ARTICLE IN PRESS

0723-2020/$ - se

doi:10.1016/j.sy

$Note: Nucl

and FJ514043;

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E-mail addr1Present addr

Systematic and Applied Microbiology 32 (2009) 460–470

www.elsevier.de/syapm

Vigna mungo, V. radiata and V. unguiculata plants sampled in different

agronomical–ecological–climatic regions of India are nodulated by

Bradyrhizobium yuanmingense$

Chinnaswamy Appunua,b,c,1, Angele N’Zoued,e,f,g,h, Lionel Moulind,e,f,g,h,Geraldine Depretb,c, Gisele Laguerreb,c,d,e,f,g,h,�

aMicrobial Genetics Laboratory, Department of Genetics and Plant Breeding, Institute of Agricultural Sciences,

Banaras Hindu University, Varanasi 221 005, IndiabINRA, UMR 1229 Microbiologie et Geochimie des Sols, F-21065 Dijon, FrancecUniversite de Bourgogne, UMR 1229, F-21065 Dijon, FrancedIRD, UMR 113 Symbioses Tropicales et Mediterraneennes, F-34398 Montpellier, FranceeINRA, UMR 113, F-34398 Montpellier, FrancefCIRAD, UMR 113, F-34398 Montpellier, FrancegSupAgro, UMR 113, F-34398 Montpellier, FrancehUniversite de Montpellier II, UMR 113, F-34398 Montpellier, France

Received 25 February 2009

Abstract

Vigna mungo, Vigna radiata and Vigna unguiculata are important legume crops cultivated in India, but little isknown about the genetic resources in native rhizobia that nodulate these species. To identify these bacteria, a corecollection of 76 slow-growing isolates was built from root nodules of V. mungo, V. radiata and V. unguiculata plantsgrown at different sites within three agro-ecological-climatic regions of India. The genetic diversity of the bacterialcollection was assessed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified DNAfragments of the 16S–23S rDNA intergenic spacer (IGS) region, and the symbiotic genes nifH and nodC. One rDNAIGS type grouped 91% of isolates, but more diversity was found at the symbiotic loci (17 symbiotic genotypes).Overall, no host plant specificity was shown, the three host plant species sharing common bradyrhizobial genotypesthat represented 62% of the collection. Similarly, the predominant genotypes were found at most sampling sites and inall agro-ecological-climatic regions. Phylogenies inferred from IGS sequencing and multi-locus sequence analysis of thednaK, glnII and recA genes indicated that all isolates but one were clustered with the Bradyrhizobium yuanmingense

species. The nifH phylogeny also grouped the different nif haplotypes within a cluster including B. yuanmingense,except for one infrequent nif haplotype which formed a new lineage within the Bradyrhizobium genus. These resultsmay reflect a long history of co-evolution between B. yuanmingense and Vigna spp. in India, while intra-species

e front matter r 2009 Elsevier GmbH. All rights reserved.

apm.2009.05.005

eotide sequence data reported are available in the GenBank database under the following accession numbers: rDNA IGS, FJ514042

nifH, FJ514062–FJ514071; dnaK, FJ514044–FJ514049 and FJ514073; glnII, FJ514056–FJ514061 and FJ514074; recA, FJ514050–

J514072.

ing author at: USC INRA 1242-UMR 113 IRD-CIRAD-SupAgro-UM2, Laboratoire des Symbioses Tropicales et Mediterra--82/J Campus de Baillarguet, F-34398 Montpellier Cedex 5, France. Tel.: +33 4 67 59 38 62; fax: +334 67 59 38 02.

ess: gisele.laguerre@supagro.inra.fr (G. Laguerre).

ess: Division of Crop Improvement, Sugarcane Breeding Institute, Coimbatore 641 007, Tamil Nadu, India.

ARTICLE IN PRESSC. Appunu et al. / Systematic and Applied Microbiology 32 (2009) 460–470 461

polymorphism detected in the symbiotic loci may be linked with the long history of diversification of B. yuanmingense

coinciding with that of its host legumes.r 2009 Elsevier GmbH. All rights reserved.

Keywords: Bradyrhizobium; Vigna spp.; Diversity; Phylogeny; Multi-locus sequence analysis; Symbiotic genes

Introduction

The genus Vigna belongs to the family Fabaceae andincludes more than 200 species distributed throughoutthe tropics [8]. Several Vigna species, especially mungbean or green gram [Vigna radiata (L.) Wilczek)], urdbean or black gram [V. mungo (L.) Hepper] and cowpea[V. unguiculata (L.) Walp], are widely cropped as grainlegumes, fodder and vegetable crops in many tropicaland subtropical developing countries with an annualworldwide production of about 20 million hectares [8].India is considered to be the primary center of origin ofgreen gram and black gram while cowpea originated inAfrica [19]. However, cowpea was probably introducedto India more than 2000 years ago, and the subcontinentis considered to be a secondary center of cowpeadiversity. India is the major producer of green gramand black gram in the world and the largest cowpeaproducer in Asia. However, average productivity is low(500–600 kg ha�1), mainly because little effort has beeninvested in breeding and in improving the managementof these crops, which are mainly grown under rain-fedconditions without nutrient input [15,19,23]. The nitro-gen requirement of these crops is fulfilled by theircapacity to establish a N2-fixing symbiosis with rhizobia.However, variability in symbiotic effectiveness of Vigna-

nodulating rhizobia has been reported [3,7,27] raisingthe question of whether indigenous nodulating rhizobialpopulations are capable of fulfilling the optimum Nrequirement.

Many tropical legumes, including cowpea and otherVigna spp., are cross nodulated by a heterogeneousgroup of slow-growing rhizobia known as ‘cowpea-miscellany’ [1], which is now classified in the genusBradyrhizobium. Although less frequent, fast-growingrhizobia have also been isolated from root nodules ofVigna spp. and classified in the genera Rhizobium,Sinorhizobium, and Mesorhizobium [9,16,38–40]. Somehost specificity was reported based on the range ofeffectiveness of cowpea bradyrhizobia [27], but cross-nodulation of Vigna spp. by bradyrhizobia from otherVigna species and various host legumes is commonlyobserved [3,4,6,17,18,38]. Recent investigations on thegenetic diversity of indigenous rhizobia nodulatingVigna spp. growing in various worldwide geographicareas did not reveal Vigna-specific rhizobial lineages andprevalence of a particular group. Cowpea bradyrhizobiaisolated in Africa [12,24,35], in China [39,40], and inBrazil [41] were identified as Bradyrhizobium elkanii,

B. japonicum, B. liaoningense, B. yuanmingense,unnamed Bradyrhizobium genospecies, or as novelBradyrhizobium lineages. Less information is availablefor indigenous rhizobia nodulating green gram andblack gram. The recent study of Zhang et al. [40] showsthat bradyrhizobia nodulating green gram in thesubtropical region of China are as genetically diverseas cowpea bradyrhizobia. Also, unidentified slowgrowers with different 16S rRNA gene haplotypes wereisolated from black gram plants growing in the south ofIndia [21], while some strains isolated from green gramand black gram plants growing in Thailand were closelyrelated to B. japonicum [38].

Despite the importance of Vigna spp. cultivation inIndia, little is known about the genetic resources innative rhizobia. High genetic diversity of Vigna-nodu-lating rhizobia may be expected in the center of origin ofthe host legumes. On the other hand, the old history ofVigna spp. cropping in India, may have led to selectionof specific rhizobial populations. In this study the aimwas to identify the indigenous rhizobia that nodulategreen gram, black gram and cowpea plants grown indifferent agro-ecological-climatic regions of India.Genetic diversity of a core collection of nodule isolateswas assessed by restriction fragment analysis of PCR-amplified DNA fragments (PCR–restriction fragmentlength polymorphism (RFLP)) of the intergenic spacer(IGS) between the 16S and 23S rRNA genes, and thesymbiotic genes nifH and nodC. For molecular taxono-mical classification, multi-locus sequence analysis ofrDNA IGS and housekeeping genes (dnaK, glnII andrecA) was chosen. These markers have been currentlyused for molecular systematics and/or estimation ofphylogenetic relationships among bradyrhizobia ratherthan the 16S rRNA gene that shows low-level poly-morphism within the Bradyrhizobium genus [32–34].Phylogenetic analysis of the symbiotic gene nifH wasalso performed because the evolutionary history of thesymbiotic genes often differs from that of core genes,which was explained by the more recent acquisition and/or lateral transfer of the symbiotic genes [11,14,20,29].

Materials and methods

Bacterial strains

The rhizobial strains used in this study were isolatedfrom root nodules of Vigna mungo, V. radiata and

ARTICLE IN PRESSC. Appunu et al. / Systematic and Applied Microbiology 32 (2009) 460–470462

V. unguiculata grown in various fields from three agro-ecological-climatic regions of India (Table 1; thesampling sites are also located on the map of Indiaincluded as supplementary material). All sampled siteshad a history of intensive Vigna spp. cultivation of morethan 100 years, with no known history of inoculation.Local Vigna spp. varieties were grown by the farmers.The varieties were the same at adjacent sites (districts),but were different between agro-ecological-climaticregions and also between distant sites within regions.

Table 1. Origin of Vigna rhizobial isolates and agro-eco-climatic c

Vigna rhizobial isolates.

Host species No. of

fields

sampled

Previous cropa Siteb

V. mungo (black

gram)

2 Black gram or green

gram

Phul

Utta

V. mungo (black

gram)

1 Green gram Rang

Raje

Andh

V. mungo (black

gram)

1 Sorghum Nalla

Dhar

Nadu

V. mungo (black

gram)

1 Green gram Santh

Tam

V. radiata (green

gram)

3 Black gram Phul

Utta

V. radiata (green

gram)

2 Cajanus cajan (red

gram)

Nadu

Cudd

Nadu

V. unguiculata 1 Green gram Rang

Raje

Andh

V. unguiculata 2 Green gram Kom

Hyde

Prad

V. unguiculata 2 Red gram Atha

Tam

V. unguiculata 1 Red gram Thiru

Kum

Tam

V. unguiculata 1 Sorghum Sulam

Krish

Nadu

V. unguiculata 1 Green gram Sant

Tam

aCrop grown in the previous season.bName of the village, district and state.cSource: Agro-ecological zones, their soil resource and cropping systems by

Use Planning, Nagpur, India; on-line publication at http://agricoop.nic.in/

sampled regions 550–1000mm; moisture index �33.3 to �66.7; mean temper

acidic, 6.1–6.5; neutral, pH 6.6–7.5; slightly alkaline, 8.0–8.5; highly alkaline

Twenty plants from at least 200m distance apart werecollected at each site during August–September 2002,and a single nodule was excised from each plant. Allnodules were surface sterilized by following the standardprotocol of Vincent [30] and crushed nodules werestreaked on yeast–mannitol agar (YMA) plates. Nobacterial growth was observed for �30% of nodules.For the remaining nodules, only plates showing theaspect of pure cultures were conserved (�70% of thecultures).

haracteristics of sampled regions and host plants of origin for

Agro-ecological-climatic region and

dominant soil characteristicsc

lpur, Varanasi,

r Pradesh

4: Hot semi-arid, hot and dry summer, cool

winter; alluvium-derived soils, loamy to

clayey, slightly alkaline

a Reddy,

ndranagar,

ra Pradesh

7: Hot semi-arid, hot and moist summer,

mild and dry winter; red and black soils

clayey, neutral to highly alkaline

mpalli,

mapuri, Tamil

8: Hot semi-arid, hot and dry summer, mild

winter; red and loamy soils, slightly acidic

iyur, Salem,

il Nadu

8

lpur, Varanasi,

r Pradesh

4

veerapattu,

alore, Tamil

8

a Reddy,

ndranagar,

ra Pradesh

7

palli,

rabad, Andhra

esh

7

ni, Pudukottai,

il Nadu

8

valanchuli,

bakonam,

il Nadu

8

alai,

nagiri, Tamil

8

hiyur, Salem,

il Nadu

8

KS Gajbhiye & C Mandal, National Bureau of Soil Survey and Land

Farm%20Mech.%20PDF/05024-01.pdf. Mean annual rainfall of the

ature 24–29 1C; length of growing period 90–150 days. Soil pH: slightly

, 49.0.

ARTICLE IN PRESSC. Appunu et al. / Systematic and Applied Microbiology 32 (2009) 460–470 463

Plant tests

All isolates were inoculated onto plant seeds toconfirm their ability to form nodules with their host oforigin. Varieties used for nodulation tests were V. mungo

T 9, V. radiata CoGG 23 and V. unguiculata Pusa 9531.Seeds were surface sterilized and germinated in sterilizedwater agar (1% w/v). The seedlings were transferred toN-free nutrient-agar slants in glass tubes (200� 38mm)and placed in a growth chamber at 28 1C with a 14–10 hphotoperiod. One-day-old seedlings were inoculatedwith 1mL of rhizobial suspension (�108 cellsmL�1).Non-inoculated treatments were included as a control.Nodules appeared 10–12 days after inoculation. Allplants that were nodulated produced red-colorednodules indicating that they were effective at nitrogenfixation. However, �40% of isolates failed to nodulate.Five weeks after inoculation, one nodule per plant wasexcised and nodule isolates were obtained as describedabove. Single colonies were picked up and maintainedon a YMA slant at 2872 1C. All cultures were

Table 2. Genotypic characterization by PCR-RFLP of bradyrhizo

Genotypes IGS RFLP

patternsaIGS typeb nifH RFLP

patternsan

1 A A A I A A A I

2 A A A I A A A I

3 A A A I A A A I

nd A A A I A A A I

nd A A A I A A A I

4 A A A I A D B I

5 A A A I A D B I

nd A A A I A D B I

nd A A A I A D B I

nd A A A I A D B I

6 A A A I A E B V

7 A A A I C A A V

8 A A A I C B A V

9 A A A I C B A V

10 A A A I C B A V

nd A A A I C B A V

11 A A A I C F A V

12 A A A I C F B I

13 A A A I C F B I

14 A A A I C F F X

15 A A A I D F D X

16 A A A I D F D X

17 A B A II A B B I

18 A B A II C A A V

19 A G A VII A D B I

20 E F E VIII A B B I

nd, not determined due to poor PCR amplification of nodC.aThe letters identify RFLP patterns obtained with AluI, CfoI, and HaeIII f

MspI for nodC. The lettering of patterns follows the classification given in ApbThe numbering of haplotypes follows the classification given in Appunu

transferred to fresh slants monthly. All nodule isolateswere slow growers, with a mean generation time of 7.8 h.

Characterization of rhizobial isolates by PCR

fingerprinting

The isolates were characterized by PCR–RFLP of therDNA IGS, the nifH and the nodC genes, as previouslydescribed [2,13], with the various restriction enzymeslisted in Table 2.

Statistical analyses

The distribution of rhizobial genotypes was statisti-cally compared between crop species and regions oforigin by exact tests of sample differentiation from dataof haplotype frequencies using the Arlequin software[22].

bia isolated from root nodules of Vigna spp.

if typeb nodC RFLP

patternsanod typeb No. of

isolates

A C V 2

D A VII 1

D C VIII 3

nd C nd 1

nd nd nd 2

V D C VIII 17

V D E IX 1

V D nd nd 1

V nd C nd 5

V nd nd nd 2

D C VIII 3

I F E XII 1

II D A VII 1

II D C VIII 17

II E E XI 1

II D nd nd 1

III D A VII 1

X A C V 3

X A E VI 1

A C V 1

I E C X 2

I E E XI 2

I A C V 4

I F E XII 1

V nd C nd 1

I A C V 1

or rDNA IGS; with CfoI, HaeIII, and MspI for nifH; with HaeIII, and

punu et al. [2] for native Indian bradyrhizobia isolated from soyabean.

et al. [2].

ARTICLE IN PRESSC. Appunu et al. / Systematic and Applied Microbiology 32 (2009) 460–470464

Sequencing and phylogenetic analysis

PCR products of the rDNA IGS and nifH werepurified using PCR purification kits (Microcon-PCR,Millipore, France or QIAquick PCR purification kit,QIAGEN S.A., France) and sequenced using PCR-producing primers. The nucleotide sequences weredetermined on both strands using the DTCS-1 kit(Beckman Coulter) and a CEQ 8000 XL sequencer(Beckman Coulter), according to the manufacturer’sinstructions, or by MacroGen Inc. (Seoul, Korea). Thesequences were checked by mapping restriction sites.Fragments of dnaK, glnII, and recA genes wereamplified by PCR using the primer pairs described byVinuesa et al. [31] and/or Stepkowski et al. [25]. ThePCR products were purified using the QIAquick PCRpurification kit and the sequences were performed byMacroGen Inc. (Seoul, Korea).

Comparisons of nucleotide sequences against thesequences in the databases were performed usingBLASTN (http://www.ncbi.nlm.nih.gov/BLAST). Mul-tiple sequence alignment was performed with Clustal W

Table 3. Distribution of bradyrhizobial genotypes among Vigna cro

Genotype V. mungo (15 isolates) V. radiata (2

IGS nifH nodC 4 7 8 4

Ph Ra Nal Sa Ph

I I V

I I VII 1

I I VIII 1 1

I I nd 1

I IV VIII 4 3

I IV IX 1

I IV nd

I V VIII 1

I VI XII 1

I VII VII 1

I VII VIII 2 2 1 2

I VII XI 1

I VII nd

I VIII VII 1

I IX V

I IX VI

I X V

I XI X

I XI XI

II II V 4

II VI XII 1

VII IV nd

VIII II V 1

Total 3 5 5 2 15

aRegions 4, 7, and 8 (see Table 1).bPh, Phullpur; Ra, Ranga Reddy; Nal, Nallampalli; Sa, Santhiyur; Nad, N

Athani.

version 1.8 [28] and manually corrected using theGeneDoc software (version 2.6.002; http://www.psc.edu/biomed/genedoc). The phylogenetic analyses wereperformed on the Phylogeny.fr platform [5]. Thephylogenetic trees were constructed by maximum like-lihood (ML) analyses with the GTR substitution modelusing PhyML [10]. Shimodaira–Hasegawa (S–H) tests ofcongruence of ML tree topologies were performed usingPAUP version 4.0b10 [26].

Results

Genetic diversity of the isolates

A core bacterial collection of 76 isolates that wereable to nodulate their host of origin and produce red-colored healthy nodules was built. Four different IGShaplotypes were identified among the 76 isolates byPCR–RFLP analysis of the rDNA IGS region (Table 2),but one of them was largely predominant, grouping91% of the isolates (Table 3). This haplotype, named

p species, agro-ecological-climatic regionsa and sampling sitesb.

3 isolates) V. unguiculata (38 isolates) Total

8 7 8

Nad Ko Ra Su Sa Th At

1 1 2

1

1 3

2 1 4

1 3 1 2 1 2 17

1 1

3 1 1 2 7

1 3

1

1

1 1 2 4 2 17

1

1 1

1

1 2 3

1 1

1 1

1 1 2

1 1 2

4

1

1

1

8 7 5 5 3 6 12 76

aduveerapattu; Ko, Kompalli; Su, Sulamalai; Th, Thiruvalanchuli; At,

ARTICLE IN PRESS

99

74

77

71

76

92

100

0.04

B. elkanii LMG 6134T(Glycine)

Bradyrhizobium sp.type XIX (V. unguiculata)

Bradyrhizobium sp. PAC53 (Pachyrhizus)

IGS type III, nif type I B. yuanmingense SR94 (Glycine)

B. yuanmingenseLMG R16434T(Lespedeza)

IGS type I, nif type II,B. yuanmingense SR69 (Glycine)

IGS type II, nif type II,B. yuanmingenseSR88 (Glycine)

Bradyrhizobium sp. PAC40(Pachyrhizus)

B. japonicum LMG 6138 T(Glycine)

B. betaeLMG 21987 T

B. canariense BC-P22 (Chamaecytisus)

B. liaoningense LMG 18230T (Glycine)

Bradyrhizobium sp. V LMG 11944 (Faidherbia)

Bradyrhizobium sp. IVLMG10726 (Faidherbia)

Bradyrhizobium sp.EC550-1(Pachyrhizus)

Bradyrhizobium sp.ORS 3257(V. unguiculata)

Bradyrhizobium sp.type VI (V. unguiculata)

IGS type VII,nif type IV, isolate SR135 (V. unguiculata)

IGS type VIII,nif type II,isolate SR43 (V. radiata)

Bradyrhizobium sp.type VIII (V. unguiculata)

Bradyrhizobium sp.ORS 3259 (V. unguiculata)

IGS type IV,nif type III,Bradyrhizobiumsp.SR83 (Glycine) 99

74

77

92

100

Fig. 1. Phylogenetic ML tree based on 657 bp-aligned nucleo-

tide sequences of the 16S–23S rDNA IGS after gap curation.

Only bootstrap probability values Z70% (over 100 replicates)

are indicated at the branching points. The scale bar indicates

substitutions per site. The two Vigna (V) isolates obtained in

the present study are shown in bold with their IGS and nifH

haplotypes. The letter ‘‘T’’ indicates the type strain of the

species. Accession numbers of sequences extracted fromT

C. Appunu et al. / Systematic and Applied Microbiology 32 (2009) 460–470 465

IGS type I, was previously identified among bradyrhi-zobia isolated from soybean growing in different regionsof India [2], and its nucleotide sequence was identical tothat of the type strain of the B. yuanmingense speciesisolated from Lespedeza cuneata in China [37]. The IGStype II was also previously identified among Indiansoybean bradyrhizobia and was found to be closelyrelated to IGS type I (97% similarity). By contrast, thetwo other IGS types constituted new haplotypes, eachrepresented by a single Vigna isolate. They were namedIGS types VII and VIII to follow the classification givenin Appunu et al. [2].

PCR–RFLP analysis of nifH and nodC fragmentsrevealed much more polymorphism, the isolates beingdistributed in 10 nif types and eight nod types, whichyielded 17 combinations of nif-nod types (Tables 2 and 3).Three nif types were predominant, types I, IV, and VII(14%, 26%, and 34% of isolates, respectively). Similarly,71% of isolates were distributed in two nod types, V andVIII, the latter being largely predominant (63% ofisolates). Overall, no correlation was found between nif

and nod classification, and also between IGS andsymbiotic type classification. Especially, the new IGS type(type VIII) shared a symbiotic type with IGS type II. Thenif types I and II were previously identified among Indiansoybean bradyrhizobia [2], but the Vigna and soybeanbradyrhizobial collections did not share nod haplotypes. Inthe previous study, it was shown that the nif types I and IIhad nucleotide sequences closely related to those ofB. yuanmingense strains from various host legumes,including Vigna bradyrhizobia isolated in China [40].

The combined data of PCR–RFLP analysis categor-ized the isolates into 20 combined IGS-nif-nod genotypes(Tables 2 and 3). However, two genotypes, (I–IV–VIII,and I–VII–VIII) were the most frequent, each grouping27% of the isolates. Notably, they were identified innodules of the three Vigna crop species, in all regions, andat most geographical sites. The global test of differentia-tion did not show statistically significant differencesbetween the genetic structure of the populations from thethree Vigna species (P40.05), but tests between all pairsof samples indicated a differentiation between thesamples from V. radiata and V. unguiculata. However,the differences might also be attributed to variation ingeographical distribution. When testing the geographicdistribution of genotypes, significant differentiation wasfound only between agro-ecological-climatic regions 4and 8 (Po0.01). The small sample sizes did not allow thebiogeographic distribution within regions to be analyzed.

GenBank are as follows: LMG 6134 , AJ279308; PAC53,

AY628088; type XIX, AY493859; SR94, EU357930; SR88,

EU357931; SR69, EU357926; LMG R16434T, AJ534605; ORS

3257, AY039018; PAC40, AY628087; type VI, AY493849;

LMG 6138T, AJ279264; BC-P22, AY386706; LMG 21987T,

AJ631967; LMG 18230T, AJ279301; LMG 11944, AJ279287;

LMG 10726, AJ279281; type VIII, AY493852; ORS 3259,

AY039019; EC550-1, AY628085; SR83, EU357927.

Phylogenic analysis of the IGS and housekeeping

genes

The PCR product of the representatives of the newIGS types (VII and VIII) were sequenced and compared

to IGS sequences available in the databases. The IGStype VII sequence was very similar to that of IGS type I(99% identity over 738 aligned bp). The sequence of IGStype VIII showed only 96% similarity with that of IGStype I (over 657 aligned bp), which, however, was thehighest score for IGS type VIII sequence comparison.The phylogenetic analysis shown in Fig. 1 confirmedthat IGS types I, II, and VII were clustered with

ARTICLE IN PRESSC. Appunu et al. / Systematic and Applied Microbiology 32 (2009) 460–470466

B. yuanmingense. This cluster also included a bradyrhi-zobial genotype, named type XIX, nodulating V.

unguiculata in Senegal (Krasova-Wade T. and Neyra

71

93

74

100

80

72

0.02

IGS-nif type IV-III, Bradyrhizobium sp. SR83 (G. m.)

Bradyrhizobium sp. ORS 278

B. liaoningense LMG 18230T

B. japonicum USDA 6T

B. japonicum USDA 110B. betae LMG 21987T

B. canariense BTA-1T

B. elkanii USDA 76T

Bradyrhizobium sp. BTAI1

B. yuanmingense CCBAU 10071T

IGS-nif type II-II, B. yuanmingense SR88 (G. m.)

IGS-nif type I-II, B. yuanmingense SR69 (G. m.)

IGS-nif type VIII-II, isolate SR43 (V. r.)

IGS-nif type VII-IV, isolate SR135 (V. u.)

IGS-nif type I-VIII, isolate SR42 (V. r.)IGS-nif type II-II, isolate SR33 (V. r.)

IGS-nif type I-VII, isolate SR1 (V. m.)IGS-nif type III-I, B. yuanmingense SR94 (G. m.)

IGS-nif type I-VI, isolate SR35 (V. r.)

IGS-nif type I-X, isolate SR119 (V. u.)

dnaK

76

)

, ( )

)

,

, ( )

100

90

100

8798

0.02

IGS-nif type IV-III, Bradyrhizobium sp. SR83 (G. m.)

Bradyrhizobium sp. ORS 278

B. liaoningense LMG 18230T

B. japonicum USDA 6T

B. japonicum USDA 110

B. betae LMG 21987T

B. canariense BTA-1T

B. elkanii USDA 76T

Bradyrhizobium sp. BTAI1

B. yuanmingense CCBAU 10071T

IGS-nif type II-II, B. yuanmingense SR88 (G. m.)

IGS-nif type I-II, B. yuanmingense SR69 (G. m.)

IGS-nif type VIII-II, isolate SR43 (V. r.)

IGS-nif type VII-IV, isolate SR135 (V. u.)

IGS-nif type I-VIII, isolate SR42 (V. r.)

IGS-nif type II-II, isolate SR33 (V. r.)

IGS-nif type I-VII, isolate SR1 (V. m.)

IGS-nif type III-I, B. yuanmingense SR94 (G. m.)

IGS-nif type I-VI, isolate SR35 (V. r.)IGS-nif type I-X, isolate SR119 (V. u.)recA

0.02

,

,,

0

d+ g+ r

000

d+ g+ r

Fig. 2. Phylogenetic ML trees based on nucleotide sequences of dnaK

and of concatenated nucleotide sequences of dnaK, glnII, and recA.

are indicated at the branching points. The scale bar indicates substit

shown in bold with their IGS and nifH haplotypes. V. m., Vigna mun

max. The letter ‘‘T’’ indicates the type strain of the species. Accession

for dnaK, SR94, EU818929; CCBAU 10071T, AY923039; SR69, E

EU818931; USDA 6T, AM168362; BTA-1T, AY923047; USDA 110,

BTAI1, CP000495; ORS 278, CU234118. For glnII, CCBAU 100

EU818934; SR83, EU818935; LMG 18230T, AY386775; BTA-1T, A

21987T, AB353733; USDA 76T, AY599117; BTAI1, CP000495;

EU818936; CCBAU 10071T, AY591566; SR88, EU818938; LMG

USDA 110, BA000040; USDA 6T, AM168341; LMG 21987T, AB3

CP000495.

M., personal communication). The IGS type VIIIformed a novel lineage that could not be ascribed toany described bradyrhizobial species.

89

10077

95

100

0.02

IGS-nif type IV-III, Bradyrhizobium sp. SR83 (G. m.)

Bradyrhizobium sp. ORS 278

B. liaoningense LMG 18230T

B. japonicum USDA 6T

B. japonicum USDA 110

B. betae LMG 21987T

B. canariense BTA-1T

B. elkanii USDA 76T

Bradyrhizobium sp. BTAI1

B. yuanmingense CCBAU 10071T

IGS-nif type II-II, B. yuanmingense SR88 (G. m.)

IGS-nif type I-II, B. yuanmingense SR69 (G. m.)

IGS-nif type VIII-II, isolate SR43 (V. r.)

IGS-nif type VII-IV, isolate SR135 (V. u.)

IGS-nif type I-VIII, isolate SR42 (V. r.)IGS-nif type II-II, isolate SR33 (V. r.)

IGS-nif type I-VII, isolate SR1 (V. m.)

IGS-nif type III-I, B. yuanmingense SR94 (G. m.)

IGS-nif type I-VI, isolate SR35 (V. r.)

IGS-nif type I-X, isolate SR119 (V. u.)

glnII

,,

,

100

100

83100

99

100

99

85

99

100

.02

IGS-nif type IV-III, Bradyrhizobium sp. SR83 (G. m.)

Bradyrhizobium sp. ORS 278

B. liaoningense LMG 18230T

B. japonicum USDA 6T

B. japonicum USDA 110B. betae LMG 21987T

B. canariense BTA-1T

B. elkanii USDA 76T

Bradyrhizobium sp. BTAI1

B. yuanmingense CCBAU 10071T

IGS-nif type II-II, B. yuanmingense SR88 (G. m.)

IGS-nif type I-II, B. yuanmingense SR69 (G. m.)

IGS-nif type VIII-II, isolate SR43 (V. r.)

IGS-nif type VII-IV, isolate SR135 (V. u.)

IGS-nif type II-II, isolate SR33 (V. r.)IGS-nif type I-VIII, isolate SR42 (V. r.)

IGS-nif type I-VII, isolate SR1 (V. m.)

IGS-nif type III-I, B. yuanmingense SR94 (G. m.)

IGS-nif type I-VI, isolate SR35 (V. r.)

IGS-nif type I-X, isolate SR119 (V. u.)

naKlnIIecA

.02.02.02

,

IGS-nif type II-II, isolate SR33 (V. r.)

,

,

naKlnIIecA

(493 aligned bp), glnII (496 aligned bp), recA (482 aligned bp),

Only bootstrap probability values Z70% (over 100 replicates)

utions per site. Vigna isolates obtained in the present study are

go; V. r., Vigna radiata; V. u., Vigna unguiculata; G. m., Glycine

numbers of sequences extracted from GenBank are as follows:

U818928; SR88, EU818930; LMG 18230T, AY923041; SR83,

BA000040; LMG 21987T, AY923046; USDA 76T, AM168363;

71T AY386780; SR94, EU818933; SR69, EU818932; SR88,

Y386765; USDA 6T, AF169582; USDA 110, BA000040; LMG

ORS 278, CU234118. For recA, SR94, EU818937; SR69,

18230T, AY591564; SR83, EU818939; BTA-1T, AY591553;

53734; USDA 76T, AM168342; ORS 278, CU234118; BTAI1,

ARTICLE IN PRESSC. Appunu et al. / Systematic and Applied Microbiology 32 (2009) 460–470 467

In order to build a more robust phylogeny for theVigna bradyrhizobia, the partial sequences of thehousekeeping genes dnaK, glnII, and recA were deter-mined for four representatives of IGS type I associatedwith various nif types and isolated from different Vigna

species, and one representative of each other IGS types.The phylogenies for each locus were analyzed and a treederived from the three pooled concatenated sequencealignments was also constructed (Fig. 2). The lattershowed a topology similar to that of the recA tree, but asignificant difference in topology with those derivedfrom individual dnaK and glnII markers based on S–Htests (Po0.05). Similarly, pairwise S–H tests revealedincongruence between the glnII tree and the two others,which could be especially explained by the phylogeneticposition of IGS type VIII, and also of B. betae, differingaccording to the marker. The IGS type VIII showed aclose relationship with B. liaoningense based on analysisof dnaK (98.5% similarity) and recA (97.5% similarity).

100

96

86

100

100

89

84

100

92

100

100

B. liao

nif type I, IGS type

B. yuanmingense C

B. jap

B. yuanmingense CC

B. yuanmingense

B. yuanmingense

B. elkanii S B. elkanii IFO

M

nif type II, IGS type

nif type IV, IGS type

nif type X, IGS type

nif type IX, IGS type

nif type VIII, IGS ty

nif type I, IGS typ

nif type V, IGS type

nif type VI, IG

nif type VII, IGS ty

nif type XI, IGnif type XI, IG

B. yuanming

Bradyrhizobium

0.050.05

Fig. 3. Phylogenetic ML tree based on 617 bp alignment of nucleotid

Z70% (over 100 replicates) are indicated at the branching points. T

obtained in the present study are shown in bold with their nifH and

species. Accession numbers of sequences extracted from GenBank

CCBAU 23230, AY934871; CCBAU 83698, EU146010; CCBAU 35

EU113228; SR69, EU357925; CCBAU 51377, EU113236; USDA 110

14791T, AB094963; BTA-1T, EU818926.

However, the glnII sequence of IGS type VIII did notshow more than 94–95% similarity with B. liaoningense

and the other sequences included in the analysis, as wellas those available in GenBank, including recentlyreleased sequences from a collection of cowpea bradyr-hizobia from southern Africa [24]. Therefore, the glnII

phylogeny, in agreement with the IGS phylogeny,identified a novel lineage within the genus Bradyrhizo-

bium. By contrast, congruence was found between thethree housekeeping gene trees in grouping IGS types I,II, and VII with B. yuanmingense, which againcorroborated the IGS data analyses.

Phylogenic analysis of nifH

The 782 bp PCR products of one representative ofeach of the various nifH haplotypes identified in thepresent study (two representatives for nif type XI) were

ningense LMG 18230T(Glycine)

I, B. yuanmingense SR66 (Glycine)

CBAU 61071 (V. radiata)

onicum USDA 110 (Glycine)

BAU 10071T(Lespedeza)

CCBAU 23230 (Desmodium)

CCBAU 83698 (Glycine)

127 (Glycine) 14791T(Glycine)

B. canariense BTA-1T(Chamaecytisus)

.loti MAFF303099 (Lotus)

I, B. yuanmingense SR69 (Glycine)

VII, isolate SR135 (V. unguiculata)

I, isolate SR119 (V. unguiculata)

I, isolate SR49 (V. unguiculata)

pe I, isolate SR42 (V. radiata)

e I, isolate SR121 (V. unguiculata)

I, isolate SR30 (V. radiata)

S type I, isolate SR35 (V. radiata)

pe I, isolate SR53 (V. unguiculata)

S type I, isolate SR50 (V. unguiculata)S type I, isolate SR125 (V. unguiculata)

ense CCBAU 35272 (V. unguiculata)

sp. CCBAU 51377 (V. unguiculata))

(

( )

)

e sequences of the nifH gene. Only bootstrap probability values

he scale bar indicates substitutions per site. Vigna (V) isolates

IGS haplotypes. The letter ‘‘T’’ indicates the type strain of the

are as follows: MAFF303099, BA000012; SR66, EU357924;

272, EU113232; CCBAU 10071T, EU818927; CCBAU 61071,

, BA000040; LMG 18230T, EU818985; S 127, DQ485701; IFO

ARTICLE IN PRESSC. Appunu et al. / Systematic and Applied Microbiology 32 (2009) 460–470468

sequenced to estimate their phylogenetic relationships.All haplotypes except nif type XI were clustered withB. yuanmingense strains (Fig. 3) and showed 100–95%similarities of sequences. The nif type XI showed only94% sequence similarity with its closest relative, nif typeVII, and did not share more than 93% sequencesimilarity with the other sequences included in thephylogenetic analysis and those found in GenBank. Thisgenotype formed a new lineage within the Bradyrhizo-

bium genus, although it was associated with an IGS typeidentical to that of the type strain of B. yuanmingense,and shared a nod haplotype with nif type VII.

Discussion

All the Indian Vigna isolates characterized in thisstudy were slow growers and were assigned to theBradyrhizobium genus based on their molecular char-acterization. In contrast to previous studies, which havegenerally reported genetic diversity at the species levelfor Vigna bradyrhizobia from different continents[12,21,24,36,39,40], it was found that 99% of the isolates(all isolates but one) from the core collection analyzed inthe present study were phylogenetically related to asingle species, B. yuanmingense, based on IGS andhousekeeping gene sequencing. This species was origin-ally created based on the description of nodule isolatesfrom Lespedeza spp. [37], but since then, nodulation ofvarious legumes, including V. radiata and V. unguicula-

ta, by indigenous rhizobia related to B. yuanmingense

has been reported [24,35,40]. However, B. yuanmin-

gense-related rhizobia isolated in the south of Africa andin the subtropical region of China represented onlyapproximately 15–29% of the Vigna isolates, and severalbradyrhizobial species coexisted at a same site. In thepresent study only three IGS haplotypes were identifiedwithin the B. yuanmingense lineage, one being highlypredominant (91% of isolates), while intra-speciesdiversity within B. yuanmingense was high in China,with seven IGS haplotypes delineated among 18 Vigna

isolates. Although our sample of isolates was smallerthan initially expected with regard to the numbers ofsites and plant species sampled, our sampling size wasfully comparable to those of Steenkamp et al. [24], andZhang et al. [40]. Therefore, increasing the samplingeffort might lead to identification of other less frequentrhizobial species, although our results clearly show thatB. yuanmingense is the predominant species at the sitesexplored in India.

Contrary to the IGS marker, high genetic polymorph-ism was found in symbiotic genes nifH and nodC

(17 symbiotic genotypes) and, therefore, within theB. yuanmingense species (also 17 symbiotic genotypesamong the IGS types related to B. yuanmingense).Therefore, the genetic diversity (categorization of the 76

isolates into 20 IGS-nif-nod genotypes) reflects mainlythe genetic polymorphism of the symbiotic genome.However, nine out the 10 nifH haplotypes identified byRFLP (95% of isolates) formed a monophyletic cluster,including B. yuanmingense strains isolated from varioushost legumes, which is consistent with phylogeniesinferred from IGS and housekeeping genes. This resultsuggests vertical acquisition of the symbiotic geneswithin the B. yuanmingense clade, although we alsofound evidence of lateral transfer of nifH from anunknown origin (no close relative was identified) toB. yuanmingense-related strains (IGS type I associatedwith nif type XI). The high nucleotide similarity amongthe symbiotic genes of the Vigna isolates is alsoconsistent with previous reports showing their abilityto cross-nodulate different Vigna species [4,38] and, inthe present study, with the lack of genetic differentiationof the populations in relation to the host species oforigin. Similarly, Zhang et al. [40] reported that mungbean and cowpea bradyrhizobia isolates from Chinashared similar symbiotic genotypes. We also found thatsome Indian Vigna bradyrhizobia showed the sameIGS–nifH haplotype combinations as Indian soybeanbradyrhizobia, IGS/nifH types I-I and II-II [2]. How-ever, the two bradyrhizobial collections did not sharenod haplotypes, which suggests some host specificity fornodulation, although cross-nodulation between Vigna

spp. and soybean has been reported [4,18,38]. Furtherinvestigation testing cross-nodulation and measuringnitrogen fixation effectiveness for the three Vigna speciesand soybean would provide useful information toattempt to link the genotypic data and the symbioticfeatures of these rhizobia.

The low genetic diversity at the species level amongthe Vigna isolates was not expected based on theprevious studies suggesting that Vigna spp. are ratherpromiscuous hosts and given that India is a center ofdiversification of the Vigna species under study. India isalso considered as a second center of diversification ofsoybean and, in contrast to the present study, werecently reported genetic diversity at the species levelamong soybean isolates by using similar sampling andanalytical approaches [2]. Furthermore, we found thatmost Vigna rhizobial genotypes were conserved acrosssites and agro-ecological-climatic regions of India. Thesampled sites had a history of intensive cultivation of theVigna crop species (more than 100 years). All togetherthese data suggest that the host legume is the mainselective force that drives genetic diversity of the micro-symbionts rather than adaptation to local environmen-tal conditions. At the evolutionary scale, our resultsmay reflect a long history of co-evolution betweenB. yuanmingense and Vigna spp. Long cultivationhistory and more recent intensive cultivation of Vigna

spp. crops may have led to selection of the most adaptedcombination of core genomes and symbiotic genotypes

ARTICLE IN PRESSC. Appunu et al. / Systematic and Applied Microbiology 32 (2009) 460–470 469

of rhizobia. The genetic diversity of nifH and nodC

observed among our isolates may also be linked to theirlong history of diversification coinciding with that oftheir host legumes.

Acknowledgements

Chinnaswamy Appunu was supported by a researchfellowship from the Indian Council of Scientific andIndustrial Research-University Grant Commission, anda short-term training fellowship from the EuropeanMolecular Biology Organization. Angele N’Zoue wassupported by a research fellowship from the FrenchInstitut de Recherche pour le Developpement. The workwas supported by Grant no. 45 from the Frenchgovernmental organization ‘‘Bureau des RessourcesGenetiques’’.

Appendix. Supplementary materials

The online version of this article contains additionalsupplementary data. Please visit doi:10.1016/j.syapm.2009.05.005.

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