NOTE / NOTE

Phenotypic, genotypic, and symbiotic diversitiesin strains nodulating clover in different soils inSpain

Martha-Helena Ramırez-Bahena, Encarna Velazquez, Felix Fernandez-Santos,Alvaro Peix, Eustoquio Martınez-Molina, and Pedro F. Mateos

Abstract: Trifolium species are the most common legumes present in wild Spanish soils; however, there are no studies todate on the diversity of rhizobia nodulating clover in Spain. Twenty strains from different Spanish soils with acidic, neu-tral, and basic pH were selected to study their genotypic, phenotypic, and symbiotic features. The results showed that theisolates were genotypically diverse, displaying 12 different DNA fingerprint patterns and also 14 different plasmid profiles.Although they have 16S rRNA gene sequences that are nearly identical to that of the type strain of Rhizobium leguminosa-rum, their recA and atpD gene sequences were phylogenetically divergent from those of R. leguminosarum referencestrains, and phenotypic divergence as well as different host ranges were also found. Although most of them nodulatedboth Trifolium and Phaseolus, only 5 strains were also able to nodulate Pisum. The results of the effectiveness analysisshowed a high variability in the symbiotic characteristics of our strains and suggested that Pisum is the more restrictivehost of this group. Interestingly, some of the Trifolium isolates showed an ability to promote growth of Pisum in the ab-sence of nodulation.

Key words: Rhizobium, Trifolium, diversity, symbiosis.

Resume : Les especes de Trifolium sont les legumineuses les plus frequentes des sols sauvages de l’Espagne. A ce jour, iln’existe cependant aucune etude de la diversite des rhizobiums des nodules du trefle en Espagne. Vingt souches provenantde differents echantillons de sols de pH acides, neutre ou basiques, en Espagne, ont ete selectionnees afin d’etudier leurscaracteristiques genotypiques, phenotypiques et symbiotiques. Les resultats ont montre que les isolats etaient diversifiesd’un point de vue genotypique, montrant 12 patrons caracteristiques d’ADN et 14 profils differents de plasmides. Quoiqueles sequences du gene de l’ARNr 16S aient ete a peu pres identiques a celle de la souche Rhizobium leguminosarum, lessequences des genes recA et atpD etaient divergentes d’un point de vue phylogenique de celles des souches de referencede R. leguminosarum. Des divergences phenotypiques et de diversite d’hotes ont aussi ete trouvees. Meme si la plupartd’entre elles formaient des nodules sur Trifolium et Phaseolus, seules 5 souches pouvaient former des nodules sur Pisum.Les resultats d’analyses d’efficacite ont montre une grande variabilite quant aux caracteristiques symbiotiques des soucheset ont suggere que Pisum soit l’hote le plus restrictif de ce groupe. Etonnamment, quelques isolats provenant de Trifoliumpouvaient promouvoir la croissance de Pisum sans former de nodules.

Mots-cles : Rhizobium, Trifolium, diversite, symbiose.

[Traduit par la Redaction]

Trifolium is an ubiquitous legume distributed in temperateand subtropical regions of the 2 hemispheres (Ellison et al.2006). The greatest species diversity is found in the Medi-terranean basin, in the northwest of America, and in thehighlands of eastern Africa. Trifolium species occur in a

wide range of habitats, including meadows and prairies,open woodlands, semi-deserts, mountains, and alpine peaks(Ellison et al. 2006). In Spain, several species of Trifoliumare common in pastures and grow as wild cover, being themost important legume genus in Spanish soils. This legume

Received 3 March 2009. Revision received 3 June 2009. Accepted 9 June 2009. Published on the NRC Research Press Web site atcjm.nrc.ca on 13 October 2009.

M.-H. Ramırez-Bahena, E. Velazquez,1,2 F. Fernandez-Santos, E. Martınez-Molina, and P.F. Mateos. Departamento deMicrobiologıa y Genetica, Universidad de Salamanca, Salamanca, Spain.A. Peix. Instituto de Recursos Naturales y Agrobiologıa, IRNASA–CSIC, Salamanca, Spain.

1Corresponding author (e-mail: evp@usal.es).2Present address: Encarna Velazquez. Departamento de Microbiologıa y Genetica, Lab 209, Edificio Departamental de Biologıa, CampusMiguel de Unamuno, 37007 Salamanca, Spain.

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commonly establishes nitrogen-fixing symbiosis with thebiovar trifolii of the fast-growing species Rhizobium legumi-nosarum, whose taxonomic status, together with those of theformer species Rhizobium phaseoli and Rhizobium trifolii,has been recently revised by Ramırez-Bahena et al. (2008).In this work, the phylogenetic analysis of the 16S rRNA,recA, and atpD genes showed that R. phaseoli is a valid spe-cies, whereas R. trifolii should be considered as a biovar ofR. leguminosarum. As a conclusion of this analysis, a newspecies named Rhizobium pisi was also proposed anddescribed. Therefore, the phylogenetic group of R. legumi-nosarum currently contains the species R. leguminosarum,R. phaseoli, Rhizobium etli, and R. pisi (Ramırez-Bahena etal. 2008).

In spite of the ecological importance of Trifolium species,as well as its great agronomic relevance as a forage legumeworldwide, there are very few studies focusing on the diver-sity of rhizobia responsible for nitrogen fixation in the rhizo-bia–clover symbiosis (Tesfaye and Holl 1999; Seguin et al.2001; Duodu et al. 2007; Liu et al. 2007). To our knowl-edge, this is the first report on the diversity of rhizobia nod-ulating Trifolium in Spain, despite that it is one of the mostabundant legumes in Spanish soils and its contribution to theglobal input of nitrogen to soil is crucial in the maintenanceof ecosystems. Therefore, this work aims to contribute to thestudy of the diversity of rhizobial strains nodulating cloverin soils with different pH and edaphic characteristics fromdiverse locations of northern Spain.

The isolation of rhizobia was performed from effectivenodules of Trifolium pratense L. (red clover) growing inthese soils on yeast mannitol agar (YMA) plates accordingto Vincent (1970). A single colony from each nodule waspicked and transferred to YMA medium. The phenotypic di-versity of the Trifolium isolates (Table 1) was analysed onthe basis of 80 characteristics commonly used in the charac-terization of rhizobia (Table 2). The temperature range forgrowth was determined by incubating cultures in YMA me-dium between 4 and 40 8C. The pH range was determined inYMA medium with a final pH between 5.0 and 10.0. Salttolerance was studied in YMA medium containing 0%–5%(m/v) NaCl. The antibiotic resistance was performed usingthe disc diffusion method on YMA medium supplementedwith 10 g/L of yeast extract. The ability to use 23 differentcarbon sources was evaluated using the basal medium ofBergersen (1961) with ammonium nitrate as the nitrogensource (0.1 g/L) and without mannitol. Assimilation of 11amino acids or related compounds was checked using thesame basal medium without ammonium nitrate. Plates con-taining the same medium with no carbon source were usedas negative controls, and bromothymol blue was added asthe pH indicator (15 mg/L). The strains were incubated at28 8C for up to 7 days. The data obtained (Table 2) werecoded in binary form, and Jaccard’s coefficient was calcu-lated to construct a similarity matrix. A dendrogram was ob-tained using the unweighted pair group method witharithmetic mean (UPGMA). It showed that our strains

formed a large group separated from the type strains of thespecies included as references, and were distributed into sev-eral subgroups with an internal similarity coefficient >75%(Fig. S1).3 Ten different phenotypes (Table 1) were foundamong our strains, with internal similarity values >90%,which was the value considered as the threshold since thisis the similarity between R. phaseoli ATCC 14482T andR. leguminosarum ATCC 1004T, currently accepted as sepa-rated species (Ramırez-Bahena et al. 2008). Similarity val-ues among these 10 phenotypes ranged from 81% to 90%,and therefore we can conclude that the strains presented ahigh phenotypic diversity. Moreover, some of them showedhigher phenotypic variability than some type strains belong-ing to different species currently accepted in the phyloge-netic group of R. leguminosarum. No relation was observedbetween the phenotype and the soil from which the strainswere isolated, since strains isolated in the same soil pre-sented different phenotypes and the same phenotype wasfound in different soils (Table 1).

The genetic diversity of the strains was analysed by ran-dom amplification of polymorphic DNA (RAPD) finger-printing, a technique that allows the differentiation amongstrains of the same rhizobial species (Dooley et al. 1993;Moschetti et al. 2005; Rivas et al. 2006; Valverde et al.2006; Iglesias et al. 2007). RAPD patterns were obtained us-ing the primer M13 (5’-GAGGGTGGCGGTTCT-3’), accord-ing to Rivas et al. (2006), with the following PCRconditions: preheating at 95 8C for 9 min; 35 cycles of de-naturing at 95 8C for 1 min; annealing at 45 8C for 1 minand extension at 75 8C for 2 min; and a final extension at72 8C for 7 min. The bands present in each profile werecoded for input into a database including all the strainsstudied, and Jaccard’s similarity coefficient was calculatedto construct the distance matrix. A dendrogram was drawnfrom the distance matrix using UPGMA. The same referencestrains used for the phenotypic characterization were in-cluded in this analysis. RAPD fingerprinting showed thatthe Trifolium isolates are genetically diverse, since 12 differ-ent patterns were displayed, which were also different fromthose of the reference strains (Table 1 and Fig. S2).3 Fromthese patterns, only pattern B was found in strains isolatedfrom different soils having a low pH. The results of themathematical analysis showed that the 12 RAPD patternshave a similarity coefficient among them, and with respectto those formed by the reference strains, <80% (Table 1and Fig. S3).3 These results imply the high genetic diversityof Trifolium strains, confirming the usefulness of RAPD pat-terns to analyze the genetic diversity of rhizobial popula-tions. The high biodiversity of strains nodulating clover innorthern Spain is congruent with that found in clover iso-lates from other European soils (Duodu et al. 2007; Seguinet al. 2001). Since it has been previously reported thatstrains showing similar RAPD patterns have identical 16SrRNA, recA, and atpD genes (Valverde et al. 2006; Iglesiaset al. 2007), these genes were analysed in a representativestrain from each RAPD group.

3 Supplementary data for this article are available on the journal Web site (http://cjm.nrc.ca) or may be purchased from the Depository ofUnpublished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ONK1A 0R6, Canada. DUD 5295. For more information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/eng/ibp/cisti/collection/unpublished-data.html.

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Table 1. Characteristics of the strains isolated in this study.

Strain Geographic localizationSoilpH

Phenotype*(>90% internalsimilarity)

RAPD genotype{

(>80% internalsimilarity)

Plasmidtype

No. ofplasmids Approximate size of plasmids (kb)

HTP01 Puerto La Hoya (Salamanca) 4.5 10 A h 6 100, 180, 210, 300, 400, 700HTP02 Puerto La Hoya (Salamanca) 4.5 9 A i 8 100, 180, 200, 250, 300, 350, 500,

1200HETP01 Hermosillo (Avila) 4.5 9 B j 5 180, 250, 300, 600, 700HETP02 Hermosillo (Avila) 4.5 10 C b 3 200, 300, 600HETP03 Hermosillo (Avila) 4.5 10 D n 4 200, 300, 450, 750HETP04 Hermosillo (Avila) 4.5 7 B b 3 200, 300, 600HETP05 Hermosillo (Avila) 4.5 10 C b 3 200, 300, 600NTP02 Nuevo Naharros (Salamanca) 5.5 7 B g 3 300, 550, 750NTP04 Nuevo Naharros (Salamanca) 5.5 7 B g 3 300, 550, 750RTP02 Riego de la Vega (Leon) 6.5 1 E a 1 700RTP05 Riego de la Vega (Leon) 6.5 1 E a 1 700ARTP01 Arapiles (Salamanca) 6.0 5 F c 2 300, 500MITP01 Miranda de Azan (Salamanca) 6.5 2 B e 4 220, 250, 600, 700MITP02 Miranda de Azan (Salamanca) 6.5 5 G l 2 250, 550MITP03 Miranda de Azan (Salamanca) 6.5 8 G k 2 280, 600PETP01 Pedrosillo (Salamanca) 8.0 3 H d 5 220, 300, 400, 700, >2000PETP03 Pedrosillo (Salamanca) 8.0 6 I l 2 250, 450PETP05 Pedrosillo (Salamanca) 8.0 4 J m 4 60, 180, 350, 500PETP06 Pedrosillo (Salamanca) 8.0 4 K l 2 250, 450HUTP01 Huelmos de Canedo (Salamanca) 8.0 10 L f 4 220, 300, 500, 750Rhizobium leguminosarum

biovar viciae USDA 2370Tna na 12 M nd nd nd

R. leguminosarum biovartrifolii ATCC 14480

na na 12 N nd nd nd

R. phaseoli ATCC 14482T Beltsville, Maryland na 13 O nd nd ndR. pisi DSM 30132T na na 11 P nd nd ndR. etli CFN42T na na 14 Q nd nd nd

Note: na, not available; nd, not determined.*Phenotype according to the grouping of strains from Fig. 2 based on 80 characteristics.{Rapid amplification of polymorphic DNA (RAPD) patterns were obtained with the primer M13.

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The amplification and sequencing of the 16S rRNA genewere carried out according to Rivas et al. (2006) and that ofrecA and atpD according to Gaunt et al. (2001). PCR ampli-fication was carried out with an AmpliTaq Gold reagent kit(Applied Biosystems Inc., Foster City, California) followingthe manufacturer’s instructions. The PCR products wereelectrophoresed in 1% agarose gels and stained with ethi-dium bromide, as mentioned earlier. The band correspond-ing to the different genes was purified directly from the gelby room temperature centrifugation using a DNA gel extrac-tion device (Millipore Corp., Beford, Massachusetts) for10 min at 5000g according to the manufacturer’s instruc-tions. The sequence reaction was performed on an ABI377sequencer (Applied Biosystems Inc.) using a BigDye termi-nator version 3.0 cycle sequencing kit as supplied by the

manufacturer. The sequences obtained were compared withthose from the GenBank using the Basic Local AlignmentSearch Tool (BLAST)N program (Altschul et al. 1990) andwere aligned using the ClustalW software (Thompson et al.1997). The distances were calculated according to Kimura’s2-parameter model (Kimura 1980). Phylogenetic trees wereinferred using the neighbour-joining method (Saitou andNei 1987). Bootstrap analysis was based on 1000 resam-plings. The MEGA 4 package (Tamura et al. 2007) wasused for all analyses.

The complete 16S rRNA gene sequences (Fig. 1) of all ourstrains and R. leguminosarum biovar trifolii ATCC 14480were 100% identical (thus only the sequences of 2 represen-tative strains were deposited in GenBank and included in thephylogenetic tree), and 99.8% identical with respect to R. le-

Table 2. Phenotypic characteristics of the strains analyzed in this study (+, indicates positive; –, indicates negative; and w, indicates weak).

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guminosarum biovar viciae USDA 2370T. Although the 16SrRNA gene is currently used to classify rhizobia (Kuykendall2005), closely related species of genus Rhizobium have beenreported to have 16S rRNA genes that are nearly identical(Valverde et al. 2006; Ramırez-Bahena et al. 2008), andtherefore the high identity values found in our strains do notimply that they correspond to the species R. leguminosarum.

In these cases, the housekeeping genes recA and atpD areuseful markers for species differentiation, as well as for ana-lysing genetic diversity within the same species (Gaunt et al.2001; Valverde et al. 2006; Santillana et al. 2008). Accord-ing to the analysis of both the recA and atpD genes (Figs. 2and 3), the Trifolium isolates form a group with the speciesR. leguminosarum including the reference strain of biovartrifolii ATCC 14480. Although strains showing differentRAPD patterns usually have different recA and atpD genesequences, our results showed that some strains with differ-

ent patterns have nearly identical housekeeping genes. Thishappened in the case of strains MITP02, NTP02, RTP05,HETP03, and HETP05, the RAPD patterns of which showedhigh similarity (70%), but also in the case of strains PETP05,HUTP01, and PETP06 with low similarity (<30%) in theirRAPD patterns. The internal identity values of the recA andatpD genes of the group formed by our strains ranged from96% to 100% and from 94% to 100%, respectively.Although there are no wide taxonomic studies of strainsfrom different rhizobial species focusing on the similaritylevels in recA and atpD genes within the same species, andtherefore it is difficult to establish the identity of rhizobialisolates based on these genes, our previous data showed thatvalues >92% in recA and atpD are found in different closelyrelated species of genus Rhizobium (Valverde et al. 2006;Ramırez-Bahena et al. 2008). Hence, some Trifolium strainscould correspond to species different from R. leguminosarum

Fig. 1. Neighbour-joining phylogenetic tree based on rrs gene sequences showing the position of representative strains isolated from cloverin Spain compared with those of related species of genus Rhizobium. Bootstrap values calculated for 1000 replications are indicated. Thebar represents 1 nt substitution per 100 nt.

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because they are in the limit for species differentiation, espe-cially strains HTP01, PETP01, and ARTP01 and those ofgroups formed by strains MITP02, NTP02, RTP05,HETP05, and HETP03, and by strains HUTP01, PETP05,and PETP06, when both genes are collectively considered.In summary, the results of the housekeeping analysis confirmthe high phylogenetic diversity of our strains even when theywere isolated from the same host and the same country.Although wider analyses of strains from different geographiclocations and from different Trifolium species are necessaryto draw conclusions, our results highlight the interest ofapplying housekeeping genes analysis to explore the phylo-genetic diversity of clover endosymbionts and their biogeo-graphy.

The analysis of the symbiotic diversity was based on theanalysis of plasmid profiles and plant–microbe interactions.For plasmid profile analysis, the strains were incubated intryptone–yeast (TY) medium at 25 8C and 180 r/min untilthe culture reached a concentration of 1 � 106 cells/mL, andthen the cells were collected by centrifuging 1.5 mL of cul-

ture at 9000g for 5 min and subjected to plasmid analysis ac-cording to Plazinski et al. (1985), with the electrophoreticconditions modified as follows: electrophoresis was per-formed at 2 V/cm for 90 min, followed by 3 V/cm for60 min, and finally 6 V/cm for 4 h. The pRmeGR4b(205 kb) and pRmeGR4a (175 kb) plasmids of Sinorhizobiummeliloti GR4 (Toro and Olivares 1986) were used as molecu-lar markers. The results obtained (Table 1 and Fig. S4)3

showed that the strains isolated carried from 1 to 8 plasmids.These results are in agreement with those of other authors re-porting the existence of complex plasmid profiles containingup to 8 plasmids in strains nodulating Trifolium (Thurmanet al. 1985; Harrison et al. 1988). According to their plasmidcontent, our strains presented high extrachromosomal ge-netic diversity with 14 different plasmid profiles. In agree-ment with the findings observed for other R. leguminosarumstrains (Thurman et al. 1985; Harrison et al. 1988; Crossmanet al. 2008), most of the plasmids detected have sizes>100 kb, except in the case of strain PETP05, which alsocontains 1 plasmid of ~60 kb. In any case, a high variability

Fig. 2. Neighbour-joining phylogenetic tree based on partial recA gene sequences showing the position of representative strains from eachrandom amplification of polymorphic DNA (RAPD) group within genus Rhizobium. Bootstrap values calculated for 1000 replications areindicated. The bar represents 1 nt substitution per 100 nt.

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in the number and size of the plasmids was observed. Thestrains isolated in Leon, Spain, were the only strains present-ing a single plasmid. Most of the strains presented 2 or 3plasmids, regardless of the pH of the soil from which theywere isolated, and only 2 strains carried 6 or 8 plasmids,which was the highest number of plasmids found in thisstudy. Although more studies are necessary to draw conclu-sions, we detected a certain relationship between the com-plexity of the plasmid profile and the pH of the soil, sincethe most complex profiles were found in strains isolatedfrom soils with extreme pH values, either acidic or alkaline.It is interesting to highlight that the average plasmid numberin strains from both pH 4.5 and pH 8 soils (mean = 4.1,SD = 1.7) compared with the isolates from pH 6–6.5 soils(mean = 2.3, SD = 1.1) showed significant differences by ttest (p = 0.039). In contrast, the difference in mean plasmidnumber between isolates from pH 4.5 (4.8) and pH 8 (3.4)was not significant (p = 0.198).

These results contrast with those of Thurman et al.(1985), who observed no correlation between the number ofplasmids and the soil pH. Therefore, it would be very inter-esting to further study the role of plasmids of Trifoliumstrains in soils with extreme conditions, since this legume isan ubiquitous plant that may be used in revegetation and

phytoremediation schemes targeting contaminated and de-graded soils (Palmroth-Marja et al. 2002; Bidar et al. 2007).

The host range of our strains was analysed using Trifo-lium repens L. ‘Huia’ (white clover; all our strains nodu-lated both T. pratense and T. repens), Phaseolus vulgaris L.‘Pinta’ (pinto bean), and Pisum sativum L. ‘Frisson’ (pea).Plants were inoculated with the isolates in modified Leonardjars, using sterile vermiculite as substrate and nitrogen-free(Rigaud and Puppo 1975) nutrient solution. The inoculatedplants were placed in a plant growth chamber with mixedincandescent and fluorescent lighting (400 mE/m2/s; 400–700 nm), programmed for a 16 h photoperiod, day–nightcycle, with a constant temperature varying from 25 to27 8C, and 50%–60% relative humidity. Non-inoculated ni-trogen-free and nitrogen-supplemented plants were used ascontrols. Five replicates per treatment were set, and plantswere harvested 6 weeks after planting. The parametersmeasured were shoot dry mass and number of nodules. Sym-biotic efficiency was determined as described by Somase-garan and Hoben (1994). Data were analyzed by one-wayanalysis of variance, and mean values compared by Fisher’sprotected least significant differences test (p £ 0.05).

Almost all strains nodulated Trifolium and Phaseolus(Table 3), but only a few strains were able to nodulate

Fig. 3. Neighbour-joining phylogenetic tree based on partial atpD gene sequences showing the position of representative strains from eachrandom amplification of polymorphic DNA (RAPD) group within genus Rhizobium. Bootstrap values calculated for 1000 replications areindicated. The bar represents 1 nt substitution per 100 nt.

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Pisum. These results suggest that Trifolium and Pisum donot correspond to the same cross-inoculation group, andconfirm the high promiscuity of Phaseolus, which wasnodulated by most of the strains isolated from clover. Theresults also indicate that Pisum is even more restrictive fornodulation than Trifolium, which is considered a restrictivehost (Broughton and Perret 1999). The results of the symbi-otic efficiency showed high diversity among the strains inthe 3 hosts analysed. These results contrast with those foundin strains isolated in Scandinavian soils by Duodu et al.(2007) that presented low symbiotic variability in spite oftheir great genotypic variability. No relation was found be-tween the plasmid number and the ability to nodulate differ-ent hosts or the symbiotic efficiency of the strains.Nevertheless, the least symbiotically efficient strain (near10%) in clover, HTP01, has a high number of plasmids,whereas the most efficient strain (near 80%), NTP04, pre-sented an intermediate number of plasmids. No relation wasfound between the pH of the soil from which the strainswere isolated and their symbiotic efficiency. Nevertheless,the strains isolated in acidic soils were generally more effi-cient than those from soils with pH neutral or basic, exceptin the case of HTP01, which was the least efficient andcame from an acidic soil. In the common bean, the mostsymbiotically efficient strains (near 80%), HETP01 andHTP02, were isolated from acidic soils and have a highplasmid number (5 and 8, respectively). Interestingly, thesestrains are much more efficient in common bean than in clo-

ver, this being a general trend in all strains studied, most ofwhich show a symbiotic efficiency near or >50% in P. vul-garis. On the contrary, only 4 strains were able to nodulatePisum, showing an efficiency <40%. Interestingly, Pisumplants inoculated with the strains RTP05, ARTP01, andPETP01 (this last strain showing statistically significant dif-ferences) isolated in neutral or basic soils presented efficien-cies >40%. These results indicate that these 3 strains canpromote the growth of Pisum by other mechanisms differentto that of nodulation, since they do not form nodules in thislegume. This result is in agreement with those obtained inother studies about the ability of Rhizobium isolated fromclover to promote the growth of non-legume plants (Yanniet al. 2001; Mishra et al. 2006), and therefore the strainsRTP05, ARTP01, and HETP01 could be good candidates tobe examined as plant growth promoting rhizobacteria ofnon-legume plants in future studies. Moreover, the resultsof this study point out the interest of linking genetic diver-sity to functional diversity in soil microbial populations,which is one of the biggest challenges in environmental mi-crobiology (Maron et al. 2007).

ReferencesAltschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman,

D.J. 1990. Basic local alignment search tool. J. Mol. Biol.215(3): 403–410. doi:10.1016/S0022-2836(05)80360-2. PMID:2231712.

Table 3. Symbiotic characteristics of the strains isolated from clover in this study.

Trifolium repens Phaseolus vulgaris Pisum sativum

Treatment Nodulated hosts NN*SDM{

(mg)SE{

(%) NN*SDM{

(mg)SE{

(%) NN*SDM{

(mg) E§ (%)HTP01 Trifolium, Phaseolus 15cd 2.70a 11 75de 796.00cd 66 0a 180.67a 24HTP02 Trifolium, Phaseolus 30g 5.84de 23 110ef 953.67de 79 0a 284.67ab 38HETP01 Trifolium, Phaseolus 9bc 9.25hi 37 28bc 982.50de 82 0a 258.25ab 34HETP02 Trifolium, Phaseolus 10bc 8.30gh 33 16ab 579.00ab 48 0a 282.67ab 36HETP03 Trifolium, Phaseolus, Pisum 20ef 7.48fg 30 48cd 783.60cd 65 45d 258.20ab 34{HETP04 Trifolium, Phaseolus 8bc 6.22ef 25 10ab 582.40ab 49 0a 297.83ab 40HETP05 Trifolium, Phaseolus 10bc 8.35gh 33 14ab 580.00ab 49 0a 283.00ab 38NTP02 Trifolium, Phaseolus, Pisum 18de 13.63k 54 111ef 455.00ab 38 2a 248.00ab 33{NTP04 Trifolium, Pisum 30g 18.99l 76 0a 529.67ab 44 33c 135.17a 18{RTP02 Trifolium, Phaseolus 8bc 5.00cd 20 57cd 788.25cd 66 0a 145.70a 19RTP05 Trifolium, Phaseolus 5ab 4.00cd 16 0a 688.00bc 57 0a 305.25bc 41ARTP01 Trifolium, Phaseolus 12cd 9.00hi 36 120ef 828.80cd 69 0a 304.60bc 41MITP01 Trifolium, Phaseolus 17de 4.27cd 17 14ab 649.50bc 54 0a 222.50ab 30MITP02 Trifolium, Phaseolus 22ef 3.93bc 16 30bc 637.20bc 53 0a 243.80ab 32MITP03 Trifolium, Phaseolus 32h 3.17bc 13 2a 629.33bc 52 0a 237.25ab 32PETP01 Trifolium, Phaseolus 7ab 7.30fg 29 150fg 754.20cd 63 0a 337.83c 45PETP03 Trifolium 15cd 11.17j 45 0a 514.67ab 43 0a 143.50a 19PETP05 Trifolium, Phaseolus, Pisum 30g 3.51bc 14 27bc 566.50ab 47 46 153.67a 20{PETP06 Trifolium, Phaseolus 42i 3.43bc 14 8a 673.17bc 56 0a 121.17a 16HUTP01 Trifolium, Phaseolus, Pisum 16de 3.43bc 14 69de 633.80bc 53 8b 280.67ab 37

Note: Values followed by the same letter are not significantly different from each other at p = 0.05 according to Fisher’s protected least significant dif-ferences test.

*NN, no. of nodules per plant (average of 5 plants).{SDM, shoot dry mass per plant (average of 5 plants).{SE, symbiotic efficiency = SDM inoculated plants/SDM non-inoculated control plants (140 ppm nitrogen as NH4NO3), where SDM is the average shoot dry

mass of 5 replicates.§E, efficiency as plant growth promoting rhizobacteria. The strains that nodulate Pisum are marked with the symbol ({) corresponding to the symbiotic

efficiency.

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Bergersen, F.J. 1961. The growth of Rhizobium in synthetic media.Aust. J. Biol. Sci. 14: 349–360.

Bidar, G., Garcon, G., Pruvot, C., Dewaele, D., Cazier, F., Douay,F., and Shirali, P. 2007. Behavior of Trifolium repens and Lo-lium perenne growing in a heavy metal contaminated field: plantmetal concentration and phytotoxicity. Environ. Pollut. 147(3):546–553. doi:10.1016/j.envpol.2006.10.013. PMID:17141383.

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