Biofilms of Bacillus subtilis as biostimulants for plant resilience to drought and flooding.

Microbial biofilms, composed of surface-attached microbial communities embedded in an extracellular polysaccharide matrix, enhance microbial resilience under environmental stress. This study investigates the role of biofilm formation by Bacillus subtilis strains in promoting stress tolerance in pak choi (Brassica rapa subsp. chinensis). We compared three strains, B. subtilis 3A1 (a biofilm-forming strain), 168 (a biofilm-deficient mutant derived from strain 3A1 harboring multiple domestication-associated mutations, notably in degQ, spo0F, sfp, and comI), and WMA1 (a native Taiwanese isolate), focusing on their ability to form biofilms on microplates and root surfaces, and their effects on plant tolerance to drought and flooding. Strains 3A1 and WMA1 exhibited significantly greater biofilm-forming ability and root colonization than strain 168. Both strains also expressed ACC deaminase activity, while strain 168 did not. Application of culture broths from 3A1 or WMA1 significantly improved plant survival and growth under drought stress or flooding stress compared to water, SYB medium, and strain 168 treatments. Following six days of drought with three days of re-irrigation, or six days of flooding, plants treated with strains 3A1 and WMA1 showed improved recovery and biomass. Physiological responses under abiotic stress were further analyzed by measuring chlorophyll fluorescence (Fv/Fm) and antioxidant enzyme activities. Pak choi treated with strains 3A1 and WMA1 maintained more stable Fv/Fm values under drought and flooding stress, indicating reduced damage to photosystem II. Moreover, catalase and superoxide dismutase activities were significantly elevated in these treatments, suggesting enhanced antioxidant defense mechanisms. We also evaluated the use of agricultural waste, particularly fruit pomace, as a sustainable culture supplement. Media enriched with fruit pomace enhanced the production of exopolysaccharides (EPS) and γ-polyglutamic acid (γ-PGA) by B. subtilis strains, which are key components of biofilm structure. Notably, γ-PGA is known for its excellent water retention capacity, which likely contributes to improved moisture availability and plant stress mitigation. Use of fruit pomace-based media further strengthening plant stress mitigation. In summary, B. subtilis strains 3A1 and WMA1 confer abiotic stress resilience in pak choi by forming root-associated biofilms, producing ACC deaminase, EPS, and γ-PGA, and enhancing antioxidant defenses while reducing damage to photosystem II. These effects are further amplified when the bacteria are cultured in fruit pomace-enriched media, highlighting the potential of integrating microbial biostimulants with agricultural waste to promote sustainable crop production and mitigate abiotic stress impacts.