Presence of natural variants of Bradyrhizobium elkanii and Bradyrhizobium japonicum and detection of Bradyrhizobium yuanmingense in Phitsanulok province

Soybean rhizobia are Gram negative bacteria that fix nitrogen in root nodules of soybeans. Selection of soybean rhizobia from present and previous soybean cultivation areas is one way to obtain efficient strains for inoculant production. At present, information on the diversity of soybean rhizobia in Thailand is scarce. The experiments aimed to isolate and characterize soybean rhizobium strains in soils from 16 subdistricts in Phitsanulok province, Thailand. Host trapping method was used to isolate bacteria from root nodules of 5 soybean cultivars grown in soils from the 16 subdistricts. Identical RAPD-PCR fingerprints revealed the 202 slow-growing isolates consisted of 121 strains. Authentication tests using 5 soybean cultivars showed all the 121 slow-growing strains had nodulated soybean roots. Four types of colony morphology on yeast extract mannitol containing congo red agar plates were obtained for all the isolated strains. Bromothymol blue (BTB) reactions on BTB agar plates revealed two types of slow-growing soybean rhizobia. Type 1 secreted alkali products after 5-day incubation, and acidic products upon prolonged incubation for another 5 days. Type 2 secreted only alkali products after 5- and 10-day incubation. Results from nucleotide sequences of 16S rDNA revealed 7, 6, and 2 strains of Bradyrhizobium elkanii, B. japonicum, and B. yuanmingense, respectively. The B. elkanii and B. japonicum strains were found to be natural variants with different RAPD-PCR fingerprints. This study is the first report on the findings of B. yuanmingense as well as natural variants of slow-growing soybean rhizobia in Thailand.