Thirty-one strains of two new genomic species (genomic species 1 and 2) of rhizobia isolated from root nodules of Phaseolus vulgaris and originating from various locations in France were compared with reference strains of rhizobia by performing a numerical analysis of 64 phenotypic features. Each genomic species formed a distinct phenon and was separated from the other rhizobial species. A comparison of the complete 16s rRNA gene sequences of a representative of genomic species 1 (strain R602spT) and a representative of genomic species 2 (strain H152T) with the sequences of other rhizobia and related bacteria revealed that each genomic species formed a lineage independent of the lineages formed by the previously recognized species of rhizobia. Genomic species 1 clustered with the species that include the bean-nodulating rhizobia, Rhizobium leguminosarum, Rhizobium etli, and Rhizobium tropici, and branched with unclassified rhizobial strain OK50, which was isolated from root nodules of Pterocurpus klemmei in Japan. Genomic species 2 was distantly related to all other Rhizobium species and related taxa, and the most closely related organisms were Rhizobium gaZegae and several Agrobacterium species. On the basis of the results of phenotypic and phylogenetic analyses and genotypic data previously published and reviewed in this paper, two new species of the genus Rhizobium, Rhizobium gallicum and Rhizobium giardinii, are proposed for genomic species 1 and 2, respectively. Each species could be divided in two subgroups on the basis of symbiotic characteristics, as shown by phenotypic (host range and nitrogen fixation effectiveness) and genotypic data. For each species, one subgroup had the same symbiotic characteristics as R. leguminosarum biovar phaseoli and R. etli biovar phaseoli. The other subgroup had a species-specific symbiotic phenotype and genotype. Therefore, we propose that each species should be subdivided into two biovars, as follows: R. gallicum biovar gallicum and R. gallicum biovar phaseoli; and R. giardinii biovar giardinii and R. giardinii biovar phaseoli.Until recently, the rhizobia that nodulate the roots of common bean, Phaseolus vulgaris, were classified in a single species, Rhizobium leguminosarum (23). This species was subdivided into three biovars differentiated by legume host specificity, and strains recovered from Phaseolus spp. nodules were assigned to R. leguminosarum bv. phaseoli. Although this biovar has long been recognized as a very heterogeneous group on the basis of a variety of phenotypic (5, 16,40,41) and genotypic (7, 15, 21, 38) criteria, it is only with the development of the molecular approach that it gave rise to new taxa. First, based on host range characteristics encoded by genes located on the symbiotic plasmid and on the organization of the symbiotic plasmid (33, 35), two subgroups were distinguished within R. Zeguminosarum bv. phaseoli. Type I strains had multiple copies of the nitrogenase reductase gene, ni&4(39), and a narrow host range (6) (which recently appeared t...

The nodC and nifH genes were characterized in a collection of 83 rhizobial strains which represented 23 recognized species distributed in the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium, as well as unclassified rhizobia from various host legumes. Conserved primers were designed from available nucleotide sequences and were able to amplify nodC and nifH fragments of about 930 bp and 780 bp, respectively, from most of the strains investigated. RFLP analysis of the PCR products resulted in a classification of these rhizobia which was in general well-correlated with their known host range and independent of their taxonomic status. The nodC and nifH fragments were sequenced for representative strains belonging to different genera and species, most of which originated from Phaseolus vulgaris nodules. Phylogenetic trees were constructed and revealed close relationships among symbiotic genes of the Phaseolus symbionts, irrespective of their 16S-rDNA-based classification. The nodC and nifH phylogenies were generally similar, but cases of incongruence were detected, suggesting that genetic rearrangements have occurred in the course of evolution. The results support the view that lateral genetic transfer across rhizobial species and, in some instances, across Rhizobium and Sinorhizobium genera plays a role in diversification and in structuring the natural populations of rhizobia.

The ability of denaturing gradient gel electrophoresis (DGGE) technique to resolve 16S rDNA products generated from two different collections of bacteria using universal 16S primers was investigated. Alignments of 16S rDNA sequences of known species of rhizobia and methanotrophs were performed in order to determine the genetic variations within a 200 bp product obtained with PCR primers which amplify the 16S rRNA encoding genes from Eubacteria. Theoretical DNA melting curves were obtained with the Melt87 program and found to correlate with the ability to resolve fragments by DGGE. In the case of the rhizobia, the inability of DGGE analysis to resolve the PCR products from closely related species was in accordance with the low polymorphism observed amongst the sequences in the amplified area. In the case of the methanotrophs, the PCR products were surprisingly difficult to resolve given the high degree of sequence polymorphism of the amplified area in some distantly related species. The difference in sequence divergence within the two groups members allowed therefore to scale the resolution ability of the DGGE technique.

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