Water availability modulates how Bacillus subtilis promotes alfalfa growth under salinity stress: coordinated physiological and rhizosphere responses

Background Soil water availability can influence plant responses to salinity and may determine the effectiveness of plant growth-promoting rhizobacteria (PGPR). However, how water availability modulates PGPR-mediated plant and rhizosphere responses under salinity stress remains unclear. A pot experiment was conducted to evaluate alfalfa (Medicago sativa L.) responses to Bacillus subtilis inoculation under two salinity levels (3.0 and 4.5 g kg − 1 ) and two soil moisture regimes (55–70% and 70–85% of field capacity). Results B. subtilis increased alfalfa dry biomass by 10.31-188.43% relative to the corresponding non-inoculated treatments. Inoculation increased catalase activity and reduced proline and malondialdehyde accumulation, indicating alleviation of physiological stress. These responses were accompanied by lower rhizosphere electrical conductivity and NH 4 + -N concentrations and generally higher soil organic matter, total nitrogen, and NO 3 − -N availability. Activities of urease, glutamine synthetase, nitrate reductase, and nitrogenase also increased following inoculation. Bacterial α-diversity did not differ significantly among treatments, whereas community structure varied with water and salinity conditions. Redundancy analysis identified nitrate reductase activity, total nitrogen, electrical conductivity, NO 3 − -N, and NH 4 + -N as important variables associated with bacterial community structure. Exploratory PLS-SEM further indicated that reduced salinity and improved rhizosphere nutrient conditions were closely associated with enhanced alfalfa growth. The most favorable overall response occurred at 3.0 g kg − 1 salinity and 70–85% field capacity. Conclusions B. subtilis improved alfalfa performance under combined water and salinity stress, but its effectiveness depended on soil water availability and salinity severity. Coordinating PGPR application with appropriate water management may therefore improve alfalfa production in salt-affected environments.