Laboratoire de Microbiologie des Sols, Centre de Microbiologie du Sol et de lEnvironnement, INRA, 17 rue Sully, BP 86510,F-21065 Dijon Cedex, France1
Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, E-18008 Granada, Spain2
Author for correspondence: Gisèle Laguerre. Tel: +33 3 80693093. Fax: +33 3 80693224. e-mail: laguerre{at}dijon.inra.fr
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ABSTRACT |
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Keywords: Rhizobium, phylogeny, nodulation gene, nitrogen fixation gene, common bean
Abbreviations: Sym genes, symbiotic genes
The GenBank accession numbers for the sequences reported in this paper are AF217261 through AF217272 for nodC and AF218126, AF275670 and AF275671 for nifH.
a Present address: Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ont., Canada N5V 4T3.
b Present address: Centre de Recherches et de Développement sur les Sols et les Grandes Cultures, Agriculture et Agroalimentaire Canada, 2560 Boul. Hochelaga, Sainte-Foy, Québec, Canada GIV 2J3.
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INTRODUCTION |
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The convergence of different rhizobia that harbour distinct nod genes to the same nodulation phenotype has been reported by comparison of phylogenies inferred from housekeeping and Sym (symbiotic) gene loci (Haukka et al., 1998 ; Wernegreen & Riley, 1999
). On the other hand, lateral gene transfer of the Sym genes appears to be the most plausible hypothesis to explain cases of phylogenetic incongruence between Sym and housekeeping genes (Martinez-Romero & Caballero-Mellado, 1996
; Young & Haukka, 1996
). Indeed, phylogenetic trees based on sequences of nod genes are generally not congruent with those based on 16S rDNA sequences, but the nod trees show some correlation with host plant range (Dobert et al., 1994
; Lindström et al., 1995
; Ueda et al., 1995
; Haukka et al., 1998
; Wernegreen & Riley, 1999
). By contrast, the phylogeny of nifH genes, which encode the dinitrogenase reductase enzyme, has been reported as closely following that of 16S rRNA genes (Hennecke et al., 1985
; Young, 1992
; Dobert et al., 1994
), despite some exceptions (Eardly et al., 1992
). However, these nifH phylogenies were based on analysis of a small number of sequences. Recently, Haukka et al. (1998)
analysed many more sequences and concluded that, for rhizobia, the phylogeny of nifH was generally not consistent with the phylogeny of 16S rRNA, but was broadly similar to that of nodA genes. This result agrees with the fact that the nod and nif genes are often tightly linked in rhizobia, and can be located on transmissible elements such as plasmids in many rhizobial species or transposon-like elements in Mesorhizobium loti (Sullivan et al., 1995
; Sullivan & Ronson, 1998
).
Although it is widely agreed that phylogenies based on stable chromosomal genes are necessary to establish a biologically meaningful rhizobial taxonomy, a proper definition of broad host range should consider the diversity of the Sym genes rather than the diversity of the species that carry them. It would thus make sense to include the characterization and the phylogenetic classification of Sym genes in the minimal standards for the description of new rhizobia as previously proposed for the 16S rRNA gene sequences (Graham et al., 1991 ). Such a classification should provide a complementary basic framework for our understanding of the Rhizobiumlegume symbiosis.
The P. vulgaris microsymbionts form a taxonomically heterogeneous group. So far, five recognized species plus two distinct 16S rDNA lineages have been described from rhizobial isolates recovered from bean nodules (Martinez-Romero et al., 1991 ; Segovia et al., 1993
; van Berkum et al., 1996
; Amarger et al., 1997
; Herrera-Cervera et al., 1999
). These species are distributed in two genera, Rhizobium and Sinorhizobium, and the potentially new genus represented by R. giardinii. Moreover, some of these species are subdivided in biovars based on the extent of host range and genetic characteristics of Sym genes (Amarger et al., 1997
; Wang et al., 1999a
). Our aim was to determine the congruence between classifications of rhizobia based on Sym genes and 16S rRNA and to estimate evolutionary relationships among rhizobia that have similar host legumes but are chromosomally diverse. Our main focus was the bean symbionts, but we have also analysed a larger collection of rhizobia. We initially developed a simple and rapid method to characterize Sym genes in rhizobia based on RFLP of PCR-amplified DNA, as previously achieved for 16S rRNA genes (Laguerre et al., 1994
, 1997
), and Sym gene loci in Rhizobium leguminosarum (Laguerre et al., 1996
). Representative Sym genotypes were selected for subsequent phylogenetic analyses based on DNA sequencing. As a nodulation gene marker, we chose the nodC gene, which is a common nod gene essential for nodulation in all rhizobial species investigated so far. This gene encodes an N-acetylglucosaminyltransferase which is involved in the first step of Nod factor assembly, and it is also a determinant of host range (reviewed by Perret et al., 2000
). In addition, the nodC sequence is relatively long, which enabled the PCR amplification of large DNA fragments to a priori ensure maximum specificity of RFLP fingerprints and maximum robustness of phylogeny inferred from nucleotide sequences. As a nitrogen fixation marker, we chose the nifH gene, for which the largest number of rhizobial sequences is available for comparison (Haukka et al., 1998
).
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METHODS |
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The cell growth conditions and the target DNA preparation were as previously described (Laguerre et al., 1997 ). The nodC DNA was amplified from 510 µl lysed cell suspension mixed with all PCR reagents: polymerase reaction buffer (Gibco-BRL); 2·5 mM MgCl2; 200 µM (each) dATP, dCTP, dTTP, dGTP; 0·40·8 µM (each) nodC primers; 0·04 U Taq DNA polymerase (Gibco-BRL) µl-1. DNA amplification was done by using a standard temperature profile including an annealing temperature of 55 °C (Laguerre et al., 1994
). The procedure for amplification of the nifH fragments was similar, except that reactions were made with 1·5 mM MgCl2, 20 µM (each) dNTP and 0·1 µM (each) nifH primers and the annealing temperature was increased to 57 °C. For a few strains, multiple nodC bands were obtained. In these cases, a small piece of agarose containing the band of the expected size was aspirated by using a hypodermic needle and used as a template in a new PCR.
Restriction pattern analysis of the PCR products with the restriction endonucleases listed in Table 1 was as previously described (Laguerre et al., 1997
).
Sequencing of nodC and nifH DNA.
All the nucleotide sequences, apart from two nifH sequences (see below), obtained in this study were determined by Genome Express (Grenoble, France). In a first attempt, crude nodC PCR products were directly sequenced on both strands by using primers nodCF and nodCI. The less degenerate primer nodCFn, which matched the same oligonucleotide sequence as nodCF, was used for PCR amplification and sequencing of crude PCR products from strains PhD12 and H251. Sequencing of nodC DNA with nodCFn and nodCI was achieved for strains H132 and Viking I only after purification of PCR products by either 70% ethanol precipitation in the presence of 0·7 M ammonium acetate or (when multiple PCR products were obtained) extraction of the nodC fragment from agarose gels by using a QIAEX II gel extraction kit (Qiagen) followed by ethanol/ammonium acetate precipitation. The sequences of the nifH fragments and the nodC fragment of strains R602sp, H152, ACCC 19665, USDA 2071 and OR191 were obtained after cloning the PCR products by using either a pT7Blue T-Vector kit (Novagen), a pGEM-T Vector kit (Promega) or a TOPO TA cloning kit (Invitrogen) according to the manufacturers instructions. The result of cloning was checked by PCR amplification by using the vector plasmid primers T7 and SP6 according to the procedure described by Novagen. The crude PCR products were directly used for sequencing with both primers T7 and SP6. Sequencing of nifH fragments of strains GR-06 and GR-X8 was done in an ABI 373 XL Stretch Sequencer (Perkin-Elmer Biosystems) using an ABI Prism BigDye Terminator kit and vector-based sequencing primers (M13 universal and reverse primers).
From all these experiments, 450750 nucleotides of each DNA strand were determined and an 808935 bp sequence of nodC fragments and a 736783 bp sequence of the nifH gene were reconstituted for each strain. Restriction site analyses of the sequences were performed by using the Bisance software (Dessen et al., 1990 ).
Phylogenetic analysis.
The sequences have been deposited in the GenBank database under accession numbers AF217261 through AF217272 for the nodC sequences and AF218126, AF275670 and AF275671 for the nifH sequences of strains R602sp, GR-X8 and GR-06, respectively.
The accession numbers of the published sequences used for comparisons were as follows. The numbers for the nodC sequences were: M13658 (R. leguminosarum bv. viciae 238), X98514 (R. tropici IIA CFN 299), X87578 (R. galegae HAMBI 1174), M11268 (S. meliloti 1021), M73699 (S. fredii USDA 257), X73362 (Sinorhizobium sp. NGR234), X52958 (M. loti NZP 2037), U53327 [Mesorhizobium sp. (Oxytropis) N33], AF105431 (Bradyrhizobium sp. SNU001), L18897 (Azorhizobium caulinodans ORS 571T). The numbers for the nifH sequences were: K00490 (R. leguminosarum bv. trifolii SU329), M55225 (R. tropici IIB CIAT 899T), M15942 (R. etli bv. phaseoli CFN 42T), M55227 (R. etli bv. phaseoli Olivia-4), AF107621 (R. etli bv. mimosae Mim2), M55226 (Rhizobium gallicum bv. gallicum FL27), M55228 [Rhizobium sp. (Medicago) OR191], Z95230 [Rhizobium sp. (Lonchocarpus) BR6001], J01781 (S. meliloti 41), M55232 (S. meliloti USDA 1002T), M55232 (S. meliloti CC2013), M55231 (Sinorhizobium medicae CC169), Z95229 (S. fredii USDA 191), Z95218 (Sinorhizobium terangae bv. acaciae ORS 1009T), SSZ95221 (Sinorhizobium saheli bv. sesbaniae ORS 609T), AE000105 (Sinorhizobium sp. NGR234), Z95224 [Sinorhizobium sp. (Acacia) HAMBI 1499], Z95212 [Sinorhizobium sp. (Leucaena) BR827], Z95213 [Sinorhizobium sp. (Prosopis) M6], Z95228 [Mesorhizobium sp. (Leucaena) INPA78B], K01620 (B. japonicum USDA 110), M16709 (Azorhizobium caulinodans ORS 571T), X51500 (Azospirillum brasilense Sp7), X03916 (Azotobacter chroococcum MCD1), X07866 (Rhodobacter capsulatus SB1003), J01740 (Klebsiella pneumoniae). The numbers for the 16S rDNA sequences were: U89831 (R. leguminosarum bv. viciae USDA 2508), X67227 (R. leguminosarum bv. trifolii ATCC 14480), U29388 (R. leguminosarum bv. phaseoli RCR3644), X67233 (R. tropici IIA CFN 299), X67234 (R. tropici IIB CIAT 899T), U38469 (R. tropici IIB CIAT 166), U28916 (R. etli CFN 42T), U28939 (R. etli TAL182), U47303 (R. etli SEMIA0430), U86343 (R. gallicum R602spT), AF008129 (R. gallicum FL27), U89817 (Rhizobium mongolense USDA 1844T), U89816 (R. mongolense USDA 1832), U89818 (R. mongolense USDA 1834), U89819 (R. mongolense USDA 1836), U89821 (R. mongolense USDA 1890), U89823 [Rhizobium sp. (Medicago) USDA 1920], X91211 [Rhizobium sp. (Medicago) OR191], U29387 [Rhizobium sp. (Phaseolus) RCR3618D], U71078 (Rhizobium hainanense I66T), D12793 (R. galegae HAMBI 540T), AF025852 (R. huautlense S02T), U86344 (R. giardinii H152T), D12783 (S. meliloti USDA 1002T), D12783 (S. medicae A321T), X67231 (S. fredii USDA 205T), X68388 (S. terangae ORS 1009T), X68390 (S. saheli ORS 609T), X67229 (M. loti NZP 2213T), U50164 (M. loti R8CS), U50165 (M. loti R88b), U50166 (M. loti ICMP 3153), D12797 (Mesorhizobium huakuii CCBAU 2609T), U07934 (Mesorhizobium ciceri UPM-Ca7T), L38825 (Mesorhizobium mediterraneum UPM-Ca36T), U71079 (Mesorhizobium tianshanense A-1BST), Y14158 (Mesorhizobium plurifarium LMG 11892T), AF041442 (Mesorhizobium amorphae ACCC 19665T), U69638 (B. japonicum USDA 6T), Z35330 (B. japonicum USDA 110), U35000 (B. elkanii USDA 76T), X87273 [Bradyrhizobium sp. (Lupinus) DSM 30140], X70405 [Bradyrhizobium sp. (Acacia) LMG 10689], X70403 [Bradyrhizobium sp. (Acacia) LMG 9966], X70404 [Bradyrhizobium sp. (Enterolobium) LMG 9980], X70401 [Bradyrhizobium sp. (Lonchocarpus) LMG 9514], Y17047 (Allorhizobium undicola LMG 11875T), X67221 (Azorhizobium caulinodans ORS 571T), X67223 (Agrobacterium tumefaciens LMG 196), X67228 (Agrobacterium rubi LMG 156T), X67225 (Agrobacterium vitis LMG 8750T), X67224 (Agrobacterium rhizogenes LMG 152).
Molecular sequence analyses were performed by using programs available in the Bisance software. Nucleotide and amino acid sequences were aligned with CLUSTAL W (Thompson et al., 1994 ). Phylogenetic trees of nodC, nifH and 16S rRNA genes were inferred by using the Phylogenetic Inference Package (PHYLIP; Felsenstein, 1989
) with neighbour-joining analyses from Kimuras (Kimura, 1980
) two-parameter nucleotide distances, and the maximum-likelihood method. Phylogenetic trees of NodC and NifH proteins were constructed using the neighbour-joining method from Dayhoff PAM distance matrix computed with the PROTDIST program of PHYLIP. Confidence in neighbour-joining trees was assessed by bootstrap analysis with the SEQBOOT and CONSENSE programs of PHYLIP.
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RESULTS AND DISCUSSION |
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Correlation between RFLP of Sym genes and host specificity
The results of the RFLP analysis of the PCR-amplified nodC and nifH fragments are given in Table 1. From the combined data with each gene, we identified 45 composite nod types and 41 nif types among the 82 and 76 strains investigated, respectively. The combination of the nod and nif gene analyses revealed 50 symbiotic (nodnif) genotypes. We concluded that both genes were highly polymorphic among species and biovars.
Intraspecies polymorphism was also detected among strains isolated from the same host legume (in R. tropici, S. meliloti, M. loti and M. huakuii) and, irrespective of the species, within previously defined biovars (viciae, trifolii, phaseoli, gallicum, sesbaniae), but differences between pairs of restriction patterns could be simply explained in terms of gain or loss of only one or two restriction sites. These results indicate that the genes are closely related.
Unclassified strains could be assigned to previously defined biovars. Thus Rhizobium sp. (Phaseolus) RCR3618D had Sym genes characteristic of bv. phaseoli. Rhizobium sp. (Leucaena) USDA 3497 could be classified into bv. gallicum, which is consistent with the ability of R. gallicum bv. gallicum to nodulate Leucaena leucocephala (Amarger et al., 1997 ).
More generally and independent of their taxonomic status, the strains sharing at least two similar nodC and/or nifH restriction patterns originated from host plants belonging to the same species or genus, or to the same known cross-inoculation group. The Rhizobium sp. (Phaseolus) strains HT2a2 and HT4c1 had nodC genes typical of R. tropici. The soybean bradyrhizobia B. japonicum and Bradyrhizobium liaoningense had identical nod and nif types. Most restriction patterns were similar in the chickpea mesorhizobia M. ciceri and M. mediterraneum. The strains of R. mongolense and Rhizobium sp. OR191 which originated from Medicago species shared four nodC restriction patterns and the ability to form nitrogen-fixing nodules with common beans (Eardly et al., 1992 ; van Berkum et al., 1998
). However, only the latter was reported to nodulate Leucaena leucocephala (Del Papa et al., 1999
). The Medicago sinorhizobia S. meliloti and S. medicae also showed two identical nodC restriction patterns, which indicates that they have closely related nodC genes. Similar nifH restriction patterns were obtained among the R. huautlense strains isolated from Sesbania herbacea and the bv. sesbaniae of S. terangae and S. saheli, showing that at least their nifH genes are closely related. However, the nodC genes of the R. huautlense strains were easily amplified by contrast to those of the Sesbania sinorhizobia. This result indicated some nucleotide differences between their nodC genes but further investigations are needed to estimate to what extent their nodulation genes differ. The five unclassified bradyrhizobia isolated from Lupinus or Ornithopus species, two legume genera that form a single cross-inoculation group (Graham, 1976
), also had closely related nifH genes.
Conversely, strains with no known common host plants shared few restriction patterns (and no more than one in pairwise comparisons). However, there were also strains among Phaseolus, Medicago, Acacia or soybean rhizobia that did not share more than one restriction pattern. The RFLP method was not suitable for obtaining more information about phylogenetic relationships between the genes. Few restriction sites in nodC and nifH gene sequences were actually conserved among species or biovars, according to the available nucleotide sequences. Therefore, it was not possible to map the restriction sites for rigorous phylogenetic analyses, and so further nucleotide sequencing was required.
Relationship of phylogeny of Sym genes to host specificity
Complete or partial sequences of the PCR-amplified nodC fragments were determined for representatives of the different RFLP nodnif types among strains that originated from Phaseolus vulgaris nodules, and for R. leguminosarum bv. trifolii M37, Rhizobium sp. OR191 from Medicago sativa, and the type strain ACCC 19665 of the recently described species M. amorphae (Wang et al., 1999b ). We also determined the sequence of the nifH fragment of R. gallicum bv. gallicum R602sp and of the Sinorhizobium sp. (Phaseolus) strains GR-06 and GR-X8. Restriction site mapping and comparisons with the experimental RFLP data confirmed the quality of the sequences. Phylogenetic analysis of nodC and nifH sequences was performed by using the neighbour-joining and the maximum-likelihood methods, which led to similar results, except for some uncertain nodes which were not supported by high bootstrap values (>85% over 500 replicates) in the nifH neighbour-joining tree.
The nodC trees were also similar to that derived from protein translation of the DNA sequences and only the neighbour-joining tree is shown in Fig. 2. The nodC phylogeny was well correlated with the host plant range. All the nodC genes but one of the Phaseolus rhizobia formed a robust cluster within which the similarity values ranged from 81·9 to 99·5%. This result suggests that these nod genes evolved from a common ancestor. R. tropici fell outside the bean symbiont cluster and the present data did not show evidence that R. tropici was more strongly associated with the bean symbionts than with other rhizobia. Each of the bvs phaseoli, gallicum and giardinii corresponded to distinct lineages or subclusters, which correlates with the differences observed in host plant range between these biovars (Amarger et al., 1997
; Sessitsch et al., 1997
). However, a close relationship was found between the nodC genes of R. gallicum bv. gallicum and R. giardinii bv. giardinii, a result that could not have been anticipated from the RFLP analysis. The similarity values were higher than 90%. Strain GR-06 formed its own lineage within the bean symbiont cluster, and consequently this strain could not be assigned to any of the previously defined biovars.
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The nifH neighbour-joining tree is shown in Fig. 3. A tree derived from the protein translation was also constructed (not shown), but the phylogenetic analysis was not reliable since the bootstrap values were generally low (<50%). Analysis of longer nucleotide sequences of nifH (738 bp aligned) improved the general robustness of nifH phylogenetic trees (not shown), but the number of rhizobial sequences available for comparison was too small for phylogenetic use. Our neighbour-joining tree was similar to those reported previously (Haukka et al., 1998
; Wang et al., 1999a
). As in the nodC tree, R. gallicum bv. gallicum formed a tight cluster with R. etli bv. phaseoli, which also included R. etli bv. mimosae Mim2. This strain was isolated from Mimosa affinis but is able to nodulate P. vulgaris and Leucaena leucocephala like strains of R. gallicum bv. gallicum (Wang et al., 1999a
). On the basis of the lack of polymorphism revealed by the RFLP analysis (Table 1
), it seems probable that the nifH genes are very similar among all the strains classified in bv. phaseoli, whatever the species. By contrast, but consistent with the RFLP data in R. gallicum bv. gallicum, the nifH nucleotide sequence of R602sp, the type strain of the species isolated in France, differed from that of the Mexican strain FL27 at 2% of nucleotide sites. This intra-biovar difference was relatively high compared to the differences observed between R602sp and R. etli bvs mimosae and phaseoli strains, which were only of 3·3 and 3·5%, respectively. The grouping of the two R. gallicum bv. gallicum strains into a subcluster was supported by an 83% bootstrap value, but not corroborated by the maximum-likelihood tree, in which the two R. gallicum strains and R. etli bv. mimosae Mim2 each formed a distinct lineage within the Phaseolus cluster. So far, almost all isolates of R. gallicum have been obtained from Europe (Amarger et al., 1997
; Sessitsch et al., 1997
; Herrera-Cervera et al., 1999
). Because of the Mesoamerican origin of bean and of R. gallicum strain FL27, Sessitsch et al. (1997)
suggested that R. gallicum might have been imported to Europe as a seed contaminant. However, taking into account the relative divergence among the Sym genes of the French and Mexican R. gallicum bv. gallicum strains, this hypothesis appears unlikely.
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The second major difference between the nodC and nifH trees concerned the Medicago strain OR191, which was not associated with the tight cluster formed by S. meliloti and S. medicae in the nifH trees. The nifH sequence of OR191 formed its own lineage. Our updated analysis confirmed the earlier results of Eardly et al. (1992) indicating that this sequence was substantially different from those of the other rhizobia (similarity values lower than 89%). Again, the data suggest that the nodC and the nifH genes did not co-evolve together in this strain. The nodC gene of OR191 could be the ancestor from which Medicago strain genes diverged. Alternatively, OR191, and also GR-06-like strains, may have acquired only the nod genes by horizontal transfer from host-specific ancestral nod genes. Since nod and nif genes are often closely linked, another hypothesis would be that additional events of gene exchange and internal genetic rearrangements might have followed the co-transfer of the nod and nif genes. High-frequency rearrangements in plasmids of rhizobial strains involving recombination among reiterated sequences have been reported (see Garcia-de los Santos et al., 1996
; Romero et al., 1998
).
Previous reports also indicated that some rhizobial strains, such as S. saheli, had different phylogenetic locations within rhizobia by comparing common nod gene and nifH evolutionary trees (Dobert et al., 1994 ; Haukka et al., 1998
). Clearly, more work is needed to assess the linkage of nod and nif genes and the rest of the symbiotic genome.
Incongruence between symbiotic types and 16S-rDNA-based classification
Our results extend previously reported evidence showing that distinct rhizobial species can share similar Sym genes, and, conversely, that distinct Sym genotypes and phenotypes can be harboured by similar genomic backgrounds as defined by 16S rDNA types. Also, we observed additional cases of incongruence between the classifications and phylogenies resulting from comparative analyses of Sym genes and 16S rRNA (Figs 13).
The 16S rDNA type of Rhizobium sp. bv. gallicum USDA 3497 matched that of the R. mongolense type strain (Table 1), though it should be noted that this type is closely related to that of R. gallicum (Laguerre et al., 1997
; van Berkum et al., 1998
; Fig. 1
). On the other hand, the nodC gene of R. mongolense was found to be close to that of the Medicago rhizobium OR191 (Table 1
) and then probably distant from those of bv. gallicum on the basis of sequence analysis of the OR191 nodC gene. Although R. mongolense strains formed nitrogen-fixing nodules on P. vulgaris (van Berkum et al., 1998
), they were not able to nodulate Leucaena leucocephala in contrast with strains in bv. gallicum.
Strains HT2a2 and HT4c1 harboured nodC genes typical of R. tropici, but their 16S rDNA type was identical to that of strain OR191 (Table 1). This 16S rDNA type differed in nine restriction sites from that of the R. tropici type strain (Laguerre et al., 1997
), and the sequence data indicated that strain OR191 does not show significant phylogenetic affinity for R. tropici or any other rhizobial lineage (Fig. 1
). Strains OR191, HT2a2 and HT4c1 may form a new Rhizobium species that would harbour different Sym genes.
The classification resulting from the Sym gene analysis fully reflected the host specificity for the sample of mesorhizobia investigated, while the 16S-rDNA-based phylogeny was irrespective of the host plant (Sullivan et al., 1996 ; Laguerre et al., 1997
; de Lajudie et al., 1998b
; Fig. 1
). In particular, the sample of four strains of M. loti examined represented three distinct 16S rDNA types intermixed with other mesorhizobia from various host legumes (Laguerre et al., 1994
, 1997
), but they had clearly closely related nodC and nifH genes.
Similarly, the samples of soybean and lupin bradyrhizobial strains investigated in this study constituted heterogeneous 16S rDNA groups that were phylogenetically intermixed with each other and with bradyrhizobia isolated from other host legumes (Laguerre et al., 1994 , 1997
). The nodC gene was polymorphic among the lupin bradyrhizobia, but the nifH gene sequences appear to be conserved within this group (Table 1
). The soybean species B. japonicum and B. liaoningense that were delineated by 16S rDNA sequence comparison (Xu et al., 1995
) harboured similar Sym genes. The B. elkanii strains had a specific Sym RFLP type, but former phylogenetic analyses had established that the common nodulation genes of B. japonicum and B. elkanii were closely related (Dobert et al., 1994
; Ueda et al., 1995
). However, further studies are necessary to investigate whether the Sym gene phylogeny is correlated with the 16S rRNA classification and host range within the Bradyrhizobium genus, which includes a wide variety of yet unclassified microsymbionts associated with many legumes.
The nod genes of the soybean symbionts S. fredii and B. japonicum have been reported as being of the same lineage (Dobert et al., 1994 ), but the result was not supported by bootstrap analysis, as confirmed by more recent phylogenetic surveys inferred from nodA (Haukka et al., 1998
) and nodC (Prévost et al., 2000
) genes. Also, the nifH genes were not found to be related in these two species (Dobert et al., 1994
; Haukka et al., 1998
; Fig. 3
). Therefore, among the soybean symbionts, the Sym gene phylogenies agree with the 16S rRNA phylogeny at the genus level.
By contrast, the nodC phylogeny was not only irrespective of the classification into species but also of the classification into genera among the Phaseolus rhizobia. Indeed, the highest similarity values (9999·5%) between the nodC genes of the Phaseolus rhizobia were found between species rather than within species (only 96·5% similarity between the two R. etli bv. phaseoli strains, and a maximum of 84·6% between biovars within R. gallicum and R. giardinii). Almost all the Phaseolus symbionts belong to the genus Rhizobium, but GR-06-like strains were classified into the genus Sinorhizobium. The 16S rDNA sequence of strain GR-06 was found to be identical to that of S. fredii (Herrera-Cervera et al., 1999 ), but their Sym genes are not closely related (Figs 2
and 3
). These results are consistent with the observation that GR-06-like strains were not able to nodulate soybean (Herrera-Cervera et al., 1999
). R. giardinii provides further evidence of incongruence between nodC and 16S-rDNA-based phylogenies, the latter indicating that R. giardinii would deserve a genus status distinct from the genera described so far (Amarger et al., 1997
), as confirmed by our updated phylogenetic tree (Fig. 1
).
Additional evidence of discrepancy between Sym gene phylogeny and 16S-rDNA-based classification of rhizobia in genera was obtained within the Medicago and the Sesbania symbionts (Table 1; Figs 1
and 2
). A similar case was reported by Wernegreen & Riley (1999)
for Rhizobium sp. strains isolated from Glycyrrhiza and Hedysarum species that had nod genes closely related to those of Medicago sinorhizobia.
Specificity of the rhizobiaP. vulgaris associations
The Phaseolus rhizobia investigated in this study constitute a representative sample of the Phaseolus symbionts described so far. For all the strains except R. tropici, the ability to establish a symbiosis in beans appears to be directed by a specific set of diversified but closely related nodulation genes. This should lead to a reconsideration of the usually accepted view that P. vulgaris is an undiscriminating host based on the diversity of its microsymbionts and the fact that most rhizobia studied so far are able to nodulate P. vulgaris when tested in laboratory assays (Martinez et al., 1985 ; Michiels et al., 1998
). Moulin et al. (2000)
indicated that nodA phylogeny gives indications on structural features of Nod factors. It is possible that the bean symbionts that have closely related nod genes produce similar Nod factors adapted for specific bean receptors. These Sym genes may confer upon the bacteria that harbour them a good competitive ability for nodule formation on beans. Furthermore, bean isolates that were effective in nitrogen fixation have been found in each species and biovar in which they are included (Young, 1985
; Martinez-Romero et al., 1991
; Segovia et al., 1993
; Amarger et al., 1994
, 1997
; Wang et al., 1999a
), except in R. giardinii (Amarger et al., 1997
). By contrast, the other rhizobia capable of nodulating bean plants as single-strain inoculants generally produce ineffective nodules (Martinez et al., 1985
; Michiels et al., 1998
). Taken together, these data suggest a certain specificity in the rhizobiabean symbiosis.
Gene transfer is probably involved in evolution of the symbiotic functions
Assuming that R. etli bv. phaseoli strains would be the symbionts that co-evolved with P. vulgaris, it has been hypothesized that they may be the original donors of the Sym plasmid in R. leguminosarum bv. phaseoli (Segovia et al., 1993 ). Likewise, R. gallicum and R. giardinii bv. phaseoli probably received their Sym plasmids from R. etli bv. phaseoli or, more plausibly, from the R. leguminosarum bv. phaseoli strains which co-existed in the fields in which the R. gallicum and R. giardinii isolates originated (Amarger et al., 1997
). The hypothesis of interspecies gene transfer is supported by the high similarity of the Sym genes among the bv. phaseoli subgroups and the co-occurrence of all these species in Europe (Geniaux et al., 1993
; Sessitsch et al., 1997
; Herrera-Cervera et al., 1999
). In the same way, the finding that the Sym genes of strains HT2a2 and HT4c1 and of R. tropici are similar although their 16S rDNA types are relatively distant, and the co-occurrence of these rhizobia in the southwest of France (Amarger et al., 1994
), argue for interspecies gene transfer. Hence, these data support the view that gene transfer would play a role in diversification and in structuring the natural populations of rhizobia, notably those nodulating P. vulgaris.
Furthermore, this work has also revealed the probable common origin of nod and nif genes among rhizobia belonging to the Rhizobium and Sinorhizobium genera that nodulate Phaseolus, Medicago and Sesbania. These findings suggest gene exchange events across genera, which does not support the view of Wernegreen & Riley (1999) that the Sym genes in rhizobial genera diverge independently. Additionally, analysis of glutamine synthetase genes also suggests transfers of genes between the rhizobial genera (Turner & Young, 2000
).
The comparison of the nod, nif and 16S rRNA phylogenies and the substantial correlation that we found between symbiotic genotypes and host plant groups taken together support the generally accepted hypothesis that lateral transfer of Sym genes and genetic rearrangements are involved in the acquisition and evolution of rhizobial symbiotic functions.
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ACKNOWLEDGEMENTS |
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This work was funded by INRA as part of AIP Microbiologie, and by MESR as part of ACC-SV no. 6. It was also financially supported by a grant from the Conseil Régional de Bourgogne.
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Received 12 July 2000;
revised 28 November 2000;
accepted 15 December 2000.