Genetic Diversity And Phylogeny Of Rhizobia Isolated From Astragalus Spp. In The West Plateau Of Sichuan

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 After the nodulation test, the phenotypic characteristics and genectic diversity of these 33 strains and 18 reference strains, totally 51 strains, were characterized by using numerical taxonomy, BOX-PCR, amplified fragment length polymorphism (AFLP),16SrDNA-RFLP, and by sequencing of rrs, gln A, glnâ…¡(house keeping genes)and nifH(symbiotic gene).76 physiological and biochemical characteristics were tested in numerical taxonomy. The results revealed that all 33 Astragalus isolates were fast-growing rhizobia. Considerable phenotypic diversity was also demonstrated. The Astragalus isolates were divided 2 large groups at 65% similarity. Most of them located in the same group and were closer to Mesorhizobium genus. Compared to the reference strains, The Astragalus isolates showed their being vulnerable in the media which contain antibiotics, and similarly in the extreme pH conditions.In BOX-PCR analysis the Astragalus isolates were divided 5 large groups at 60% similarity. Most novel strains located in 3 groups. High genomic diversity of the Astragalus isolates was demonstrated. The strains obtained in the two different altitudes and hosts were not separated respectively.16S rDNA PCR-RFLP analysis also revealed considerable genomic diversity.33 Astragalus isolates have 9 rrs genotypes and most of them belonged to genotype A. The results also discovered that the rrs fingerprints of the novel isolates were more similar to the reference strains of Mesorhizobium genus.In AFLP analysis, the Astragalus isolates were divided 3 large groups at 65% similarity.20 of 33 Astragalus rhizobia formed three small distinct clades, with others dispersed into different groups with the reference strains. As same as the results of BOX-PCR, AFLP also revealed rich genomic diversity among the Astragalus rhizobia. On the hand the AFLP dendrogram showed the fingerprints of Astragalus rhizobia are different obviously from the recognized reference strains. Phylogenetic analysis of the rrs gene showed the relationships among the defined reference species were similar to those in previously reported trees. The representative isolates were members of the genus Mesorhizobium. Three of the six isolates formed a sister clade to Mesorhizobium loti and Mesorhizobium ciceri, whereas the other three formed a sister clade to a clade harboring the species Mesorhizobium huakuii, Mesorhizobium plurifarum, Mesorhizobium septentrionale and Mesorhizobium amorphae, indicating the existence of two new species.In the phylogenetic tree based on glnA gene sequences, Genus Mesorhizobium formed a well-separated clade. Strains SCAU2 and SCAU9 clustered with Mesorhizobium sp. MAFF303099, forming a sister clade to M. huakuii. Strains SCAU7, SCAU13 and SCAU27 formed a sister clade to M. ciceri and M. loti. The topology of the glnA tree was different from that of the rrs tree, with the positions of strains SCAU11 and SCAU13 swapped in relation to the other Astragalus isolates. The glnâ…¡tree was similar to glnA tree. Analysis of glnA and glnâ…¡confirmed the rrs phylogenies for four strains, but incongruences were tested.nifH Sequences of the Astragalus strains were all closely related and formed a well-defined, monophyletic clade among sequences from described species of Mesorhizobium. And the results showed host specifity existed in the evolution of nifH of Astragalus rhizobia.The incongruent phylogenies of the genes studied suggest that horizontal gene transfer and recombination shape mesorhizobial populations in the gene center of the host plants.