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Title: RHIZOBIA ISOLATED FROM ASTRAGALUS, OXYTROPIS AND ONOBRYCHIS SPP.: ESTIMATEDPHYLOGENY BY ANALYSIS OF MAPPED RESTRICTION SITE POLYMORPHISM (MRSP) WITHIN16S RRNA GENES/DETERMINATION OF GENOMIC DIVERSITY BY PCR DNA FINGERPRINTING

Author
item LAGUERRE, GISELE - INRA, FRANCE
item Van Berkum, Peter
item AMARGER, NOELLE - INRA, FRANCE
item PREVOST, DANIELLE - AGCANADA, CANADA

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/14/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Rhizobia are important soil bacteria with application in agricultural production because they establish symbioses with legume crop plants. These symbioses benefit agricultural production because biological nitrogen fixation enhances the efficiency of growing crops under low input sustainable agricultural management practices. A problem in management of biological nitrogen fixation is that among the rhizobia there is considerable genetic variation in the capacity for nitrogen fixation and traits affecting plant-microbe interaction. Genetic characterization of rhizobia permits their positive identification both in nature and in the laboratory. Phylogeny of all bacteria is estimated from sequences of the small subunit ribosomal (SSU rRNA) genes. Although SSU rRNA gene sequencing methods are available, they are expensive and laborious limiting the number of analyses. We present a method based upon a database of variations in the SSU rRNA gene restriction sites to obtain preliminary phylogenetic information rapidly and at low-cost. The rhizobia of three legumes had wide phylogenetic diversity and some were closely related to the symbionts of soybean. This information will benefit scientists.

Technical Abstract: We have improved PCR-RFLP analysis of rhizobial 16S rDNA by constructing a database of mapped restriction sites in this gene of bacteria in the family Rhizobiaceae. This approach is more reliable because it eliminates the problem associated with RFLP analysis of scoring restriction fragments of identical molecular size together regardless of whether they are corresponding regions of the gene. The method was applied to the analysis of 44 rhizobial isolates from Astragalus, Oxytropis and Onobrychis spp. which originated from different geographic locations. The isolates were distributed into 14 16S rDNA types and by comparison to reference strains were clustered into three major groups corresponding to three genera. Most of the isolates were classified within the genus of which R. loti is the type species. Five of these types were associated with different genomic species nodulating Lotus spp. and Cicer arietinum. Three Astragalus strains were classified as Bradyrhizobium, one similar to B. elkanii and another to B. japonicum. Six isolates were classified within the genus Rhizobium. PCR DNA fingerprinting with repetitive sequences (rep-PCR) revealed a high level of diversity within single 16S rDNA types. The 44 strains were distributed into 34 rep groups. Rhizobial classification was independent of geographic origin and host plant affinity.