DNA fingerprinting of soybean rhizobia isolated from nodules of soybeans inoculated with biofertilizer NA7 in Nam Moub subdistrict, Nan province

Abstract

Soybean rhizobia nodulate soybean roots and convert atmospheric nitrogen to ammonia which is utilized by soybeans for growth. The first criterion in the development of soybean rhizobium biofertilizers is selection of soybean rhizobia strains which could compete with indigenous soybean rhizobia in nodulating soybean roots. There is no DNA fingerprints quality control in the production of rhizobium biofertilizers available in the market. Rhizobium biofertilizer NA7 had previously been produced at the lab scale by mixing strain NA7 with peat at the ratio of 2x10 [superscript 8] cells per gram. Soybean seeds cv. CM60 mixed with the biofertilizer were planted in a 15x24 m[superscript 2]experimental plot in 2007/2008 in Nam Moub subdistrict, Nan province. The aim of the thesis is to determine nodulation efficiency of NA7 by isolating bacteria from root nodules of soybean plants after one month cultivation in the experimental plot. DNA fingerprints of the isolates were obtained by RAPD-PCR using either RPO1 or CRL-7 as the primer. Out of the 198 root nodule isolates, 147 were fast-growers and 51 isolates were slow-growers. Since soybean rhizobium strain NA7 was a slow-grower, DNA fingerprints of the 51 slow-growing isolates were obtained. Comparisons of DNA fingerprints showed strain NA7 in the biofertilizer nodulated 13 out of the 51 isolates which made up 6.6% of nodule occupancy. The average number of soybean rhizobia in soil samples from the experimental plot in Nam Moub subdistrict was determined by the Most Probable Number (MPN) to be 4x10[superscript 4] cells per gram soil. Seven soybean rhizobium strains (NA7, NM22-8, NM22-11, NM22-13, NM22-15, NM22-25, and NM22-30) were identified by polyphasic taxonomy. Strain NM22-25 was found to be identical to strain NA7 which was found to be Bradyrhizobium elkanii. Strains NM22-11, NM22-13, and NM22-15 were found to be Bradyrhizobium elkanii while strains NM22-8 and NM22-30 were found to be Bradyrhizobium japonicum.