Evaluation of rhizobacteria as non-rhizobial inoculants for mung beans

Transcription

1 AJCS 5(13): (211) ISSN: Evlution of rhizocteri s non-rhizoil inoculnts for mung ens Sf Akhtr nd Bshrt Ali* Deprtment of Microiology nd Moleculr Genetics, University of the Punj, Quid-e-Azm Cmpus, Lhore- 5459, Pkistn *Corresponding uthor: shrt.li.mmg@pu.edu.pk Astrct Rhizocteri ssocited with nturl plnt settings were evluted s non-rhizoil inoculnts for Vign rdit (L.). Five cteril strins of Providenci, Bcillus nd Alcligenes gener were screened for 1-minocyclopropne-1-croxylte (ACC) deminse, uxin production nd phosphte soluiliztion. Highest ACC-deminse ctivity ws exhiited y Bcillus pumilus Sol-1 (43 nmol h - 1 ), Alcligenes sp. Ml-4 (39 nmol h -1 ) nd Providenci vermicol Am-2 (377 nmol h -1 ). Auxin production y rhizocteri showed significnt positive correltion (up to r =.965; P =.1) with incresing L-tryptophn concentrtions. Bcteril ACCdeminse ctivity significntly enhnced root length (up to 5%) nd numer of roots (up to 47%), over control. On the other hnd, L-tryptophn dependent uxin production showed significnt negtive nd positive correltion for root length (up to r = -.992; P =.1) nd numer of roots (up to r =.979; P =.1), respectively. In pot trils, Alcligenes sp. Ml-4 recorded mximum increse for shoot length (57%), shoot fresh weight (85%), shoot dry weight (96%), numer of pods (64%) nd seeds weight (19%). The results showed tht Alcligenes sp. Ml-4 could e used s iofertilizer to enhnce the vigor nd yield of leguminous plnts. Keywords: Bcteril uxin; Nturl plnt settings; Non-rhizoil inoculnts; Rhizocteri; Vign rdite. Arevitions: ACC-1-minocyclopropne-1-croxylte; CFU-colony forming units; IAA-indole-3-cetic cid; PGPR-plnt growth promoting rhizocteri; SE-stndrd error. Introduction Plnt growth promoting rhizocteri (PGPR) is group of free-living cteri tht colonize the rhizosphere nd exert eneficil impct on plnt helth nd soil fertility. PGPR cn directly fcilitte the plnt growth in severl wys such s syntheses of phytohormones, siderophores production, soluiliztion of minerl phosphte nd synthesis of the enzyme ACC-deminse (Rdddi et l., 28; Ali et l., 21). The gseous plnt hormone ethylene prticiptes in the regultion of numer of developmentl processes in plnts. ACC is n immedite precursor of ethylene tht my e exuded from plnt roots. For mny plnts urst of ethylene is required to rek seed dormncy. However, fter seed germintion, sustined high level of ethylene inhiits root elongtion. It hs een reported tht PGPR contin n enzyme ACC-deminse tht hydrolyses ACC into mmoni nd α-ketoutyrte (Penrose nd Glick, 23; Glick, 25). PGPR hve the potentil to eliminte the inhiitory effects of higher ethylene concentrtions nd fcilitte the formtion of longer roots (Contesto et l., 28). Severl reports hve indicted tht inocultion of plnts with rhizocteri contining ACC-deminse incresed growth nd yield of plnts (Shhroon et l., 27; Zhir et l., 29; Siddikee et l., 211). In recent yers, rhizocteri contining ACCdeminse ctivity hs een used to llevite deleterious effects of ethylene under stressed conditions of high slt content, hevy metls, flooding nd drought (Grichko nd Glick, 21; Zhir et l., 28; Koo et l., 21; Ndeem et l., 21). Indole-3-cetic cid (IAA) represents one of the most extensively studied nd undnt type of uxin in plnts. Like plnts, IAA is lso quntittively the most undnt phytohormone secreted y rhizocteri. Synthesis of IAA y plnt ssocited cteri is proly one of the most importnt cuse for improving growth nd yields of vrious crops (Ali et l., 29, ). For instnce, in cse of Azospirillum, it is generlly greed tht uxin production rther thn nitrogen fixtion, is the mjor fctor responsile for the stimultion of rooting nd enhnced plnt growth (Bloemerg nd Lugtenerg, 21). A cterium my directly ffect plnt growth nd development using ny one or more of these mechnisms. Since mny PGPR possess severl of these trits, cterium my utilize different trits to fcilitte plnt growth (Rdddi et l., 28; Gulti et l., 29; Mehnz et l., 21). Effect of rhizocteri with uxin production, ACC-deminse ctivity nd phosphte soluiliztion is well documented in literture. However, the rhizocteri exhiiting these growth promoting trits simultneously hve not een evluted in detil. Moreover, little is known out the plnt growth promoting potentil of Alcligenes nd Providenci strins ssocited with the rhizosphere of nturl plnt settings. Especilly, the strins of Alcligenes nd Providenci gener hve not een evluted s non-rhizoil inoculnts for leguminous plnts. Therefore, in present work we compred Alcligenes, Providenci nd Bcillus strins for their growth promoting effects on Vign rdit (L.) Wilczek. Results 16S rrna gene sequencing The sequences of 16S rrna gene were nlyzed y comprison with sequences in GenBnk through BLAST ( After comprison, 1723

2 strins Am-2, Am-6 ssocited with the rhizosphere of A. viridis showed 98 nd 99% similrity with Providenci vermicol nd Bcillus pumilus, respectively. Strins Ml-4 nd Nic-2 isolted from the rhizosphere of M. tricuspedtum nd N. plumginifoli, respectively, hd mximum similrity of 99% with genus Alcligenes. Strin isolted from S. nigrum (Sol-1) showed homology of 99% with B. pumilus. The sequences from strins Am-2, Am-6, Ml-4, Nic-2 nd Sol-1 hve een deposited in the GenBnk under HQ161774, HQ161775, HQ161776, HQ nd HQ ccession numers, respectively (Tle 1). ACC-deminse Activity Strins showed vrile potentil for ACC-deminse ctivity (Fig 1). B. pumilus Sol-1 isolted from the rhizosphere of S. nigrum showed highest ACC-deminse ctivity (43 nmol h -1 ). On the other hnd, P. vermicol Am-2, B. pumilus Am-6, Alcligenes sp. Ml-4 nd A. feclis Nic-2 expressed 377, 234, 39 nd 16 nmol h -1 ACC-deminse ctivity, respectively. Auxin production y rhizocteri Anlysis of culture superntnts reveled tht cteril strins produced vrile mounts of uxin when grown in the sence nd presence of different concentrtions of L- tryptophn (Fig 2). In the sence of L-tryptophn, P. vermicol Am-2, Alcligenes sp. Ml-4 nd A. feclis Nic- 2 did not showed uxin production. However, the ddition of L-tryptophn (5 to 5 µg ml -I ) to L-roth medium enhnced the uxin production severl folds compred to non-mended medium. For instnce, Providenci vermicol Am-2 (r =.965; P =.1), Bcillus pumilus Am-6 (r =.814; P =.5) nd Alcligenes sp. Ml-4 (r =.947; P =.1), A. feclis Nic-2 (r =.97; P =.1) nd B. pumilus Sol-1 (r =.931; P =.1) showed significnt positive correltion with incresing L-tryptophn concentrtions. The most ctive IAA producers were Providenci vermicol Am-2, Bcillus pumilus Am-6 nd Alcligenes sp. Ml-4 tht produced 46, 45 nd 75 μg IAA ml -I respectively t 5 μg ml -I L-tryptophn. Phosphte soluiliztion Phosphte soluiliztion ility of rhizocteri ws determined qulittively y streking cteri on Pikovsky gr medium. Out of five cteril strins, P. vermicol Am-2, B. pumilus Am-6 nd B. pumilus Sol-1 were found positive for phosphte soluiliztion (Fig 3). Biossys Inocultion of seeds with rhizocteri exhiiting ACCdeminse ctivity stimulted root growth in mjority of the tretments (Fig 4). For root length, significnt increses of 5%, 4%, 4% nd 3%, respectively, were recorded with P. vermicol Am-2, Alcligenes sp. Ml-4, B. pumilus Sol-1 nd A. feclis Nic-2. Increses in numer of roots were lso oserved with P. vermicol (33%) nd Alcligenes sp. (47%). Significnt positive correltion (r =.93; P =.5) ws oserved etween cteril ACC-deminse ctivity nd root length. However, non-significnt correltion ws oserved with numer of roots. Effect of cteril uxin on root growth of V. rdit ws lso evluted in the presence of different concentrtions of L-tryptophn (Fig 5). Generlly, strins showed inhiitory effect on root length with incresing concentrtions of L-tryptophn (Fig 5). For instnce, P. vermicol Am-2 nd Alcligenes sp. Ml-4 showed 5% reduction in root length. Mximum root inhiition (62%) ws recorded with A. feclis Nic-2 t 5 µg ml -I L-tryptophn. However, cteril strins significntly stimulted numer of roots t 1 (122%), 2 (136%), 3 (122%), 4 (25%) nd 5 (22%) µg ml -I L-tryptophn (Fig 5). Alcligenes sp. Ml-4 ws the most effective to enhnce numer of roots nd showed 122%, 136% nd 25% increses t 1, 2, nd 4 µg ml -I L-tryptophn, respectively, over control. Highly significnt negtive correltions etween L-tryptophn concentrtions nd root length were recorded with P. vermicol Am-2 (r = -.942; P =.1), B. pumilus Am-6 (r = -.896; P =.5), A. feclis Nic-2 (r = -.992; P =.1) nd B. pumilus Sol-1 (r = -.888; P =.5). On the other hnd, numer of roots showed highly significnt positive correltions with P. vermicol Am-2 (r =.915; P =.5), B. pumilus Am-6 (r =.979; P =.1), Alcligenes sp. Ml-4 (r =.892; P =.5), A. feclis Nic-2 (r =.927; P =.1) nd B. pumilus Sol-1 (r =.982; P =.1). Plnt growth Bcteril inocultions showed vrile responses for different vegettive nd yield prmeters t full mturity (Tle 2). Significnt increses in shoot length were oserved with Alcligenes sp. Ml-4 (57%), B. pumilus Sol-1 (28%) nd B. pumilus Am-6 (25%) s compred to wter treted control. Mximum increses in shoot fresh weight were recorded with Alcligenes sp. Ml-4 (85%), B. pumilus Sol-1 (67%) nd B. pumilus Am-6 (44%). On the other hnd, shoot dry weight showed 96% increse with Alcligenes sp. Ml-4. In cse of numer of pods, significnt increses of 64%, 43% nd 35% were recorded with Alcligenes sp. Ml-4, A. feclis Nic-2 nd B. pumilus Sol-1, respectively, over control. Similrly, Alcligenes sp. Ml-4 nd B. pumilus Am-6 showed 19% nd 8% increses in seeds weight, respectively. However, mrginl improvements in seed weight were oserved with P. vermicol Am-2 nd A. feclis Nic-2. Significnt positive correltion etween cteril uxin production (t 5 µg ml - I L-tryptophn) nd seed weight (r =.857; P =.5) ws oserved. Shoot length lso showed highly significnt positive correltion with shoot fresh weight (r =.941; P =.1), shoot dry weight (r =.989; P =.1) nd numer of pods (r =.424; P =.5). Discussion Present work demonstrted the ility of newly isolted strins of Alcligenes, Providenci nd Bcillus gener from the rhizosphere of nturl plnt settings. The results showed tht strins exhiited uxin production, ACC-deminse nd phosphte soluiliztion simultneously. Colorimetric nlysis confirmed tht rhizocteri vried in their ility for uxin production in the sence nd presence of L- tryptophn. However, cteril efficiency for uxin production enhnced severl folds when medium ws mended with L-tryptophn. It is evident from highly significnt positive correltion (r =.814; P =.5 to r =.965; P =.1) etween cteril uxin production nd L- tryptophn concentrtions. Our results re in greement with previous findings tht incresing concentrtions of L- tryptophn lso stimulted cteril uxin production (Ahmd et l., 28; Ali et l., 29). Tretment of V. rdit seeds with rhizocteri exhiiting ACC-deminse ctivity significntly enhnced the root length (up to 5%) 1724

3 Tle 1. Identifiction of rhizocteri y 16S rrna gene sequencing. Strins Plnt Identified s Accessions Am-2 Amrnthus viridis L. Providenci vermicol Am-2 HQ Am-6 A. viridis L. Bcillus pumilus Am-6 HQ Ml-4 Mlvstrum tricuspedtum A. Gry Alcligenes sp. Ml-4 HQ Nic-2 Nicotin plumginifoli Viv. A. feclis Nic-2 HQ Sol-1 Solnum nigrum L. B. pumilus Sol-1 HQ Tle 2. Effect of cteril inocultions on vegettive nd yield prmeters of V. rdit (L.). Strins Vegettive growth prmeters Yield prmeters Shoot length (cm) Shoot fresh weight (g) Shoot dry weight (g) Numer of pods/ plnt Weight of 1 seeds (g) Control P. vermicol Am B. pumilus Am c Alcligenes sp. Ml c 2. d c A. feclis Nic c B. pumilus Sol cd Men of six replictes (3 plnts). Different letters within sme column indicte significnt difference etween tretments using Duncn s multiple rnge test (P=.5). nd numer of roots (up to 47%), over wter treted control. Root length showed positive correltion (r =.93; P =.5) with cteril ACC-deminse ctivity. The iologicl ctivity of cteril IAA ws demonstrted y its inhiitory effect on root length nd increse in lterl root numers. Doelere et l. (22) reported the root prolifertion nd increse in root dry weight upon inocultion with Azospirillum rsilense s compred to control plnts. Similrly, inocultion of seeds with incresing inoculum concentrtion of wild type A. rsilense Sp245 resulted in strong inhiition of root length nd increse in root hir formtion (Spepen et l., 28). Inocultion of whet nd mung ens with uxin producing rhizocteri hs een shown to result in the reduction of root length nd increse in lterl root numer (Ali et l., 29, ). Sttisticl nlysis of dt reveled tht mjority of the cteril inocultions significntly enhnced vegettive nd yield prmeters under nturl environmentl conditions. The percentge increses in shoot length, shoot fresh weight, shoot dry weight, numer of pods nd seed weight were up to 57%, 85%, 96%, 64% nd 19%, respectively. PGPR inoculted mung en plnts hve shown to improve shoot length, fresh weight, numer of pods nd seed weight (Ali et l., 29). It hs lso een reported tht inocultion of V. rdit with rhizospheric isoltes significntly enhnced the plnt growth under nturl environmentl conditions in pot trils (Shhroon et l., 26; Ali et l., 21). The improved plnt growth due to PGPR inocultion on different crop plnts hs lso een reported y severl workers. Fisl nd Hsnin (26) demonstrted tht B. cereus inocultions significntly enhnced growth nd yield of V. rdit under in vitro nd wire house conditions. In nother study, PGPR strins when co-inoculted with Rhizoium increses the vegettive growth nd grin yield of V. rdit (Rz et l., 24). Mterils nd methods Isoltion of cteril strins Bcteril strins were isolted from the rhizosphere of Amrnthus viridis, Mlvstrum tricuspedtum, Nicotin plumginifoli nd Solnum nigrum. One grm rhizosphere soil ws thoroughly mixed in 99 ml of utoclved glss- distilled wter to mke suspensions (Cppuccino nd Shermn, 22). Suspensions were serilly diluted (1-2, 1-4 nd 1-6 ) nd 1 µl plted on L-gr pltes. Pltes were incuted t 37 C for 24 h. After incution, thirty cteril colonies showing prolific growth were picked nd purified y mny rounds of restreking on L-gr pltes. Finlly, five rhizocteril strins showing plnt growth promoting trits were selected (Tle 1) nd mintined y trnsferring them to L-gr slnts. DNA extrction, PCR mplifiction nd 16S rrna gene sequencing Genomic DNA ws otined from cteril cultures grown in L-roth for 24 h, t 37 o C, using AquPure genomic DNA isoltion kit (BIO-RAD) in ccordnce with the instructions of mnufcturer. The 1.5 k DNA frgment contining 16S rrna gene ws mplified using forwrd primer 27f (5'- AGAGTTTGATCCTGGCTCAG-3') nd reverse primer 1522r (5'-AAGGAGGTGATCCA(AG)CCGCA-3') (Johnson, 1994). PCR mplifiction ws performed y using 5 µl of Drem Tq TM Green PCR Mster Mix (Ferments) with.5 µg of chromosoml DNA templte nd.5 µm of ech primer. The rection mixtures were incuted in thermocycler Primus 96 (PeQL, Erlngen, Germny) t 94 o C for 5 min nd pssed through 3 cycles: denturtion for 2 s t 94 o C, primer nneling for 2 s t 5 o C nd extension t 72 o C for 2 min. Finl extension ws crried out t 72 o C for 5 min. The mplified products were purified using QIAquick Gel Extrction Kit (QIAGEN) nd sequenced using 27f nd 1522r primers y ABI PRISM-31 Genetic Anlyzer (Applied Biosystems, USA). ACC-deminse ssy ACC-deminse ctivity of rhizocteri ws ssessed y quntifying mmoni lierted y the hydrolysis of ACC. Induction of ACC-deminse ctivity in rhizocteri ws ccomplished following the method of (Penrose nd Glick, 23). Bcteril cultures were grown in 25 ml L-roth y incuting overnight in n oritl shker t 12 rev min -I t 37 o C. Bcteril iomss ws hrvested y centrifugtion (8 g) for 1 min t 4 o C. Cells were wshed with 5 ml DF slts miniml medium nd re-suspended in 7.5 ml DF slts miniml medium supplemented with 3 mm ACC to induce 1725

4 Auxin (μg ml -I ) ACC-deminse (nmol h -1 ) the ACC-deminse ctivity. The tues were incuted in n oritl shker t 12 rev min -I t 3 o C for 24 h. After incution, superntnt ws removed nd the cells were wshed with 5 ml DF slts miniml medium. Finlly, cteril cells were suspended in 7.5 ml DF slts miniml medium in fresh culture tue contining 3 mm ACC s mentioned ove. The cteril cultures were incuted for 1 h with shking t 12 rev min -I t 3 o C. After 1 h, cultures were centrifuged t 8 g nd lierted mmoni ws mesured in superntnt s descried in Ngtsu nd Ygi (1966). Auxin production Auxin production y cteril strins ws determined in the presence nd sence of precursor L-tryptophn. Different concentrtions of L-tryptophn were used to evlute the in vitro uxin production y plnt ssocited cteri. Strins were grown in 1 ml L-roth medium in 25 ml Erlenmeyer flsks supplemented with filter sterilized solution of L- tryptophn in different concentrtions such s, 5, 1, 2, 3, 4 nd 5 µg ml 1. The flsks were inoculted in triplicte with 5 µl of cteril cell suspension djusted to 1 7 CFU ml -I. All inoculted flsks were incuted t 37 C for 72 h t 12 rev min -I. After incution, cells were removed y centrifugtion t 23 g for 15 min (Sigm 2-5). One ml of superntnt ws tken nd mixed with 2 ml of Slkowski regent s mentioned previously (Ali nd Hsnin, 27). The contents in test tues were llowed to stnd for hlf n hour for color development. The intensity of color ws mesured t 535 nm y Aqurius UV/Visile Doule Bem Spectrophotometer (CECIL CE 72). Stndrd curve ws drwn for comprison to determine uxin production in cteril culture superntnt. Phosphte soluiliztion Phosphte soluiliztion ility of plnt ssocited cteri ws determined qulittively y streking strins on Pikovsky gr pltes (Pikovsky, 1948). The presence of clering zone round cteril growth fter one week incution period t 3 o C ws used s indictor for positive phosphte soluiliztion c Am-2 Am-6 Ml-4 Nic-2 Sol-1 Strins Fig 1. ACC-deminse ctivity of rhizocteri. Brs represents men ± SE of three replictes. Different letters on rs indicte significnt difference etween tretments, using Duncn s multiple rnge test (P=.5) c Am-2; r =.965** Am-6; r =.814* Ml-4; r =.947** Nic-2; r =.97** Sol-1; r =.931** L-Tryptophn (μg ml -I ) Fig 2. Effect of different L-tryptophn concentrtions on uxin production y rhizocteri. The results shown re representtive of three repetitions of the experiment. Brs t different points indicte SE for ech tretment. Vlue r indictes highly significnt positive correltion etween L- tryptophn nd cteril uxin production. ** (P =.1), * (P =.5). d Seed steriliztion nd preprtion of inoculum Certified seeds of V. rdit were otined from Punj Seed Corportion, Lhore, Pkistn. Helthy seeds were surfce sterilized with.1% HgCl 2 for 5 minutes, with continuous shking followed y repeted wshing for 3-4 times in sterilized glss-distilled wter. For preprtion of cteril suspensions, cteril strins were grown in 1 ml L-roth overnight t 37 C. Bcteril cells from cultures were hrvested y centrifugtion t 23 g s mentioned ove. Cells were wshed nd re-suspended in sterilized glssdistilled wter. To ensure the equl cell popultion of ech cteril strin in the suspension, opticl density ws mesured y spectrophotometer t 6 nm. Opticl density of cultures ws djusted with sterilized glss-distilled wter, to finl concentrtion of 1 7 CFU ml -1. Petri dish iossys Effect of cteril ACC-deminse ctivity nd uxin production on root growth of V. rdit ws demonstrted in the sence nd presence of L-tryptophn, respectively. Petri dishes with two filter ppers were utoclved nd soked Fig 3. Phosphte soluiliztion y rhizocteri. Arrows indicte the clering zones round cteril growth. ()- P. vermicol Am-2; () - B. pumilus Sol

5 Numer of roots Root length (cm) Root growth c c RL Am-2 Am-6 Ml-4 Nic-2 Sol-1 Control Strins NR c c with 1 ml sterilized distilled wter. Five sterilized seeds were plced in ech petri dish nd experiment ws repeted three times under xenic conditions. Two ml sttionry phse cultures grown in the presence of, 5, 1, 2, 3, 4 nd 5 µg ml -I L-tryptophn in L-roth were poured. To oserve the effect of cteril ACC-deminse on root growth, two ml cteril cultures grown in the sence of L- tryptophn were pplied. Wter treted seeds were used s control nd ll petri pltes were incuted in Verstile Environmentl Test Chmer (MRL-35H; Snyo, Osk, Jpn). After 1 dys, effect of cteril cultures on root length nd numer of roots ws oserved. Fig 4. Effect of cteril ACC-deminse ctivity on root growth of V. rdit. Brs represents men ± SE of three replictes (15 plnts). Different letters on rs indicte significnt difference etween tretments, using Duncn s multiple rnge test (P=.5). Arevitions: RL, root length; NR, numer of roots. Am-2; r = -.942** Am-6; r = -.896* Ml-4; r = Nic-2; r = -.992** Sol-1; r = -.888* Control L-Tryptophn (μg ml -1 ) (A) Pot trils Sterilized seeds of V. rdit were inoculted with single cteril culture suspensions djusted to the finl concentrtion of 1 7 CFU ml -I s descried ove. For control tretment, seeds were dipped in sterilized glss-distilled wter for 15 minutes. Seeds were sown to depth of 1cm in erthen pots (3 3 cm) filled with 1 Kg of grden soil. Six pots for ech cteril strin s well s control were used. Initilly, 15 seeds in ech pot were plnted. Pots were moistened immeditely fter sowing. Arrngement of the pots ws mde in completely rndomized design in the wire house under mient light nd temperture. After complete emergence of seedlings, plntlets were thinned out to 1 plnts per pot. Plnts were irrigted regulrly nd growth of plnts ws oserved dily. After six weeks, 5 plnts were removed from ech pot to record fresh nd dry weight of plnts. After mesurements of fresh weight, plnts were dried in electric oven t 8ºC for 24 h nd dry weight of inoculted nd non-inoculted plnts ws tken. Hrvesting of plnts ws crried out t full mturity to record vegettive nd yield prmeters. Sttisticl nlysis Am-2; r =.915* Am-6; r =.979** Ml-4; r =.892* Nic-2; r =.927** Sol-1; r =.982** Control For ll experiments, the dt were sujected to sttisticl nlysis using softwre SPSS 16 progrm (SPSS lnc., Chicgo, IL). Dt were sujected to nlysis of vrince (ANOVA) nd mens seprted using Duncn s multiple rnge test (P=.5). The correltion coefficients etween cteril uxin production nd L-tryptophn concentrtions s well s etween cteril growth trits nd plnt growth prmeters were lso clculted (P =.1 or P =.5) L-Tryptophn (μg ml -1 ) Fig 5. Effect of different L-tryptophn concentrtions on root growth of V. rdit. ()- Root length, ()- Numer of roots. Vlue r in ech figure indictes significnt correltion etween L-tryptophn concentrtions nd root growth. ** (P =.1), * (P =.5). (B) Conclusion In conclusion, rhizocteri exhiiting multiple plnt growth promoting trits enhnced vegettive nd yield prmeters of V. rdit under nturl environmentl conditions. Bcteril ACC-deminse ctivity significntly enhnced root growth under xenic conditions. Incresing L-tryptophn concentrtions showed inhiitory effect on root length ut enhnced lter root numers tht indicted the production of high concentrtions of uxin y rhizocteri

6 The present study suggested tht Alcligenes sp. Ml-4, B. pumilus Am-2 nd B. pumilus Sol-1 cn e used s crop enhncer nd iofertilizers. Hence, PGPR strins from nturl plnt settings hror eneficil trits tht cn e pplied s non-rhizoil inoculnts to enhnce the growth nd yield of leguminous plnts. References Ahmd F, Ahmd I, Khn MS (28) Screening of freeliving rhizospheric cteri for their multiple plnt growth promoting ctivities. Microiol Res 163: Ali B, Hsnin S (27) Efficcy of cteril uxin on in vitro growth of Brssic olerce L. World J Microiol Biotechnol 23: Ali B, Sri AN, Ljung K, Hsnin S (29) Auxin production y plnt ssocited cteri: impct on endogenous IAA content nd growth of Triticum estivum L. Lett Appl Microiol 48: Ali B, Sri AN, Ljung K, Hsnin S (29) Quntifiction of indole-3-cetic cid from plnt ssocited Bcillus spp. nd their phytostimultory effect on Vign rdit (L.). World J Microiol Biotechnol 25: Ali B, Sri AN, Hsnin S (21) Rhizocteril potentil to lter uxin content nd growth of Vign rdit (L.). World J Microiol Biotechnol 26: Bloemerg GV, Lugtenerg BJJ (21) Moleculr sis of plnt growth promotion nd iocontrol y rhizocteri. Curr Opin Plnt Biol 4: Cppuccino JG, Shermn N (22) In: Microiology: A Lortory Mnul, sixth ed. Person Eduction, Signpore. Contesto C, Desrosses G, Lefoulon C, Ben G, Borel F, Gllnd M, Gmet L, Vroquux F, Tourine B (28) Effects of rhizocteril ACC deminse ctivity on Aridopsis indicte tht ethylene medites locl root responses to plnt growth-promoting rhizocteri. Plnt Science 175: Doelere S, Croonenorghs A, Thys A, Ptcek D, Okon Y, Vnderleyden J (22) Effect of inocultion with wild type Azospirillum rsilense nd A. irkense strins on development nd nitrogen uptke of spring whet nd grin mize. Biol Fertil Soils 36: Fisl M, Hsnin, S (26) Growth stimultory effect of Ochroctrum intermedium nd Bcillus cereus on Vign rdit plnts. Lett Appl Microiol, 43: Glick BR (25) Modultion of plnt ethylene levels y cteril enzyme ACC deminse. FEMS Microiol Lett 251:1-7. Grichko VP, Glick BR (21) Ameliortion of flooding stress y ACC deminse-contining plnt growthpromoting cteri. Plnt Physiol Biochem 39: Gulti A, Vys P, Rhi P, Ksn RC (29) Plnt growthpromoting rhizosphere-competent Acinetocter rhizosphere strin BIHB 723 from the cold deserts of Himlys. Curr Microiol 58: Johnson JL (1994) Similrity nlysis of rrnas. In Methods for generl nd moleculr cteriology (ed. Gerhrdt P, Murry RGE, Wood WA, Krieg NR, Eds.), pp Am Soc Microiol Wshington, D.C. Koo S, Hong SH, Ryu HW, Cho K (21) Plnt growthpromoting trit of rhizocteri isolted from soil contminted with petroleum nd hevy metls. J Microiol Biotechnol 2: Mehnz S, Big DN, Lzrovits G (21) Genetic nd phenotypic diversity of plnt growth promoting rhizocteri isolted from sugrcne plnts growing in Pkistn. J Microiol Biotechnol 2: Ndeem SM, Zhir ZA, Nveed M, Asghr HN, Arshd M (21) Rhizocteri cple of producing ACCdeminse my mitigte slt stress in whet. Soil Sci Am J 74: Ngtsu T, Ygi K (1966) A simple ssy of monomine oxidse nd D-mino cid oxidse y mesuring mmoni. J Biochem 6: Penrose DM, Glick BR (23) Methods for isolting nd chrcterizing ACC deminse-contining plnt growthpromoting rhizocteri. Physiol Plnt 118:1-15. Pikovsky RI (1948) Moiliztion of phosphorus in soil in connection with virl ctivity of some microil species. Mikroiologiy 17: Rdddi N, Cherif A, Boudous A, Dffonchio D (28) Screening of plnt growth promoting trits of Bcillus thuringiensis. Ann Microiol 58: Rz W, Akhtr MJ, Arshd M, Yousf S (24). Growth, nodultion nd yield of mungen (Vign rdit L.) s influenced y coinocultion with Rhizoium nd plnt growth promoting rhizocteri. Pk J Agri Sci 41: Shhroon B, Arshd M, Zhir ZA (26) Effect of plnt growth promoting rhizocteri contining ACCdeminse on mize (Ze mys L.) growth under xenic conditions nd on nodultion in mung en (Vign rdit L.). Lett Appl Microiol 42: Shhroon B, Jmro GM, Zhir ZA, Arshd M, Memon KS (27) Effectiveness of vrious Pseudomons spp. nd Burkholderi cryophylli contining ACC-deminse for improving growth nd yield of whet (Triticum estivum L.). J Microiol Biotechnol 17:

7 Siddikee MA, Glick BR, Chuhn PS, Yim WJ, S T (211) Enhncement of growth nd slt tolernce of red pepper seedlings (Cpsicum nnuum L.) y regulting stress ethylene synthesis with hlotolernt cteri contining 1- minocyclopropne-1-croxylic cid deminse ctivity. Plnt Physiol Biochem 49: Spepen S, Doelere S, Croonenorghs A, Vnderleyden J (28) Effects of Azospirillum rsilense indole-3-cetic cid production on inoculted whet plnts. Plnt Soil 312: Zhir ZA, Munir A, Asghr HN, Shhroon B, Arshd M (28) Effectiveness of rhizocteri contining ACC deminse for growth promotion of pes (Pisum stivum) under drought conditions. J Microiol Biotechnol 18: Zhir AZ, Ghni U, Nveed M, Ndeem SM, Asghr HN (29) Comprtive effectiveness of Pseudomons nd Serrti sp. contining ACC-deminse for improving growth nd yield of whet (Triticum estivum L.) under slt-stressed conditions. Arch Microiol 191: