Metagenome-mapping method discriminates native and inoculant populations of soybean bradyrhizobia in agricultural soil
1Graduate School of Life Sciences, Tohoku University, 2National Institute for Agro-Environmental Sciences
Legume productivity in an agricultural field could be improved by inoculation with effective rhizobia. However, field inoculation has often been unsuccessful because of the presence in soil with native strains that compete with the introduced inoculants. This is termed “competition problem” for successful use of rhizobial inoculants. Conventional methods to monitor the behavior of inoculants are time-consuming, because many rhizobial isolates from legume nodules should be tested for DNA polymorphisms. In this study, we developed a metagenome-mapping method that can discriminate indigenous and inoculant populations of soybean bradyrhizobia in agricultural soils. Two sets of inoculant mixtures, C110 and F110, were prepared from 65 and 55 isolates of USDA110 lineage with effective N2-fixation and N2O reductase gene of Bradyrhizobium dizoefficiense in Japan. In particular, F110 was composed of re-isolates from C110 via passage of soybean plants grown in an Andosol field of Tsukuba. In pots filled with the soil, soybeans were inoculated at 107 or 1010 cells per seed. Native bradyrhizobia were nodulated in uninoculated control. MiSeq analysis of bacteroid DNA from the root nodules generated about one million DNA sequence reads. The cleaned reads were mapped on concatenated genome sequences of B. diazoefficiens USDA110T, B. japonicum USDA6 T and B. elkanii USDA76 T by CLC Workbench. Redundancy matrix was calculated from the normalized redundancy for all genes on the reference genomes, and visualized by principal components analysis (PCA). Native populations of bacteroids were cleary descriminated from bacteroids of the inoculated treatments (C110 and F110), which moved towards the positions of inoculants C110 and F110 on PCA plot, respectively. Interstingly, the distance between F110 and bacteroids by F110 inoculation was very short on PCA plot rather than C110 series, suggesting successful breeding of F110 inoculant that adapts to the soil conditions.