Instituto de Recursos Naturales y Agrobiología, IRNASA-CSIC, c/Cordel de Merinas 40-52, 37008 Salamanca, SpainUnidad Asociada Universidad de Salamanca-CSIC ‘Interacción Planta-Microorganismo’, Salamanca, SpainDepartment of Biology, Faculty of Sciences and Techniques, University Abdelmalek Essaadi, Tangier, MoroccoDepartamento de Microbiología y Genética, Lab. 209, Universidad de Salamanca, Edificio Departamental de Biología, Campus M. Unamuno, Salamanca, SpainLaboratorio de Estudios Ambientales, Instituto de Zoología Tropical, Facultad de Ciencias, Universidad Central de Venezuela, Caracas 1041-A, Apartado 47.058, Venezuela
a r t i c l e i n f o
rticle history:eceived 14 January 2013eceived in revised form 5 March 2013ccepted 15 March 2013
eywords:hizobiaradyrhizobiumentrosemaiodiversity
a b s t r a c t
Centrosema is an American indigenous legume that can be used in agroecosystems for recovery of acidicand degraded soils. In this study, a Centrosema-nodulating rhizobial collection of strains isolated in a pooracid savanna soil from Venezuela was characterized, and the members of the collection were comparedto other Centrosema strains from America. The analysis of the rrs gene showed that the strains nodulatingCentrosema in American countries were closely related to different species of the genus Bradyrhizobium.However, the analysis of the atpD and recA genes, as well as the 16S–23S ITS region, showed that theyformed several new phylogenetic lineages within this genus. The Venezuela strains formed three lineagesthat were divergent among themselves and with respect to those formed by Centrosema strains isolatedin other countries, as well as to the currently described species and genospecies of Bradyrhizobium. In
hylogeny addition, the symbiotic genes nodC and nifH carried by Centrosema-nodulating strains were analyzed forthe first time, and it was shown that they belonged to three new phylogenetic lineages within Bradyrhi-zobium. The nodC genes of the Centrosema strains were divergent among themselves and with respectto the genistearum and glycinearum symbiovars, indicating that Centrosema is a promiscuous legume.According to these results, the currently known Centrosema-nodulating strains represent several newputative species and symbiovars of the genus Bradyrhizobium.
ntroduction
Centrosema is a leguminous genus of voluble climbing herbs orubshrubs within the tribe Phaseolae that includes 34 species, allf them native to America. Most species are widely distributedn the savanna and forests of tropical regions (Penteado et al.,996). Centrosema is a very nutritive pasture legume occurringaturally in arid tropical ecosystems, and many species of thisenus become well adapted to very diverse tropical environmentsncluding highlands, dried or seasonally flooded areas, as wells acidic soils showing low fertility (Keller-Grein et al., 2000;
Please cite this article in press as: M.-H. Ramírez-Bahena, et al., Centrosemaand symbiovars of Bradyrhizobium in various American countries, Syst. App
chultze-Kraft et al., 1990). In addition, Centrosema has been stud-ed recently in phytoremediation schemes (Nwaichi et al., 2011)nd investigated as a medicinal plant for its likely antiproliferative
∗ Corresponding author at: Instituto de Recursos Naturales y Agrobiología,RNASA-CSIC, c/Cordel de Merinas 40-52, 37008 Salamanca, Spain.el.: +34 923219606.
activity in leukemia (Mani and Lakshmi, 2010). Due to its impor-tance, several studies on the biology and agronomic performanceof Centrosema species have been conducted in several America(Keller-Grein et al., 2000; Reátegui et al., 1985; Sousa et al., 2011),Africa (Odeyinka et al., 2008) and Asia (Humphreys et al., 1990)countries, as well as in Australia (Schultze-Kraft et al., 1997). Somespecies, such as Centrosema molle or Centrosema macrocarpum, arecommonly cultivated as pastures in agricultural savanna ecosys-tems in Venezuela because of their property for increasing thenitrogen content in soils. These legumes are combined with othercrops in tropical agroecosystems where acid soils with poor fertil-ity occur, and they act as plant covers that provide protein andenergy sources for animals in the drought season, which is themost critical season for the production of forage (Alguacil et al.,2010).
Although Centrosema has been widely studied for its proper-
is a promiscuous legume nodulated by several new putative speciesl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.03.007
ties as a forage legume (Navas et al., 2011; Sousa et al., 2011)and studies on Centrosema-rhizobia-fungi interactions have beenconducted recently in Brazil (Matias et al., 2009) and Venezuela(Navas et al., 2011), little attention has been given to date to the
Centrosema strains CMVU02, CMVU20 and CMVU30 that are
ARTICLEYAPM-25544; No. of Pages 9
M.-H. Ramírez-Bahena et al. / Systematic
hizobial endosymbionts. There are no studies concerning thetrains nodulating this legume in Venezuela where Centrosema isommonly cultivated, and only seven strains isolated from differententrosema species have been reported in some studies togetherith strains nodulating different legumes (Menna et al., 2009;
arker, 2003; Vinuesa et al., 2005b). These strains isolated fromentrosema pubescens, Centrosema plumbieri and Centrosema sp.
n Brazil, Colombia and Panama were classified within the genusradyrhizobium (Menna et al., 2009; Parker, 2003; Vinuesa et al.,005b).
The genus Bradyrhizobium currently contains 17 speciesEuzéby, J.P. LPSN – List of Prokaryotic Names with Standing inomenclature, http://www.bacterio.cict.fr/index.html) and they
eem to be the predominant organisms establishing symbiosis withmerican legumes (Menna et al., 2009; Ormeno-Orrillo et al., 2011;arker, 2003). Also, this genus includes several strains not isolatedrom nodules, such as Bradyrhizobium betae and Bradyrhizobiumriomotense isolated from tumors of the non-legume Beta vulgarisnd the legume Entada koshunensis, respectively (Islam et al., 2008;ivas et al., 2004), Bradyrhizobium denitrificans that was previouslyamed Blastobacter denitrificans isolated from water (Van Berkumt al., 2006), and Bradyrhizobium oligotrophicum (formerly namedgromonas oligotrophica) isolated from a rice paddy soil (Ramírez-ahena et al., 2013). Moreover, this genus contains photosynthetictrains, such as BTAi1, ORS278 and ORS285 that nodulate the trop-cal legume Aeschynomene whose genomes have been completelyequenced (Nzoué et al., 2009). From the strains that have not beensolated from nodules, as well as the photosynthetic bradyrhizobia
entioned, the nodulation genes have been sequenced to date onlyrom strains B. iriomotense EK05T and ORS285 (Chaintreuil et al.,001; Islam et al., 2008).
Bradyrhizobium has been proposed to be divided into two groupsccording to the rrs gene and ITS region analyses designated Ind II by Menna et al. (2009). However, the analysis of the ITSegion performed in this study showed that B. denitrificans andhe Bradyrhizobium photosynthetic strains clustered into a differentroup which we have named group III following the same nomen-lature. The Centrosema strains included in the work of Mennat al. (2009) mostly belong to group II regardless the species ofhis legume and the American country from which they were iso-ated. However, the number of Centrosema strains analyzed toate is still too low, and further studies are required in ordero identify the biodiversity of strains nodulating this legume inmerica.
Therefore, the objective of this study was to carry out a bio-iversity analysis of strains nodulating Centrosema in Venezuela,or which their core and symbiotic genes were studied. Severalutatively novel species and symbiovars of Bradyrhizobium wereetected and this is discussed.
aterials and methods
trains and nodulation experiments
Plants of Centrosema molle and C. macrocarpum were useds trap plants in an acid soil (pH 4.99) from Guarico State inenezuela, where savanna ecosystems are widespread. The rhizo-ial strains were isolated from the Centrosema nodules accordingo the method of Vincent (1970). In order to confirm the nodula-ion capacity of the strains, infectivity tests were conducted in a
Please cite this article in press as: M.-H. Ramírez-Bahena, et al., Centrosemaand symbiovars of Bradyrhizobium in various American countries, Syst. Ap
rowth chamber umder controlled conditions using sterile vermi-ulite as substrate. The Centrosema seeds were surface disinfectednd seedlings were inoculated as described by Ramírez-Bahenat al. (2009b).
PRESSpplied Microbiology xxx (2013) xxx– xxx
RAPD fingerprinting
RAPD patterns were obtained as previously described (Rivaset al., 2006) using the primer M13 (5′-GAGGGTGGCGGTTCT-3′) andthe GoTaq Flexi DNA polymerase (Promega) kit. PCR conditionsand electrophoresis were performed as described by Faghire et al.(2012). A dendrogram was constructed based on the matrix gen-erated using the UPGMA method and the Pearson coefficient withBionumerics version 4.0 software (Applied Maths, Austin, TX).
Analysis of rrs, atpD, recA, nodC and nifH genes, and the 16S–23Sintergenic spacer (ITS)
The rrs gene was amplified and sequenced according to Rivaset al. (2007a), and the ITS region as described by Peix et al. (2005).The recA and atpD genes were amplified and sequenced as describedby Gaunt et al. (2001) and Vinuesa et al. (2005b). The nodC and nifHgenes were amplified with the primers and conditions describedby Laguerre et al. (2001) and Velázquez et al. (2010), except forstrains CMVU04 and CMVU44 whose nodC genes were amplifiedand sequenced with the following primers designed in this study:nodCRB1F (5′-GGCVAASAAYGTBGGAAAGCGCAAGGCGCAGATCG-3′) and nodCRBIR (5′-AGCGNAGYTGCTGNCGHAGTATGGYC-3′). PCRamplifications were performed with a REDExtract-N-Amp PCR Kit(Sigma) or GoTaq Flexi DNA polymerase kit (Promega) follow-ing the manufacturers’ instructions. The bands corresponding tothe different genes were purified and sequenced as described inFaghire et al. (2012). The sequences obtained were compared tothose held in GenBank by using the BLASTN program (Altschulet al., 1990). They were aligned by using Clustal W software(Thompson et al., 1997). Kimura’s two-parameter model (Kimura,1980) was used to infer phylogenetic trees with the maximum like-lihood method (Felsenstein, 1981) using MEGA5 software (Tamuraet al., 2011). Confidence values for nodes in the trees were gen-erated by bootstrap analysis using 1000 permutations of the datasets.
Results and discussion
RAPD fingerprinting analysis
A total of 44 Centrosema nodulating strains were isolated(Table 1) and analyzed by RAPD fingerprinting that allowed dif-ferentiation between strains of the same rhizobial species, forwhich this technique provides an estimation of the genetic diver-sity (Dooley et al., 1993; Faghire et al., 2012; Iglesias et al., 2007;Moschetti et al., 2005; Rivas et al., 2006; Valverde et al., 2006). Sev-eral different RAPD profiles were obtained that were distributedinto seven groups with similarity percentages lower than 85% (Fig. 1and Table 1), from which representative strains were selected forgene sequence analysis.
Analysis of the rrs gene
The current phylogenetic classification of rhizobia is predom-inantly based on rrs gene sequences and thus the identificationof nodule isolates should also be based on this gene. In Bradyrhi-zobium, as mentioned before, two well-differentiated groups,designated I and II by Menna et al. (2009), have been defined accord-ing to the 16S rRNA gene.
is a promiscuous legume nodulated by several new putative speciespl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.03.007
representative of RAPD groups V, VI and VII, respectively, belongto Bradyrhizobium group II and have identical rrs gene sequences,for which only the sequence of strain CMVU02 is shown in thephylogenetic tree (Fig. 2). The closest type strain of CMVU02 is
CMVU30, CMVU47 C. macrocarpum Venezuela This study VII II
Reference strainsSEMIA 690 C. pubescens Brazil Menna et al. (2009) nd IISEMIA 6146 Centrosema sp. Brazil Menna et al. (2009) nd IISEMIA 6424 C. pubescens Brazil Menna et al. (2009) nd IISEMIA 6425 C. pubescens Colombia Menna et al. (2009) nd IISEMIA 6368 C. plumieri Colombia Menna et al. (2009) nd ICp5.3 C. pubescens Panama Parker (2003) nd IICIAT 3101 C. plumieri Colombia Vinuesa et al.
(2005b)nd I
n
BCVIf2fmtCmf
RgCtBo
d =no data.
radyrhizobium jicamae PAC68T with 99.8% identity. As for theentrosema strains previously isolated in America, most of theenezuelan strains isolated in this study belonged to rrs group
I within the genus Bradyrhizobium, with strain Cp5.3 isolatedrom C. pubescens in Panama being the most divergent (Parker,003). Two strains isolated in Brazil, SEMIA 690 and SEMIA 6424rom C. pubescens, were close to Venezuelan strains and to B. jica-ae PAC68T isolated from Pachyrhizus erosus in Honduras. Finally,
wo strains, SEMIA 6146 and SEMIA 6425, isolated in Brazil andolombia from Centrosema sp. and C. pubescens, respectively, wereost closely related to Bradryhizobium pachyrhizi PAC48T isolated
rom P. erosus in Costa Rica (Ramírez-Bahena et al., 2009a).The strains CMVU44, CMVU43 and CMVU04, representative of
APD groups I, II and III, respectively, belonged to Bradryhizobiumroup I and showed identical rrs gene sequences, so again the
Please cite this article in press as: M.-H. Ramírez-Bahena, et al., Centrosemaand symbiovars of Bradyrhizobium in various American countries, Syst. App
MVU04 sequence was the only one included in the phylogeneticree (Fig. 2). This strain showed 100% identity with respect toradyrhizobium daqingense CCBAU 15774T, but since the sequencef the latter strain has only 1300 nucleotides it could not be
concluded if this group of strains represented by CMVU04 (RAPDgroups I, II and III) was classified in this species.
Strain A9, representative of RAPD group IV, also belonged to rrsgene group I and formed a separate branch clustering with a largegroup comprising several species of Bradyrhizobium, all of themshowing less than 99.7% identity in the rrs gene. In this group, therewas also a Centrosema plumieri strain from Colombia (SEMIA 6368),but it was phylogenetically divergent to our Centrosema strains(Fig. 2).
In summary, the rrs gene sequences of the strains from groupsI and II had identity values higher than 99% with respect to thoseof the validly described species of Bradyrhizobium. This is a com-mon finding in this genus that contains many species with rrsgene sequences with identities higher than 99%. However, highidentities of the rrs gene (even 100%) in the case of the genus
is a promiscuous legume nodulated by several new putative speciesl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.03.007
Bradyrhizobium does not imply that they belong to the same species(Chahboune et al., 2011; Ramírez-Bahena et al., 2009a), and the ITSfragment offers a better picture in support of the diversity of thisgenus.
4 M.-H. Ramírez-Bahena et al. / Systematic and Applied Microbiology xxx (2013) xxx– xxx
ing Pe
A
egt2Iwaasbtfisy2tL
Fig. 1. Dendrogram obtained for the strains of Centrosema us
nalysis of ITS sequences
The ITS sequence analysis has been reported to be a more pow-rful tool than rrs gene analysis for species delineation within theenus Bradyrhizobium, in which ITS sequence similarities higherhan 95.5% indicate a genospecies level relatedness (Rivas et al.,004; Willems et al., 2003). Menna et al. (2009) showed that theTS analysis allows the differentiation of two groups congruently
ith rrs gene analysis. However, when the species B. denitrificansnd photosynthetic bradyrhizobia are considered in this analysis
third group is obtained (Fig. 3), including the photosynthetictrains BTAi1, ORS278 and ORS285. Nevertheless, these bacteriaelong to different subgroups that suggest the existence of morehan one species within ITS group III. Strains BTAi1 and B. denitri-cans LMG 8443T have identical ITS sequences and are the samepecies previously shown by Nzoué et al. (2009) after MLST anal-
Please cite this article in press as: M.-H. Ramírez-Bahena, et al., Centrosemaand symbiovars of Bradyrhizobium in various American countries, Syst. Ap
sis of Aeschynomene-nodulating strains (Nzoué et al., 2009). ORS78 and ORS 285 with 98.2% identity in the ITS region were likelyo be the same species, which would be different to B. denitrificansMG 8443T with 94.8% identity.
arson’s coefficient and UPGMA analysis of the RAPD profiles.
The Centrosema strains CMVU44, CMVU43 and CMVU04, repre-sentative of RAPD groups I, II and III, respectively, formed a clusterwithin group I that contained the same strains obtained by rrsgene analysis. The closest related strain was B. daqingense CCBAU15774T with more than 99% identity, although the ITS fragment ofthis strain had a 15 nucleotide insert in the ITS sequence that wasabsent in the Centrosema strains (between nucleotides 684 and 699considering the sequence of strain CCBAU 15774T).
Strain A9, representative of RAPD group IV, was also phyloge-netically divergent to all the currently described Bradyrhizobiumspecies, with the type strain of B. yuanmingense being its clos-est relative with approximately 96% identity of the ITS sequencesafter a pairwise analysis. Although SEMIA 6368 isolated from C.plumieri in Colombia branched into ITS group I it had a sequencephylogenetically very divergent to those of the Venezuela iso-lates, with identity values lower than 93%. Strains from both
is a promiscuous legume nodulated by several new putative speciespl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.03.007
Venezuela and Colombia had identities of lower than 96% betweenthem and with respect to all described species of Bradyrhizo-bium, and therefore they could represent three different novelspecies.
ig. 2. Maximum likelihood phylogenetic rooted tree based on rrs gene sequenceroups. Bootstrap values calculated for 1000 replications are indicated. Bar, 1 nt subsolated in this study are shown in bold.
In the case of the Centrosema strains clustering within group II,he ITS analysis showed that they branched together with SEMIA424 isolated from C. pubescens in Brazil. This cluster was close to
second group containing B. pachyrhizi PAC48T and strain SEMIA425 isolated from Centrosema pubescens in Colombia. A pairwisenalysis of sequences of CMVU02 and PAC48T showed an identityalue of 96.2%. Although this value was higher than the estimatedut-off value for species delineation within the genus Bradyrhi-obium, the existence of different inserts in the ITS fragments ofhese strains (data not shown) makes their classification ambiguousn the species B. pachyrhizi, unlike Colombian strain SEMIA 6425,
hich was clearly identified as this species, with 98.9% identity forhe ITS sequence.
Therefore, in spite of the closeness of the rrs genes of Cen-rosema isolates to the current Bradyrhizobium species, the analysisf the ITS fragment suggested they could represent several putativeovel species. Nevertheless, for this purpose, the analysis of house-eeping genes is necessary, since their usefulness in delineation ofradyrhizobium species has been well established (Chahboune et al.,011, 2012; Chang et al., 2011; Islam et al., 2008; Ramírez-Bahenat al., 2009a, 2013; Vinuesa et al., 2005a; Zhang et al., 2012).
nalysis of recA and atpD housekeeping genes
recA and atpD are two housekeeping genes that have beenequenced in all Bradyrhizobium species, as well as in two Cen-rosema strains isolated in America, CIAT 3101 (Vinuesa et al.,
Please cite this article in press as: M.-H. Ramírez-Bahena, et al., Centrosemaand symbiovars of Bradyrhizobium in various American countries, Syst. App
005b) and SEMIA 6146 (Menna et al., 2009). Since the ITS frag-ent contains large non-coding regions it showed the highest
ariability among Bradyrhizobium strains and is therefore very use-ul for selecting different strains for housekeeping gene analysis.
ing the taxonomic affiliation of the strains representative of the different RAPDion per 100 nt. Accession numbers from GenBank are given in brackets, and strains
Therefore, the two more divergent strains were selected from eachITS group found in this study for the analysis of concatenated recAand atpD genes. The results showed that they belonged to differentbranches divergent to those formed by strains CIAT 3101 and SEMIA6146 previously isolated from Centrosema in America, as well as tothe currently described species of the genus Bradyrhizobium (Fig. 4).The tree topologies (provided as supplementary material) obtainedfor separate recA and atpD gene phylogenies were congruent withthe concatenated tree (Figs. S1 and S2, respectively).
Strains CMVU02 and CMVU30, which had slightly different ITS,formed a branch related to B. pachyrhizi and Bradyrhizobium elkanii,although the identities were lower than 96% for both atpD and recAgenes, respectively, with respect to these two species. Strain SEMIA6146 also belonged to this group, although it is probably a differentspecies since it had almost 95% identity in both genes.
Strains CMVU04 and CMVU44, which also had slightly differentITS, formed a branch related to the recently described species B.daqingense with identities lower than 96% and 97% for the atpD andrecA genes, respectively.
Finally, strain A9 formed a lineage unrelated to the cur-rently described species of Bradyrhizobium, although the recentlydescribed species B. arachidis was its closest relative with identitiesclose to 96% for both the recA and atpD genes.
Considering that identities lower than or close to 95% for recAand atpD genes are shown by different Bradyrhizobium species(Chahboune et al., 2011, 2012; Ramírez-Bahena et al., 2009a;Rivas et al., 2004), the strains isolated from Centrosema nodules
is a promiscuous legume nodulated by several new putative speciesl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.03.007
in Venezuela probably belonged to three new species of Bradyrhi-zobium.
As was previously mentioned, the recA and atpD genes areonly available for two strains previously isolated from Centrosema,
Bradyrhizobium huanghuaiha iens e CCBAU 23303T (HQ428043)
Bradyrhizobium daqingense CCBAU 15774T (HQ231312)
Bradyrhizobium sp. CMV U04 (KC247 118)
Bradyrhizobium sp. CMV U43 (KC247 116)
Bradyrhizobium sp. CMVU44 (KC247121)
Bradyrhizobium elkan ii USDA 76T (AB509379 )
Bradyrhizobium lab labi CCBAU 23086T (GU433583)
Bradyrhizobium jicamae PAC68T (AY628094)
Bradyrhizobium pachy rhiz i PAC48T (AY628092)
Bradyrhizobium sp. SEMI A 6425 (FJ39 1134)
Bradyrhizobium sp. SEMI A 6146 (FJ391096)
Bradyrhizobium sp. CMV U20 (KC247 117)
Bradyrhizob ium sp. CMV U02 (KC247 119)
Bradyrhizobium sp. CMV U30 (KC247120)
Bradyrhizobium sp. SEMI A 6424 (FJ39 1127)
Bradyrhizobium deni tri ficans LMG 8443T (AJ279318)
Bradyrhizobium sp. B TAi -1 (AJ534588)
Bradyrhizobium sp. O RS285 (AJ279297)
Bradyrhizobium sp. O RS278 (CU234 118)
91
91
100
98
92
98
67
70
95
66
96
769667
90
80
74
0.05
Fig. 3. Maximum likelihood phylogenetic rooted tree based on 16S–23S rRNA internal transcribed spacer (ITS) sequences showing the taxonomic affiliation of the strainsr licatioa
S2eaatistwfCBoas
tits
A
p
epresentative of the different RAPD groups. Bootstrap values calculated for 1000 repre given in brackets, and strains isolated in this study are shown in bold.
EMIA 6146 isolated from Centrosema sp. in Brazil (Menna et al.,009) and CIAT 3101 isolated from C. plumieri in Colombia (Vinuesat al., 2005b). From this latter strain these are the only genes avail-ble in the databases and it has been classified into genospecieslpha by Vinuesa et al. (2005a). However, despite the fact thathis strain clustered with strain BC-C1, which is also classifiedn genospecies alpha, they could be different species since theyhowed 95% and 93% identity in the atpD and recA genes, respec-ively. Strain SEMIA 6146 also formed a divergent branch clusteringith B. elkanii and B. pachyrhizi but with identities close to 95%
or both atpD and recA. These results therefore indicated thatIAT 3101 and SEMIA 6146 belonged to undescribed species ofradyrhizobium and it will hence be convenient to revise the tax-nomic status of genospecies alpha by performing phenotypicnd hybridization tests in order to describe the correspondingpecies.
Collectively the core genes analyzed in this study showedhat the Centrosema-nodulating strains isolated in Amer-ca studied to date constituted several new lineages withinhe genus Bradyrhizobium that may represent several novelpecies.
Please cite this article in press as: M.-H. Ramírez-Bahena, et al., Centrosemaand symbiovars of Bradyrhizobium in various American countries, Syst. Ap
nalysis of symbiotic genes
Despite the fact that some core genes have been sequencedreviously in a few strains isolated from Centrosema in American
ns are indicated. Bar, 5 nt substitution per 100 nt. Accession numbers from GenBank
countries, there are no available sequences of symbiotic genes forthe rhizobia nodulating this legume. Therefore, this work is the firstreport on the nodC and nifH genes carried by strains nodulatingCentrosema and, therefore, on the degree of promiscuity for thishost. The nodC gene encoding a chitin synthase is related to the hostrange of rhizobia and the degree of promiscuity of the hosts (Iglesiaset al., 2007; Laguerre et al., 2001; Perret et al., 2000; Rivas et al.,2007b; Roche et al., 1996; Zurdo-Pineiro et al., 2009). Therefore,currently, the nodC gene is commonly used to define symbiovars(García-Fraile et al., 2010; Iglesias et al., 2007; León-Barrios et al.,2009; Mnasri et al., 2007; Rivas et al., 2007b; Rogel et al., 2011;Vinuesa et al., 2005a). The nifH gene encodes for the dinitrogenasereductase of the nitrogenase complex and, like nodC, usually existsas a single copy in the strains of Bradyrhizobium whose genome hasbeen sequenced to date. The nifH gene has been found in all photo-synthetic strains, but the nodC gene has only been shown in strainORS285 (Figs. 5 and 6).
The analysis of the nodC gene showed that the five strainsisolated from Centrosema studied in this work belonged to three dif-ferent phylogenetic lineages (Fig. 5). Strains CMVU02 and CMVU30had 99.4% identity between them, and formed a group related toB. elkanii USDA 76T and B. pachyrhizi PAC48T, although the identity
is a promiscuous legume nodulated by several new putative speciespl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.03.007
values were lower than 82%. Strains CMVU04 and CMVU44, withidentical nodC gene sequences, were related to B. iriomotense EK05T
but only with 85.6% identity. Finally, strain A9 occupied a branchrelated to several species, some recently described as B. daqingense
Brady rhizo bium deni tri ficans LMG 8443T (FM253196, FM253153)
Brady rhizo bium sp. B TAi -1 ( NC_009485)
Bradyrhizo bium ol igot rophicum LMG 10732T (JQ619231, JQ619232)
99
99
99
90
99
98
84
71
68
84
91
95
76
50
70
50
0.01
F D gend cated.b
aw
t
Fcs
ig. 4. Maximum likelihood phylogenetic tree based on concatenated recA and atpifferent RAPD groups. Bootstrap values calculated for 1000 replications are indirackets, and strains isolated in this study are shown in bold.
Please cite this article in press as: M.-H. Ramírez-Bahena, et al., Centrosemaand symbiovars of Bradyrhizobium in various American countries, Syst. App
nd B. arachidis, nodulating soybean and peanut, respectively, butith identity values lower than 87% (Fig. 5).
The analysis of the nifH gene showed results congruent withhose of the nodC gene since our strains also clustered into three
BradyrhBradyrh Bradyrh Bradyrh
BradyBr
Bradyrhizo bium BradBrad
Bradyrhizo bBradyrhizo b
Bradyrhizo bium lab labi CCBAU
Bradyrhizo bium cy tisi CTA Bradyrhizo bium
Bradyrhizo bium ri fens Bradyrhizo bium geno
Bradyrhizo bium japon Bradyrhizobium gen
Bradyrhizo bium jicamae PAC68T (AB
Bradyrhizo bium ir iomotens e EK05T (A
Bradyrhizobium sp. CMVU44 (KC247130)
Bradyrhizobium sp. CMV U04 (KC247132)
B100
99
88
98
70
10057
86
88
80
99
86
100
0.05
ig. 5. Maximum likelihood phylogenetic tree based on nodC gene sequences showing talculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. Accession
hown in bold.
e sequences showing the taxonomic affiliation of the strains representative of the Bar, 1 nt substitution per 100 nt. Accession numbers from GenBank are given in
is a promiscuous legume nodulated by several new putative speciesl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.03.007
divergent phylogenetic lineages (Fig. 6). Strains CMVU02 andCMVU30 carried identical nifH genes and formed a divergent groupwith respect to Bradyrhizobium strains isolated from other legumes.Strains CMVU04 and CMVU44 also showed identical nifH gene
izobium huanghuaihaiense CCBAU 23303T (HQ231507)
izobium daqingense CCBAU 15774T (HQ231326)
izobium japonicum USDA 110 ( NC_004463 )
izobium japonicum USDA 6T (AB354632 )
rhizo bium yuanmingen se CCBAU 10071T (AB354633)
adyrhizob ium sp. A9 (KC247134)
arach idis CCBAU 05 1107T (HM107267)
yrhizobium sp. CMVU02 (KC247131)
yrhizob ium sp. CMV U30 (KC247133)
ium elkan ii LMG 6134T (AB354631)
ium pachy rhizi PAC48T (HQ588110)
23086T (GU433565)
W11T (EU597844)
canar iens e BTA-1T (AJ560653)
e CTA W71T (EU597853)
sp. beta BRE-1 (FJ499319)
icum BGA-1 (EF694757)
osp. alpha BC-C1 (AJ560654)
573869)
B301000)
rady rhizo bium sp. O RS285 (AF284858)
he position of representative strains from different RAPD groups. Bootstrap valuesnumbers from GenBank are given in brackets, and strains isolated in this study are
Bradyrhizobium yuanmingen se CCBAU 10071T (EU8 18927)
Bradyrhizobium sp. A9 (KC247139)
Bradyrhizobium daqingense CCBAU 15774T (HQ231323)
Bradyrhizobium huanghuaihaiens e CCBAU 23303T (HQ231551)
Bradyrhizobium liaoningense LMG18230T (EU8 18925)
Bradyrhizobium japon icum USDA 110 (K01620)
Bradyrhizobium japon icum USDA 6T (HM047126)
100
100
100
99
69
99
76
68
99
94
66
62
99
0.02
F ing thc ssions
sBo
sgsotctwobparh
stnhsh
t
A
pirr
ig. 6. Maximum likelihood phylogenetic tree based on nifH gene sequences showalculated for 1000 replications are indicated. Bar, 2 nt substitution per 100 nt. Accehown in bold.
equences and they were also phylogenetically divergent to otherradyrhizobium species, as also occurred in the case of the nifH genef strain A9 (Fig. 6).
Therefore, the analysis of the symbiotic genes nodC and nifHhowed that our Centrosema strains belonged to three phylo-enetically divergent lineages representing three new putativeymbiovars that should be defined when more strains from eachne are available. Since legumes nodulated by strains belongingo different symbiovars are considered as promiscuous hosts, itan be concluded that Centrosema is highly promiscuous, at leasthe species C. molle and C. macrocarpum. This legume is classifiedithin the tribe Phaseolae that, according to the analysis of symbi-
tic genes, contains other highly promiscuous legumes nodulatedy bradyrhizobia, such as Pachyrhizus whose endosymbionts B.achyrhizi and B. jicamae belong to divergent phylogenetic groups,nd Glycine since B. elkanii was divergent to B. japonicum and to theecently described soybean endosymbionts Bradyrhizobium huang-aihainense CCBAU 23303T and B. daqingense CCBAU15774T.
In summary, the analysis of the core and symbiotic genes oftrains isolated from Centrosema in American countries showedhat it is a promiscuous legume nodulated by different putativeew species and symbiovars of Bradyrhizobium. Since this legumeas an American origin it will be very interesting in the future totudy whether it is able to nodulate on other continents where itas never been cultivated.
Accession numbers for sequences are recorded in the phylogenicrees (Figs. 2–6).
cknowledgments
This research was funded by the AECID Spanish-Venezuelan
Please cite this article in press as: M.-H. Ramírez-Bahena, et al., Centrosemaand symbiovars of Bradyrhizobium in various American countries, Syst. Ap
rojects ref. A/021330/08 and A/023939/09 from the Spanish Min-stry of External Affairs and Cooperation (MAEC). MHRB was theecipient of a JAE-Doc researcher contract from the Consejo Supe-ior de Investigaciones Científicas (CSIC) cofinanced by ERDF.
e position of representative strains from different RAPD groups. Bootstrap values numbers from GenBank are given in brackets, and strains isolated in this study are
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.syapm.2013.03.007.
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