Antifungal effects of pulsed light (PL) on Alternaria alternata isolated from cheese: cellular disruption and gene expression responses

Alternaria alternata is a mycotoxin-producing spoilage fungus frequently isolated from caciocavallo cheese samples. Its growth threatens cheese quality and safety in dairy systems. Pulsed light (PL) technology is a non-thermal method that has shown promise in controlling spoilage microorganisms, but its effects on fungal physiology and molecular response remain insufficiently understood. This study aimed to evaluate the antifungal potential of PL against A. alternata by investigating its effects on mycelial growth, spore germination, cellular damage, and gene expression. Fungal strains were cultured on cheese agar medium (CAM) and potato dextrose agar (PDA) and treated with 30 or 120 pulses using a Xenon X-100 system. Mycelial growth and spore inhibition were measured, and cell damage was visualized using confocal microscopy. Gene expression changes in 22 target genes involved in metabolism, stress response, and virulence were analyzed using RT-qPCR at several post-treatment time points (0, 1, 6, 24, 48 hpt ). PL significantly inhibited mycelial growth (22–37%) and spore germination (78–83%) in a dose-dependent manner on both media. Confocal microscopy revealed membrane damage, mitochondrial and nuclear disruption, ROS/RNS accumulation, and lipid and chitin reorganization. Gene expression analysis showed immediate downregulation of key genes such as PKS, CHIT, and ALM1, followed by later adaptive upregulation in stress- and repair-related genes (PP2A, CAL, TES). These findings indicate that PL induces oxidative stress and cellular injury, triggering compensatory gene responses. PL is an effective non-chemical method to inhibit A. alternata in cheese through both physical disruption and transcriptional modulation. Its dual action at structural and genetic levels supports its use as a sustainable antifungal technology in dairy preservation.