Polyphasic Systematics Of Rhizobia Isolated From Astragalus Luteolus And Astragalus Ernestii In Sichuan,China

In our previous study, Thirty-three rhizobial strains were isolated from the root nodules of Astragalus luteolus and Astragalus ernestii growing on the west plateau at two different altitudes in Sichuan province, China.28strains were classified in the genus Mesorhizobium by using amplified16S rDNA restriction analysis (ARDRA), amplified fragment length polymorphism (AFLP), BOX-PCR fingerprinting,16S rRNA gene sequence analysis and other polyphasic taxonomical methods. The results revealed that the two strains SCAU7T and SCAU27exhibited different distinct characteristics from the known species in Mesorhizobium. Therefore, the taxonomic position of these two strains within the genus Mesorhizobium were further analyzed by comparison of their16S rRNA gene sequences, housekeeping gene (atpD, recA and gin II) sequences, symbiotic gene (nodC and nifH) sequences, fatty acid profiles, phenotypic characteristic determination and DNA-DNA hybridization in the present study.In the phylogeny tree based on16S rDNA gene sequences, the two rhizobial strains formed a distinctive16S rRNA gene types within the genus Mesorhizobium and had some hereditary distance with known species in Mesorhizobium. So it was possible that strains SCAU7T and SCAU27were novel species different from whole type strains in the genus Mesorhizobium.The phylogenetic trees based on the partial sequences of housekeeping genes and16S rRNA gene of SCAU7T and SCAU27had good consistency. However, the reference strains are not the same location in different phylogenetic trees. The results indicated that16S rDNA sequence of Mesorhizobium may have gene transfer and gene recombinant events.In the phylogeny tree based on multilocus sequence analysis of housekeeping genes, the sequence similarities between SCAU7T (SCAU27) and M. ciceri were lower than97%, So it could make sure that the novel group(SCAU7T and SCAU27) of isolates was distinct from previously described species in Mesorhizobium. Symbiotic gene sequence analysis showed that nodC gene and nifH gene p hylogenetic trees were different from housekeeping genes. The analysis showed t heir evolutionary history and host plant ranges. Comparison of phylogenies with those derived from housekeeping genes may reveal events of lateral gene trans fer among rhizobial symbiotic genes.The novel group synthesized16:0,17:0,18:0,17:0iso,17:1ω8c,18:1ω7c and19:0cyclo ω8c fatty acids, and lacked20:3co6,9,12c and summed feature2(12:0, unknown ECL10.928,16:1iso1/14:03OH). Thus the novel group could be placed in the genus Mesorhizobium. The strains of the novel group could be distinguished from all related Mesorhizobium species because they contained higher concentration of16:0and18:1ω9c.The DNA-DNA relatedness between the strains SCAU7T and SCAU27was83.0%, indicating that they were members of the same genomic species. The DNA-DNA relatedness values of strain SCAU7T with M. loti NZP2213T, M. ciceri USDA3383T and M. shangrilense CCBAU65327T were41.1%,48.8%and23.4%, respectively. Given the recommended threshold value of70%DNA-DNA relatedness for the definition of a species, these results indicate that the strains should be considered as representing a novel species.A series of phenotypic and genotypic tests indicated that the novel group of isolates was distinct from previously described species. Therefore, we propose that strains SCAU7T and SCAU27represent a novel species, Mesorhizobium sangaii sp. nov., with strain SCAU7T as the type strain.