The Study Of Genetic Diversity And Phylogeny Of Soybean Rhizobia In China

Fifty-two soybean rhizobia isolated from root nodules of 9 soybean cultivars of soil samples collected from seven different sites in China were studied by phenotypic characteristics, host specificity , 16S rDNA PCR-RFLP, 16S rDNA IGS PCR-RFLP, RAPD, REP-ERIC-PCR and DNA-DNA hybridization. They were compared with twenty-three type strains representing Sinorhizobium fredii, Bradyrhizobium japonicum and other Rhizobium type species. The diversity and phylogeny of fifty-two strains were carefully studied. The results indicated that twenty-nine strains were identified to belog to Sinorhizobium fredii and twenty-three strains to Bradyrhizobium japonicum within fifty-two strains tested. The results of phenotypic characteristics, plasmid patterns, host specificity, 16S rDNA PCR-RFLP, 16S rDNA IGS PCR-RFLP, RAPD and REP-ERIC-PCR revealed the high diversity within strains tested.Phenotypic characteristics results indicated that twenty-nine fast-growing strains could be distinguished with Sinorhizobium xinjiangensis CCBAU110 and twenty-three slow-growing strains with Bradyrhizobium elkanii USDA76 respectively. But little differences could be observed within fast-growing strains or slow-growing strains tested. Twenty-nine fast-growing strains were all grouped with Sinorhizobium fredii USDA205 and twenty-three slow-growing strains were all grouped with Bradyrhizobium japonicum USDA6 and USDA110. Similarities of some slow-growing strains were related with geographical sampling sites, the similarities of all other strains did not correlate with the geographical sampling site and soybean cultivars. All strains tested could be divided by host specificity into two types. One could nodulate the soybean cultivars tested and showed wider host range, the other only nodulate limited soybean cultivars. Plasmid could not be detected from twenty-three slow-growing strains and twenty-nine fast-growing strains were found containing 1-4 plasmids by Echardt's method. The correlation of plasmid type and host specificity were reported.Results of 16S rDNA PCR-RFLP and 16S rDNA completed sequence analysis revealed that twenty-nine strains of fast-growing rhizobia were clustered with Sinorhizobium fredii USDA205 and Sinorhizobium xinjiangensis CCBAU110. Similarly, twenty-three strains of slow-growing rhizobia were highly related with Bradyrhizobium japonicum and Bradyrhizobium liaoningensis. There were only 2bp differences between 16S rDNA completed sequence of strains BA1 and Bradyrhizobium liaoningensis.The results of IGS PCR-RFLP, RAPD and REP-ERIC-PCR revealed even greater diversities within fast-growing and slow-growing strains tested. Fourty clusters werefound using three enzymes in IGS PCR-RFLP analysis. While the results of RAPD and REP-ERIC-PCR fingerprinting revealed more diversities than IGS PCR-RFLP. There were distinct differences among IGS PCR-RFLP, RAPD, REP-ERIC-PCR. In dendrogram constructed using IGS PCR-RFLP, the strains tested were mainly grouped according to cultivars origins. They were correlated well with the geographical sites according to the analysis of RAPD and REP-ERIC-PCR. RAPD and REP-ERIC-PCR were more efficient in detecting minor differences among closely related strains tested.Results of DNA-DNA hybridization were corresponded with that of IGS PCR-RFLP, RAPD and REP-ERIC-PCR which confirmly strongly that all fast-growing strains tested should belong to the Sinorhizobium fredii and all slow-growing strains tested belong to Bradyrhizobium japonicum.