Diversity and distribution of indigenous soybean-nodulating rhizobia in the Okinawa islands, Japan

We investigated the genetic diversity and field distribution of indigenous soybean-nodulating rhizobia in the Okinawa islands, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of their 16S rDNA regions and 16S - 23S rDNA internal transcribed spacer (ITS) regions. We collected rhizobia from four sites on Okinawa Island, one on Miyako Island and one on Ishigaki Island, isolating 360 strains from soybean cultivars with three Rj-genotypes, Akishirome or Bragg (non-Rj), CNS (Rj(2)Rj(3)) and Hill or Fukuyutaka (Ri(4)), planted with soil from each field site. Phylogenies of all isolates were analyzed by PCR-RFLP with Bradyrhizobium and Sinorhizobium USDA strains as reference strains. The PCR-RFLP analysis of 16S rDNA and the ITS regions revealed the dominance of Bradyrhizobium elkanii strains in acidic Okinawa soils, Miyako soil and Ishigaki soil, and the dominance of Sinorhizobium fredii strains in alkaline Okinawa soils. To detect phaAB genes, which are components of K+ efflux systems similar to Na+/H+ antiporters involved with alkaliphilic traits, we conducted Southern hybridization with a phaAB gene probe. Specific signals were observed in all representative S. fredii strains isolated from Okinawa alkaline soils, but not in any representative Bradyrhizobium strains.