Multilocus sequence analysis of genes in soybean rhizobia isolated from Nongkula Subdistrict, Phitsanulok province

Abstract

Soybean rhizobia are bacteria in soybean root nodules which are able to convert atmospheric nitrogen to ammonia for soybeans to assimilate for growth. At present, there has been an annual decline in soybean cultivation areas and Thailand imports approximately 85% of local soybean consumption resulting in a trade deficit and in an opportunity loss for sustainable maintenance of soil quality. The aims of this research were to identify slow-growing soybean rhizobia from root nodules of soybean cultivar Chiangmai 2 grown in a 15 x 24 sq.m. experimental plot in Nongkula subdistrict, Phitsanulok province. Methods included RAPD-PCR fingerprinting with either RPO1 or CRL-7 as the primer, grouping slow-growing bacterial isolates with identical RAPD-PCR fingerprints into the same strains, constructing dendrograms from RAPD-PCR fingerprints, and identification of 5 selected soybean rhizobia by Multilocus Sequence Analysis (MLSA) of 16S rDNA, dnaK, nifH, glnII and recA. Experimental results showed 116 slow-growing bacterial isolates were obtained. Identical RAPD-PCR fingerprints showed 116 slow-growing bacterial isolates were 43 strains. Authentication tests with soybean seeds (Glycine max cv. CM2, CM60, ST1, ST2, ST3, SJ4, SJ5 and Sri Samrong1) revealed all the 43 strains were soybean rhizobia. BLAST results of glnII revealed the 5 soybean rhizobial strains could be grouped into two groups with strains NKL09216, NKL09231, NKL09666 and NKL09693 were found to be Bradyrhizobium yuanmingense while strain NKL09273 was found to be B. elkanii. MLSA using glnII yielded the same results as obtained from the BLAST program while MLSA from dendrograms constructed from sequences of the remaining four genes and concatenated sequences of the 5 genes could not identify the 5 soybean rhizobial strains into different species. They were found to be related to Bradyrhizobium elkanii, B. japonicum, B. liaoningense, and B. yuanmingense.