Bacillus subtilis reprograms host transcriptome and rhizosphere microbiome via systemic signaling to confer alkaline stress tolerance 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 revealed that B. subtilis triggered systemic signaling underlying alkaline tolerance. Further, FeEDDHA partially reduced alkaline stress symptoms but did not fully restore nodulation. In contrast, B. subtilis enhanced siderophore availability and improved nodulation, leading to stronger symbiotic recovery than inorganic Fe alone. This indicates that nodulation recovery is driven primarily by B. subtilis-mediated stimulation of Rhizobium leguminosarum, not by Fe availability alone. This is further supported by in vitro co-culture experiments that showed increased growth of both R. leguminosarum and B. subtilis, pointing to their complementary interactions that promote mutual fitness under alkaline 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). Furthermore, B. subtilis reshaped the rhizosphere by restoring microbial community structure and enriching beneficial taxa such as Pseudomonas, Pseudorhizobium, and Chaetomium, which may act as helper microbes to promote pea survival under alkalinity. Taken together, microbial interventions such as B. subtilis offer an effective strategy to boost legume tolerance to alkaline soils.