Effects of Different Rhizobacteria on Nodulation and Nitrogen Fixation in Alfalfa (Medicago sativa) at Suboptimal Root Zone Temperatures

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1 Amerin-Eursin J. Agri. & Environ. Si., 3 (0): 30-34, 203 ISSN IDOSI Pulitions, 203 DOI: /iosi.ejes Effets of Different Rhizoteri on Noultion n Nitrogen Fixtion in Alflf (Meigo stiv) t Suoptiml Root Zone Tempertures 2 O. Younesi, A. Mori n M.R. Chihi Deprtment of Agronomy n Plnt Breeing, Fulty of Agriulture, University of Tehrn, Irn 2 Deprtment of Agronomy n Plnt Breeing, Fulty of Agriulture, Ysouj University, Ysouj, Irn Astrt: Co-inoultion of plnt growth promoting rhizoteri (PGPR) with Sinorhizoium meliloti hs een shown to inrese legume noultion n nitrogen fixtion t suoptiml soil tempertures. In this work, three rhizoteri (, Azototer n ) o-inoulte with S. meliloti were teste for their ility to reue the negtive effets of low root zone temperture (RZT) on lflf (Meigo stiv L.) noultion n nitrogen fixtion. Three RZTs were teste: 25 (optiml), 5 (somewht inhiitory) n 0 C (very inhiitory). The results of this stuy inite tht noultion ws inhiite uner suoptiml root zone tempertures. At low temperture PGPR strins inrese the numer of noules forme n the mount of fixe nitrogen when o-inoulte with S. meliloti, ut the stimultory strins vrie with tempertures. Rhizoium n o-inoultion moerte the negtive effets of low tempertures on noultion n nitrogen fixtion in lflf plnts. Key wors: Alflf, Azototer Sinorhizoium meliloti Suoptiml root zone tempertures. INTRODUCTION in non-legume rops suh s potto, rish, sugr eet, whet n nol [8, 9]. Co-inoultion stuies with PGPR Bteri of the genus Rhizoium re ple of n Sinorhizoium meliloti hve lso emonstrte infeting the roots of host plnts, resulting in the inrese lflf plnt root n shoot weight, see yiel, evelopment of novel orgns lle noules, where plnt vigour, noultion n nitrogen fixtion [0, ]. iologil N2-flxtion tkes ple [, 2]. Biologil nitrogen Similrly, n inrese in grin yiel, noule ry mtter n fixtion is sustinle lterntive to inustril nitrogen nitrogense tivity were lso otine in hikpe fixtion whih is ssoite with severl environmentl inoulte with mixture of rsilense n prolems [3]. Rhizoium strins [2]. Grimes n Mount [3] foun tht In res with suoptiml root zone tempertures puti strin (M), whih h een (RZTs) erly in the growing seson, nitrogen fixtion y selete s potentil iologil ontrol gent, mrkely legume rops is mjor limittion to rop yiel [4] s inrese Rhizoium noultion of en in fiel soils. nitrogen fixtion is more sensitive thn ry weight to RZT Polonenko et l. [9] foun similr effets of ertin [3]. Stuies of low RZT effets on nitrogen fixtion y rhizoteri (primrily pseuomons fiuoresent) on soyen n other sutropil legumes hve inite noultion of soyen roots y B. jponium. tht low RZT ereses oth noultion n noule Numerous stuies hve therefore inite tht funtion [5, 6]. Infetion n erly noule evelopment o-inoultion of rhizoium n ertin PGPR, n proesses re most sensitive to low RZT []. positively ffet symioti nitrogen fixtion y enhning Certin rhizosphere miroorgnisms, olletively oth root noule numer or mss [4] n inresing referre to s plnt growth promoting rhizoteri nitrogense tivity [5]. However, these stuies hve ll (PGPR), n inrese plnt growth, evelopment n yiel een onute using optiml RZT n to te, there hs Corresponing Author: O. Younesi, Deprtment of Agronomy n Plnt Breeing, Fulty of Agriulture, University of Tehrn, Irn. 30

2 Am-Eurs. J. Agri. & Environ. Si., 3 (0): 30-34, 203 een no etile investigtion of the effet of PGPR on temperture without shking. The inoulum ws then noultion n nitrogen fixtion t ifferent RZTs. oole to the orresponing root temperture n 2 ml Therefore, the present stuy ws onute to etermine were pplie y pipette to the rooting meium t the se whether PGPR oul inrese lflf noultion n of the plnt. nitrogen fixtion t suoptiml n optiml RZTs n whether the PGPR oul inrese nitrogen trnsport from Experimentl Design n Dt Colletion: The root noules to the plnt shoot. experiment ws onute using ompletely rnomize split-plot esign with three replitions. The min-plot MATERIALS AND METHODS units onsiste of three RZTs, 25 C [optiml temperture for lflf noultion n nitrogen fixtion], 5 C Plnt Mterils: See of the lflf (Meigo stiv) [somewht inhiitory], or 0 C [very inhiitory]. v. Bmi ws surfe sterilize in soium hypohlorite Comintions of S. meliloti n PGPR strins (2% solutions ontining 4 ml Tween 20) n then (, Azototer n ) forme the rinse with istille wter severl times [6]. Then sees su-units. A ontrol (S. meliloti) ws inlue in eh ple in sterilize trys of 2 50 m n 6 m eep, fille experiment. Plnts were hrveste one month fter sowing with vermiulite n llowe to germinte. Ten-y-ol n noule numer n ry weight, shoot n root ry seelings were trnsferre to litre pots, ontining weight, the nitrogen onentrtion of ifferent plnt 950 ml of sterilize sn n turfe (2: v:v) mixture. tissues (noules, shoots n roots) n nitrogense The plnts were grown in growth hmer (Conviron enzyme tivity mesure y etylene reution metho In. Winnipeg, Mnito) with y/night tempertures of [9] were mesure. 25 C n 6:8 h photoperio. The pots were then sele to the ottom of plsti tnks (68 42 m) n RZTs Sttistil Anlysis: Results were nlyse sttistilly (± 0.5 C) were ontrolle y irulting oole wter for nlysis of vrine (ANOVA) using SAS softwre roun the pots, with eight pots in eh tnk. A hole [20]. When nlysis of vrine showe signifint rille in the ottom of eh pot n through the ottom tretment effets, the lest signifint ifferenes test of the tnk llowe the pots to rin when wtere. Plnts (LSD) ws pplie to mke omprisons mong the mens were limtize for 24 h prior to inoultion. Plnts were t the 0.05 level of signifine. then wtere with moifie Hogln's solution [6], in whih C(NO 3) 2n KNO 3were reple with mm RESULTS CCl 2,mM K2HPO 4 n mm KH2PO 4, to provie nitrogen-free solution. Prior to eh wtering the e Noultion: The effets of the three teste PGPR strins Hogln's solution ws juste to the tretment RZTs. on noule numer vrie with RZT. At 0 C RZT, Azototer n i not inrese noule Inoulum n Inoultion: Sinorhizoium meliloti ws numer when ompre with the ontrol without PGPR, ulture in yest extrt mnnitol roth [] in 250 ml ut the tretment ontining P. fluoresene inrese flsks shken t 25 rpm t room temperture (20-23 C). noule numer in this experiment (Tle ). At 5 C RZT S. meliloti hs een shown to perform well over rnge n Azototer of the PGPR inrese of tempertures [6]. Three PGPR strins were use in the noule numer in plnts (Tle ). At optiml RZT (25 C), experiment. All the PGPR strins were ulture in only o-inoultion of S. meliloti with P. fluoresene meium [9] in 250 ml flsks shken t 250 inrese noule numer ompre with the ontrol rpm t room temperture (20-23 C). After rehing without PGPR. PGPR inrese not only noule numer, sttionry phse ( for S. meliloti n.5 for PGPR), ut lso noule ry weight per plnt t oth suoptiml oth S. meliloti n PGPR were suulture. At log n optiml RZT. For exmple, P. fluoresene inrese phse, (3 for S. meliloti n for PGPR), the ell the totl noule weight per plnt t 0 C RZT, with these ensity of S. meliloti ws juste with istille wter to inreses eing prtilly ue to inrese noule size 8 n pproximtely 0 ells ml [8] n eh PGPR strin (inite y the higher verge weight per noule) n ws juste with istille wter to n A 400 vlue giving prtilly to inrese noule numer (Tle ). Also, P. n pproximte ell ensity of 0) ells ml. Before fluoresene inrese totl noule weight per plnt t inoultion, roth ontining S. meliloti ws e to the the optiml (25 C) RZT in this experiment. Noule ry sme volume of the pproprite PGPR strin n the weight ws not ffete y o-inoultion with mixture ws llowe to stn for more thn 30 min t room t 0 n 5 C, wheres it ws inrese y 3

3 Am-Eurs. J. Agri. & Environ. Si., 3 (0): 30-34, 203 Tle : The effet of o-inoultion of S. meliloti with plnt growth promoting rhizoteri on lflf noultion n nitrogen fixtion t three RZT. Shoot Root ry Totl noule Totl noule Plnt N ry weight weight numer weight onentrtion ARA per plnt Strins (g orgn ) (g orgn ) (plnt ) (mg DW plnt ) (mg g ) (mmol C2H 4 plnt h ) 0 C root zone temperture Azototer C root zone temperture Azototer C root zone temperture Azototer Mens within the sme olumn n experiment were nlyse y n ANOVA protete LSD test. Tle 2: The effet of o-inoultion of B. jponium with plnt growth promoting rhizoteri on nitrogen onentrtions of i?erent lflf tissues n nitrogen onentrtion rtios for noule : shoot, noule : root n shoot : root t three RZT. Nitrogen onentrtions (mg g ) Nitrogen onentrtion rtios Strins Noule Shoot Root Noule : shoot Noule : root Shoot : root 0 C root zone temperture Azototer C root zone temperture Azototer C root zone temperture Azototer Mens within the sme olumn n experiment were nlyse y n ANOVA protete LSD test Azototer t 0 C). Noule ry weight ws not with Azototer, inrese 45.9 ompre with ontrol influene y o-inoultion Azototer n plnts in the reserh. At 5 C RZT the nitrogen with S. meliloti t 25 C RZT (Tle ). At onentrtion of plnt tissues ws ffete only y optiml RZT (25 C), only o-inoultion of S. meliloti with o-inoultion of S. meliloti with P. fluoresene. P. fluoresene inrese noule ry weight ompre However, sine plnt ry mtter ws inrese y P. with the ontrol without PGPR. fluoresene, totl plnt nitrogen fixe ws inrese (Tle 2). At the optiml RZT for lflf plnt, ll PGPR Nitrogen Fixtion: At 0 C RZT, plnt nitrogen ws strins inrese totl plnt fixe nitrogen in this reserh inrese y P. fluoresene n Azototer strins in (Tle 2). this experiment. The inrese ws prtly ue to n Nitrogense (etylene reution) tivity y plnts inrese in plnt tissue nitrogen onentrtion n prtly inoulte with P. fluoresene inrese t three RZTs. ue to n inrese in plnt totl ry mtter, whih, for For the plnts o-inoultion with Azototer or plnts inoulte with P. fluoresene inrese 45.9 Nitrogense tivity inrese only t ompre with ontrol plnts n for plnts inoulte optiml (25 C) RZT. 32

4 Am-Eurs. J. Agri. & Environ. Si., 3 (0): 30-34, 203 Nitrogen Distriution: In this experiment, noule, shoot i not inrese noule numer n n root nitrogen onentrtions were ssye weight t three RZT, ut i inrese plnt nitrogen seprtely. The ifferenes of nitrogen onentrtion rtio onentrtion. Only P. fluoresene use oth of noule to shoot tissues etween tretments inite inrese noultion n fixe nitrogen per plnt. A tht the pplie tretments vrily ffete nitrogen possile explntion of these oservtions is tht the trnsfer from root-noules to shoot tissues. For exmple, PGPR inrese overll plnt growth whih les to plnts inoulte with P. fluoresene h higher inrese nitrogen emn, whih, in turn, les to nitrogen onentrtion rtio of noule to shoot tissues ontrolle inrese in noule numer n /or size when ompre to plnts reeiving or [2]. Azototer t 0 RZT. At 25 RZT, the plnt noule / Zhng et l. [] emonstrte the existene of liner shoot n shoot/ root nitrogen onentrtion rtios were reltionship etween RZT n the time from inoultion not ifferent mong PGPR tretments or when ompre with B. jponium to the onset of nitrogen fixtion to ontrol plnts reeiving only S. meliloti (Tle 2). etween 25 n C RZTs. The results from experiment showe tht the nitrogen onentrtions of plnt shoots DISCUSSION n whole plnts grown t 0 n 5 C were lower thn those t 25 C. These results gree with those of Inoultion of lflf plnts with PGPR strins previous stuy []. together with S. meliloti proue wie rnge of effets, In summry, the results of this stuy inite tht whih vrie mong strins of PGPR n over RZTs. some of the PGPR strins teste inhiite spets of Some PGPR strins stimulte noule formtion n /or lflf nitrogen fixtion, the effets of the stimultory nitrogen fixtion, while others i not ffet these strins vrie with RZT in wy tht ws unique to eh proesses. At 0 C RZT, P. fluoresene inrese plnt strin, (3) some strins ffete only noultion or noule numer n plnt noule weight, wheres nitrogen fixtion,wheres others ffete oth noultion i not exhiit ny effet on noule numer n nitrogen fixtion. Overll some PGPR stimulte the n ry weight. At 25 C RZT, only o-inoultion of S. S. meliloti -lflf symiosis, ut the nture of these meliloti with P. fluoresene inrese plnt noule effets vrie mong PGPR. Inoultion with PGPR my numer n noule weight when ompre with the e le to improve lflf nitrogen fixtion n yiel in ontrol without PGPR. Azototer ws effetive on res with ool spring soil tempertures, ut it will e noule numer only t 25 C. Also, Noule ry weight ws neessry to sreen the PGPR strins t the require not ffete y o-inoultion with Azototer t 5 n temperture for the esire effets. 25 C, wheres it ws inrese t 0 C. These results inite tht the physiologil effets of PGPR were REFERENCES ltere y temperture. These oservtions suggeste tht the PGPR intene for use s noultion stimultors. Hungri, M. n G. Stey, 99. Moleulr signls in ifferent res shoul e sreene for n selete exhnge etween host plnts n rhizoi: Bsi oring to previling onitions, inluing RZT. spets n potentil pplition in griulture. Soil Previous stuies hve shown tht o-inoultion of Biol. Biohem., 29: sp. (inluing P. fluoresens n P. 2. Sheng, C. n J.E. Hrper, 99. Shoot versus root puti) with B. jponium inrese noule formtion signl involvement in noultion n vegettive n weight of soyen plnts in ifferent rooting mei growth in wil type n hypernoulting soyen (soil n perlite mix) [9]. Grimes n Mount [3] reporte genotypes. I. Loliztion of infetile root ells. tht soil pseuomon inhiite R. phseoli on gr Plnt Physiol., 3: mei, ut stimulte its ility to form noules on P. 3. Dte, R.A. n D. Rtliff, 989. Growth, noultion ulgris grown in the fiel. It is not ler, however, tht the n nitrogen flxtion in Stylosnthes: effet of sme effets woul our in soil. In the sme wy, our ifferent root tempertures n two shoot experimentl results, se on plnts grown in sterilize tempertures. Experimentl Agriultur. 25: Turfe n sn rooting meium, remin to e teste 4. Mtthews, D.J. n P. Hyes, 982. Effet of root uner fiel onitions. The enhnement of noule zone temperture on erly growth, noultion n numer n/or size y PGPR ws not lwys relte to nitrogen fixtion in soy ens. J. Agri. Si. Cm., inrese nitrogen fixtion, for exmple strin 98:

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