Rhizobacteria isolated from a metal-polluted area enhance plant growth in zinc and cadmium-contaminated soil

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1 Int. J. Environ. Si. Tehnol. (215) 12: DOI 1.17/s z ORIGINAL PAPER Rhizoteri isolted from metl-polluted re enhne plnt growth in zin nd dmium-ontminted soil S. I. A. Pereir L. Bros P. M. L. Cstro Reeived: 23 Septemer 213 / Revised: 28 Mrh 214 / Aepted: 1 My 214 / Pulished online: 4 June 214 Islmi Azd University (IAU) 214 Astrt In this work, we evluted the ility of rhizoteri isolted from metl-ontminted site to produe plnt growth-promoting sustnes, suh s indole-3- eti id (IAA), mmoni, hydrogen ynide, siderophores nd extrellulr enzymes, to soluilize phosphte nd for 1-minoylopropne-1-roxylte (ACC)-deminse tivity, while ssessing their pity to enhne Trifolium repens growth. The rhizoteri tht etter performed were used s ioinoulnts for plnts grown in zin- nd dmium-spiked soils. Rhizoteri were inoulted onto white lover seedlings growing in 25 nd 5 mg kg -1 of zin nd in 1 nd 3 mg kg -1 of dmium-spiked soils nd plnt trits determined. The ville nd exhngele rhizosphere metl onentrtions were nlyzed. Twenty rhizoteri stimulted white lover growth under xeni onditions. Overll, strins Rhodoous erythropolis EC 34, Ahromoter sp. 1AP2 nd Miroterium sp. 3ZP2 inresed lover iomss in the sene nd in presene of metls; however, in soils with 25 mg kg -1 of zin, teril inoultion did not enhne plnt iomss. Biomss enhnement my e relted to the multiple teril promoting trits, espeilly the prodution of high levels of IAA nd siderophores nd the tivity of ACC-deminse. To the est of our knowledge, this is the first study reporting the potentil of R. erythropolis to inrese plnt growth in metl-ontminted soils. In generl, strins Miroterium sp. 3ZP2 nd Arthroter sp. EC 1 enhned the ville nd S. I. A. Pereir L. Bros P. M. L. Cstro (&) CBQF - Centro de Biotenologi e Quími Fin - Lortório Assoido, Esol Superior de Biotenologi, Universidde Ctóli Portugues/Porto, Ru Dr. António Bernrdino Almeid, Porto, Portugl e-mil: plstro@porto.up.pt exhngele metl onentrtions in rhizosphere showing their potentil to improve phytoremedition effiieny. This work lerly indites tht rhizoteri re promising for the development of ioinoulnts useful for phytoremedition of polluted soils. Keywords Hevy metls Plnt growth promotion Rhizoteri White lover Introdution Soil ontmintion with hevy metls hs eome one of the most severe environmentl hzrds. Anthropogeni tivities suh s mining, smelting, omustion of fossil fuel nd sewge sludge hve polluted extensive res throughout the world (Tsezos 29). High levels of metls not only derese soil miroil tivity nd rop prodution, ut lso threten humn helth sine they re le to enter the food hin, inresing the risk of trnsfer to food produts (Boyd 21). Biologil remedition of polluted sites hs reeived muh ttention worldwide s method for improving soil qulity y removing metls from soils (Mrques et l. 29). Phytoremedition is mong these tehniques offering signifintly more enefits thn onventionl lenup tehnology (Pilon-Smits 25). However, the slow growth nd low iomss of plnts in metl-ontminted soils my limit the effiieny of phytoremedition (Li nd Rmkrishn 211; M et l. 211). Moreover, the metl iovilility in rhizosphere soil is onsidered to e nother ritil ftor tht determines the effiieny of metl trnslotion nd phytostiliztion proess (M et l. 211). In reent yers, severl hemil mendments, suh s EDTA, hve een used to enhne either phytoextrtion

2 2128 Int. J. Environ. Si. Tehnol. (215) 12: or phytostiliztion proess (Brruti et l. 21). However, heltors my e toxi to plnts nd to soil miroorgnisms (Evngelou et l. 27; Ultr et l. 25). A promising lterntive is the utiliztion of plnt miroe intertions to improve the effiieny of phytoremedition, ltering the moility nd iovilility of metls in soils (Glik 21; M et l. 211; Rjkumr et l. 21). The results of severl studies hve demonstrted tht plnt-ssoited miroorgnisms ontriute to the uptke of metls y plnts nd improve plnt growth under metl exposure (Andrezz et l. 21; Dell Amio et l. 28; M et l. 29; Mdhiyn et l. 27; Sheng nd Xi 26). Thus, the intertions etween plnts nd enefiil rhizosphere miroorgnisms my e key element of ny phytoremedition tehnology (Andrezz et l. 21; Glik 21). Among the rhizosphere miroorgnisms involved in hevy metl phytoremedition, the plnt growth-promoting rhizoteri (PGPR) deserve speil ttention. PGPR re heterogeneous group of rhizospheri teri le to ggressively olonize plnt roots nd improve the extent or qulity of plnt growth (Kloepper nd Shroth 1978). Plnt growth-promoting rhizoteri elerte nutrient vilility nd ssimiltion nd suppress the deleterious effets of phytopthogeni miroorgnisms. PGPR n stimulte plnt growth y diret mehnisms whih inlude the iologil nitrogen fixtion, the prodution of phytohormones (uxins, gierellins nd ytokines), mmoni (NH 3 ) nd siderophores nd the soluiliztion of phosphte (Gupt et l. 22; Vessey 23; Zidi nd Khn 27). Moreover, some PGPR hve the enzyme 1-minoylopropne-1-roxylte (ACC)-deminse, whih hydrolyze ACC, the immedite preursor of ethylene in plnts, lowering ethylene level nd thus its inhiitory effet on plnt growth (Glik 25). The indiret promotion ours when the rhizoteri derese or prevent the deleterious effets of pthogeni miroorgnisms (Penrose nd Glik 23). This proess is omplished y the prodution of ntgonisti sustnes suh s ntiiotis; ntifungl metolites (e.g., hydrogen ynide HCN); nd extrellulr enzymes (Glik 21; vn Loon 27). Trifolium repens is one of the most importnt forge legume speies worldwide, nd it hs een used s n inditor plnt in severl studies due to its high sensitivity to orgni nd inorgni ompounds (Ain et l. 26; Vsseur et l. 1998). However, most of the studies on T. repens growing in metl-polluted res hve een foused on the symioti nitrogen fixtion (Broos et l. 24; Cstro et l. 23; Chudri et l. 28), nd little ttention hs een pid to the effet of PGPR on the growth of white lover in metl-ontminted soils. The ojetives of this study were to test the ility of severl PGPR previously isolted from hevy metl-ontminted site in Estrrej, Northern Portugl, to produe plnt growthpromoting sustnes, suh s indole-3-eti id (IAA), NH 3, HCN, siderophores nd extrellulr enzymes (lipses, ellulses, proteses, petinses), to soluilize phosphte nd ACC-deminse tivity nd to ssess the PGPR potentil to promote Trifolium repens growth under xeni onditions. The rhizoteri tht etter performed were seleted to evlute their effet on the enhnement of white lover growth promotion in plnts grown in metlspiked soils, s possile ioinoulnts to ssist phytoremedition proesses. Zin (Zn) nd dmium (Cd) were the hosen metls to rry out this study sine Zn is present t very high onentrtions in Estrrej region nd Cd is often ssoited with Zn in metl-polluted soils. Mterils nd methods Bteril strins The teril strins used in this study were indigenous from hevy metl-ontminted site loted in Northern Portugl Esteiro de Estrrej. The site hs long history of metl ontmintion, due to the industril tivity in the surrounding re. The onentrtions of hevy metls in the re reh hzrdous levels. Averge levels of 835 mg P kg -1, 66 mg Hg kg -1, 3,62 mg Zn kg -1,26mgCrkg -1,37mg Ni kg -1 nd 16,8 mg Fe kg -1 were mesured for the site (Oliveir et l. 21). In previous work, we rried out n extensive hrteriztion nd identifition through 16S rrna, of the miroil ommunities presented in the rhizosphere of severl thriving plnts growing in this re. Prtil sequene of the 16S rrna gene of teril strins ws otined using universl primers 27F (5 -GAG- TTTGATCCTGGCTCAG-3 ) nd 1492R (5 -ACCTTGT- TACGACTT-3 ). The vlues of 16S rrna gene sequene similrity etween the teril strins nd the losest reltive were generlly high (97 1 %) (Pires 21). For the present work, we seleted 32 teril strins sed on their high tolerne to metls (Zn nd Cd) in tryptise soy gr (TSA) medium. In generl, these isoltes tolerte up to 5 mg l -1 of Cd nd 1, mg l -1 of Zn (Pires 21). Evlution of plnt growth promotion ilities Prodution of indole-3-eti id (IAA) The prodution of IAA ws determined ording to the method of Gordon nd Weer (1951) with slight modifitions. Bteri were grown overnight in tryptise soy roth (TSB) nd then septilly olleted y entrifugtion t 9, rpm for 1 min. The teril pellet ws then

3 Int. J. Environ. Si. Tehnol. (215) 12: inuted t 3 C for 48 nd 72 h with 3 ml of phosphte uffer (ph 7.5) with gluose (1 %) nd 2 ml of L-tryptophn (1 %). After inution, 2 ml of 5 % trihloroeti id nd 1 ml of.5 M CCl 2 were dded nd the mixture ws entrifugted t 9, rpm for 1 min. The superntnt (5 ll) ws mixed with 3 ll of slper solution (2 ml of.5 M FeCl 3 nd 98 ml of 35 % perhlori id). The sorne of pink olor developed fter 3 min of inution in the drk ws red t 535 nm. The experiments were repeted three times, nd IAA determintions were mde in triplite. ACC-deminse ssy The ACC-deminse tivity of ell-free extrts ws determined y monitoring the mount of -ketoutyrte generted y the enzymti hydrolysis of ACC (Sleh nd Glik 21). For determining ACC-deminse tivity, the teril ell pellets were wshed twie in 5 ml of.1 M Tris HCl, ph 7.6. Then pellets were suspended in 5 ll of.1 M Tris HCl, ph 8.5, nd 5 ll of toluene ws dded to the ell suspension nd vortexed for 3 s. Two hundred miroliter of the toluenized ells ws pled in miroentrifuge tue, nd 2 ll of.5 M ACC ws dded. The suspension ws vortexed nd inuted t 3 C for 3 min. Following the ddition of 1 ml of.56 M HCl, the mixture ws vortexed nd entrifuged for 1 min t 1, rpm t room temperture. Two hundred nd fifty milliliter of the superntnt ws mixed together with 2 ll of.56 M HCl. Thereupon, 75 ll of the 2,4-dinitrophenylhydrzine (DNPH) regent (.2 % DNPH in 2 M HCl) ws dded, nd the mixture ws inuted t 3 C for 3 min. Following the ddition of 5 ll of 2 M NOH, the sorne of the mixture ws mesured y using spetrophotometer t 54 nm. The tivity of ACCdeminse ws lulted using lirtion urve of pure -ketoutyrte (.5 lm). The experiments were repeted three times, nd ACC-deminse determintions were mde in triplite. Ammoni, siderophores nd HCN prodution For ssessing the ility to produe mmoni, fresh ultures were inoulted into 1 ml of peptone wter nd inuted for 48 h t 3 C. Then, 5 ll ofnessler s regent (Sigm-Aldrih) ws dded to eh tue. The ssy ws repeted three times. Development of rown to yellow olor ws positive test for mmoni prodution (Cppuino nd Shermn 1992). Bteril isoltes were sreened for the prodution of siderophores on the hrome zurol S (CAS) gr medium, s desried y Shwyn nd Neilnds (1987). Development of yellow to ornge hlo round the teril growth fter inution for 48 h t 3 C ws onsidered positive result for siderophore prodution. Four pltes were mde for eh strin. The prodution of HCN y teril isoltes ws determined y mending TSA medium with 4.4gl -1 of glyine. Strins were streked on the modified pltes, nd Whtmn filter pper No.1 soked in 2 % sodium ronte prepred in.5 % piri id solution ws pled on the top of eh of the pltes. Pltes were inuted t 3 C for4dysfterwhih development of ornge to red olor indited HCN prodution (Ahmd et l. 28). Four replites were mde for eh teril strin. Phosphte soluiliztion The phosphte soluilizing tivity of the strins ws nlyzed in Institute Phosphte medium (NBRIP) mended with.5 % of trilium phosphte. The strins were grown t 3 C for 1 dys (Nutiyl 1999). The presene of lering hlo round teril olonies is inditive for positive phosphte soluiliztion. Four replites were mde for eh teril strin. Prodution of extrellulr enzymes Bteril strins were grown overnight in 1 ml of TSB t 3 C. The ulture ws then entrifuged t 9, rpm for 1 min. The superntnt ws olleted nd used s enzyme rude extrt. Protese prodution ws tested using nutrient gr medium mended with 1 % of skim milk. After 3 dys of inution t 3 C, ler zone round the ells indited positive proteolyti tivity (Smiert nd Krieg 1994). Cellulse prodution ws determined in roxymethylellulose (CMC) gr medium (per liter:.5 g KH 2 PO 4,.25gMgSO 4 7H 2 O, 2 g CMC disodium slt, 15 g gr). After 5 dys of inution t 3 C, CMC pltes were flooded with n queous solution of Congo red (1 % w/v) for 15 min. The Congo red solution ws then poured off, nd the pltes were flooded with 1 M NCl for 15 min. A ler hlo ws onsidered s positive for ellulse prodution (Gupt et l. 212). Petinse prodution y the teril strins ws determined s desried y Ctteln et l. (1999). Briefly, M9 medium gr ws mended with 1 g l -1 petin, 2 g l -1 gluose plus 1.2 g l -1 yest extrt. Pltes were inuted 3 dys t 3 C pltes nd then flooded with Lugol s iodine solution for 1 min. Cler hlos round the olonies were onsidered s positive for petinse prodution. The prodution of lipses ws ssessed in LB-modified gr medium supplemented with 2.gl -1 of CCl 2 nd1gl -1 of Tween-2, Tween-4 ndtween-8(smiertndkrieg1994). All ssys were performedintriplite.

4 213 Int. J. Environ. Si. Tehnol. (215) 12: Influene of PGPR on T. repens growth under xeni onditions This experiment ws rried out to sreen the PGPR ility to promote T. repens growth under xeni onditions. T. repens seeds were surfe-sterilized y exposing them 3 s to 95 % ethnol nd 1 min to.5 % (v/v) NOCl. Then, seeds were wshed six times with sterilized distilled wter. Seeds were germinted in wter gr pltes (15 %) for 24 h. Bteril strins were grown in TSB roth overnight t 3 C, fter whih they were wshed nd resuspended in sline solution (.85 % NCl) to otin finl onentrtion of 1 8 CFU ml -1. White lover seedlings with 1 dy of growth were pled in flsks with Jensen s gr medium (per liter:.8 g CCl 2,.2 g K 2 HPO 4,.2 g MgSO 4 7H 2 O,.1 g NCl,.1 g FeCl 3,.5 g KNO 3,1ml of mironutrient solution whih omposition is: per liter, 2.8 g H 3 BO 3, 1.83 g MnSO 4 4H 2 O,.1 g ZnCl 2,.91 g CuSO 4 7H 2 O,.2 g (NH 4 ) 6 Mo7O 2 7H 2 O) nd then inoulted with 1 ml of suspension of eh teril strin. One milliliter of sterilized sline solution ws dded to the ontrol tretment. The plnts were kept in ontrolled growth hmer (12 h photoperiod, 3 lmol m -2 s -1 photosynthetilly tive rdition, 22 C temperture, 75 % reltive humidity). Hrvest ourred 6 weeks fter the eginning of the experiment. Entire plnts were wshed with distilled wter nd divided in roots nd shoots, fter whih root elongtion nd shoot elongtion were registered. The dry iomss of the plnts ws determined fter oven drying t 7 C for 48 h. Seedling length ssy in presene of Zn nd Cd Bsed on the PGP trits nd the ility to inrese T. repens growth under xeni onditions, four teril strins (Rhodoous erythropolis EC34; Ahromoter sp. 1AP2; Miroterium sp. 3ZP2 nd Arthroter sp. EC1) were seleted for exploring their potentil to promote seedling elongtion in the presene of metls. Seleted strins were grown in TSB medium for 24 h t 3 C onn oritl shker. Cells were olleted y entrifugtion, wshed twie with sline solution (.85 % NCl) nd suspended in sterile sline solution to otin the finl inoulum density of 1 8 CFU ml -1. Two milliliter of teril suspensions or sterile sline solution (uninoulted ontrol) ws dded to Petri dishes with filter pper. Bteril suspensions nd sterile sline solution were supplemented with, 25, 5 nd 1, mg Zn l -1 nd, 1, 3, 5 nd 1 mg Cd l -1 supplied s ZnCl 2 nd CdCl 2 2.5H 2 O (Sigm), respetively. Trifolium repens vr. Hui seeds were surfe-sterilized nd septilly pled on wetted filter pper. Seedlings length ws mesured fter inution of the losed Petri dishes for 7 dys t 22 C (Belimov et l. 25). The vigor index ws determined s follows: % of germintion 9 seedling length (Luo et l. 211). Pot experiments: T. repens growth under Zn nd Cd exposure The experiments for studying the effet of the teril strins Rhodoous erythropolis EC34; Ahromoter sp. 1AP2; Miroterium sp. 3ZP2 nd Arthroter sp. EC1 on T. repens growth in the presene of different onentrtions of Zn nd Cd were onduted in pots (5 m dimeter nd 6 m high) ontining non-spiked or metlspiked soil. The soil used in this study ws n griulturl soil from northern Portugl, nd the physiohemil properties hve een desried in detil y Mrques et l. (213). The soil ws milled (2 mm) nd sterilized y steming (12 C for 7 min on two onseutive dys nd dried in n oven t 4 C for 4 dys). After steriliztion, the soil ws rtifiilly ontminted with queous solution of ZnCl 2 or CdCl 2 2.5H 2 O to hieve finl onentrtions of 25 nd 5 mg Zn kg -1 nd 1 nd 3 mg Cd kg -1 dry soil. Soil moisture ontent ws mintined t 6 % of the wter holding pity y dding deionized sterile wter. Spiked soils were pled for period of 6 weeks in the greenhouse undergoing 3 yles of sturtion with wter nd ir drying, fvoring the distriution of the metls throughout the entire soil mss nd llowing the soil to reh equilirium etween ville nd orgni mtter sored frtions (Blylok et l. 1997). Tretments in the greenhouse inluded ontrol (nonspiked soil), 25 nd 5 mg Zn kg -1, 1 nd 3 mg Cd kg -1 spiked soil nd five types of inoultion: no teri B (uninoulted ontrol), B1 (EC 34), B2 (1AP2), B3 (3ZP2) nd B4 (EC 1). T. repens vr. Hui seeds were surfe-sterilized nd germinted in pots with 1 g of the tested soils. Five pots were used for eh tretment. Eh pot reeived six seeds, fter germintion, seedlings were redued to four per pot. For inoultion of the seedlings, teril strins were grown overnight in TSB medium t 3 C. Cells in the exponentil phse were hrvested y entrifugtion t 9, rpm for 1 min, wshed twie with sterile sline solution (.85 % NCl) nd entrifuged gin. Bteril inoulum ws prepred y resuspending pelleted ells in sterile sline solution to get n inoulum density of. 1 8 CFU ml -1. Two dys fter seedling emergene, the pots were inoulted with 2 ml of suspension of eh teril strin. Two milliliter of sline solution (B) ws lso dded to the ontrol tretment pots (uninoulted). Pots were pled in ontrolled growth room (12 h photoperiod, 45 lmol m -2 s -1 photosynthetilly tive rdition, C temperture rnge, 6 75 % reltive humidity rnge) nd were wtered dily. After 1 weeks, plnts were

5 Int. J. Environ. Si. Tehnol. (215) 12: removed from the pots nd the plnt surfe ws thoroughly wshed with tp nd deionized wter. Then, root elongtion nd shoot height were registered. Plnt dry iomss ws determined fter shoots, nd roots were ovendried t 7 C for 72 h. The exhngele ethylenediminetetreti id (EDTA) extrtle nd ville mmonium ette (NH4-A) extrtle Zn nd Cd frtions were determined for the soils t the end of the experiment. The exhngele nd ville Zn nd Cd frtions were determined using flme tomi sorption spetrosopy (FA-AAS) (Wlling et l. 1989) in Unim 96 spetrophotometer (Wlthm, USA). Sttistil nlysis All sttistil nlyses were performed using the SPSS 18. softwre pkge (SPSS In., Chigo, IL, USA). The dt were nlyzed through nlysis of vrine (ANOVA). To detet the sttistil signifine of differenes (P \.5) etween mens, the Dunn nd Dunnett s tests were performed. Results nd disussion Plnt growth-promoting rhizoteri hve gined worldwide importne nd eptne for soil remedition strtegies nd for griulturl purposes (Shrn nd Nehr 211). PGPR my use enefiil effets on plnt helth nd growth, through vriety of mehnisms nmely through the inrese of nutrient vilility nd ssimiltion from the environment, providing the plnt with hormones nd growth ftors or suppress disese-using miroes (Vessey 23). In the present study, teril strins isolted from metl-ontminted site showed PGP trits nd the ility to enhne T. repens growth under xeni onditions. Those trits llowed us to selet four rhizoteri, R. erythropolis EC 34, Ahromoter sp. 1AP2, Miroterium sp. 3ZP2 nd Arthroter sp. EC 1, with the ility to enhne white lover growth in Zn- nd Cd-spiked soils, through the inrese of root nd shoot iomss, nd ility to moilize metls in rhizosphere soils, thus with potentil to improve the effiieny of phytoremedition of metl-ontminted soils. PGP trits nd white lover growth promotion under xeni onditions Twenty teril strins signifintly inresed T. repens root iomss prodution nd/or shoot nd root elongtion when ompred to ontrol uninoulted plnts (Fig. 1). The root dry iomss of white lover vried from 3.7 mg (plnts inoulted with strin EC 6) to 1.9 mg (plnts inoulted with strin EC 34) (Fig. 1) nd root nd shoot elongtion from 4. m (plnts inoulted with strin EC 19) to 14.5 m (plnts inoulted with strin 3ZP2) nd from 5.8 m (plnts inoulted with strin EC 29) to 1.3 m (plnts inoulted with strin EC32), respetively (Fig. 1, ). Among the twenty teril strins whih inresed white lover growth, the gener Arthroter, Rhodoous, Miroterium nd Pseudomons gener were well represented. The positive impt of the Pseudomons strins on plnt growth hs een reported y severl uthors, while Arthroter, Rhodoous nd Miroterium strins re not so frequently reported s plnt growth-promoting rhizoteri. Dell Amio et l. (28) showed tht the inoultion of Brssi npus seeds with P. fluoresens ACCD strin used 64 % inrese of shoot fresh iomss. Similrly, the Pseudomons sp. Ps29C strin tested y Rjkumr nd Freits (28) inresed the fresh weight of Indin mustrd shoots y 22 %. It hs een reported tht rhizoteri indues plnt growth y ontriuting to the host plnt endogenous pool of phytohormones, suh s IAA (Khlid et l. 24; Ptten nd Glik 22). A low level of IAA promotes primry root elongtion, wheres high level stimultes lterl nd dventitious root formtion (Xie et l. 1996). In this study, ll strins were le to synthesize IAA in the presene of the preursor L- tryptophn (Fig. 2). The IAA mounts produed y strins rnged from 2.4 to 63.5 nd from 1.4 to 8.1 mg l -1, fter 48 nd 72 h, respetively (Fig. 2, ). Strins S3X (7.81 mg l -1 ) nd EC1B (8.5 mg l -1 ) elonging to Pseudomons nd Mesorhizoium gener, respetively, produed the highest mounts of IAA. Suh high levels of IAA prodution hve een reported efore. Frin et l. (212) reported tht 78 teril isoltes derived from Brssi npus rhizosphere showed IAA levels ove 1 mg l -1 nd t lest one strin from Pseudomons genus produed mg l -1 of IAA. M et l. (29) lsoshowedthtpsyhroter strin produed 11 mg l -1 nd severl Billus strins produed more thn 77 mg l -1 of IAA. In this study, severl isoltes (1AP2, EC 34, 3ZP2 nd EC 1) with high ility to produe IAA promoted white lover root iomss whih suggests reltion etween oth prmeters. There results were orroorted y Mrques et l. (21) who reported positive orreltion etween IAA-produing rhizoteri nd Ze mys root iomss promotion nd y Khlid et l. (24) who showed tht teril strins whih produed the highest mount of IAA lso inresed growth of whet. Promotion of root growth is one of the mjor mrkers y whih the enefiil effet of PGPR is mesured (Glik et l. 1995). Rpid estlishment of roots is dvntgeous for young seedlings s it inreses their ility to nhor themselves to the soil nd

6 2132 Int. J. Environ. Si. Tehnol. (215) 12: Root 12 Biomss (mg) () Control 3ZP2 1ZP3 EC 16 EC 29 EAL EC 39 3AP1 EAMR EC 19 EC 1 EC 28 EC 33 ECX 16 3C5 EC 32 1C3 1A2 EC 34 EC1B 3A12 3A1 1ZP4 1C2 EC 6 EAR EC 8 S3X EAPC8 EDP 28 1AP2 1ZP7 EC Root 14 length (m) () Control 3ZP2 1ZP3 EC 16 EC 29 EAL EC 39 3AP1 EAMR EC 19 EC 1 EC 28 EC 33 ECX 16 3C5 EC 32 1C3 1A2 EC 34 EC1B 3A12 3A1 1ZP4 1C2 EC 6 EAR EC 8 S3X EAPC8 EDP 28 1AP2 1ZP7 EC 2 12 Shoot 1 length (m) () Control 3ZP2 1ZP3 EC 16 EC 29 EAL EC 39 3AP1 EAMR EC 19 EC 1 EC 28 EC 33 ECX 16 3C5 EC 32 1C3 1A2 EC 34 EC1B 3A12 3A1 1ZP4 1C2 EC 6 EAR EC 8 S3X EAPC8 EDP 28 1AP2 1ZP7 EC 2 Fig. 1 Influene of PGPR inoultion on root () dry iomss (mg) nd on root () nd shoot () elongtion (m) of Trifolium repens plnts growing under xeni onditions. Results re expressed s men ± SE (n = 3 5). A one-wy ANOVA ws performed for eh plnt setion. The test results re shown with the test sttisti s: *signifint t the level P \.5; **signifint t the level P \.1; ***signifint t the level P \.1 ording to the Dunnett s test. The F vlue of ANOVA for root iomss is F = (P \.1) nd for root elongtion nd shoot elongtion re F = (P \.5) nd F = (P \.5), respetively to otin wter nd nutrients from their environment, thus enhning their proilities for survivl (Ptten nd Glik 22). 1-Aminoylopropne-1-roxylte-deminse tivity ws deteted in ll rhizoteri (Fig. 3). The strin EAR from Lysiniillus genus reorded the highest

7 Int. J. Environ. Si. Tehnol. (215) 12: h ***F(B) = ***F(T) = h ***F(BxT) = IAA (mg l -1 ) d d d, d d d d d,d d d g,,d e,,d,, g f f d () 3ZP2 1ZP3 EC 16 EC 29 EAL EC 39 3AP1 EAMR EC 19 EC 1 EC 28 EC 33 ECX 16 3C5 EC 32 1C3 1A2 EC 34 EC1B 3A12 3A1 1ZP4 1C2 EC 6 EAR EC 8 S3X EAPC 8 EDP 28 1AP2 1ZP7 EC h k 8 j 7 6 IAA (mg l -1 ) ,h g,h f, d,e,,d,d,d,e d,e d,e e,f h e,f, e,f,d,, i e,f g,h,d,e () 3ZP2 1ZP3 EC 16 EC 29 EAL EC 39 3AP1 EAMR EC 19 EC 1 EC 28 EC 33 ECX 16 3C5 EC 32 1C3 1A2 EC 34 EC1B 3A12 3A1 1ZP4 1C2 EC 6 EAR EC 8 S3X EAPC 8 EDP 28 1AP2 1ZP7 EC2 Fig. 2 Prodution of indole eti id (IAA) (mg l -1 ) y teril strins fter 48 h () nd 72 h () of inution. Results re expressed s men ± SE (n = 6 12). A two-wy ANOVA ws performed to determine the influene of the time of inution (48 nd 72 h) on IAA prodution. The test results re shown with the test sttisti for eh se (B teril isolte, T time of inution, B 9 T teril isoltes 9 time of inution intertion). ***Signifint t the level P \.1. A one-wy ANOVA ws performed for eh time of inution. Mens for the sme time of inution with different letters re signifintly different from eh other (P \.5) ording to the Dunn test. For 48 nd 72 h of inution, the F vlues of one-wy ANOVA re F = (P \.1) nd F = (P \.1), respetively. IAA ws never deteted in ontrol ultures whih were thus not onsidered for this sttistil nlysis ACC-deminse tivity (6.87 nmoles -ketoutyrte g -1 h -1 ) followed y strin EAPC8 (5.66 nmoles -ketoutyrte g -1 h -1 ) from Pseudomons genus. It ws possile to estlish positive trend etween plnt trits nd ACC-deminse, sine severl isoltes with high ACC-deminse tivity (EC 1, EC 32, EAPC8 nd EC 2) enhned white lover growth y inresing root iomss or shoot elongtion. Our results re in greement with Shhzd et l. (21), who showed tht rhizoteri hving ACC-deminse were highly effetive in improving root nd shoot length of hikpe plnts. Ctteln et l. (1999) lso reported tht ACC-deminse rhizoteri used erly growth in soyen. Ammoni prodution is nother ommon trit in tested rhizoteri (Tle 1). NH 3 prodution y teril isoltes my enhne plnt growth, whih ws lso seen y Mrques et l. (21) tht demonstrted positive orreltion etween teril mmoni prodution nd root nd shoot elongtion of Ze mys plnts. Only 2 strins (ECX16 nd 3C5) elonging to Arthroter genus showed ility to soluilize phosphte. The ility of rhizoteri to onvert insolule phosphorous into solule forms is n importnt trit in PGPR for inresing plnt yields (Chen et l. 26).

8 2134 Int. J. Environ. Si. Tehnol. (215) 12: ACC (nmoles α -ketoutyrte g -1 h -1 ) g,h e, g,h,h i e,, i h,i h,i,d,h d,e,f e, i,,d, i,,d k j,d,d i 3ZP2 1ZP3 EC 16 EC 29 EAL EC39 3AP1 EAMR EC 19 EC 1 EC 28 EC 33 ECX16 3C5 EC 32 1C3 1A2 EC 34 EC1B 3A12 3A1 1ZP4 1C2 EC 6 EAR EC 8 S3X EAPC8 EDP28 1AP2 1ZP7 EC2 Fig. 3 ACC-deminse tivity (nmoles -ketoutyrte g -1 h -1 )of sreened isoltes. Results re expressed s men ± SE (n = 4 8). Mens with different letters re signifintly different from eh other (P \.5) ording to the Dunn test. The F vlue of one-wy ANOVA is F = (P \.1) Siderophore prodution ws positive in ll tested strins (Tle 1). More thn 55 % of the isoltes showed strong or very strong prodution of siderophores, while only 5.4 % (EAL nd 1ZP7) were onsidered wek siderophore produers. Correltions etween siderophore prodution nd plnt trits were not signifint, proly due the sene of phytopthogeni miroorgnisms sine the plnt experiment ws performed in septi onditions. Severl studies demonstrted tht siderophore-produing rhizoteri improve plnt growth y inresing iron vilility to plnts or y limiting iron for phytopthogeni orgnisms (Shu nd Sindhu 211). Mrques et l. (21) showed tht teril siderophore prodution influened plnt trits, evidening positive orreltions with nutrients ssimiltion (N nd P) nd Z. mys growth. Hydrogen ynide prodution is nother possile wy y whih rhizoteri my suppress plnt pthogens. In the present study, 92 % of teril isoltes were le to produe HCN. Isoltes elonging to Arthroter, Pseudomons, Mesorhizoium nd Shewnell were mong the est HCN-produing strins (Tle 1). PGPR my lso synthesize extrellulr enzymes suh s, ellulses, petinses, proteses nd lipses. These teril trits will onfer signifint dvntge in the presene of phytopthogens, sine their ell wlls will e degrded y the extrellulr enzymes nd their deleterious effets suppressed. In the present work, generlly rhizoteri were le to produe lipse nd more thn 6 % of rhizoteri produed protese, while ellulse nd petinse tivity were restrited to few teril strins (Tle 1). The strins EAL (Billus sp.) nd EC 6 (Alligenes sp.) showed positive results for ll tested extrellulr enzymes, showing their potentil to e used on ioontrol of plnt pthogens. Correltions etween these teril trits nd plnt prmeters were not signifint, whih my e due to the septi onditions of the ssy. However, these PGP trits should e tken into onsidertion when onsidering field pplitions, s plnt resistne will e potentilly inresed if the ssoited teri produe these ompounds. Influene of PGPR inoultion on plnt growth in presene of Zn nd Cd Bsed on their PGP trits nd on growth promotion of T. repens under xeni onditions, four hevy metl-tolernt rhizoteri B1 (Rhodoous erythropolis EC 34), B2 (Ahromoter sp. 1AP2), B3 (Miroterium sp. 3ZP2) nd B4 (Arthroter sp. EC 1) were seleted for pot experiments. These rhizoteri showed multiple PGP trits, suh s the prodution of high levels of IAA nd siderophores nd ACC-deminse tivity. Strins B1 nd B3 gretly enhned root iomss y 112 nd 82 % nd root elongtion y 44 nd 46 %, respetively, while strins B2 nd B4 inresed white lover iomss y 19 nd 86 %, respetively (Fig. 1). Some of these rhizoteri were le to improve seedling length of T. repens in the sene nd in the presene of different levels of Zn nd Cd ontmintion (Tle 2); however, some signifint differenes were oserved etween tretments with different teril strins. The rhizoteri tht etter performed were strins B1 nd B4 for Zn-treted seeds, whih inresed seedling growth y 64 nd 96 %, respetively, in the presene of 25 mg l -1 of Zn nd strins B1, B2 nd B4 for Cd-treted

9 Int. J. Environ. Si. Tehnol. (215) 12: Tle 1 Bteril strins used nd in vitro sreening for NH 3, siderophore, HCN nd extrellulr enzymes prodution (ellulse, petinse, protese, lipse) nd phosphte soluiliztion Isolte Closest reltives (GenBnk ession numer) Grm retion NH 3 Siderophore HCN P soluiliztion Extrellulr enzymes Cellulse Petinse Protese Lipse T2 T4 T8 3ZP2 Miroterium sp. (KC128917)??????? - - -????? 1ZP3 Miroterium oxydns (KC128898)?????? - - -???? EC 16 Miroterium oxydns (KC128933)?????? - - -??? - EC 29 Miroterium oxydns (KC128945)?????? ??? EAL Billus sp. (KC12955)????? -??????? EC 39 Billus sp. (KC128952)??????? - - -????? 3AP1 Billus megterium (KC12895)?????? - -?????? EAMR Billus sutilis (KC12956)??????? - -?????? EC 19 Billus weihenstephnensis (KC128935)????????? - - -????? EC 1 Arthroter sp. (KC128928)???????? - - -?? - - EC 28 Arthroter sp. (KC128944)??????? - -?????? EC 33 Arthroter sp. (KC128948)??????? -? - -? - - ECX 16 Arthroter sp. (KC1295)???????? - -??? 3C5 Arthroter sp. (KC12899)???????? EC 32 Arthroter romi (KC128947)????????? - - -???? 1C3 Rhodoous sp. (KC128893)??????? - -? -??? 1A2 Rhodoous wrtislviensis (KC128881)??????? ??? EC 34 Rhodoous erythropolis (KC128949)?????? - - -????? EC1B Agroterium tumefiens (KC128919) -???? - - -? -? - - 3A12 Mesorhizoium sp. (HQ54554) -??????? - -?????? 3A1 Chryseoterium plustris (EU36967) -????? -? -???? 1ZP4 Sphingoterium sp. (HQ54555) -????? ??? 1C2 Cuprividus sp. (HQ54556) -????? ??? EC 6 Alligenes sp. (KC128924) -????? -??????? EAR Lysiniillus fusiformes (KC12971) -??????? - - -????? EC 8 Pseudomons sp. (KC128926) -???????? - -? -? - - S3X Pseudomons fluoreseins (KC12953) -???????? - - -????? EAPC 8 Pseudomon sputid (KC12966) -???????? - -? -??? EDP 28 Pseudomons retns (KC12945) -?????? - -? -??? 1AP2 Ahromoter sp. (KC128889) -?????? - -? -?? - 1ZP7 Vriovx prdoxus (KC12891) -?? ????? EC 2 Shewnell lti (KC12892) -????????? - - -???? - Negtive,? positive/wek,?? intermedite,??? strong,???? very strong prodution

10 2136 Int. J. Environ. Si. Tehnol. (215) 12: Tle 2 Seedling length (mm) nd vigor index (seedling length 9 % germintion) of T. repens vr. Hui seeds uninoulted (B) nd inoulted (B1 EC 34; B2 1AP2; B3 3ZP2; B4 EC 1) nd grown in the sene or presene of Zn (, 25, 5, 1, mg l -1 ) nd Cd (, 1, 3, 5, 1 mg l -1 ) Tretment Zn (mg l -1 ) Seedling length (mm) Vigor index , , B ± ± ±.34, 3.4 ±.2 4,36.7 ± ± ± ± B ± ± ± ±.23 4,355.3 ± ± ± ± 21. B ± ± ± ±.28 4,42.8 ± ± ± ± 5.61 B ± ± ± ±.1 2,858.4 ± ± ± ± 9.7 B ± ± ± ±.9 2,791.7 ± ± d ± ± F = *** F = 18.41*** F = 8.255*** F = *** F = *** F = *** F = *** F = *** F(M) = 6, *** F(M) = 7,99.649*** F(B) = *** F(B) = *** F(M 9 B) = *** F(M 9 B) = *** Cd (mg l -1 ) Seedling length (mm) Vigor index B ± ± ± ± ±.2 4,36.7 ± ± ± ± ± 18.97, B ± ± ± ± ±.32 4,355.3 ± ,78.9 ± ± ± ± 29.1 B ± ± ± ± ±.15 4,42.8 ± ,351.4 ± ± ± ± 11.9 B ± ± ± ± ±.29, 2,858.4 ± ,2.1 ± 55.17, ± ± ± B ± ± ± ± ±.23 2,791.7 ± ,94.5 ± ± ± ± F = *** F = *** F = 3.59* F = NS F = 9.595*** F = *** F = *** F = 3.534** F = NS F = 2.879*** F(M) = 4, *** F(M) = 4, *** F(B) = *** F(B) = *** F(M 9 B) = *** F(M 9 B) = *** Results re expressed s men ± SE (n = 18 24). One-wy ANOVA ws performed for eh metl onentrtion. Mens in the sme olumn with different letters re signifintly different from eh other (P \.5) ording to the Dunn test. A two-wy ANOVA ws performed to determine the influene of metl onentrtion nd of teril tretment in seedling length nd vigor index. The test results re shown with the test sttisti for eh se (M metl onentrtion, B teril tretment, M 9 B metl 9 teril tretments intertion) NS nonsignifint t the level P \.5; * signifint t the level P \.5; ** signifint t the level P \.1; *** signifint t the level P \.1

11 Int. J. Environ. Si. Tehnol. (215) 12: Biomss (mg) Root NS F(M)= ***F(B)= 1.62 *F(MxB)= B B1 B2 B3 B4 Biomss (mg) Shoot, ***F(M)= ***F(B)= *F(MxB)= B B1 B2 B3 B4 () 25 Zn soil (mg kg -1 ) 5 () 25 5 Zn soil (mg kg -1 ) Biomss (mg) , Root,,, ***F(M)= ***F(B)= ***F(MxB)= 3,17, B B1 B2 B3 B4 Biomss (mg) Shoot,, ***F(M)= ***F(B)= 2.73 ***F(MxB)= 1.78, B B1 B2 B3 B4 () 1 Cd soil (mg kg -1 ) 3 (d) 1 Cd soil (mg kg -1 ) 3 Fig. 4 Trifolium repens shoot nd root dry iomss (mg) when exposed to different Zn (, ) nd Cd (, d) onentrtions (mg kg -1 ) in the soil. The error r represents the SE (n = 5 6). Two-wy ANOVA ws performed to determine the influene of soil Zn nd Cd onentrtion nd of teril tretment (B uninoulted; B1 EC 34; B2 1AP2; B3 3ZP2; B4 EC 1) in roots nd shoots iomss. The test results re shown with the test sttisti for eh se (M soil metl onentrtion; B teril tretment; M 9 B metl 9 teril tretments intertion). NS nonsignifint t the level P \.5; *signifint t the level P \.5; **signifint t the level P \.1; ***signifint t the level P \.1. One-wy ANOVA ws performed for eh metl onentrtion in the soil. Mens for the sme metl onentrtion with different letters re signifintly different from eh other (P \.5) ording to the Dunn test. For the root tissues, the F vlues of one-wy ANOVA re F = (P \.1), F = 1.24 (P [.5) nd F = (P \.1) for, 25 nd 5 mg Zn kg -1 spiked soils, respetively, nd F = 9.15 (P \.1) nd F = 4.67 (P \.5) for 1 nd 3 mg Cd kg -1 spiked soils, respetively. For the shoot tissues, the F vlues of onewy ANOVA re F = (P \.1), F = (P [.5) nd F = (P \.1) for, 25 nd 5 mg Zn kg -1 spiked soils, respetively, nd F = (P \.1) nd F = 6. (P \.1) for 1 nd 3 mg Cd kg -1 spiked soils, respetively seeds, whih inresed seedling growth in the presene of 3 mg l -1 of Cd. In generl, the results otined for vigor index orroorte the results otined for the seedling length in the presene of oth metls. Belimov et l. (25) lso showed tht severl metl-tolernt rhizoteri were ple of stimulting root elongtion of B. june seedlings either in the presene or sene of toxi Cd onentrtions. The detrimentl effets of metls on plnt growth were lso redued y teril inoultion, s reveled y the performne of the plnts in Zn- nd Cd-spiked soils (Figs. 4, 5). The metl onentrtions used in this study re within the rnge of those onsidered s of onern y the Duth stndrds (trgets of 2 mg Cd kg -1 nd 14 mg Zn kg -1 nd intervention levels of 12 mg Cd kg -1 nd 72 mg Zn kg -1 ) or y the Cndin Soil Qulity Guidelines (1.4 to 22 mg Cd kg -1 nd 2 to 36 mg Zn kg -1 ) nd the ommon totl metl ontent in unpolluted-soils whih is etween 1 nd 3 mg kg -1 for Zn nd elow 1 mg kg -1 for Cd (Kt Pendis nd Pendis 1992). In generl, inresing metl onentrtion in the soil exerts severe effet on root growth (El-Tye et l. 26). In the present work, inresing metl ontmintion did not redue white lover iomss nd growth (Fig. 4); the study of Mnier et l. (29) lso showed tht the root development of white lover plnts ws not signifintly ffeted y Zn ( mg kg -1 ) nd Cd ( mg kg -1 ) ontmintion in soils.

12 2138 Int. J. Environ. Si. Tehnol. (215) 12: length (m) () ***F(M)= 8.21 Root NS F(B)= ***F(MxB)= 4.35,,,, B B1 B2 B3 B Zn soil (mg kg -1 ) height (m) () Shoot Zn soil (mg kg -1 ) **F(M)= ***F(B)= NSF(MxB)= 1.88 B B1 B2 B3 B4 length (m) ,,,, Root NS F(M)= NS F(B)= *F(MxB)= B B1 B2 B3 B4 height (m) Shoot NS F(M)=.1225 ***F(B)= NS F(MxB)= 1.79 B B1 B2 B3 B4 () 1 3 Cd soil (mg kg -1 ) (d) 1 3 Cd soil (mg kg -1 ) Fig. 5 Trifolium repens shoot nd root elongtion (m) when exposed to different Zn (, ) nd Cd (, d) onentrtions (mg kg -1 ) in the soil. The error r represents the SE (n = 15 25). Two-wy ANOVA ws performed to determine the influene of soil Zn nd Cd onentrtion nd of teril tretment (B uninoulted; B1 EC 34; B2 1AP2; B3 3ZP2; B4 EC 1) in roots nd shoots elongtion. The test results re shown with the test sttisti for eh se (M soil metl onentrtion, B teril tretment; M 9 B metl 9 teril tretments intertion). NS nonsignifint t the level P \.5; *signifint t the level P \.5; **signifint t the level P \.1; ***signifint t the level P \.1. One-wy ANOVA ws performed for eh metl onentrtion in the soil. Mens for the sme metl onentrtion with different letters re signifintly different from eh other (P \.5) ording to the Dunn test. For the root tissues, the F vlues of one-wy ANOVA re F = 3.16 (P \.5), F = 8.92 (P \.1) nd F =.963 (P [.5) for, 25 nd 5 mg Zn kg -1 spiked soils, respetively, nd F = 1,517 (P [.5) nd F = (P \.5) for 1 nd 3 mg Cd kg -1 spiked soils, respetively. For the shoot tissues, the F vlues of one-wy ANOVA re F = (P \.1), F = (P [.5) nd F = 2.57 (P [.5) for, 25 nd 5 mg Zn kg -1 spiked soils, respetively, nd F = (P [.5) nd F = 7.98 (P \.1) for 1 nd 3 mg Cd kg -1 spiked soils, respetively In sene of metls, teril inoultion promoted white lover iomss prodution nd growth (Figs. 4, 5). Strins B1, B2 nd B3 enhned root iomss y 64, 53 nd 64 % (Fig. 4) nd shoot iomss y 74, 62 nd 83 %, respetively (Fig. 4). Shoot height ws lso improved y the inoultion with strins B1 nd B2 (Fig. 5, ). These results were lso oserved in Zn- nd Cd-treted plnts whih showed n enhnement of root nd shoot iomss when rhizoteri were inoulted if ompred to uninoulted plnts. Inoultion of strins B1 nd B3 enhned root iomss y 29 nd 94 % nd shoot iomss y 62 nd 12 %, respetively, t 5 mg Zn kg -1 (Fig. 4). Similr results were otined in Cd-treted plnts, the rhizoteri tht etter performed were strins B2 nd B3, whih promoted root iomss in oth Cd onentrtions. Strins B1, B2 nd B3 lso improved shoot iomss y 83, 137 nd 66 % in the presene of 1 mg Cd kg -1 nd y 39, 58 nd 56 %, respetively, in plnts treted with 3 mg Cd kg -1 (Fig. 4d). Plnt height in metl-spiked soils ws lso promoted y the inoultion with strin B1 in 25 mg Zn kg -1 -exposed plnts nd strins B1, B2 nd B3 in 3 mg Cd kg -1 -treted plnts (Fig. 5). The four seleted rhizoteri produed high levels of IAA ([31 mg l -1 ) nd exhiited ACC-deminse tivity (Figs. 2, 3), suggesting n involvement of these PGP trits on plnt growth promotion under metl exposure. Belimov et l. (25) showed positive orreltion etween in vitro ACC-deminse tivity of the

13 Int. J. Environ. Si. Tehnol. (215) 12: Tle 3 The ville nd exhngele Zn nd Cd onentrtions (mg kg -1 ) in uninoulted (B) nd inoulted soils (B1 EC 34; B2 1AP2; B3 3ZP2; B4 EC 1) t the end of experiment Tretment Zn (mg kg -1 ) 25 5 Aville Exhngele Aville Exhngele Aville Exhngele B.71 ± ± ± ± ± ± B1.53 ±.3, ± ± ± ± ± 6.84 B2.5 ±.16, ± ± ± ± ± B3.8 ±.5, ± ± ± ± ± 2.47 B4.37 ± ± ± ± ± ± 12.72, F = 7.311** F = *** F = 3.976* F = 14.35** F = 15.71*** F = 42.62*** Cd (mg kg -1 ) 1 3 Aville Exhngele Aville Exhngele Aville Exhngele B \.2 \ ± ± ±.16, 2.93 ±.52 B1 \.2 \ ± ± ±.2, 2.43 ±.45 B2 \.2 \ ± ± ± ± 1.6 B3 \.2 \ ± ± ±.89, ±.72 B4 \.2 \ ±.3 d 7.48 ± ± ±.38 F = *** F = *** F = 7.73** F = *** Results re expressed s men ± SD (n = 4). A one-wy ANOVA ws performed for eh onentrtion. Mens in the sme olumn with different letters re signifintly different from eh other (P \.5) ording to the Dunn test NS nonsignifint t the level P \.5; * signifint t the level P \.5; ** signifint t the level P \.1; *** signifint t the level P \.1 teri nd their stimulting effet on root elongtion of B. june seedlings in the presene of Cd. In ft, ACCdeminse is of gret importne when plnts fe high levels of metls, sine the presene of these elements is normlly enountered s stress nd plnts respond y synthesizing more levels of ethylene, whih in turn results in redued plnt growth. Although numer of studies hve demonstrted tht teril inoultion in the rhizosphere of plnt speies my inrese the effiieny of phytoremedition, sine plnt growth nd survivl in metl-ontminted soils re improved (He et l. 29; M et l. 29; Mdhiyn et l. 27; Vivs et l. 26) to the est of our knowledge, this is the first reserh tht reports on the potentil of R. erythropolis (B1) to inrese plnt growth in Zn- nd Cdontminted soils. The proess of metl uptke nd their umultion y different plnts depends on the onentrtion of the ville metls in soils (Gupt nd Sinh 26). The ville nd exhngele metl onentrtions in rhizosphere soils determined t the end of the experiment re shown in Tle 3. Aville nd exhngele Zn onentrtions rnged from.37 to 77.5 mg Zn kg -1 nd from to mg Zn kg -1, respetively, in the different Zn soil onentrtions nd etween 1.45 nd 6.7 mg Cd kg -1 nd from 5.73 to mg Cd kg -1 in the different Cd onentrtions. In most ses, the inoultion with strins B3 nd B4 signifintly enhned the ville nd exhngele Zn nd Cd onentrtions in rhizosphere soils. For ville metl onentrtions, strin B4 showed etter performne while for exhngele onentrtions, strin B3 seems to hve more pronouned effet on metl moility in soils. Whiting et l. (21) lso reported tht rhizoteri inoultion inresed wter-solule Zn onentrtion in soils nd onsequently enhned Zn umultion in shoots of Thlspi erulesens. Aou-Shn et l. (23) lso showed the importne of PGPR on Ni soluiliztion in soils. Among the vrious metolites produed y PGPR, the siderophores ply signifint role in metl moiliztion in soils, s these ompounds produed y PGPR omplexes with ivlent metl ions tht n e ssimilted y plnts (Crrillo-Cstñed et l. 23; Whiting et l. 21). Our results highlight the potentil of inoulting plnts with siderophore-produing rhizoteri to further improve their phytoextrtion effiieny.

14 214 Int. J. Environ. Si. Tehnol. (215) 12: Conlusion The urrent study onluded tht in vitro sreening of rhizoteri trits nd growth promotion under xeni onditions n e n effiient tool to selet rhizoteri to use s ioinoulnts in stress environmentl situtions. Inoultion of T. repens plnts with seleted PGPR signifintly enhned plnt growth in the presene nd sene of Zn nd Cd. The positive influene of PGPR inoultion on plnt growth in metl-ontminted soils my e relted to the multiple PGP trits of teril strins. However, it is not possile to determine extly whih teril mehnisms hve more pronouned impt in given plnt miroe ssoition. PGPR my positively influene plnt growth in degrded environments using more thn one trit, suggesting tht plnt growth stimultion is the net result of multiple mehnisms. The present work lerly indites tht some PGPR, suh s R. erythropolis EC 34, Ahromoter sp. 1AP2 nd Miroterium sp. 3ZP2 my e used s ioinoulnts nd onsequently onstitute n ttrtive lterntive to the hemil mendments used to improve phytoremedition effiieny. Aknowledgments This work ws supported y Ntionl Funds from FCT Fundção pr Ciêni e Tenologi through projets PTDC/AGR/CFL/111583/29 nd PEst-OE/EQB/LA16/213. S.I.A. Pereir wish to knowledge reserh grnt from FCT (Ref. SFRH/BPD/65134/29) nd Fundo Soil Europeu (Progrm Operionl Potenil Humno (POPH), Qudro de Referêni Estrtégio Nionl (QREN)). Referenes Aou-Shn RA, Angle JS, Delorme TA, Chney RL, vn Berkum P, Mowd H, Ghnem K, Ghozln HA (23) Rhizoteril effets on nikel extrtion from soil nd uptke y Alyssum murle. New Phytol 158: Ahmd F, Ahmd I, Khn MS (28) Sreening of free-living rhizospheri teri for their multiple plnt growth promoting tivities. 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