Natural food flavour (E)-2-hexenal, a potential antifungal agent, induces mitochondria-mediated apoptosis in Aspergillus flavus conidia via a ROS-dependent pathway

Natural food flavour (E)-2-hexenal, a green leaf volatile, exhibits potent antifungal activity on Aspergillus flavus, but its antifungal mechanism has not been fully elucidated. In this study, we evaluated (E)-2-hexenal-induced apoptosis in A. flavus conidia and explored the underlying mechanisms of action. Evidence of apoptosis in A. flavus conidia were investigated by methods including fluorescent staining, flow cytometry, confocal laser scanning microscope, and spectral analysis. Results indicated that 4.0 μL/mL (minimum fungicidal concentration, MFC) of (E)-2-hexenal application induced early markers of apoptotic cell death in A. flavus conidia with a rate of 38.4% after 6 h exposure. Meanwhile, typical hallmarks of apoptosis, such as decreased mitochondrial membrane potential (MMP), activated metacaspase activity, fragmented DNA, mitochondrial permeability transition pore (MPTP) opening and cytochrome c (Cyt C) release from mitochondria to the cytosol were also confirmed. Furthermore, intracellular ATP levels were reduced by 63.3 ± 3.6% and reactive oxygen species (ROS) positive cells increased by 31.1 ± 3.1% during A. flavus apoptosis induced by (E)-2-hexenal. l-Cysteine (Cys), an antioxidant, could strongly block the excess ROS generation caused by (E)-2-hexenal, which correspondingly resulted in a significant inhibition of MPTP opening and decrease of apoptosis in A. flavus, indicating that ROS palys a pivotal role in (E)-2-hexenal-induced apoptosis. These results suggest that (E)-2-hexenal exerts its antifungal effect on A. flavus conidia via a ROS-dependent mitochondrial apoptotic pathway. •(E)-2-hexenal-induced mitochondria-mediated apoptosis in A. flavus was demonstrated.•Excess ROS was generated during (E)-2-hexenal-induced A. flavus conidia apoptosis.•Elimination of ROS reduced MPTP opening and prevented apoptosis in A. flavus conidia.•(E)-2-hexenal induced apoptosis in A. flavus conidia via a ROS-dependent pathway.