Diversity of soybean rhizobia in 16 subdistricts of Phitsanulok province

Abstract

Soybean rhizobia are Gram negative bacteria that fix nitrogen in root nodules of soybeans. At present, there is not much information on polyphasic taxonomy of soybean rhizobia in Thailand. The aim of the experiments is to isolate and characterize, by polyphasic taxonomy, soybean rhizobium strains in soils from 16 subdistricts in Phitsanulok province. Host trapping method was used to isolate bacteria from root nodules of 5 soybean cultivars grown in soils from the 16 subdistricts. A total of 340 isolates were purified and categorized into fast- and slow-growers based on their visible growth on yeast extract mannitol with 0.25 μg.ml-1 Congo red agar plates. The results indicated there were 138 and 202 isolates of fast- and slow-growers, respectively. Identical RAPDPCR fingerprints revealed the 202 slow-growing isolates consisted of 121 strains. Authentication tests employing 5 soybean cultivars showed all the 138 fast-growing isolates did not nodulate soybean roots. Thus, they were not fast-growing soybean rhizobia. On the contrary, all the 202 slowgrowing isolates were found to nodulate soybean roots. Therefore, they were slow-growing soybean rhizobia. Bromthymol blue (BTB) reactions on BTB agar plates showed there were two types of slowgrowing soybean rhizobia. Type 1 secreted alkali product(s) after 5-day incubation then secreted acidic product(s) upon prolonged incubation for another 5 days. Type 2 secreted only alkali product(s) after 10-day incubation. Results from nucleotide sequences of 16S rDNA of randomlyselected 20 slow-growing strains revealed 12 strains (STB8, STB119, STB120, STB147, STB173, STB176, STB179, STB185, STB220, STB238, STB245 and STB327) were related to B. ellkanii. 6 strains (STB30, STB54, STB67, STB96, STB250 and STB310) were closely related to Bradyrhizobium japonicum, and 2 strains (STB169 and STB264) were related to B. yuanmingense. The results indicated that nodY sequences and results on utilization/non-utilization of 95 carbon and nitrogen sources could not be used to identify soybean rhizobium strains. In addition, dendrograms constructed from DNA fingerprints of the 121 soybean rhizobium strains revealed genetic diversity. This research is the first report on the findings of B. yuanmingenseas as well as natural variants of soybean rhizobia in Thailand.