Symbiotic Effectiveness And Phylogeny Of Rhizobia Isolated From Root Nodules Of Faba Bean (Vicia Faba L.) In Sichuan,China

Legume-Rhizobia symbiosis could reduce the dinitrogen into ammonia with little environmental pollution which plays an important role in sustainable agricultural. Exploration on the rhizobial resource and biodiversity is still required because the microsymbionts of very few legume genera have been characterized in the symbiotic nitrogen fixation study. In this research, we studied the symbiotic effectiveness, genetic diversity and phylogeny of rhizobia isolated from root nodules of faba bean (Vicia faba L.) in three parts of Sichuan (Hilly areas, Chengdu plain and Panxi areas) based on hydroponics,16S rDNA and IGS RFLP and multilocus sequence analysis of housekeeping genes and symbiotic genes.A total of 54 rhizobial strains were isolated from faba bean root nodules in 21 counties of Sichuan hilly areas. Only six strains increased the shoot dry mass of the host plant significantly (P< 0.05) which could be regarded as the high efficient strains. Based on the cluster analysis of combined 16S rDNA and intergenic spacer region (IGS) PCR-RFLP (CACAI), the strains were divided into 31 genotypes in 11 groups, indicating a high degree of genetic diversity among the strains. The sequence analysis of three housekeeping genes (atpD, glnll and recA) and 16S rDNA indicated that the strains represented R. anhuiense, R. sophorae, R. mesosinicum, A.tumefaciens and three potential new species related to Rhziobium and Agrobacterium, among which R. anhuiense was the dominant species in this area. The strains representing five Rhizobium species were divided into two distinct nodC and nifH genotypes. However, the phylogeny of housekeeping genes and symbiotic genes was not congruent, implying that the strains had been shaped by vertical evolution of the housekeeping genes and lateral evolution of the symbiotic genes.Thirty-one nodulating rhizobial strains were collected from root nodules of faba bean cultivars in Chengdu Plain. Nine strains could significantly improve the shoot dry mass of the host which could be considered as the potential high efficient isolates.Thirty one strains obtained the same type of ARDRA while 13 types of IGS RFLP. Phylogenetic analysis based on 16S rDNA showed the strains in this area were related to Rhizoibum. These strains were assigned as R. anhuiense and one new potential species related to Rhizobium based on multilocus sequence analysis of three housekeeping genes (atpD, glnll and recA). Symbiotic gene sequence analysis indicated the strains gained 5,4 and 3 genotypes of nifH, nodC and nodD respectively which showed a great diversity.65 strains were isolated from root nodules of faba bean(Vicia faba L.) from 12 counties of Panxi area.11 out of the 65 strains could significantly (P?0.05) improve the plant shoot dry mass of the host comparing to that of non-inoculation treatment. And the isolates obtained a total of 9,25 and 26 genotypes of ARDRA, IGS-RFLP and CACAI (Cluster Analysis of Combined ARDRA and IGS-RFLP) respectively which showed a high genetic diversity. According to the 16S rDNA based phylogenetic tree, the strains were classified into two distinct clusters related to genera Rhizobium and Agrobacterium. To further clarify the genetic relationship, multilocus sequence analysis (MLSA) of concatenated sequences of three housekeeping genes (atpD, glnll and recA) was constructed.65 strains were clustered into 9 clades representing R. anhuiense, R. fabae, R. vallis, R. sophorae, A. tumefaciens and four potential new species related to Rhizobium and Agrobacterium. R. anhuiense (representing 41 isolates) was the dominant species in this area. An amount of 4 and 3 genotypes of nifH and nodC were distinguished from the nine species. And the incongruence of housekeeping genes and symbiotic genes indicated the faba bean rhizobia in this area had undergone lateral gene transfer and gene recombination.Overall, faba bean rhizobial strains in Sichuan area had a great genetic diversity representing R. anhuiense, R. sophorae, R. mesosinicum, R. fabae, R. vallis, A. tumefaciens and 4 potential new species related to Rhizobium and 2 related to Agrobacterium, and the dominant symbiomints of faba bean in Sichuan is R. anhuiense. The distribution of faba bean rhizobia in different parts of Sichuan had a common as well as regional feature. The special geographic and environmental characters and lateral gene transfer of symbiotic genes made the faba bean rhizobia more diverse in this area.