Novel rhizobia exhibit superior nodulation and biological nitrogen fixation even under high nitrate concentrations

Legume-rhizobium symbiosis leads to the formation of nitrogen-fixing root nodules. However, externally applied chemical nitrogen fertilizers (nitrate and ammonia) strongly inhibit nodule formation and nitrogen fixation. Here, we isolated several rhizobial strains exhibiting a superior nodulation and nitrogen fixation with soybean at high nitrate concentrations. The nodulation of soybean symbiont Bradyrhizobium diazoefficiens USDA110 was significantly inhibited at 12.5 mM nitrate; however, three isolates (NKS4, NKM2 and NKTG2) were capable of forming nitrogen-fixing nodules, even at 20 mM nitrate. These isolates exhibited higher nodulation competitiveness and induced larger nodules with higher nitrogen-fixation activity than USDA110 at 5 mM nitrate. Furthermore, these isolates induced more nodules than USDA110 even in nitrate-free conditions. These isolates had a distant lineage within the Bradyrhizobium genus; though they were relatively phylogenetically close to Bradyrhizobium japonicum, their morphological and growth characteristics were significantly different. Notably, in the presence of nitrate, expression of the soybean symbiosis-related genes (GmENOD40 and GmNIN) was significantly higher and expression of GmNIC1 that is involved in nitrate-dependent nodulation inhibition was lower in the roots inoculated with these isolates in contrast with inoculation of USDA110. These novel rhizobia serve as promising inoculants for soybeans cultivated in diverse agroecosystems, particularly on nitrate-applied soils.