Noëlle Amarger

Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts

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 Phaselous 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.

Rhizobium gallicum sp. nov. and Rhizobium giardinii sp. nov., from Phaseolus vulgaris Nodules

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 Pterocarpus 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 galegae 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.

Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes.

ABSTRACT 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 bean-nodulating 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.

Competition for nodule formation between effective and ineffective strains of Rhizobium meliloti

Abstract Ineffective mutants of four effective strains of Rhizobium meliloti were isolated and tested for their ability to compete with effective parents or antibiotic resistant mutants in the formation of nodules on Medicago sativa . In 5 out of the 6 cases studied, ineffective mutants were no different to effective strains of the same origin in their competitive ability. A difference in selection for infection by the host plant was observed between equally effective strains as well as between ineffective strains. Except for one pair of strains, the more-competitive effective strains (resistant or not to antibiotics) had the same origin as the more-competitive ineffective strains. For such strains the ability to compete with other strains to form nodules was a characteristic of each parent strain. Competitiveness was independent of effectiveness and had been retained during mutation.

Rhizobium tropici nodulates field-grown Phaseolus vulgaris in France

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.

Comparison of geographically distant populations of Rhizobium isolated from root nodules of Phaseolus vulgaris

Seventy‐two rhizobial strains were isolated from the root nodules of french beans (Phaseolus vulgaris). They were sampled from two geographically distant field populations and 18 additional different sites in France. They were characterized by a) plasmid profiles, (b) RFLP analysis of total cellular DNA using various chromosomal and symbiotic gene probes (including nifH from Rhizobium etli bv. phaseoli) and c) their ability to nodulate a potential alternative host, L. leucocephala. Over half of the isolates were ascribed to Rhizobium leguminosarum bv. phaseoli on the basis of the hybridization analysis, the possession of multiple copies of nifH and their inability to nodulate L. leucocephala. The remaining isolates belonged to 2 groups which were shown to be genomically distinct from R. leguminosarum bv. phaseoli, R. etli bv. phaseoli and R. tropici. Most members of these two groups shared with R. tropici the ability to nodulate L. leucocephala and, for isolates of only one of these groups, the presence of one copy of nif H. Members of each of the 3 taxa were widely distributed in France and circumstantial evidence of pSym transfer between them was shown. R. leguminosarum bv. phaseoli and one of the two novel groups co‐occurred within the two geographically distant populations. Individual genotypes were conserved between them. The finding of a third taxon at various other locations indicated additional diversity among rhizobia nodulating beans.

Strain of Rhizobium lupini determines natural abundance of 15N in root nodules of Lupinus spp

Abstract Discrepancies between French and Australian results in measurement of the natural enrichment of 15 N in nodules of Lupinus spp were due to the strains of Rhizobium lupini used. The Australian inoculant strain, WU425, produced nodules enriched in 15 N above values for the entire plants, on all lines of L. luteus and L. albus tested. The total nitrogen and the natural abundance of 15 N in nodules produced by the French strain, LL13, were much lower. In tests in Australia and in France with L. luteus inoculated with 15 other strains of R. lupini from diverse origins, 9 produced nodules with elevated 15 N abundance, but of these 8 were less than with strain WU425. Most strains producing 15 N-enriched nodules were slower-growing on yeast extract-mannitol agar; most faster-growing strains produced little or no nodular 15 N enrichment. For strains with 15 N-enriched nodules, enrichment increased with increasing nodule N·plant −1 , and shoot nitrogen tended to be depleted in 15 N. There was no relationship between symbiotic effectiveness of strains (mg N 2 fixed·plant −1 ) and 15 N enrichment of nodules.

Selection of Rhizobium strains on their competitive ability for nodulation

The shoot dry weight of alfalfa inoculated with an effective strain of Rhizobium meliloti mixed with an ineffective strain in different ratios was found to be directly proportional to the log of the number of effective nodules. Consequently the comparison of the shoot dry weight of plants inoculated with a mixture of effective and ineffective strains with the shoot dry weight of plants inoculated with the effective strain should allow the estimation of the relative competitiveness of the effective strains. To check this. the competitiveness of 14 antibiotic-resistant strains of R. leguminosarum was evaluated in this way and compared with the ability of the strains to form nodules when inoculated to seeds of Vicia faba planted in a soil containing indigenous R. leguminosarum. The percentage of recovery of the inoculum strains in the nodules of field-grown fababeans was positively correlated with the competitiveness of the strains as estimated by the greenhouse test. This simple way of evaluating the nodulating competitiveness of strains of rhizobia being indicative of their competitive behaviour with indigenous rhizobia in the field could therefore be useful for screening a large number of strains for competitiveness.

Semiselective medium for isolation of Rhizobium leguminosarum from soils

Abstract A medium which controlled the growth of soil bacteria without inhibiting Rhizobium leguminosarum strains and other fast- and slow- growing rhizobia was developed. This medium, MNBP, included antibiotics (penicillin G and bacitracin) which inhibit the growth of Gram-positive bacteria, fungicides (cycloheximide and benomyl) and also pentachloronitrobenzene in order to control actinomycetes. Colony counts of total bacteria from different soils were reduced by 88–95% on this medium, compared to those obtained on a nutrient-rich agar medium, MGYF, commonly used for culture of rhizobia. The numbers of both Gram-positive and Gram-negative bacteria were lowered on medium MNBP. When used in conjunction with a complementary selection step (inability to grow on Luria-Bertoni agar medium) and a specific identification method (colony blot hybridization with a biovar-specific DNA probe), this medium allowed the isolation of a population of 66 R. leguminosarum bv. viciae from a soil sample. The effectiveness of the isolation procedure was found to be satisfactory because the number of R. leguminosarum bv. viciae isolated agreed with that estimated by indirect plant infection counts.

Structural determination of the lipo-chitin oligosaccharide nodulation signals produced by Rhizobium giardinii bv. giardinii H152

Rhizobium giardinii bv. giardinii is a microsymbiont of plants of the genus Phaseolus and produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis. We report here the structures of seven lipochitooligosaccharide (LCO) signal molecules secreted by R. giardinii bv. giardinii H152. Six of them are pentamers of GlcNAc carrying C 16:0, C 18:0, C 20:0 and C 18:1 fatty acyl chains on the non-reducing terminal residue. Four are sulfated at C-6 of the reducing terminal residue and one is acetylated in the same position. Six of them are N-methylated on the non-reducing GlcN residue and all the nodulation factors are carbamoylated on C-6 of the non-reducing terminal residue. The structures were determined using monosaccharide composition and methylation analyses, 1D- and 2D-NMR experiments and a range of mass spectrometric techniques. The position of the carbamoyl substituent on the non-reducing glucosamine residue was determined using a CID-MSMS experiment and an HMBC experiment.