Pulsed light induces antifungal activity, oxidative stress, and transcriptional reprogramming in Alternaria alternata isolated from cheese.

Alternaria alternata is a major spoilage fungus of dairy products and a potential producer of mycotoxins. This study evaluated Pulsed Light (PL) as a non-thermal strategy to control A. alternata FCS26 strain isolated from Italian Caciocavallo cheese and investigated its physiological and transcriptional stress responses. To the best of our knowledge this is the first report on the anti-fungal mechanism of PL against A. alternata. In vitro, treatments with 30 PL (11.46 J cm-2) and 120 PL (45.84 J cm-2) differentially inhibited fungal structures: spore germination was reduced by 77% and 83%, respectively, while mycelial growth was inhibited by up to 22% and 37%, respectively. PL treatment also extended the lag phase and decreased cell viability as confirmed by confocal microscopy. Furthermore, PL pronounced oxidative and nitrosative stress, as evidenced by the early accumulation of Reactive Oxygen (ROS) and Nitrogen (RNS) Species and the modulation of antioxidant enzymes. Superoxide dismutase (SOD) and peroxidase (POD) showed sustained enzymatic responses under the 30-pulse treatment, whereas the 120-pulse treatment induced strong early activation followed by late disruption of POD and catalase (CAT) activity, consistent with severe redox imbalance. Reverse transcription-quantitative PCR revealed broad transcriptional modulation, with consistent downregulation of genes encoding polyketide synthase, chitinase, and dedh DEAD/DEAH box helicase-like protein. In situ studies showed that PL significantly reduced fungal severity on Caciocavallo cheese during refrigerated storage. In fact, the application of 120 pulses yielded the strongest effect, decreasing fungal growth severity by about 80% compared to the untreated control. Overall, PL effectively inhibited A. alternata, supporting its potential for fungal control, shelf-life extension and improved dairy safety.