Ployphasic Taxonomic Study Of Novel Species Of Rhizobia Isolated From Lespedeza Floribunda

During the study on the genetic diversity of rhizobia isolated from the root nodules of leguminous plant Lespedeza floribunda growing in Shangluo City, Shaanxi Province by analyzing the PCR-RFLP and phylogeny of 16S rDNA, the strains of GenotypeⅠand GenotypeⅡ, with CCNWSX0884 and CCNWSX0906 as the representative strains, were found to be distinctively different from all the known Rhizobium species, indicating that the two groups of rhizobia are possibly two novel species. In this study, ployphasic taxonomic technologies including phylogenetic analysis, phenotypic characterization, rep-PCR fingerprint and DNA relatedness measurement were employed to identify the exact taxonomic status of these two groups of rhizobia strains.According to the result of the phylogenetic analysis of 16S rDNA, the 4 strains of GenotypeⅠhave identical 16S rDNA sequences, with a similarity of 100% to the newly published novel species R. vallis within the genus Rhizobium. The phylogenetic analysis of housekeeping genes recA, atpD and glnA showed that these 4 strains are markedly different from the published strains of the species R. vallis and they also formed a separate branch in the phylogenetic trees of all the sequenced housekeeping genes. The results of the phenotypic tests also revealed that these 4 strains are more tolerant to unfavorable conditions when compared with the known strains of R. vallis sp.. In the nodulation test, CCNWSX0884 nodulated Lepedeza floribunda and Melilotus officinalis, but no nodule formed on the other leguminous plants tested.The 2 strains of 16S rDNA GenotypeⅡ, CCNWSX0906 and CCNWSX0914, have a difference of 2 bp in their 16S rDNA sequences, and are most closely related to R. tibeticum CCBAU 85039T phylogenetically, with a 16S rDNA similarity of 99.17%. In the phylogenetic tree of recA, atpD and glnA, these 2 strains are also most closely related to R. tibeticum CCBAU 85039T, but they formed a separate cluster. The results of all the phylogenetic analysis confirmed that 16S rDNA GenotypeⅡis possibly a novel species of rhizobia.In the dendrogram based on numerical taxonomy, the similarity between CCNWSX0906 and CCNWSX0914 was nearly 90%, and the similarity between the novel group and any of the reference strains are lower than 80%. The result of the nodulation test and PCR amplification of nodA, nodC and nifH genes revealed that both CCNWSX0906 and CCNWSX0914 do not carry any symbiotic gene and are unable to induce any nodule formation.BOX-PCR fingerprint showed that there are differences among the strains of the novel group and the reference strain. The similarity between CCNWSX0906 and CCNWSX0914 was above 87% and the similarity between the novel group and the reference strain R. tibeticum CCBAU 85039T was about 30%. The result of DNA G+C mol% measurement suggested that the novel group belongs to the genus Rhizobium. The DNA relatedness between CCNWSX0906 and R. tibeticum CCBAU 85039T was 36.21%.All the above listed results proved that the 4 strains of 16S rDNA GenotypeⅠare new strains of Rhizobium vallis sp. while the 2 strains of 16S rDNA GenotypeⅡconstituted a novel species of Rhizobium gen..