Integrated Phenotyping Reveals Trait-Specific Bacillus Modulation of Soybean Water-Deficit Responses

Abstract

Water deficit restricts soybean (Glycine max (L.) Merr.) performance by reducing leaf expansion, water status, carbon assimilation, photosystem II activity, and leaf optical properties. Here, soybean plants were grown under four fixed irrigation volumes corresponding to 100, 50, 25, and 0% of the substrate field-capacity reference volume (W100, W50, W25, and W0), with or without rhizosphere-applied Bacillus subtilis + Bacillus amyloliquefaciens before water restriction. Plant growth, relative water content (RWC), thermal and VIS–NIR–SWIR optical traits, photosynthetic pigments, A–PPFD and A–Ci gas exchange, chlorophyll a fluorescence, OJIP transients, microscopy, and principal component analysis were integrated. Two-way ANOVA showed that water availability was the dominant source of variation, whereas Bacillus and water × Bacillus effects were trait dependent. Bacillus produced a severity-dependent phenotype, with the clearest whole-plant benefit under W50: leaf area increased from 526.3 ± 32.9 to 620.9 ± 45.3 cm2 plant–1 and total biomass from 5.63 ± 0.23 to 6.89 ± 0.28 g plant–1; total chlorophyll also increased by 63.2–93.5% across Bacillus-treated water regimes. However, A–PPFD-derived Amax declined from 13.66 ± 1.57 μmol CO2 m–2 s–1 under W100 to near-zero values under W0 and W0B. In contrast, A–Ci traits were more responsive: Vcmax increased by 106.9% in W100B and showed a 74.7% numerical, nonsignificant increase in W50B (p = 0.058), while W0B showed higher Fv/Fm relative to W0 under severe dehydration. PCA separated treatments across the first two components (51.79%) mainly along a drought-severity axis and secondarily by Bacillus-associated photochemical and structural shifts. These findings indicate that rhizosphere-applied Bacillus acts as a trait-specific modulator of soybean drought physiology, with growth benefits most evident under intermediate water restriction.