Mixed Enterobacter and Klebsiella bacteria enhance soybean biological nitrogen fixation ability when combined with rhizobia inoculation

Biological nitrogen fixation is a key process for nitrogen geochemical cycling and plant‒microbe interactions. At present, little is known about the possible effects of nonnodulating microbes on rhizobia nodulation in soils. Here, we show that Enterobacter sp. Z1 and Klebsiella sp. Z2 significantly improved soybean growth and nitrogen fixation in the presence of Bradyrhizobium diazoefficiens USDA110. Soil metabolomic analysis showed that the application of mixed strains Z1 and Z2 significantly increased the levels of flavonoids, indoleacetic acid (IAA), salicylic acid (SA), and taurine in rhizosphere soil. The addition of IAA, SA, or taurine increased nitrogenase activity, nodule production, and chlorophyll content in soybean. Flavonoids were the determinant of the interaction between soybeans and rhizobia. Moreover, strains Z1 and Z2 produced an abundance of IAA and SA, thus increasing the expression of flavonoid synthesis-related genes and eventually increasing the accumulation of flavonoids. Both strains Z1 and Z2 exhibited two metabolic pathways of taurine, and they catalyzed taurine to produce acetyl-CoA, a key flavonoid synthesis substrate in soybeans. This study revealed the mechanism by which rhizosphere microorganisms mediated soybean-rhizobium interactions, thus promoting soybean nitrogen fixation.