Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava beannodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.

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.

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR-amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to their biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.

Little is known about factors that affect the indigenous populations of rhizobia in soils. We compared the abundance, diversity and genetic structure of Rhizobium leguminosarum biovar viciae populations in soils under different crop managements, i.e., wheat and maize monocultures, crop rotation, and permanent grassland. Rhizobial populations were sampled from nodules of pea- or vetch plants grown in soils collected at three geographically distant sites in France, each site comprising a plot under long-term maize monoculture. Molecular characterization of isolates was performed by PCR-restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer as a neutral marker of the genomic background, and PCR-restriction fragment length 0polymorphism of a nodulation gene region, nodD, as a marker of the symbiotic function. The diversity, estimated by richness in types and Simpson's index, was consistently and remarkably lower in soils under maize monoculture than under the other soil managements at the three sites, except for the permanent grassland. The highest level of diversity was found under wheat monoculture. Nucleotide sequences of the main rDNA intergenic spacer types were determined and sequence analysis showed that the prevalent genotypes in the three maize fields were closely related. These results suggest that long-term maize monoculturing decreased the diversity of R. leguminosarum biovar viciae populations and favored a specific subgroup of genotypes, but the size of these populations was generally preserved. We also observed a shift in the distribution of the symbiotic genotypes within the populations under maize monoculture, but the diversity of the symbiotic genotypes was less affected than that of IGS types. The possible effect of such changes on biological nitrogen fixation remains unknown and this requires further investigation.

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.