml. Drie extrts were either reonstitute in eionize wter. 2.7 Antifungl Ativity Assy of Cynoteri Extrts The ntifungl tivity ws teste y using the filter pper is iffusion metho [17] employing 3
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 24 hours ultures of the ove mentione orgnisms. Petri ishes (9 m in imeter) ontins 15 ml of PDA meium were ivie into two equl hlves, the first hlf ws inoulte with isk (0.5 m in imeter) of pthogeni fungi, iniviully n the seon hlf ws inoulte with isk (0.5 m in imeter) sturte with 250 µg/ml of eh rue extrt [18]. The perentge of inhiition of the pthogen ws lulte. Eh tretment ws replite three times with five pltes per replition. The perentge reution (Mr) of olony imeter y eh extrt ws ompute s following Inhiition (%) = (M1-M2)/M1 100 Where: M1=Colony imeter in the untrete meium (ontrol) n M2=Colony imeter in the trete meium. 2.8 Proution of IAA The proution of IAA y the Cynoteri strins ws etermine y olorimetri mesurement t 530 nm using Slkowski s regent. Cynoteri strins were grown uner shking (120 rpm) for 2 ys t 30ºC in BG11 roth meium supplemente with tryptophn (1 mg ml 1 ) s IAA preursor. After inution, the ells were entrifuge (3,000 rpm for 10 min t 4 C) n 1 ml of superntnt ws omine with 2 ml of Slkowski s regent (150 ml of 95-98% H 2 SO 4, 7.5 ml of 0.5 M FeCl 3 6H 2 O, n 250 ml istille wter) n inute for 30 min t room temperture. The quntifition of IAA ws rrie out using stnr urve with known onentrtions of pure IAA (Sigm Alrih, Co.). 2.9 Estimtion of Totl Phenoli Content Totl phenoli (TP) ontents were etermine y the spetrophotometri metho [19]. In rief, 0.5 ml of eh extrt ws me up to 3 ml with istille wter, n then mixe with 0.5 ml of Folin-Ciolteu s phenol regent. After 5 min, 2 ml of 2% N 2 CO 3 solution were e to the mixture n thoroughly mixe. The mixture ws kept t 30 C for 60 min in rk ple, n then the sorne ws reore t 650 nm. The TP ws etermine from extrpoltion of lirtion urve tht ws onstrute y stnr onentrtions of glli i solution. Estimtion of phenoli ompouns ws rrie out in triplite. The TP ws expresse s milligrms of glli i equivlents (GAE) per g of rie smple. 2.10 Determintion of Totl Flvonoi Content Totl flvonoi (TF) ws etermine y olorimetri metho s esrie y Zhishen et l. [20]. A 0.5 ml of eh extrt ws me up to 1 ml with methnol. Afterwrs 0.4 ml of istille wter ws e followe y 0.3 ml of 5% NNO 2 solution n the mixture ws left for 5 min. Therefter, 0.3 ml of (10%) AlCl 3 solution ws e n llowe to stn for 6 min. Two ml of (1 M) NOH solution ws e to the mixture n the finl volume ws juste to 10 ml with istille wter. The mixture ws thoroughly shken n llowe to stn for 15 min. Asorne of the retion mixture ws re t 510 nm. The onentrtions of totl flvonois were etermine s queretin equivlents (mg/g of ry weight). 2.11 Protese Ativity Protese tivity ws etermine y moifie proeure 15, 16 using 2% sein in 0.2 M ronte uffer (ph 10) s sustrte. Csein solution (0.5 ml) with n equl volume of suitle ilute enzyme solution ws inute t 40ºC. After 10 min the retion ws terminte y ition of 1 ml of 10% trihloroeti i. The mixture ws entrifuge, superntnt ws tken to 5 ml of 0.44 M N 2 CO 3 n 1 ml of two-fol ilute Folin-Ciolteu regent ws e. After 30 min the olor evelope ws re t 660 nm ginst regent lnk prepre in the sme mnner. Tyrosine serve s the referene stnr. The optil ensity of these solutions ws mesure. 2.12 Greenhouse Experiment Pthogeni isoltes of R. solni, F. solni, M. phseolin inoulum ws prepre s esrie efore for use in pthogeniity test. Plsti pots (30 m imeter) were pke with sterilize sny ly soil infeste with fungl inoul t the rte 3% (w/w), seven ys efore plnting. Lupine isinfeste sees were soke in the solution of eh extrt (methnol, etone n wter) for 12 h, then sown in infeste pots t rte 5 sees pot -1. Also, in ontrol tretment, lupine sees soke in wter for the sme time n seeing in infeste soil with the pthogen t the sme rte. Five pots were use per tretment s replites. Perentges of mping-off n root rot severity were reore fter 30 n 90 ys from plnting, respetively. 4
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 2.13 Uner Fiel Conitions The experiments were rrie out in fiel nturlly infeste with the sul orgnisms of mping-off n root rot of lupine lote t the experimentl frm of El-Khrg Agriulture Sttion, New Vlley governorte uring two suessive winter growing sesons (2014/15 n 2015/16). Selete see smples of lupine v. Giz 1 were trete following ipping metho. The sees were ippe in eh lgl extrts with the sme onentrtion (250 µg/ml) for 12 hour in lge previously prepre extrts (Aetone, methnol n wter). All tretments were rrnge in omplete rnomize lok esign with three plots s replites. A plot ws 3 3.5 m with five rows; sees were sown in hills (2 sees hill-1 n 20 m prt). In the ontrol tretment, lupine sees were soke in wter for the sme time n sown with the sme metho. Perentges of mping-off n root rot isese inine were lulte s well s some vegettive growth n yiel prmeters, i.e. plnt height (m), numer of rnhes, pos n sees per plnt, weight of 100 sees (see inex) n totl yiel per fen were reore. Also, protein perentge ontent in sees ws reore using the metho of Jkson [21]. 2.14 Sttistil Anlysis All experiments were performe twie. Anlyses of vrine were rrie out using MSTAT-C progrm version 2.10 [22]. Lest signifint ifferene (LSD) ws employe to test for signifint ifferene etween tretments t P 0.05 [23]. 3. RESULTS 3.1 Isoltion n Ientifition of the Cusl Orgnisms Isoltion trils resulte 19 ifferent fungl isoltes otine isoltes were sreene mirosopilly for R. solni (8 isoltes), F. solni (6 isoltes), n M. phseolin (3 isoltes) s well s 2 isoltes not ientifie. 3.2 Pthogeniity Tests Results presente in Fig. 1 show tht ll teste isoltes le to infet roots of lupine using mping-off n root rot symptoms thus reue the survive plnts. Rhizotoni solni isolte LR3 reore the highest presentge of mping-off isese eing 85% followe R. solni y isolte LR2 (65%) then F. solni isolte LR10 (50%), while R. solni isolte LR4 gve the lowest mping-off perent (10%). On the other hn, ll teste isoltes use root rot/wilt rnging etween 10.25-55.36%. Fusrium solni isoltes LR11 n LR9 followe y M. phseolin isolte LR16 n R. solni isolte LR8 use the highest root rot severity, wheres reore 55.36, 55.00, 52.25 n 50.36%, respetively. While, isolte LR18 using the lowest perentge of root rot isese. In generl, R. solni isoltes LR3 n LR2, F. solni isolte LR9 n LR11, n M. phseolin isolte LR16 were the more virulent ones. 3.3 Antifungl Ativity Dt present in Tle 1 show tht ll solvents extrt of the four ynoteri resulte in signifint inhiitory effet of the growth of the teste pthogeni fungi. The ynoteri O. grhii reore the highest suppresse of the liner growth of ll teste pthogeni followe y N. musorum, while the lest inhiition effet of ll the teste fungi were reore in se of A. spheri. The reution in the liner growth of the teste fungi ws iffere epening of type of solvent extrt. In generl, etone extrt of ll teste ynoteri suppresse liner growth of the teste pthogeni fungi more thn methnol n wter extrts. Aetone extrt of O. grhii reore the highest suppresse of ll teste fungi, while methnol extrt showe moerte inhiite effet of ll fungl speies n wter extrt ws the lowest ones. On the other hn, M. phseolin ws more sensitive to ynoteri extrts thn R. solni n F. solni. 3.4 Pot Experiment Dt present in Tle 2 show tht the four teste ynoteri le to reue signifintly mping of n root rot iniene use y R. solni, F. solni n M. phseolin ompre with hek tretment. The reution of mpingoff n root rot use y pthogeni fungi were epens minly on the type of the ynoteri speies, type of solvent use n the teste fungl speies. Osilltori grhii ws gve the highest reution of mping-off n root rot ompre with the other teste ynoteri in 5
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 ll pthogeni teste fungi followe y N. musorum. Also, etone extrt of ll ynoteri teste ws more effetive for ontrolling mping off n root rot iseses in lupine plnts n methnol extrts were moerte, while wter extrts were the lest ones in this respet. In generl, etone extrt of O. grhii reore the highest reution of mping-off n root rot use y ny pthogeni teste fungi. Dmping-off % Root rot% %Disese severity 90 80 70 60 50 40 30 20 10 0 1CH 2CH 3CH 4CH 5CH 6CH 7CH 8CH 9CH 10CH 11CH 12CH 13CH 14CH 15CH 1CH 17CH 18CH 19CH Isoltes Fig. 1. Pthogeniity tests of soil orne fungi isolte from lupine roots uner the greenhouse onitions. Different letters inite signifint ifferenes mong tretments within the sme olor olumn oring to lest signifint ifferene test (P 0.05). Perentges of mping- 90 ys off were reore 30 ys fter plnting, while root rot isese inex ws etermine fter plnting 2.5 Methnol Aetone Wter 2 IAA (mg/g.wt) 1.5 1 0.5 0 N. musorum O. grhii S. pltenis A. spheri Axis Title Fig. 2. IAA proution in lgl ulture filtrtes of ynoteri strins. Vlues in the olumn followe y ifferent letters inite signifint ifferenes mong tretments oring to LSD test t 0.05. Brs inite the stnr evition 6
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 Tle 1. Antifungl tivity of ynoteri extrts ginst pthogeni fungi isolte from lupine plnts Teste ynoteri Solvents Inhiition (%) extrt R. solni F. solni M. phseolin Men Nosto musorum Methnol 41.25 45.47 50.14 45.62 Aetone 48.67 52.25 58.36 53.09 Wter 31.42 35.26 42.15 36.28 Men 40.45 44.33 50.22 - Osilltori grhii Methnol 45.09 45.09 45.09 45.09 Aetone 50.23 50.23 50.23 50.23 Wter 35.90 35.90 35.90 35.90 Men 43.74 43.74 43.74 - Spirulin pltensis Methnol 26.14 30.24 35.45 30.61 Aetone 33.12 35.36 42.15 36.88 Wter 16.59 17.36 19.09 17.68 Men 25.28 27.65 32.23 - Anen spheri Methnol 30.14 37.25 39.36 35.58 Aetone 38.36 43.56 48.76 43.56 Wter 28.57 32.14 37.59 32.77 Men 32.36 37.65 41.90 - LSD t 0.05 for: Teste ynoteri (A) = 2.96 Solvents extrt (B) = 3.02 Fungi (C) = 2.85 Intertion (A B C) = 6.58 3.5 Seonry Metolite Proution y Cynoteri Extrts The proution of inole eti i (IAA), totl phenol n flvonoi ontents n protese enzyme s seonry metolites in the extrts of N. musorum, O. grhii, S. pltensis n A. spheri is shown in Figs. (2-5). The otine t inite tht O. grhii n N. musorum were more proution of ll seonry metolites thn A. spheri n S. pltensis. Aetone extrt of ll ynoteri ws inrese proution of IAA, totl phenols n flvonoi ontents n protese enzyme thn the other solvent extrts. 3.6 Fiel Experiment 3.6.1 Effet of ynoteri on mping-off n root rot iniene uner fiel onitions Dmping-off n root rot severity were signifintly reue ue to soking the lupine sees in ll the teste ynoteri extrts efore sowing ompre with hek tretment in oth growing sesons (Tle 3). In this regr, the extrt of O. grhii ws the highest effiient in reution of mping-off n root rot severity in oth growing sesons while the extrt of S. pltensis ws the lowest one for ontrolling mping off n root rot. On the other hn, etone extrt of ny ynoteri types ws more effetive for ontrolling mpingoff n root rot iseses in lupine plnt thn methnol or wter extrts. Aetone extrt of O. grhii reore the highest reution of mping-off n root rot wheres reue mping-off from 29.5 n 32.5% in hek tretment to 4.19 n 5.51% in oth growing sesons respetively. 3.7 On Growth n Yiel Prmeters Dt present in Tle 4 inite tht lupine sees trete with ynoteri extrts signifintly improve ll growth n yiel prmeters viz. plnt height (m), numer of rnhes plnt -1 n yiel omponents viz. No. of pos plnt -1, No. of see plnt -1, see inex (gm), totl yiel fe. -1 (kg), ompre with untrete sees in oth growing sesons (2014-15 n 2015-16). The enhnement in growth n yiel prmeters were vrie epening with ynoteri type n type of solvent extrt. Cynoteri O. grhii reore highest the verge growth n yiel prmeters in oth growing sesons followe y N. musorum n A. spheri. While, S. pltensis extrts gve the lowest inrese in plnt growth n yiel prmeters. Aetone extrt of ll ynoteri improve plnt growth n inrese yiel 7
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 prmeters more thn methnol n wter extrts. In generl, etone extrt of O. grhii reoe the highest plnt height (120.25 n 128.36 m) n No. of rnhes (7.25 n 7.29 rnh plnt -1 ) in oth growing sesons, respetively. Also, this tretment reore the highest yiel prmeters viz. No. pos plnt -1 (22.14 n 24.33 po plnt -1 ), No. sees (66.96 n 69.63 po plnt -1 ), see inex (35.26 n 35.29 gm 100 sees -1 ), n totl yiel fe. -1 (899.36 n 910.23 Kg fe. -1 ) in oth growing sesons respetively. On the other hn, ll ynoteri extrts inrese protein ontents in sees ut were slight inrese ompre with untrete sees (ontrol). Methnol Aetone Wter Totl phenoli (mg/g.wt) 30 25 20 15 10 5 0 N. musorum O. grhii S. pltenis A. spheri Cynoteri Fig. 3. Totl phenols ontents in the ynoteri ulture extrts of ynoteri strins. Vlues in the olumn followe y ifferent letters inite signifint ifferenes mong tretments oring to LSD test t 0.05. Brs inite the stnr evition Methnol Aetone Wter Totl flvonoi (mg/g.wt) 30 25 20 15 10 5 0 N. musorum O. grhii S. pltenis A. spheri Cynoteri Fig. 4. Totl phenols ontents in the ynoteri ulture extrts of ynoteri strins. Vlues in the olumn followe y ifferent letters inite signifint ifferenes mong tretments oring to LSD test t 0.05. Brs inite the stnr evition 8
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 Methnol Aetone Wter Protese tivity (IU ml -1 ) 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 N. musorum O. grhii S. pltenis A. spheri Cynoteri Fig. 5. The tivity of protese enzymes in the lgl ulture extrts of ynoteri strins (IU ml -1 ). Vlues in the olumn followe y ifferent letters inite signifint ifferenes mong tretments oring to LSD test t 0.05. Brs inite the stnr evition Tle 2. Effet of soking lupine sees in ynoteri extrts on mping-off n root rot iseses uner rtifiil infetion with Rhizotoni solni, Fusrium solni n Mrophomin phseolin in pots Teste ynoteri Solvents extrt % Dmpingoff R. solni F. solni M. phseolin % % % % Root Dmpingoff Root rot Dmping-off rot % Root rot Nosto musorum Methnol 30.00 14.90 10.0 18.30 15.0 20.33 Aetone 25.00 11.47 5.0 12.14 10.0 15.02 Wter 45.00 18.25 10.0 25.30 20.0 27.36 Men 33.33 14.87 8.33 18.58 15.00 20.90 Osilltori grhii Methnol 25.00 10.30 5.0 10.47 5.0 12.14 Aetone 15.00 7.25 0.0 7.56 0.0 8.47 Wter 40.00 14.58 5.0 18.47 15.0 18.75 Men 26.67 10.71 3.33 12.17 6.67 13.12 Spirulin pltensis Methnol 30.00 19.36 20.0 20.36 15.0 28.5 Aetone 35.00 15.47 15.0 17.45 15.0 21.14 Wter 50.00 22.47 25.0 28.36 25.0 32.47 Men 38.33 19.10 20.00 22.06 18.33 27.37 Anen spheri Methnol 25.0 15.47 10.0 17.24 10.0 18.25 Aetone 20.0 13.25 5.0 15.47 10.0 15.47 Wter 45.0 20.14 15.0 25.32 20.0 22.3 Men 30.00 16.29 10.00 19.34 13.33 18.67 Control 70.0 30.0 35.0 50.42 35.0 45.09 LSD t 0.05 for: Dmping-off Root rot Teste ynoteri (A) = 3.96 1.96 Solvents extrt (B) = 3.24 2.32 Fungi (C) = 2.85 2.09 Intertion (A B C) = 7.89 5.48 4. DISCUSSION Severl soil-orne fungi ttk lupine plnts uring its vrious growth stges from seeling till mturity using mping-off n root rot iseses. The present results inite tht mping-off n root rot iseses re inite y the soil orne fungi, Rhizotoni solni, 9
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 Fusrium solni n Mrophomin phseolin. These results re in greement with these otine y Ael-Monim et l. [3]. The mngement of soil-orne plnt pthogens is prtiulrly omplex euse these orgnisms live in or ner the ynmi environment of the rhizosphere n n frequently survive long perio in soil through the formtion of resistnt survivl strutures. Severl ommerilly ville prouts hve shown signifint isese reution through vrious mehnisms to reue pthogen evelopment n isese. Plnt iseses nee to e ontrolle to mintin the qulity n unne of foo, fee, n fier proue y growers roun the worl. However, the environmentl pollution use y exessive use n misuse of grohemils, s well s fer mongering y some opponents of pestiies, hs le to onsierle hnges in people s ttitues towrs the use of pestiies in griulture. Aquire resistne y using ioti gents s iologil ontrol seems to e one of lterntives to sustitute for, or t lest to erese the use of fungiies in plnt isese ontrol. Exessive n improper use of pestiies inluing fungiies presents mene to the helth of humn, niml n environment [24]. The present stuy ws plnne minly to investigte the possiility of minimizing the infetion y mping-off n root rot iseses of lupine using the extrts of four ynoteri s iologil ontrol gents. In this stuy, solvents extrt of the four ynoteri viz. Nosto musorum, Osilltori grhii, Spirulin pltensis n Anen spheri resulte in signifint inhiitory effet of the growth of the teste pthogeni fungi in vitro. The etone extrt of O. grhii ws more effetive for suppresse of ll teste pthogeni fungi thn the others. On the other hn, the otine t showe tht lupine sees trete with ynoteri extrts reore the highly protetion ginst infetion with soil orne pthogens more thn untrete sees uner greenhouse n fiel onitions n signifintly improve plnt growth n yiel prmeters uner fiel onitions. The solvent extrts of ll ynoteri more tive for reuing mping- Tle 3. Effet of soking lupine sees in ynoteri extrts on mping-off n root rot iseses uner fiel onition uring 2014-15 n 2015-16 growing sesons Teste ynoteri Solvents 2014-15 2015-16 extrt % mping-off % root rot % mping-off % root rot Nosto musorum Methnol 8.69 6.75 11.03 7.94 Aetone 6.55 5.19 9.19 6.12 Wter 12.58 8.26 15.54 9.73 Men 9.27 6.73 11.92 7.93 Osilltori grhii Methnol 6.99 4.66 9.19 5.49 Aetone 4.19 3.28 5.51 3.87 Wter 11.18 6.6 14.7 7.77 Men 7.45 4.85 9.80 5.71 Spirulin pltensis Methnol 12.36 8.76 14.03 10.32 Aetone 9.78 7.00 9.86 8.25 Wter 14.23 10.17 18.38 11.98 Men 12.12 8.64 14.09 10.18 Anen spheri Methnol 10.23 12.03 16.19 14.02 Aetone 8.25 9.12 13 12.05 Wter 17.02 14.36 19.54 16.23 Men 11.83 11.84 16.24 14.10 Control 29.5 22.9 32.5 21.03 LSD t 0.05 for: Dmping-off Root rot Teste ynoteri (A) = 1.96 1.02 Solvents extrt (B) = 2.12 1.32 s (C) = NS NS Intertion (A B C) = 5.21 3.26 10
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 Tle 4. Effet of soking lupine sees (v. Giz 1) in ynoteri extrts on plnt growth n yiel prmeters uner fiel onitions uring 2014-15 n 2015-16 growing sesons See inex (gm) Totl yiel Teste Filtrte Plnt height (m) No. of No. of pos No. of see % ynoteri extrts rnhes plnt -1 plnt - 1 plnt -1 fe. -1 (Kg) Protein 2014-15 2015-16 2014-15 2015-16 2014-15 2015-16 2014-15 2015-16 2014-15 2015-16 2014-15 2015-16 2014-15 2015-16 Nosto musorum Methnol 90.35 95.23 6.42 6.52 16.35 18.23 48.23 52.36 31.45 31.25 715.36 726.36 37.23 37.33 Aetone 105.36 109.32 6.96 7.02 18.25 20.12 55.42 58.36 32.14 32.25 755.36 766.21 37.36 37.25 Wter 88.41 92.04 5.56 6.01 14.36 15.23 43.56 45.02 31.25 31.36 686.25 692.35 36.96 36.89 Men 94.71 98.86 6.31 6.52 16.32 17.86 49.07 51.91 31.61 31.62 718.99 728.31 37.18 37.16 Osilltori grhii Methnol 102.36 105.36 7.02 7.25 18.69 19.99 56.38 59.03 33.36 33.35 825.36 836.35 38.23 38.36 Aetone 120.25 128.36 7.25 7.29 22.14 24.23 66.96 69.63 35.26 35.29 899.36 910.23 38.35 38.42 Wter 95.36 104.32 6.09 6.28 16.58 18.23 50.25 53.27 31.02 31.22 755.25 766.32 37.58 37.29 Men 105.99 112.68 6.79 6.94 19.14 20.82 57.86 60.64 33.21 33.29 826.66 837.63 38.05 38.02 Spirulin pltensis Methnol 82.36 88.02 4.96 5.03 14.25 15.26 43.26 46.69 30.25 30.7 523.36 539.36 36.56 36.59 Aetone 88.36 92.54 5.23 5.29 16.35 17.36 50.02 54.06 30 30.25 556.23 569.08 37.02 36.09 Wter 75 78.23 4.56 4.86 14.02 15.02 44.26 48.36 29.89 30 505.36 515 36.05 36.09 Men 81.91 86.26 4.92 5.06 14.87 15.88 45.85 49.70 30.05 30.32 528.32 541.15 36.54 36.26 Anen spheri Methnol 96.09 99.02 6.02 6.22 16.35 17.36 50.02 54.36 31.12 31.25 582.23 596.36 36.59 36.65 Aetone 102 108.26 6.53 6.85 18.29 19.86 56.23 60.14 32.15 32.02 612.32 625.35 36.99 36.92 Wter 84.23 89.25 5.32 5.83 15 16.02 46.03 48.23 30.25 30.42 555.02 566.36 35.63 35.41 Men 94.11 98.84 5.96 6.30 16.55 17.75 50.76 54.24 31.17 31.23 583.19 596.02 36.40 36.33 Control 65.23 67.25 4.25 4.55 10.42 11 30.16 32.23 28.01 28.06 445.23 452.36 34.21 33.36 LSD t 0.05 for: Teste Cynoteri (A) = 5.26 0.95 1.52 2.63 1.52 11.25 2.01 Filtrte extrts (B) = 4.59 0.84 1.66 3.02 NS 10.19 1.69 s (C) = NS NS NS NS NS NS NS Intertion (A B C) = 9.26 NS 2.26 5.26 NS 25.06 NS 11
Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 off n root rot more thn wter extrt. Aetone extrt of ll ynoteri more effetive thn the others either uner greenhouse or fiel onitions espeilly in se of O. grhii. In ition, the effiieny of the ynoteri strins ws positively orrelte with the ontent of inole eti i (IAA), totl phenols n flvonois ompouns n protese enzyme s seonry metolites in the extrts of N. musorum, O. grhii, S. pltensis n A. spheri, where the trete sees with the etone extrt of O. grhii n/or N. musorum were more effiient in eresing the infetion y soil orne pthogens in greenhouse n in fiels s well s inresing plnt growth n yiel omponents ompre with the other ynoteri. These results re in onformity with the otine t y De Cire et l. [25] who reporte tht extrellulr prouts from N. musorum re promising s iologil ontrol of soyen seeling mping-off. Kulik [11] mentione tht filtrtes or ell extrts from ynoteri were pplie to sees s protetnts ginst mping-off fungi suh s Fusrium sp., Pythium sp. n R. solni. On the other hn, the potentil tivity of ynoteri to inhiit ertin soil orne iseses oul e ttriute to proue wie rnge of plnt growth regultors suh s sisi i, ethylene, jsmoni i, uxin n ytokinin- like sustnes s well s the ytokinin isopentenyl enine [26]. The ntifungl tivity hs een ue to the presene of plnt iotive ompouns, i.e totl phenoli ompouns, totl sponins n lklois in the lgl ulture filtrtes, employe s nturl efense mehnisms ginst pthogeni teri, fungi, viruses n pests [27]. The pity of inole eti i iosynthesis hs een foun in free-living n symioti ynoteri of the gener Nosto, Chlorogloeopsis, Clothrix, Pletonem, Gloeothee, Anen, Cylinrospermum n Anenopsis [28]. The tivity of protese enzymes in the ynoteri filtrtes revele tht hyrolyti enzymes my ontriute to e the fungiil tivity of the ynoteril strins, esies other iotive ompouns, inluing inole eti i [9]. Mny ynoteri re known to relese vrious kins of iologilly tive sustnes like proteins, vitmins, rohyrtes, mino is, polyshries n phytohormones tht funtion s eliitor moleules to promote plnt growth n help them to fight ginst ioti n ioti stress. These metolites proue y the ynoteri ffet the gene expression of the host plnts n therey ring out qulittive n quntittive hnges in the phytohemil omposition of the plnts [29]. 5. CONCLUSION Cynoteri known s ioontrol gents ring out inue systemi resistne (ISR) fortifying the physil n mehnil strength of the ell wll n hnging physiologil n iohemil retion of the host leing to synthesis of efense hemils ginst the pthogens. Suh hnges n le to enhne plnt growth, improve yiels n qulity of proue. Although inution of systemi resistne hs een stuie y ifferent investigtors, little is known out the mehnisms of nitrogen fixing ynoteri in helping plnts to overome ioti stress inue y the pthogens. COMPETING INTERESTS Authors hve elre tht no ompeting interests exist. REFERENCES 1. Mknikiene Z. Effet of genotype on see yiel in lupine (Lupinus luteus L., Lupinus ngustifolius L.) n resistne to fungl isese (Colletotrihum linemuthinuim Br. Cv., Fusrium oxysporum). Biologij. 2001;3:27-29. 2. El-Shrkwy RMI. Utiliztion of ntioxints n plnt extrts in ontrolling wilt isese of lupine (Lupines termis Forsk). J. Plnt Prot. n Pth., Mnsour Univ. 2014;5(12):1139 1153. 3. Ael-Monim MF, Gr MR, El-Gntiry SM, Sht MN, El-Bn AM. Pthologil n physiologil stuies on root rot isese in white lupine (Lupinus termis Forsk). Interntionl J. of Agri. Si. 2014; 4(9):261-267. 4. El-Awi FA, Ismil IA, Essmt NEA. Evlution of lupine entries n fungiies ginst root rot n wilt isese of lupin. 8 th Congress of the Egyptin Phytopthol., Ciro. 1997;393-401. 12
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Ael-Monim et l.; BMRJ, 16(2): 1-14, 2016; Artile no.bmrj.27883 phytohormone inole-3-eti i y ynoteri. Plnt. 2002;215:229-238. 29. Singh S. A review on possile eliitor moleules of ynoteri: Their role in improving plnt growth n proviing tolerne ginst ioti or ioti stress. J. of Applie Miroiology. 2014;117:1221 1244. 2016 Ael-Monim et l.; This is n Open Aess rtile istriute uner the terms of the Cretive Commons Attriution Liense (http://retiveommons.org/lienses/y/4.0), whih permits unrestrite use, istriution, n reproution in ny meium, provie the originl work is properly ite. Peer-review history: The peer review history for this pper n e esse here: http://sieneomin.org/review-history/15629 14