The clovers that comprise Trifolium genus are naturalized in the subtropical and tropical zones in China. They are valuable bioresources as important green manures and pasture grass, which contribute biologically fixed dinitrogen (N_2) and provide nutrition to farming systems. However, there are very few effective strains available for inoculant production and there is little information available about symbiotic rhizobia in Chinese legume clover root nodules. In this study, 139 root nodule bacteria were isolated from two clover species (Trifolium repens and Trifolium pretense) growing in the subtropical and temperate regions of China, 16S rRNA gene sequence analysis, BOX-PCR, whole cell protein SDS-PAGE, and nodulation tests were performed to characterize these strains.The results showed that phenotypic and genetic diversities among 139 isolates were large, with 83 protein patterns and 66 BOXAIR profiles, respectively. The rhizobial strains were first divided into two large phenotypic protein groups. The sequencing strains representing the two groups were related to Rhizobium leguminosarum USDA2370~T and R. sophorae CCBAU03386~T and had 99.6%-100% similarities. The phylogeny specificity of the rhizobia from clover was elucidated, while showed a large variation in the fingerprints of the phenotypes and genotypes and genetic diversity was high (revealed by Shannon diversity index, H'). The rhizobial isolates from subtropical regions, such as Anhui Province, Yunnan Province and Hubei Province, had higher diversities than those from temperate areas, such as Hebei Province and Shanxi Province, which could be used to identify rhizobial strains from clover and screen efficient inoculum strains. A number of diverse rhizobial strains had been identified and a pool of currently available clover rhizobial strains were increased. This would ultimately increase the likelihood of identifying more efficient strains suited for developing a successful inoculation strategy for the production of white clover.
