Abstract

Litsea cubeba essential oil (LCEO) exhibits potent antimicrobial activity against various pathogens; however, its efficacy against Penicillium crustosum in blueberries and the underlying mechanisms remain unclear. Chemical composition analysis identified citral (42.66%), d -limonene (9.68%), and longifolene (7.32%) were the major constituents of LCEO. At a concentration of 1 μL/mL in potato dextrose agar (PDA), LCEO completely inhibited the in vitro growth of P. crustosum , suppressed conidiogenesis, and induced mycelial damage. Furthermore, a 30-min exposure to LCEO reduced cell-wall fluorescence, enhanced alkaline phosphatase activity, and increased membrane fluorescence and lipid alterations. Increased leakage of electrolytes, proteins, and nucleic acids further confirmed that the cellular barrier of P. crustosum was compromised during the early stages of treatment. Gene expression analyses revealed that LCEO suppressed genes involved in chitin biosynthesis, downregulated genes related to ergosterol synthesis-related genes, and upregulated genes associated with hydrolases. The increase in reactive oxygen species (ROS) within 60 min, followed by a decline in mitochondrial membrane potential by 120 min, suggests a cascade of events in which cell barrier damage triggers oxidative stress and mitochondrial dysfunction, ultimately leading to hyphal damage and developmental defects. In vivo application of 2 × MFC LCEO significantly improved blueberry quality, reducing decay, lower weight loss, and respiration rates, while increasing firmness, flavonoid content, and anthocyanin levels. These findings highlight LCEO's potential as a biofungicide and clarify its multitarget antifungal mechanism, supporting its use for reducing postharvest blueberry losses.