Prolonged biocontrol priming sensitizes Aspergillus flavus to cinnamaldehyde via metabolic trade-offs

The control of Aspergillus flavus and its highly carcinogenic secondary metabolite, aflatoxin B₁ (AFB₁), remains a major challenge in global agriculture and food safety. In this study, we propose a novel sequential hurdle strategy that combines priming with the biocontrol bacterium Pantoea agglomerans FL1 followed by low-dose cinnamaldehyde (CA) stress, and we further elucidate its underlying mechanisms. Phenotypic analyses revealed that prolonged FL1 priming for 7 days increased the susceptibility of A. flavus upon subsequent CA exposure. Although macroscopic radial growth remained unaffected, asexual sporulation and AFB₁ production significantly reduced. Data-independent acquisition proteomics demonstrated that this uncoupling is consistent with metabolic trade-offs hypothesis. Under compounded stress, prolonged-primed A. flavus exhibited proteomic patterns consistent with altered carbon utilization, including enrichment of proteins related to lipid remodeling and branched-chain amino acid metabolism, together with reduced abundance of aflatoxin biosynthetic proteins. In parallel, prolonged priming was associated with mitochondrial dysfunction and proteasome stress, which may drive the fungus into a state of oxidative exhaustion and structural fragility. Crucially, re-exposure assays confirmed that this prolonged biotic stress caused stable and cumulative suppression without inducing adaptive resistance. Collectively, these findings demonstrate that prolonged biocontrol priming depletes the stress-buffering capacity of A. flavus, rendering it metabolically vulnerable to subsequent mild chemical stressors. This provides a sustainable strategy for mitigating mycotoxigenic fungi in food systems.