The Study Of Diversity And Phylogeny Of Rhizobia Isolated From Thermopsis In Northwest China

Thirty-five rhizobial strains isolated from root nodules of Thermopsis lanceolata,Thermopsis kaxgarica and Thermopsis licentiana in northwest China. Based on the numerical taxonomy, the fingerprint of 3 gene fragments were studied by using PCR-RFLP. Apparently, the results revealed a high genetic diversity among these Thermopsis rhizobia.Analysis of 105 physiological and chemical characteristis of all tested strains. The results indicated all strains could grow in high salinity level and in low temperature circumstance. Most rhizobial strains have high stress resistance. For example, 60% of all strains can grow on YMA medium with some higher concentration of antibiotics(300μg/Ml), higher concentration of salt (3% NaCl)or higher alkaline environment. The result of numerical taxonomy showed that all the test strains were divided into 6 groups at the level of 92.4% by numerical taxonomy. Respectively, The groupⅢandⅣmay be a new group because in this cluster there were no reference strains included.Based on the numerical taxonomy, the fingerprint of 3 gene fragments were studied by using PCR-RFLP. The PCR-RFLP result of 16S rDNA showed that all the test strains were belonged to Mesorhizobium,Sinorhizobium,Rhizobium and Agrobacterium. In addition, representative strains CCNWGS0011 and CCNWGS0010-1 of five strains formed a distinct monophyletic line within the 16S rDNA sequence phylogenetic tree, which indicated that maybe there are putative novel species. High sequence similarity was also determined between CCNWGS0022 and R. giardinii on the 98.9% similarity level. CCNWGS0021-2 and CCNWGS0021-1 more close to Aurantimona altamirensis S21BT which was first isolated from the marine environment belong to family'Aurantimonadaceae'within the order Rhizobiales. At present, there are no members of this family from terrestrial environments. The sequence difference between them is 5 bp.Carried out PCR amplification analysis and study for their common nodulation gene nodA and nitrogen fixing gene nifH. All the strains from both Mesorhizobium,Sinorhizobium and Rhizobium phylogenetic branch got nodA PCR products and nifH PCR products, however, strains belonging to Agrobacterium phylogenetic branch did not get any nodA PCR product and nifH PCR products. The results of nodA PCR-RFLP and nifH PCR-RFLP analysis revealed rhizobia isolated from Thermopsis have diverse common nodulation gene nodA and nif H. There are 8 different nodA PCR-RFLP genotypes and 9 different nifH PCR-RFLP genotypes among tested strains having 10 different 16S rDNA PCR-RFLP genotypes. However , the diversity of nodA gene and nifH gene are variable in different rhizobial species , for S. meliloti 3 different nodA PCR-RFLP genotypes and 3 different nifH PCR-RFLP genotypes were found among the ten strains having the same 16S rDNA PCR-RFLP genotypes , while for M. sp. the same nif H PCR -RFLP genotypes were found among four strains having the same 16S rDNA PCR-RFLP genotypes. In addition, the same nod A PCR-RFLP and nifH PCR-RFLP genotypes were found in different 16S rDNA PCR-RFLP genotyes belonging to different rhizobial species. The results indicate that nodA gene and nifH gene have probably transferred between different rhizobial species.