Research in Microbiology 160 (2009) 297e306www.elsevier.com/locate/resmic

Multilocus sequence analysis of Brazilian Rhizobium microsymbiontsof common bean (Phaseolus vulgaris L.) reveals unexpected

taxonomic diversity

Renan Augusto Ribeiro a,b, Fernando Gomes Barcellos a,b, Fabiano L. Thompson c,Mariangela Hungria a,b,*

a Embrapa Soja, Cx. Postal 231, 86001-970 Londrina, Parana, Brazilb Universidade Estadual de Londrina, Department of Microbiology, Cx. Postal 60001, 86051-990 Londrina, Parana, Brazil

c UFRJ, Center of Health Sciences, Institute of Biology, Cx. Postal 68011, 21944-970 Rio de Janeiro, Rio de Janeiro, Brazil

Received 25 August 2008; accepted 13 March 2009

Available online 3 May 2009

Abstract

The diazotrophic bacteria collectively known as ‘‘rhizobia’’ are important for establishing symbiotic N2-fixing associations with manylegumes. These microbes have been used for over a century as an environmentally beneficial and cost-effective means of ensuring acceptableyields of agricultural legumes. The most widely used phylogenetic marker for identification and classification of rhizobia has been the 16S rRNAgene; however, this marker fails to discriminate some closely related species. In this study, we established the first multilocus sequence analysis(MLSA) scheme for the identification and classification of rhizobial microsymbionts of common bean (Phaseolus vulgaris L.). We analyzed 12Brazilian strains representative of a collection of over 850 isolates in addition to type and reference rhizobial strains, by sequencing recA, dnaK,gltA, glnII and rpoA genes. Gene sequence similarities among the five type/reference Rhizobium strains which are symbionts of common beanranged from 95 to 100% for 16S rRNA, and from 83 to 99% for the other five genes. Rhizobial species described as symbionts of common beanalso formed separate groups upon analysis of single and concatenated gene sequences, and clusters formed in each tree were in good mutualagreement. The five additional loci may thus be considered useful markers of the genus Rhizobium; in addition, MLSA also revealed broadgenetic diversity among strains classified as Rhizobium tropici, providing evidence of new species.� 2009 Elsevier Masson SAS. All rights reserved.

Keywords: Biological nitrogen fixation; Phaseolus vulgaris; Multilocus sequence analysis; Phylogeny; Rhizobium tropici; 16S rRNA

1. Introduction

The diazotrophic bacteria collectively known as ‘‘rhizobia’’are capable of nodulating and establishing symbiotic N2-fixingassociations with many plant species of the Leguminosaefamily. Based on polyphasic analyses of phenetic and geneticproperties, rhizobia are currently categorized into six genera e

* Corresponding author at: Embrapa Soja, Cx. Postal 231, 86001-970 Lon-

drina, Parana, Brazil. Tel.: þ55 4333716206; fax: þ55 4333716100.

E-mail addresses: renanribeiro83@hotmail.com (R.A. Ribeiro), fgbarcel@

yahoo.com.b (F.G. Barcellos), fabiano.thompson@biologia.ufrj.br (F.L.

Thompson), hungria@cnpso.embrapa.br (M. Hungria).

0923-2508/$ - see front matter � 2009 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.resmic.2009.03.009

Allorhizobium, Azorhizobium, Bradyrhizobium, Meso-rhizobium, Sinorhizobium and Rhizobium [18,37,67]. Never-theless, taxonomic changes have been proposed: first, sincephylogenetic analysis based on the 16S rRNA gene shows thatRhizobium, Agrobacterium and Allorhizobium are closelyrelated, they should be joined into a single genus, Rhizobium[74]; second, all Sinorhizobium species should be reclassifiedinto the genus Ensifer [73]. In addition, some diazotrophicsymbiotic bacteria isolated in recent years have been classifiedinto non-traditional rhizobial genera, included in both thealpha-Proteobacteria (Methylobacterium, Devosia) and in thebeta-Proteobacteria (Burkholderia, Cupriavidus) (¼Ralstonia,¼Wautersia) classes [18,37,67].

298 R.A. Ribeiro et al. / Research in Microbiology 160 (2009) 297e306

The usefulness of the 16S rRNA gene as a molecular markerfor assessing phylogeny and taxonomy of prokaryotes has beenbroadly demonstrated [18,27,63,66,70e72], and the gene hasalso been applied to rhizobial taxonomy [18,37,51,67,68,75,76].However, the high level of conservation documented in the 16SrRNA [21,22,42,65,69], and reports that genetic recombinationand horizontal gene transfer may also occur among 16S rRNAgenes [22,62] suggest that this marker has shortcomings. On thebasis of these observations, as well as to minimize their effects,other genes with a faster evolution rate than the 16S rRNA, butconserved enough to retain genetic information, have beenproposed as alternative phylogenetic markers [38,51,54,55].Requisites are that these genes should be both broadly distrib-uted among taxa and also be present in single copies withina given genome, and the current consensus is that at least fivegenes are necessary for reliable taxonomic classification[22,54,59,77]. Applying this approach to bacterial taxonomyand elucidation of phylogenetic relationships, multilocussequence analysis (MLSA) e using sequences of multipleprotein-coding genes for genotypic characterization of diversegroups of prokaryotes e has been proposed [22]. MLSA hasbeen used in studies with several genera of prokaryotes,including Burkholderia, Bacillus, Vibrio, Mycobacterium andEnsifer [22,38,59,60]. Expectations are that these analyses willhave a positive impact on how we perform taxonomic andbiodiversity studies [60].

Common bean (Phaseolus vulgaris L.) represents the mostimportant source of protein for low-income populations in LatinAmerica and in Africa, and Brazil is its largest producer andconsumer worldwide [13]. It is generally accepted that there aretwo major centers of genetic diversification of common bean, theMesoamerican (Mexico, Colombia, Ecuador and northern Peru,probably the primary center), and the Andean center (southernPeru to northern Argentina). Although wild relatives of commonbeans are not indigenous to Brazil, beans have been cultivatedthere throughout recorded history [11,17,20,32,33]. Thesymbionts of common bean were first classified as Rhizobiumphaseoli, based on the cross-inoculation-group concept [16]. In1984, using a numerical taxonomy approach, they were reclas-sified into a new species, Rhizobium leguminosarum, whichcontains three biovars, named after their main host species e bv.viciae (Pisum sativum), bv. trifolii (Trifolium spp.) and bv.phaseoli (common bean) [31]. Advances in molecular biologytechnology as well as isolation of new strains from various partsof the world led to the description of several differences amongcommon-bean rhizobia, such that they were pooled into twomain groups, denominated type I and type II [9,39,40,48]. In1991, a new species, Rhizobium tropici, was described for thetype II strains, with two subgroups, type IIA and type IIB [41].Segovia et al. [50] then proposed that some rhizobia isolatedfrom American soils and initially classified as type I should bereclassified into the new species Rhizobium etli, and two newspecies, Rhizobium gallicum and Rhizobium giardinii, weredescribed by Amarger et al. [5], the latter forming non-N2-fixingnodules. Finally, a new biovar of Ensifer (¼Sinorhizobium)meliloti characterized by tolerance to salinity was isolated froma Tunisian oasis, and termed biovar mediterranense [46].

Common bean is promiscuous in its symbiotic relationships[44] and its widespread and long-term cultivation in Brazilprobably explains the large and diverse populations of itsrhizobial symbionts detected in surveys. All described Rhizo-bium species have been found in Brazilian soils except for R.gallicum, as well as species of other genera such as Meso-rhizobium and Ensifer and putative new species [23,25,26,28e30,45,47,57]. However, despite the socioeconomic importanceof the legume in Brazil and the broad diversity of the rhizobialpopulation, taxonomic and phylogenetic knowledge of indig-enous isolates remains limited. The development of MLSApresents an opportunity to improve the identification ofrhizobia in general and to determine the phylogenetic rela-tionships among common-bean rhizobia, with emphasis onstrains of R. tropici indigenous to Brazil.

2. Material and methods

2.1. Strains, culture conditions and DNA extraction

A total of 18 common-bean rhizobial strains were analyzedin this study, 12 indigenous to Brazil (detailed informationgiven in Table 1) and six type/reference common beanRhizobium strains, as follows: R. tropici strain CIAT 899T

(type B) (¼USDA 9030; ¼ATCC 49672; ¼UMR1899; ¼TAL1797; ¼HAMBI 1163; ¼CM01; ¼SEMIA 4077; DSM 11418;BR 322), R. tropici type A strain CFN 299 (¼USDA 9039;¼LMG 9517; ¼UMR1026; ¼CENA 183), and R. etli bv.phaseoli strain CFN 42T (¼USDA 9032; ATCC 51251; DSM11541) were supplied by Dr. Esperanza Martınez-Romero(Centro de Ciencias Genomicas, Cuernavaca, Mexico). R.leguminosarum bv. phaseoli USDA 2671 (¼RCR 3644) wasprovided by Dr. Peter van Berkum (USDA, Beltsville, MD,USA). R. giardinii bv. giardinii strain H152T (¼USDA 2914)and R. gallicum bv. gallicum strain R602spT (¼USDA 2918)were provided by Dr. Noelle Amarger (INRA, Dijon, France).Strains were purified on yeast extract-mannitol agar (YMA)medium [64] containing Congo red (0.00125%) and stockswere prepared on YMA and kept at e80 �C (under 30% ofglycerol) for long-term storage and at 4 �C as source cultures.

Total genomic DNA of each strain was extracted frombacterial batch cultures grown in YM broth until late expo-nential phase (109 cells mL�1). Extraction of DNA was per-formed as described before [34], and purification andmaintenance of stocks were as described by Menna et al. [42].

2.2. Genes and primers of MLSA

In addition to 16S rRNA, the following genes were used inthis study: recA, dnaK, gltA, glnII and rpoA. Primers foramplification of 16S rRNA genes, recA, dnaK, gltA and glnIIwere obtained from the literature and are listed in Table 2.Considering the chromosome of R. etli (4,381,608 pb),complementary genes were positioned as follows (50e30 direc-tion): recA (2,422,674), dnaK (153,088), gltA (2,007,997), glnII(3,250,540) and rpoA (1,781,361).

Table 1

Strains used in this study.

Strain Geographic origina Reference Species name

Type/reference strains

CFN 299 Brazil [41] Rhizobium tropici type A

CIAT 899T Colombia [41] R. tropici type B

USDA 2671 USA Dr. P. van Berkum (USDA) R. leguminosarum bv. phaseoli

CFN 42T Mexico [50] R. etli bv. phaseoli

R602T France [5] R. gallicum bv. gallicum

H152T France [5] R. giardinii bv. giardinii

Brazilian indigenous strains

H 12 Planaltina (DF, Cerrados) [45] R. tropici

H 20 Planaltina, (DF, Cerrados) [45] R. tropiciH 52 Planaltina, (DF, Cerrados) [45] R. tropici

72 Sto Antonio (PE, northeast) [25] Not well defined

74 Sto Antonio (PE, northeast) [25] Not well defined

77 Sto Antonio (PE, northeast) [25] Not well defined

206 Francisco Alves (PR, southern) [25] Not well defined

209 Francisco Alves (PR, southern) [25] Not well defined

233 Francisco Alves (PR, southern) [25] R. tropiciPRF 35 Pitanga (PR, southern) [28] R. tropici

PRF 81 Londrina (PR, southern) [28] R. tropici

CPAO 29.8 Dois Irm~aos

do Buriti (MS, Cerrados)

[47] R. tropici

a In Brazil, listed as District (State, Region).

299R.A. Ribeiro et al. / Research in Microbiology 160 (2009) 297e306

To design primers for amplification and sequencing of therpoA gene, corresponding sequences derived from thefollowing complete genome sequences were used (accessionnumbers of the GenBank Data Library in parentheses): R. etlistrain CFN 42 (CP000133); R. leguminosarum bv. viciae strain3841 (AM236080); and chromosomes of E. meliloti strain1021 (AL591787), and E. medicae strain WSM419(CP000738). The sequences obtained were compared using theprogram CLUSTAL_X [61] to identify conserved regions andthe designed primers were named RRrpoA (Table 2). Theapproximate size of each gene and the size of the amplifiedfragments are also listed in Table 2.

To obtain the complete sequence of 16S rRNA, five reac-tions were carried out as described previously [42], whereas,for the other genes, only two reactions were needed (forward

Table 2

Primers used for PCR amplification and sequencing analysis of the DNA.

Gene size (zbp) Primer Sequence (50e30)

16S rRNA (1500) fd1 AGAGTTTGATCC

Y2 CCCACTGCTGCC

362f CTCCTACGGGAG

786f CGAAAGCGTGGG

1203f GAGGTGGGGATG

rD1 AAGGAGGTGATC

dnaK (1900) dnaK1466F AAGGARCANCA

dnaK1777R TASATSGCCTSRC

recA (1090) recA6F CGKCTSGTAGAG

recA555R CGRATCTGGTTG

gltA (1290) gltA428F CSGCCTTCTAYC

gltA1111R GGGAGCCSAKCG

glnII (1040) TSglnIIf AAGCTCGAGTAC

TSglnIIr SGAGCCGTTCCA

rpoA (1010) RRrpoAf GGAAATCGCCAT

RRrpoAr GGAAATCGCCAT

a Size used in this study considering only complete aligned sequences.

and reverse). Each reaction contained, in a volume of 50 mL:dNTPs (300 mM of each); PCR-buffer (Tris-base 20 mM, pH8.4 and KCl 50 mM); primers (15 pmol of each); Taq DNApolymerase (1.0 U); and DNA (20 ng). The conditions of PCRamplification were as described before for the followinggenes: 16S rRNA [42], dnaK [55], recA [19], gltA [38], andglnII [56]. For PCR amplification of the rpoA gene, an initialcycle of denaturation at 95 �C for 2 min was followed by 35cycles of denaturation at 94 �C for 45 s, annealing at 55 �C for45 s and extension at 72 �C for 2 min; the amplificationfinished with a final extension cycle of 72 �C for 5 min. AllPCR reactions were carried out on an MJ Research Inc. PTC200 thermocycler. For purification of PCR products, the Qia-quick PCR purification kit (Qiagen) was used according to themanufacturer’s instructions. The sample concentration was

Size (bp)a Reference

TGGCTCAG 1422 [66]

TCCCGTAGGAGT [76]

GCAGCAGTGGGG [42]

GAGCAAACAGG [42]

ACGTCAAGTCCTC [42]

CAGCC [66]

GATCCGCATCCA 305 [55]

CRAGCTTCAT

GAYAAATCGGTGGA 498 [19]

ATGAAGATCACCAT

AYGACTC 644 [38]

CCTTCAG

ATCTGGCTCGACGG 1041 [56]

GTCGGTGTCG

CAAGATGG 637 This study

CAAGATGG

300 R.A. Ribeiro et al. / Research in Microbiology 160 (2009) 297e306

verified by electrophoresis of 2 mL PCR products on 1%agarose gel and staining with ethidium bromide.

Sequencing reactions were carried out in 96-well-full-skirt-PCR microplates. Purified PCR products of each bacteriumculture (80 ng per reaction) received a mixture of 3 mL of dye(DYEnamic ET terminator reagent premix for the Mega-BACE, Amersham Biosciences), and 3 pmol of each primer.The same program was used for all primers, as follows:denaturation at 95 �C for 2 min; thirty cycles of denaturationat 95 �C for 10 s, 50 �C for 4 s, and extension at 60 �C for4 min; final soak at 4 �C. Sequencing was performed ona MEGA BACE 1000 (Amersham Biosciences) capillarysequencer according to the manufacturer’s instructions.

High-quality sequences obtained for each strain wereassembled into contigs using the programs Phred [14], Phrap(www.phrap.org) and Consed [24]. Sequences confirmed in the 30

and 50 directions were submitted to the GenBank database (http://www.ncbi.nlm.nih.gov/blast) to seek significant alignments.Accession numbers given to the genes are listed in Table 3.

2.3. Phylogenetic analysis

Multiple alignments for each gene were performed withCLUSTAL_X version 1.83 [61] using all sequences obtained inthis study in addition to sequences obtained from completegenomes of nine rhizobial strains (accession numbers of theGenBank Data Library in parentheses): Azorhizobium caulino-dans strain ORS571T (AP009384); Bradyrhizobium japonicumstrain USDA 110 (BA000040.2); Bradyrhizobium sp. strainORS278 (CU234118); Bradyrhizobium sp. strain BTAi1T

(CU234118); Ensifer (¼Sinorhizobium) medicae strainWSM419 (CP000738); E. meliloti strain 1021 (AL591787);Mesorhizobium loti strain MAF 303099 (AP002994 toAP003017); R. leguminosarum bv. viciae strain 3841(AM236080 to AM236086); and Rhizobium (¼Agrobacterium)tumefasciens strain C58 (AE007869). In addition, the following

Table 3

GenBank/EMBL/DDBJ accession numbers for the sequences obtained in this stud

Strain 16S rRNA dnaK recA

CFN 299 EU488741 EU488773 EU488

CIAT 899T EU488752 EU488764 EU488

USDA 2671 EU488755 EU488763 EU488

CFN 42T EU488751 EU488768 EU488

R602T EU488748 EU488772 EU488

H152T EU488750 EU488769 EU488

H 12 EU488743 EU488760 EU488

H 20 EU488740 EU488759 EU488

H 52 EU488756 EU488765 EU488

72 EU488754 EU488767 EU488

74 EU488746 EU488766 EU488

77 EU488745 EU488771 EU488

206 EU488744 EU488774 EU488

209 EU488747 EU488770 EU488

233 EU488749 EU488758 EU488

PRF 35 EU488753 EU488761 EU488

PRF 81 EU488742 EU488762 EU488

CPAO 29.8 EU488739 EU488757 EU488

sequences were added for single genes (16S rRNA and recA): forthe 16S rRNA, Rhizobium hainanense strain CCBAU 57015T

(U71078); Rhizobium sullae strain IS 123T (Y10170); Rhizobiumdaejeonense strain L61T (AY41343); Rhizobium cellulosilyticumstrain ALA10B2T (DQ855276); Rhizobium rhizogenes strainLMG 152 (X67224); Rhizobium pisi strain DSM 30132T

(AY509899); Rhizobium galegae strain ATCC 43677T

(D11343); Rhizobium huautlense strain SO2T (AF025852);Rhizobium multihospitium strain CCBAU 83401T (EF035074);Rhizobium lusitanum strain P1-7T (AY738130); and Ensifer fre-dii USDA 205T (AB433353). For the recA gene, the sequenceswere: R. cellulosilyticum strain ALA10B2T (AM286428);R. rhizogenes strain LMG 150T (CP000628); R. pisi strain DSM30132T (DQ431676); R. galegae strain ATCC 43677T

(AM182127); R. huautlense strain SO2T (AM182128); R. lusi-tanum strain P1-7T (DQ431675); and E. fredii USDA 205T

(AB433353). Caulobacter crescentus strain CB15T (AE005673)was used as the outgroup. Phylogenetic trees were generatedusing MEGA version 3.1 [36] with default parameters, K2Pdistance model [35], and the Neighbor-joining algorithm [49].Parsimony-informative characters were also estimated in theMEGA program. Statistic support for tree nodes was evaluatedby bootstrap analyses [15] with 1000 samplings. The similarityfor each gene and concatenated genes was calculated using theUPGMA algorithm (unweighted pair-group method, with arith-metic mean) [52]. Congruencies among each gene and theconcatenated genes were also analyzed using Pearson productmoment correlations with the Bionumerics program (AppliedMathematics, Kortrijh, Belgium, version 4.6).

3. Results and discussion

3.1. MLSA

We used the MLSA approach to analyze 12 common-beanrhizobia indigenous to Brazil, chosen to represent large groups

y.

gltA glnII rpoA

817 EU488806 EU488777 EU488845

815 EU488803 EU488791 EU488833

811 EU488800 EU488784 EU488837

824 EU488809 EU488776 EU488844

823 EU488805 EU488785 EU488840

819 EU488793 EU488778 EU488829

814 EU488804 EU488788 EU488838

816 EU488799 EU488792 EU488839

828 EU488808 EU488775 EU488834

818 EU488810 EU488783 EU488830

825 EU488807 EU488790 EU488832

813 EU488795 EU488786 EU488831

822 EU488794 EU488782 EU488846

820 EU488796 EU488779 EU488835

821 EU488797 EU488780 EU488841

826 EU488801 EU488787 EU488842

827 EU488802 EU488789 EU488836

812 EU488798 EU488781 EU488843

301R.A. Ribeiro et al. / Research in Microbiology 160 (2009) 297e306

of similar strains and selected from a collection of over 850isolates obtained from thirty-three states in three regions of thecountry: the northeast (close to Ecuador, high temperatures, low-fertility soils), the Cerrados (savannah, acidic soils, hightemperatures and a yearly dry season of about 7 months), and thesouthern (milder temperatures and more fertile soils) regions[25,28,45,47]. In addition to 16S rRNA, genes chosen for theMLSAwere recA, dnaK, gltA, glnII and rpoA, as they conformedto requirements established for the technique, i.e., single copiesof each in the chromosome and evenly distributed in the genome[22,54,59,77]. As a comparison, we obtained sequences forgenes of six type/reference Rhizobium symbionts of commonbean, in addition to genes obtained from nine complete rhizobialgenomes, and 16S rRNA and recA genes of 11 and seven type orreference rhizobial strains, respectively.

Neighbor joining (NJ) trees were built for each gene (Fig. 1),as well as for the concatenated sequences of all six genes(Fig. 2). Using the alignment with CLUSTAL_X, five type/reference strains representative of the five described commonbean Rhizobium species type strains (CIAT 899T, CFN 42T,H152T, R602T and USDA 2671) had 83e99%, 78e91%, 86e94%, 89e97% and 85e91% gene-sequence similarities forrecA, dnaK, glnII, rpoA and gltA, respectively; the estimatedpercentage for the 16S rRNA ranged from 92 to 99%, thusindicating that the five additional locus studied can be consid-ered as useful alternative markers for the genus Rhizobium.

The strains clearly formed separated clusters in each genetree, except for some differences in analyses of gltA (Fig. 1). Thegroupings obtained in each of the trees were in good agreement(Figs. 1 and 2) and high congruences between the genes wereshown in correlation analysis considering strains from this studyand those with the complete genomes available (Fig. 3).

Strains within group I formed a separated clade, includingR. tropici strains, with high bootstrap values (58e100%) in thetrees built with each of the six genes, with the lowest value forgltA (Fig. 1). For the concatenated tree, group I (R. tropici) wasconfirmed with 100% bootstrap support (Fig. 2). Within thisgroup, the highest gene sequence similarity occurred with the16S rRNA and rpoA genes, ranging from 95 to 100%, but highvalues were also obtained for recA, gltA, dnaK and glnII, withsimilarities estimated at 91e99%, 89e100%, 89e100% and91e100%, respectively (data estimated with CLUSTAL_X).Within the R. tropici group, in all trees except with gltA, threeclear subgroups were formed (Fig. 1), and were confirmed witha bootstrap of 100% on the concatenated tree (Fig. 2). Thesubgroups of R. tropici can be described as follows: the first(IA) included strains 77, CPAO 29.8 and CFN 299 (R. tropicitype A); the second (IB) included strains PRF 35 and H 52; andthe third (IC) included strains H 20, H 12 and CIAT 899T

(R. tropici type B). Two other Brazilian strains, PRF 81(¼SEMIA 4080) e effective in fixing N2 and officially rec-ommended for the production of commercial inoculants inBrazil [28,29] and strain 233 were either in a separatedsubcluster or were linked to the subcluster of type-B strains,and in general were responsible for lowering the bootstrapvalues of the trees. Therefore, a main conclusion from ourstudy is that, except for H 12 and H 20, the Brazilian strains

classified currently as R. tropici e on the basis of pheneticproperties, host range and 16S rRNA sequences e areconsiderably different from CIAT 899T and represent putativenew species. Indeed, the results from this study add moreevidence to reports of wide genetic variability within R. tropiciwhich has been reported since the first study describing thespecies [41], with the statement that R. tropici comprises atleast three genomic species. Furthermore, emphasis should begiven to results showing low DNAeDNA hybridization values(36%) between type A and type B strains [41]. Differencesamong R. tropici strains positioned in the three different groupswere also reported based on phenetic and genetic properties,including variability in 16S and 23S rRNA genes as well as inconserved and repetitive regions of the genome [28,47].

R. etli and R. leguminosarum strains were clustered togetherin all trees except for the dnaK gene (Fig. 1), with goodbootstrap support (100%) on the concatenated tree. The twoother common bean Rhizobium species, R. gallicum andR. giardinii, were not clustered with any of the other strains inthis study in any of the trees (Figs. 1 and 2). These resultsconfirmed those of Silva et al. [51] in phylogenetic studiesshowing trees built with glnII and atpD.

Except for the tree with the recA gene, a strongly supportedmonophyletic group (98e100% of bootstrap) was formed withstrains 72, 74, 209 and E. meliloti and E. medicae. Incomparison to E. fredii strain USDA 205T, when all genesexcept for rpoA were concatenated and used for comparisonvia CLUSTAL_W, similarities were 99% for strain 74, 97%for strain 72 and 88% for strain 206 (data not shown). Also, onthe 16S rRNA tree, strains 72, 74 and 209 form a supportedcluster with E. fredii, but in the recA tree, strain 72 was notclustered with Ensifer (Fig. 1). It is noteworthy that pheneticand genetic properties indicate that Ensifer spp. strains serveas microsymbionts of the common bean in Brazil [25,58], anda new biovar symbiont of the common bean, E. meliloti bv.mediterranense has been isolated from Tunisian soil [46].

3.2. Evolutionary aspects related to R. tropici, R. etliand R. leguminosarumin in Brazil

The origin of R. tropici was first suggested to be SouthAmerica [41], with Brazil being a strong candidate and repre-senting the source of most strains isolated thus far[6,25,28,30,43,45,47,57,58]. Another hypothesis is that R. tro-pici arose from the Andes region (CIAT 899 was isolated inColombia) and adapted well to Brazilian soils and environ-mental conditions. The presence of R. tropici nodulating legu-minous plants in Europe and Africa [4,7] could be explained bymigration via trade. However, as wild beans are not found inBrazil [11, 17], R. tropici could be a microsymbiont of otherindigenous host legumes. Possible candidates are species of thegenera Mimosa and Gliricidia, reported to establish effectivesymbioses with R. tropici under field conditions [42]. However,R. tropici has also been reported to be a microsymbiont oflegumes indigenous to Mexico (Gliricidia) [1] and Africa(Bolusanthus and Spartium) [10]; nevertheless, information onranges of distribution of R. tropici is missing from those studies.

Fig. 1. Phylogenetic tree based on the dnaK, glnII, gltA, recA, rpoA and 16S rRNA sequences of Brazilian indigenous and type/reference strains of common bean

rhizobia. Accession numbers are described in Material and methods and Table 3, and Caulobacter crescentus was used as an outgroup strain. The tree was

generated using MEGA version 3.1 with default parameters, K2P distance model and the Neighbor-joining algorithm, with 1000 samplings in bootstrap analyses.

302 R.A. Ribeiro et al. / Research in Microbiology 160 (2009) 297e306

Fig. 1. (continued)

303R.A. Ribeiro et al. / Research in Microbiology 160 (2009) 297e306

Therefore, the evidence that R. tropici is indigenous to Brazil isstrong but not conclusive, and one main conclusion arising fromour results is that the broad diversity within strains currentlyclassified as R. tropici indicates new species.

R. etli is the dominant microsymbiont in both the Meso-american and Andean centers of origin. It is also the predomi-nant symbiont of wild beans in northwestern Argentina[2,3,8,50]. Segovia et al. [50] hypothesized that when seedscontaining R. etli bv. phaseoli were introduced into Europe, thesymbiotic plasmid may have been transferred to R. legumino-sarum; later on, the same process may have occurred fromR. leguminosarum bv. phaseoli to R. gallicum bv. phaseoli andR. giardinii bv. phaseoli [5]. R. etli is also found in Brazilian soils[25,26,57], possibly as a result of cultivation of the legume forcenturies; interestingly, R. leguminosarum is also widelydisseminated in Brazilian soils [6,23,45,53], and was detected inColombia [12], which was suggested to be a third center ofgenetic diversification of the common bean [20]. Despite severalreports of isolation of R. leguminosarum and R. etli in Braziliansoils [6,23,25,26,45,53,57], clear-cut rhizobial strains showingphenetic properties resembling those of R. tropici, with emphasison the capacity to nodulate both common bean and Leucaenaspp. and tolerance to high temperatures [25,28,29], wereconfirmed by MLSA as belonging to R. tropici in this study.

3.3. Concluding remarks

The feasibility of improving evolutionary inferences andinformation about population genetics of Rhizobium usingseveral genes was highlighted in an important study performedby Silva et al. [51] in which the phylogeny of species andbiovars was better defined by using three housekeeping (rrs,glnII and atpD) and two symbiotic genes (nifH and nodB). Inaddition, the concept of MLSA has been successfully appliedto studies with several genera of prokaryotes, including Bur-kholderia, Bacillus, Vibrio, Mycobacterium and Ensifer[22,38,59,60]; both Burkholderia and Ensifer comprisesymbiotic diazotrophic bacteria species.

Broad genetic diversity was observed among common beanrhizobial strains indigenous to Brazilian soils, and the MLSAmethod effectively established phylogenetic relationships withtype and reference strains. As suggested for other species [38],MLSA is clearly a valuable alternative to 16S rRNA sequenceanalysis and DNAeDNA hybridization for elucidation of thetaxonomy of rhizobia. Strains currently classified as R. tropici arefound in abundance in a variety of Brazilian acid soils and areprobably the most important tropical microsymbionts ofcommon bean due to their genetic stability, effectiveness in fixingN2 and tolerance to environmental stress [28,29]. In addition, the

Fig. 2. Phylogenetic tree of concatenated genes dnaKþ glnIþ gltAþ recAþ rpoAþ 16S rRNA. Strains and accession numbers are as described in Fig. 1. The tree

was generated using MEGA version 3.1 with default parameters, K2P distance model and the Neighbor-joining algorithm, with 1000 samplings in bootstrap

analyses.

304 R.A. Ribeiro et al. / Research in Microbiology 160 (2009) 297e306

genetic diversity reported in this study lends strong support to thesuggestion that many strains classified today as R. tropici mightfit into new species. Polyphasic taxonomic work is under way inour laboratory in order to confirm this hypothesis.

rpoA

multilocus

dnaK

gltA

glnII

recA

16S rRNA

Fig. 3. Congruence of trees using single or concatenated genes of 12 indige-

nous Brazilian rhizobial strains, and reference/type strains of common bean

rhizobia with complete genomes available. Congruence was estimated using

the Bionumerics program with the UPGMA algorithm.

Acknowledgments

The work was partially supported by CNPq/MCT, projectsCulture Collections (552393/2005-3 and MAPA 577933/2008-6),PROTAX (563960/05-1), and Research Productivity (300698/2007-0). F.L. Thompson is grateful for grants from CNPq, IFS,and FAPERJ. The authors thank Ligia M.O. Chueire, PamelaMenna and Jesiane S.S. Batista (Embrapa Soja) for assistance inseveral steps of this work and Allan R.J. Eaglesham for helpfuldiscussion in the manuscript.

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