Screening of Bradyrhizobium ottawaense with High N₂O-reducing Activity from Soybean Nodules in Japan

Bradyrhizobium ottawaense has prospects as an environmentally friendly inoculant for soybean farming because of its higher N₂O reductase (N₂OR) activity than that of B. diazoefficiens. To examine high N₂O-reducing B. ottawaense, we performed a PCR anal-ysis of nosZ genes in 8,640 soybean nodules from 68 fields in Japan. Of 384 PCR-positive nodules, we obtained 90 isolates of bradyrhizobia with B. ottawaense-type nosZ, derived exclusively from 18 fields in Gunma and Osaka prefectures. Of 77 monophyletic isolates, 73 had significantly higher N₂OR activity than B. diazoefficiens USDA110. Another 13 isolates‍ ‍from Osaka were phylogenetically placed outside of the B. ottawaense clade with B. liaoningense or B. betae, 8 of which also exhibited significantly higher N₂OR activity than B. diazoefficiens USDA110. An anal-ysis of nopP gene sequences revealed amino acid sequence variations in the NopP effector protein among these high N₂O-reducing isolates, with the NopP_USDA122 type being one of the variations identified. The NopP-mediated symbiotic incompatibility of soybean host plants may eliminate nodulation by indigenous bradyrhizobia and facilitate inoculant nodulation to reduce N₂O emissions. Therefore, 90 isolates and their observed NopP types are‍ ‍potentially important resources for N₂O mitigation. Furthermore, the dense geographical map of Bradyrhizobium species based on Internal Transcribed Spacer-Restriction Fragment Length Polymorphisms (ITS-RFLP) of the 16S-23S rRNA gene from 8,640 nodules revealed the recent northward expansion of B. elkanii to central Japan potentially due to global warming. This change in indigenous soybean bradyrhizobia is important for application strategies of bradyrhizobial inoculants under field conditions.