Isolation and characterization of Bradyrhizobium elkanii as a root nodule symbiont of red sword bean Canavalia gladiata var. gladiata

ABSTRACT Bacterial strains were isolated from root nodules of red sword bean (Canavalia gladiata var. gladiata) cultivated in Shizuoka, Japan, in order to elucidate the taxonomy of the symbionts. Of the 52 bacterial isolates, 28 strains were identified as the genus Bradyrhizobium and 10 as Rhizobium, based on the nucleotide sequences of 16S rRNA genes. Nine Bradyrhizobium isolates, which were phylogenetically selected from the 28 strains, exhibited nucleotide sequences of ITS regions that were 99.9% or 100% identical with known B. elkanii strains. These nine strains shared more than 70% similarity with B. elkanii USDA 76 T or USDA 94 in DNA-DNA hybridization analysis, indicating that the strains are B. elkanii. In a nodule formation experiment using red sword bean seeds treated with mercury chloride solution, all of the plants which were inoculated with each of the B. elkanii strains (the isolate TI06 or MI08, or the type strain USDA 76 T) formed round-type root nodules (234–664 nodules per plant), while no nodules were observed in control plants that were cultivated without inoculating bacterial strains. The bacterial strains, which were isolated from the obtained nodules, exhibited nucleotide sequences of the ITS regions that were identical to those of the corresponding inoculated strains. The root nodules formed in the experiment exhibited acetylene-reducing activity, suggesting the nitrogen-fixation activity of the nodules. We thus conclude that B. elkanii is a root nodule symbiont of red sword bean.