Search

The analysis of nod genes and 16S rRNA gene regions, Nod factors, and nodulation abilities of Brady rhizobium strains isolated from tropical Thai Vigna species is reported. A total of 55 Bradyrhizobium strains isolated from two cultivated and six wild Vigna species growing in central and northern Thailand were evaluated. Thai Vigna spp. Bradyrhizobium strains showed higher levels of nod gene RFLP diversity compared with Thai soybean Brady rhizobium strains or temperate strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Analysis of the 16S rRNA gene region using selected strains also suggests a high genetic diversity of the Thai Vigna-Bradyrhizobium association. Based on thin-layer chromatography analysis, Nod factors produced by tropical Thai Vigna spp. Brady rhizobium strains are more diverse than temperate Japanese and US strains of B. japonicum and B. elkanii. Thai Vigna spp. Bradyrhizobium strains showed variation in nodulation ability and affinity, estimated by the number of normal nodules versus green nodules in an inoculation study. There are some Bradyrhizobium-host combinations that could not form any nodules, suggesting that some genetic differentiation has evolved in their host range. However, most of the Thai Vigna spp. Bradyrhizobium strains formed nodules on the cultigens soybean (Glycine max), mungbean (Vigna radiata), azuki bean (Vigna angularis), and cowpea (Vigna unguiculata). This is the first study on Bradyrhizobium strains associated with a range of cultivated and wild Vigna and reveals that these Bradyrhizobium strains are diverse and may provide novel sources of useful variation for the improvement of symbiotic systems.

To elucidate the phylogenetic relationships between Thai soybean bradyrhizobia and USDA strains of Bradyrhizobium, restriction fragment length polymorphism (RFLP) analysis using the nifDK gene probe and sequencing of the partial 16S rRNA gene were performed. In our previous work, Thai isolates of Bradyrhizobium sp. (Glycine max) were separated clearly from Bradyrhizobium japonicum and Bradyrhizobium elkanii based on the RFLP analysis using the nodDYABC gene probe. RFLP analysis using the nifDK gene probe divided 14 Thai isolates and eight USDA strains of B. japonicum into different groups, respectively, but categorized into the same cluster. All of seven strains within these Thai isolates had the same sequence of the partial 16S rRNA gene, and it was an intermediate sequence between those of B. japonicum USDA 110 and B. elkanii USDA 76T. Furthermore, three USDA strains of B. japonicum, USDA of (B. japonicum ATCC 10324T), USDA 115 and USDA 129, had the same partial 16S rRNA gene sequence that seven Thai isolates had. These results suggest that Thai isolates of Bradyrhizobium sp. (Glycine max) are genetically distinct from USDA strains of B. japonicum and B. elkanii, but also indicate a close relationship between Thai isolates and USDA strains of B. japonicum.

To determine the taxonomic relationship between Thai soybean bradyrhizobia and soybean bradyrhizobia from other regions, a total of 62 Bradyrhizobium strains were isolated in Thailand. The genetic diversity of the strains was examined with reference to 46 Japanese and 15 USDA strains. The degree of sequence divergence in and around common nod gene regions of the 123 strains was estimated by restriction fragment length polymorphism analysis using the Bradyrhizobium japonicum USDA 110 common nodDYABC gene probe. The phylogenetic grouping of the strains resulted in four major clusters. Cluster 1 comprised the Japanese and USDA strains, which originated in temperate regions, whereas clusters 3 and 4 comprised the tropical Thai strains. Cluster 1 strains comprised the DNA homology groups I and Ia, and hence, were classified as B. japonicum. Cluster 2 strains were in the DNA homology group II, and hence, were classified as Bradyrhizobium elkanii. Clusters 3 and 4 strains, however, did not correspond to any known DNA homology groups. These results indicate that Thai soybean bradyrhizobia are distantly related to B. japonicum and B. elkanii.