Isolation and characterization of diazotrophic bacteria from the surface-sterilized roots of some legumes

A total of 251 bacteria were isolated using a nitrogen-free, malate-containing, semi-solid media(JNFb)from the surface-sterilized roots of eight species of legumes：Arachis hypogea, Astragalus sinicus, Crotalaria juncea, Glycine max, Medicago sativa, Phaseolus vulgaris, Pisum sativum, Sesbania cannabina, Trifolium incaratum, Trifolium pratens, and Trifoloum repens. Thirty-one isolated bacteria showed an acetylene reduction activity(ARA)of 2.6 to 450 nmol h-1 culture-1 when grown in the JNFb media. No diazotroph was isolated from Crotalaria juncea, Sesbania cannabina, and Trifolium incarnatum. Analysis of the 16S rRNA gene sequence and physiological characteristics of the 31 isolates showed that these isolates were. Agrobacterium radiobacter, A. tumefaciens, Azospirillum lipoferum, Bradyrhizobium elkanii, Burkholderia cepacia, Frateuria aurantia, Klebsiella oxytoca, K. pneumoniae, Rhizobium gallicum, R. sp., Starkeya novella, and Xantobacter flavus. The isolates were classified into three groups by the pattern of ARA expression. Three representative strains of the A. lipoferum, X. flavus, and K. oxytoca groups were examined for the effect on the growth and the nitrogen content of red clover, kidney bean, and soybean. Inoculation of these plants with these strains had no effect on the growth and nitrogen content of all plants.