Fe3+ Promotes Biofilm Formation and Biocontrol Efficacy of Bacillus amyloliquefaciens GZY63 via Transcriptomic and Metabolic Reprogramming

Camellia oleifera , an important woody oil crop, is highly susceptible to anthracnose caused by Colletotrichum spp. Bacillus amyloliquefaciens GZY63, an endophytic bacterium from fruits of Ca . oleifera , exhibits strong potential as a biocontrol agent. This study investigated the influence of ferric iron (Fe 3+ ) on the functional traits of GZY63, including biofilm formation, motility, growth, and antifungal activity. Fe 3+ supplementation significantly drove a concentration-dependent increase in biofilm biomass, reaching a plateau at 100 μM with a four-fold enhancement compared to the control. Transcriptomic profiling revealed extensive Fe 3+ -responsive reprogramming, affecting approximately 58% of expressed genes, with key biofilm matrix genes such as tasA and sipW upregulated by 194- and 88-fold, respectively. Metabolomic analysis further identified 1,462 differentially accumulated metabolites, highlighting Fe 3+ -driven shifts in metabolic pathways associated with energy metabolism and the induction of antimicrobial compounds (e.g., resveratrol and posaconazole) that support robust biofilm formation. Consistent with these molecular changes, Fe 3+ -enhanced biofilm formation promoted stable colonization of host tissues and significantly reduced the disease incidence (DI) of anthracnose in greenhouse assays using the susceptible cultivar CL18. Together, our results demonstrate that Fe 3+ acts as a key environmental cue that coordinately regulates transcriptional and metabolic networks underlying biofilm formation in B. amyloliquefaciens , thereby strengthening biofilm-mediated biocontrol activity. This study provides mechanistic insight into iron-dependent biofilm regulation and highlights nutrient modulation as an effective strategy to optimize Bacillus biofilms for plant protection.