Development of primers for identification of fast-or slow-growing bacteria isolated from soybean root nodules

Abstract

Soybean rhizobia which fix nitrogen in root nodules of soybeans could be grouped into three groups, the fast-growers, the slow-growers, and soybean rhizobia with variable generation times. Tradition method for detecting soybean rhizobia is time consuming. In this research multiplex PCR reaction will be used to detect the presence of slow-and fast-growers based on the information that fast-growers lack nodY and the sizes of nodD1 of fast slow-growers differ. In this research 525 bacterial isolates were obtained from root nodules of seven soybean cultivars grown separately in soil samples from 15 subdistricts in Phitsanulok province. Identical RAPD-PCR fingerprints using either RPO1 or CRL-7 as the primer of 105 isolated revealed they constituted 66 strains with 9 strains of fast-growers and 57 strains of slow-growers. Multiplex PCR using forward and reverse primers of nodY and nodD1 genes could be used to detect the presence of fast-and of slow-growing soybean rhizobia as follows: fast-growing soybean rhizobial DNA yielded either 1300 bp or a combination of 500, 700, and 1500 bp while slow-growing soybean rhizobia yieded either 340 bp, or 317 bp with 340 bp, or 317 bp with 340 bp and 657 bp, or 317 bp with 657 bp, or 340 bp with 657 bp. The developed multiplex PCR was found to be specific for soybean rhizobia because when DNAs of Agrobacterium tumefacians TISTR 507, Xanthomonas campestris TISTR 786 and Proteus vulgaris were used in the reactions, different or no PCR products were found. However, the developed multiplex PCR could not be used to distinguish certain groups of soybean rhizobia (fast-growing D11, D301, D384) and slow-growing soybean rhizobia (D291 and D481), because the 317 bp fragments formed in multiplex PCR reactions were found to have identical nucleotide sequence. In order to detect if the five strains are fast-or slow-growing soybean rhizobia, it is nessary to carry out PCR reactions using DNA of each of the five strains as target DNA. If no PCR product is obtained when nodY is used as the primers, or 1300 bp or a combination of 500, 700, 1300 bp is obtained when nodD is used as the primer, the target DNA belongs to a fast-growing soybean rhizobium. On the other band, if 340 bp PCR product is obtained when nodY is used as the primer, and 317 bp PCR product is obtained when nodD1 is used as the primer, the target DNA belongs to a slow-growing soybean rhizobium.