Forty-eight strains representing the eight recognized Rhizobium species, two new Phaseolus bean Rhizobium genomic species, Bradyrhizobium spp., Agrobacterium spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa. The classification of the root-and stem-nodulating bacteria of legumes formerly based mainly on symbiotic characteristics is in constant evolution. Since 1988 two new genera, Sinorhizobium and Azorhizobium, have been proposed (3, 7). The number of species within the genus Rhizobium increased from four to eight with the addition of Rhizobium tropici (29), R. galegae (27), R. huakuii (4), and R. etli (37) to the previously recognized species, R. meliloti, R. leguminosarum, R. loti (20), and R. fredii (35). Within the genus Bradyrhizobium, a new species of slow-growing soybean microsymbionts, Bradyrhizobium elkanii, has been proposed in addition to B. japonicum (22). However, other groups of J?Jizobium strains which do not cluster with the existing species have been identified. This is the case for tree rhizobia (15, 45), group B of R. meliloti (9), and two Rhizobium genomic species (11, 24) and some other genotypes nodulating Phaseolus vulgaris (8, 10, 29, 32). As more knowledge is acquired and new isolates are studied, new species are discovered and the former species are split. Consequently, more and more leguminous plants (Glycine, Lupinus, and Lotus spp. [20]) appear to be nodulated by rhizobia belonging to different genera or to different Rhizobium and Bradyrhizobium species. For instance, three species, R. leguminosarum bv. phaseoli, R. etli bv. phaseoli, and R. tropici, two new Rhizobium genomic species (24), and other unclassified genotypes (29, 32) have been isolated from nodules of P. vulgans. The ability of legumes to be nodulated by several Rhizobium species might well appear to be the rule rather than the exception. Symbiotic performance, which has been the most practical trait, although not absolute, for differentiation between species is thus less and less usable. There is therefore a more and more pressing need for an easy and rapid method which would enable rhizobiologists, and especially ecologists, to identify the rhizobia they are working with.

Two hundred and eighty seven isolates of Rhizobium nodulating Phaseolus vulgaris L. were sampled in France from four geographically distant field populations. They were characterized by their colony morphology and by plasmid profiles. A representative sample was further characterized: a) by the ability of each isolate to nodulate a potential alternative host Leucaena leucocephala and to grow on specific media, and b) by RFLP analysis of PCR amplified 16S rRNA genes. On the basis of their phenotypic and genetic characteristics the isolates could be assigned either to Rhizobium leguminosarum bv phaseoli, or to R. tropici. The two species co-occurred at three sites. R. leguminosarum bv phaseoli represented 2%, 4%, 72% and 100% of the population at the four different sites. Eighteen and 22 different plasmid profiles were identified within R. tropici and R. leguminosarum bv phaseoli, respectively. Some of them were conserved between distant geographical regions. The fact that R. tropici was found in France shows that this species is not limited to tropical regions and gives additional evidence of the multi-specific nature of the Phaseolus microsymbiont, even over a geographically limited area.