Bacillus subtilis reprograms the host transcriptome and rhizosphere microbiome with effects consistent with systemic responses to alkaline stress in garden pea

Soil alkalinity severely limits legume growth, but the role of Bacillus subtilis in alkaline stress tolerance remains unclear in garden pea. We found that multiple garden pea genotypes inoculated with B. subtilis under alkaline stress showed host-specific improvements in growth parameters. Mechanistic analysis conducted on Sugar Snap showed improved nodulation, mineral status, and photosystem efficiency, while split-root assays showed responses consistent with systemic effects of B. subtilis in alkaline tolerance. Further, FeEDDHA partially reduced alkaline stress symptoms but did not fully restore nodulation. In contrast, B. subtilis increased rhizosphere Fe-chelating activity and improved nodulation, leading to stronger symbiotic recovery than inorganic Fe alone. This suggests that factors associated with B. subtilis inoculation, beyond Fe availability alone, may contribute to the observed recovery of nodulation. This is further supported by in vitro co-culture experiments showing enhanced growth of R. leguminosarum in the presence of B. subtilis under alkaline conditions, indicating potential microbial compatibility for coping with stress. RNA-seq analysis identified 958 upregulated and 1,134 downregulated genes in roots inoculated with B. subtilis under alkaline conditions. The upregulated genes were mostly involved in the sugar-mediated symbiotic association (SWEET and GLUT), pH homeostasis (cation/H+ exchanger and ATPase), and nutrient assimilation (Ammonium transporter and Zn/Fe permease). Microbial community analysis revealed that B. subtilis significantly altered bacterial alpha diversity under alkaline stress, whereas fungal alpha diversity remained unaffected. Further, B. subtilis reshaped the rhizosphere microbial community and enriched taxa such as Pseudomonas, Pseudorhizobium, and Chaetomium, which were potentially associated with responses to alkaline stress. Taken together, microbial interventions such as B. subtilis offer an effective strategy to boost legume tolerance to alkaline soils.