Systematic investigation of aflatoxigenic Aspergillus flavus inhibition: Integrating essential oils-based formulation...

Aspergillus flavus is among the most resilient fungi, causing significant losses in the quantity and quality of agricultural food commodities. It often produces aflatoxin B₁ (AFB₁), a Class 1 human carcinogen that poses a serious threat to human and animal health. The present study investigated the potential of a chemically characterized essential oil-based formulation, ZTC (a synergistic mixture of Zingiber officinale (ZOEO), Trachyspermum ammi (TAEO), and Coleus aromaticus (CEO)) against the growth and aflatoxin B₁ production by the toxigenic species Aspergillus flavus. The ZTC formulation demonstrated in vitro and in situ antifungal effects (0.4 μL/mL) and aflatoxin inhibition (0.3 μL/mL) against A. flavus. At the cellular level, ZTC disrupted ergosterol biosynthesis, compromised membrane integrity, impaired mitochondrial potential, and altered redox homeostasis. Integrated transcriptomic and biochemical analyses indicated coordinated downregulation of glycolysis, the TCA cycle, ATP production, and sterol and fatty acid biosynthesis. This led to energy deficiency, membrane destabilization, induction of oxidative stress, and inhibition of aflatoxin B₁ biosynthesis by suppressing gene expression and disrupting key enzymes, thereby reducing AFB₁ at sublethal concentrations. The findings highlight the multi-target mechanism of action of the developed formulation, ZTC, against fungal growth and aflatoxin B1 contamination, and recommend it as a promising candidate for eco-friendly and sustainable antifungal agents.