Phylogeny And Diversity Of Rhizobia Isolated From Alhagi, Astragalus And Robinia In Arid And Semiarid Region Of Northwestern China

The rhizobial strains isolated from the nodules of Alhagi sparsifolia, Astragalus sp. and Robinia pseudoacacia growing in the arid and mid-arid regions of northwest of China were characterized by the genetic and phenotypic methods, including analysis of 16S rDNA, nodC, nodA, nifH genes PCR-RFLP and their sequences, G+Cmol%, the DNA-DNA hybridization, the whole fatty acids and four housekeeping genes.Thirty-three rhizobia were isolated from the legume Alhagi sparsifolia growing in the Gansu and Xinjiang provinces. They fell into three groups by restriction analysis of their 16S rDNA, and these, together with dnaK, dnaJ, recA and glnA genes, were sequenced from representative isolates to assess their taxonomic position by phylogenetic analysis. The bacteria in each group belonged to different lineages that might represent three different Mesorhizobium species, two of which form a novel clade very distinct from other species in the genus. The strains belonging to genospeciesⅠandⅡshared less than 97.8% with defined Mesorhizobium species, 96.0% with Sinorhizobium species and 93.1% with Rhizobium species.Four nodA types, five nodC types and five nifH types were obtained through the PCR-RFLP analysis of symbiotic genes. Sequencing anylysis showed that the strains corresponding to genospeciesⅠandⅡharboured closely related nodA, nodC and nifH genes forming new lineages. They fell into Mesorhizobium group. The strains from Xinjiang were specific to geographical site and host plants, however strains from Gansu had high diversity of symbiotic gene types.CCNWXJ12-2 and CCNWXJ40-4 belong to Mesorhizobium because they contain 16 : 0, 17 : 0, 17 : 0 iso, 18 : 0, 19 : 0 cyclo 8c and summed feature 3, without 20 : 3 6, 9, 12cis. Both of them contain 18:1ω7c except all defined Mesorhizobium species. The difference between CCNWXJ12-2 and CCNWXJ40-4 is that the forward has 17 : 0 cyclo, 17 : 1 8c and 20 : 2 6, 9c, or the later has 18 : 1 9c. Therefore, they are two different novel species.For the strains of the genospeciesⅠ, their Tm is 78.64℃, G+Cmol% is 62.06%, DNA-DNA hybridization (<45%). The strains of the genospeciesⅡ, their Tm is 78.82℃, G+Cmol% is 62.06%, DNA-DNA hybridization (<43.3%). The DNA homology between CCNWXJ12-2 and CCNWXJ40-4 is 37.87%, which suggests they are two different new species.A total of 55 isolates from root nodules of 12 different Astragalus species growing in four provinces in northwestern China, covering 31 sampling sites, were characterized by PCR-RFLP analysis of 16S rDNA and symbiotic genes. Eleven genotypes were identified by sequence analysis of 16S rDNA, they belong to Agrobacterium, Mesorhizobium, Sinorhizobium, Mesorhizobium and Rhizobium related strains, eight of them were Mesorhizobium-related species, accounting for 54.5% of all isolates, and genospeciesⅪconsisting of 20 isolates is a biggest group, they belong to Agrobacterium tumefaciens and all of them are from Xinjiang.Eighteen nodA types, eighteen nodC types and fourteen nifH types were attained through the PCR-RFLP analysis of symbiotic genes. The nodC gene phylogeny placed them into Sinorhizobium, Mesorhizobium and Rhizobium branches, most Mesorhizobium-related strains shared the similar symbiotic genes independence of their original host plants and sites, it indicated that genes transfer occurred within genus or genera, which played an important role in diversification of rhizobia and in adapting to an introducing environment. The presence and absence of Astragalus rhizobial species in upstream or downstream suggested that their distribution should be influenced by irrigation of Yellow.Thirty three isolates from Robinia pseudoacacia were characterized using RFLP and sequencing of 16S rRNA, nodA, nodC and nifH genes. Their 16S rRNA gene patterns and sequences placed them in three clades: 85% of isolates were related to the Mesorhizobium mediterraneum/temperatum group, while the remainders were similar either to M. amorphae or to Sinorhizobium meliloti. However, despite their diverse taxonomic positions, the nodA, nodC and nifH genes phylogenies indicated that these R. pseudoacacia symbionts share similar symbiosis genes, implying gene transfers and a degree of host specificity. Comparison of R. pseudoacacia symbiotic diversity in native and other invaded areas suggests that most Chinese symbionts may not have arrived with the seed but were local bacteria that acquired specific symbiotic genes from American native rhizobia.