New Method of Denitrification Analysis of Bradyrhizobium Field Isolates by Gas Chromatographic Determination of 15 N-Labeled N 2

To evaluate the denitrification abilities of many Bradyrhizobium field isolates, we developed a new 15 N-labeled N 2 detection methodology, which is free from interference from atmospheric N 2 contamination. 30 N 2 ( 15 N 15 N) and 29 N 2 ( 15 N 14 N) were detected as an apparent peak by a gas chromatograph equipped with a thermal conductivity detector with N 2 gas having natural abundance of 15 N (0.366 atom%) as a carrier gas. The detection limit was 0.04% 30 N 2 , and the linearity extended at least to 40% 30 N 2 . When Bradyrhizobium japonicum USDA110 was grown in cultures anaerobically with 15 NO 3 − , denitrification product ( 30 N 2 ) was detected stoichiometrically. A total of 65 isolates of soybean bradyrhizobia from two field sites in Japan were assayed by this method. The denitrification abilities were partly correlated with filed sites, Bradyrhizobium species, and the hup genotype.