Bacillus velezensis and Sinorhizobium meliloti Form a Functional Complementary Alliance to Alleviate the Impact of Salt Stress on Alfalfa Growth

Abstract

Salt stress is a prevalent abiotic stress worldwide, which markedly inhibits crop growth and triggers yield losses. In this study, salt-tolerant plant-growth-promoting rhizobacteria of Cerasus humilis—Bacillus pumilus and B. velezensis, which possess nitrogen-fixing, phosphate-solubilizing, and indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing traits—were co-inoculated with Sinorhizobium meliloti. The effects of these bacterial combinations on alfalfa (Medicago sativa L.) were systematically evaluated during seed germination and plant growth under salt stress simulated using NaCl, Na2SO4, NaHCO3, and Na2CO3 at varying intensities. The results showed that under salt stress, inoculation significantly increased the seed germination rate by 11.33–41.33%. Pot experiments further revealed that inoculation significantly enhanced symbiotic nitrogen fixation efficiency in alfalfa and effectively maintained K+/Na+ homeostasis (K+ concentration increased by 4.23–125.34%, while Na+ concentration decreased by 7.15–102.09%). Concurrently, inoculation upregulated antioxidant enzyme activities and promoted the accumulation of non-enzymatic antioxidants, thereby significantly reducing reactive oxygen species levels. Moreover, inoculation substantially increased the content of osmoregulatory substances such as proline and soluble protein; proline accumulation surged more than fivefold (39.97–551.05%) compared with the non-inoculated control, effectively alleviating cellular dehydration. Through these multi-pathway regulations mediated by Bacillus sp. and S. meliloti, the inhibitory effect of salt stress on alfalfa growth was significantly mitigated, with dry weight increasing by 45.9–92.4%. Principal component analysis indicated that inoculation with the B. velezensis–S. meliloti microbial combination was the most promising strategy for promoting alfalfa growth and alleviating salt stress. The results provide a theoretical basis for developing microbial fertilizers and establishing alfalfa pastures in saline lands, thereby promoting their sustainable utilization.