Combined Effect of Bacillus subtilis and Arbuscular Mycorrhizal Fungus Rhizophagus irregularis on Rhizosphere Microflora of Achnatherum splendens Under Drought Stress

The efficacy of microbial inoculants in enhancing plant drought tolerance is context-dependent, varying with drought severity and the dynamic interactions within the plant–soil microbe system. We conducted a two-factor pot experiment using Achnatherum splendens under three moisture regimes (well-watered (control), moderate drought, and severe drought) and four inoculation treatments (sterile inoculum (control), Bacillus subtilis (BS), arbuscular mycorrhizal fungi (AMF), and their co-inoculation (MIX)). The resulting 12 treatment combinations were each replicated four times. Drought stress significantly reduced both shoot and root biomass, disrupted redox homeostasis by altering antioxidant enzyme activities and osmolyte accumulation, and induced pronounced shifts in rhizosphere microbial community composition and structure. Microbial inoculation modified osmolyte accumulation and antioxidant enzyme activity, with effects varying in magnitude and direction across moisture regimes and physiological indicators, and was generally associated with elevated soil nutrient availability and extracellular enzyme activities, though the magnitude of these effects was modulated by moisture regime. The MIX treatment showed the strongest positive responses for a subset of plant physiological and soil biochemical indicators. Bacterial and fungal co-occurrence networks exhibited divergent structural responses to inoculation and drought, with network complexity (e.g., connectivity and modularity) differentially shaped by both factors. Mantel tests and partial least squares path modeling revealed that under well-watered and moderate drought conditions, soil physicochemical properties were the primary drivers of microbial alpha and beta diversity; in contrast, under severe drought, osmotic adjustment emerged as the dominant physiological determinant of biomass maintenance. Collectively, these results reveal that the functional outcomes of microbial inoculation are contingent upon abiotic context, particularly drought intensity. This study provides a mechanistic basis and preliminary empirical evidence for subsequent field validation and further development of moisture-matched microbial intervention strategies for the ecological restoration of drought-vulnerable A. splendens ecosystems.