Chemotaxonomy of some nitrogen-fixing bacteria isolated from the Rhizosphere of rice (oryza sativa L.) Grown in Thailand

Abstract

R15, R17 and R25 are strains of N₂-fixing bacteria isolated from the rhizosphere of rice grown at Rangsit adn Tapra during 1979-1981. To determine the genera of these bacteria, their biochemical properties were compared with standard reference strains Klebsiella oxytoca 1301 from Japan, Azospirillum lipoferum FS, A. lipoferum 34H, Pseudomonas H8 and P. KLH76 from the Philippines. The DNA base composition when determined by melting temperature (Tm) method and DNA sequence homology when analyzed by reassociation experiment indicated that the three unknown strains should be classified in two distinct groups; R1 5 and R1 7 were Klebsiella-liked (mol % G + C, 52- 56 %) but R25 was most fitted in Azospirillum sp. (mol % G + c, 69-71 %). Several biochemical properties together with growth condition and acetylene reduction activity under specific conditions also supported that R15 and R17 were Klebsiella-liked strains because of their ability to produce indole, to utilize malonate as sole carbon source and positive test to Voges-Proskauer reaction. As for R25, which requires biotin for growth and forms pink-colony when grown on nutrient agar plate whereas it forms yellow-colony on rich agar plate, together with the ability to grown in NFb semisolid medium supplied with 0.5% glucose in place of malate, thus R25 is identified as A. lipoferum strain according to all results previously mentioned. In addition, glucose was the preferential carbon source for growth and N₂-fixation under either aerobic or microaerobic condition of both K. R15 and K. R17 in contrast to A. R25 that malate was preferred for growth and N₂-fixation under microaerobic condition only. The other charcters of K. R15 and K. R17 were that they harbored at least one plasmid which were larger than the plasmid of A. R25. Both K.R15 arid K.R17 were SDS and salt tolerant and also resisted to ampicillin, kanamycin, streptomycin and sulfanilamide while A. R25 resisted to ampicillin and sulfanilamide only. These results also indicated that R15/R17 and R25 belong to the distinct genus.