Mustard Root Exudates Modulate Early <i>Bacillus</i> spp. Colonization Under Osmotic Stress

Successful colonization of plant roots is vital for optimizing rhizobacterial benefits, but osmotic stress can significantly impact bacterial colonization. In this study, mustard seedlings were grown under no stress and osmotic stress (20% PEG 6000) conditions, and root exudates (RE_C and RE_OS, respectively) were collected. We analyzed how osmotic stress alters root exudate metabolites, and their impact on motility, biofilm formation, hydrolytic enzyme production and changes in cell surface components of rhizobacteria Bacillus sp. MRD-17 and B. casamancensis MKS-6. Chemical analysis revealed that RE_OS contained higher levels of sugars, organic acids (salicylic and succinic), fatty acids (octadecanoic), and phenolics/flavonoids (quercetin, genistein, dihydrodaidzein). MKS-6 exhibited higher motility in medium supplemented with root exudates from osmotic-stressed plants (RE_OS) compared to control exudates (RE_C), whereas MRD-17 showed no significant motility towards root exudates but exhibited a larger colony diameter in RE_OS than RE_C. Both rhizobacteria formed biofilms and moved toward root exudates with higher cfu/mL in RE_OS. Osmotic stress and root exudates altered rhizobacterial cell surface components and significantly affected cell wall protein expression. Osmotic stress influenced the rhizobacterial production of cell-wall hydrolytic enzymes. Seedlings inoculated with the rhizobacteria showed increased defense enzyme activities (peroxidase and polyphenol oxidase). The changes in root exudate components were positively correlated with colonization parameters. These findings indicate that osmotic stress-induced modifications in mustard root exudates enhance colonization traits of the rhizobacteria, highlighting their potential for drought stress mitigation through bioformulation development.