Enhanced biological nitrogen fixation and nodulation in alfalfa through the synergistic interactions between Sinorhizobium meliloti and Priestia aryabhattai.

Nitrogen fertilizer is crucial for agricultural output. However, prolonged overuse has resulted in nitrate leaching and potential soil acidification. Research on microbial fertilizer has become essential to enhance soil conditions and minimize nitrogen fertilizer usage. In alfalfa cultivation, research on efficient compound microbial agents remains limited, therefore this study concentrates on the investigation of dual microbial combinations. In the screening process, black soil was utilized with alfalfa plants as samples to identify a strain of rhizobacteria, Sinorhizobium meliloti LMGL3-1, exhibiting nitrogen-fixing capabilities, and Priestia aryabhattai (Bacillus aryabhattai) YJHT21, demonstrating phosphorus-solubilizing abilities. In addition, they were able to produce indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid deaminase (ACCd). The S. meliloti strain was demonstrated to have the ability to symbiotically associate with the alfalfa variety Longmu 806, resulting in the formation of effective nodules containing leghemoglobin, thereby enabling the plants to thrive in the absence of nitrogen fertilizer application. Here, we discovered that the inoculation of phosphorus-solubilizing P. aryabhattai enhanced alfalfa growth and the nitrogenase activity of S. meliloti (P < 0.0001). Comparing with no nitrogen fertilizer control, the two-bacteria complex culture made an extreme increase in chlorophyll (P < 0.0001) of alfalfa. The protein content and dry weight of alfalfa were also increased (P < 0.01 and P < 0.001, respectively). Additionally, it also increased the total nitrogen content of the black soil. Moreover, the incorporation of P. aryabhattai resulted in a significant increase in flavonoid production within the root system of alfalfa plants (P < 0.0001). Consequently, under the influence of the inducer extracted from the root system of quantitatively analyzed plants, the rhizobacteria exhibited enhanced production of metabolites associated with the Nod factor cluster. This study demonstrates that the interaction between S. meliloti and P. aryabhattai significantly enhanced biological nitrogen fixation, providing a theoretical foundation for the development of eco-friendly biofertilizer as an alternative to chemical fertilizer.