Autoregulation of nodulation interferes with impacts of nitrogen fertilization levels on the leaf-associated bacterial community in soybeans

The diversities leaf-associated bacteria on nonnodulated (Nod^-), wild-type nodulated (Nod⁺), and hypernodulated (Nod⁺⁺) soybeans were evaluated by clone library analyses of the 16S rRNA gene. To analyze the impact of nitrogen fertilization on the bacterial leaf community, soybeans were treated with standard nitrogen (SN) (15 kg N ha^-1) or heavy nitrogen (HN) (615 kg N ha^-1) fertilization. Under SN fertilization, the relative abundance of Alphaproteobacteria was significantly higher in Nod^- and Nod⁺⁺ soybeans (82% to 96%) than in Nod⁺ soybeans (54%). The community structure of leaf-associated bacteria in Nod⁺ soybeans was almost unaffected by the levels of nitrogen fertilization. However, differences were visible in Nod^- and Nod⁺⁺ soybeans. HN fertilization drastically decreased the relative abundance of Alphaproteobacteria in Nod^- and Nod⁺⁺ soybeans (46% to 76%) and, conversely, increased those of Gammaproteobacteria and Firmicutes in these mutant soybeans. In the Alphaproteobacteria, cluster analyses identified two operational taxonomic units (OTUs) (Aurantimonas sp. and Methylobacterium sp.) that were especially sensitive to nodulation phenotypes under SN fertilization and to nitrogen fertilization levels. Arbuscular mycorrhizal infection was not observed on the root tissues examined, presumably due to the rotation of paddy and upland fields. These results suggest that a subpopulation of leaf-associated bacteria in wild-type Nod⁺ soybeans is controlled in similar ways through the systemic regulation of autoregulation of nodulation, which interferes with the impacts of N levels on the bacterial community of soybean leaves.