Bacillus subtilis reprograms host transcriptome and rhizosphere microbiome via systemic signaling to confer alkaline stress tolerance in garden pea

Soil alkalinity severely impairs legume growth. While plant-associated microbiomes can ameliorate stress, the role of Bacillus subtilis under alkaline stress remains unclear in garden pea. In this study, alkaline stress reduced Fe, Mn, and N content, photosystem efficiency, and biomass accumulation. Inoculation with B. subtilis restored micronutrient balance and improved photosynthetic parameters across genetically diverse pea cultivars. The split-root assay demonstrated that BS inoculation induces systemic signaling, which underlies alkaline tolerance in pea. Furthermore, co-culture experiments showed increased growth of both Rhizobium leguminosarum and B. subtilis , pointing to their complementary interactions that promote mutual fitness under alkaline stress. RNA seq analysis identified 958 and 1134 differentially expressed genes upregulated and downregulated, respectively, in the roots inoculated with B. subtilis under alkaline conditions. The upregulated genes were mostly involved in the sugar-mediated symbiotic association ( SWEET , GLUT ), pH homeostasis ( cation/H+ exchanger , ATPase ), and nutrient assimilation ( Ammonium transporter , Zn/Fe permease ). Alkalinity mitigation was further assisted by increased expression of oxidoreductases, ferritin-like protein, and other redox-related genes, reflecting improved antioxidant defense and Fe storage under high pH conditions. Amplicon sequencing revealed lower richness and diversity of rhizosphere bacterial and fungal communities under alkaline stress. However, B. subtilis inoculation led to enrichment of beneficial taxa, such as Pseudomonas, Pseudorhizobium, Pseudallescheria, and Chaetomium, which may act as helper microbes to support nutrient cycling and enhance pea survival under alkalinity. These findings suggest that B. subtilis functions as a microbiome inducer, working synergistically with helper microbes to promote resilience in garden peas under alkaline stress.