Community shifts of soybean stem-associated bacteria responding to different nodulation phenotypes and N levels

The diversity of stem-associated bacteria of non-nodulated (Nod ⁺), wild-type nodulated (Nod⁺⁺) and hypernodulated (Nod ) soybeans were evaluated by clone library analyses of the 16S ribosomal RNA gene. Soybeans were dressed with standard nitrogen (SN) fertilization (15 kg N ha -1) and heavy nitrogen (HN) fertilization (615 kg N ha^-1). The relative abundance of Alphaproteobacteria in Nod soybeans (66%) was smaller than that in Nod and Nod soybeans (75-76%) under SN fertilization, whereas that of Gammaproteobacteria showed the opposite pattern (23% in Nod⁺ and 12-16% in Nod^- and Nod⁺⁺ soybeans). Principal coordinate analysis showed that the bacterial communities of Nod^- and Nod ⁺⁺ soybeans were more similar to each other than to that of Nod ⁺ soybeans under SN fertilization. HN fertilization increased the relative abundance of Gammaproteobacteria in all nodulation phenotypes (33-57%) and caused drastic shifts of the bacterial community. The clustering analyses identified a subset of operational taxonomic units (OTUs) at the species level in Alpha- and Gammaproteobacteria responding to both the nodulation phenotypes and nitrogen fertilization levels. Meanwhile, the abundance of Betaproteobacteria was relatively constant in all libraries constructed under these environmental conditions. The relative abundances of two OTUs in Alphaproteobacteria (Aurantimonas sp. and Methylobacterium sp.) were especially sensitive to nodulation phenotype and were drastically decreased under HN fertilization. These results suggested that a subpopulation of proteobacteria in soybeans is controlled in a similar manner through both the regulation systems of plant-rhizobia symbiosis and the nitrogen signaling pathway in plants.