Water Regime Determines Dose-Dependent Growth and Biomass Allocation Responses of Soybean Seedlings to Bacillus subtilis and Bacillus amyloliquefaciens

Water limitation during soybean (Glycine max (L.) Merr.) establishment restricts leaf expansion and root–shoot development, whereas microbial biostimulants may modify these responses only within particular moisture contexts. We tested whether a mixed-species suspension of Bacillus subtilis and Bacillus amyloliquefaciens produced water-regime-dependent dose responses in a controlled-environment tray-cell experiment. The seedlings received a single rhizosphere-directed application of 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.75 or 2.0 mL of undiluted inoculum per cell before exposure for 15 days to full irrigation (W100), 50% or 25% of the W100 replacement volume (W50 and W25), or no irrigation (W0). Each of the 44 treatment combinations comprised six independent biological replicates (264 cells). Two-way ANOVA detected significant water-regime, dose and interaction effects for most thermal, growth, biomass and allocation traits, with water regime as the dominant source of variation. Averaged across doses, relative to W100, W0 increased the leaf-surface temperature by 8.8% and reduced the root length by 44.3%, shoot length by 25.4%, stem diameter by 38.2%, leaf area by 72.3%, total dry mass by 20.1% and specific leaf area by 70.4% on average. The concurrent 17.3% increase in the leaf mass fraction indicated the retention of proportional leaf dry-matter investment despite strongly restricted surface expansion. The bacterial response was non-linear and water-regime-specific. Within W0, 0.05 mL increased root dry mass by 116.7% and total dry mass by 39.3% relative to the untreated W0 control. The highest integrated mean z-score occurred at 0.05 mL under W0, 0.2 mL under W25, 1.2 mL under W50 and 0.2 mL under W100. Correlation analysis identified coordinated trait covariation, whereas hierarchical clustering and principal component analysis separated a warm, low-expansion W0 phenotype from a cool, high-vigour W100 phenotype. These findings provide a quantitative experimental basis for selecting candidate pre-drought doses for seedling-stage screening. Validation across soils, application timings, formulation persistence and reproductive-stage yield are required before commercial recommendation.