Clove Essential Oil as an Antifungal Agent and Putative Dual-Action Antifungal Mechanism: Experimental Validation and Computational Insights into Orthosteric and Allosteric Modulation of Chitin Synthase I in Penicillium Species

Fungal contamination during postharvest storage causes significant food losses, particularly due to Penicillium expansum and Penicillium brevicompactum, highlighting the need for sustainable antifungal alternatives. This study evaluated the antifungal potential of clove essential oil (Syzygium aromaticum) against P. expansum and P. brevicompactum by integrating in vitro assays with in silico analyses. Minimum inhibitory concentrations (MICs) were determined, and effects on fungal growth, membrane integrity, and spore germination were assessed. Molecular docking and molecular dynamics simulations were performed to evaluate the affinity and stability of the five most abundant GC–MS compounds that met predefined ProTox-II toxicity criteria (categories 5–6; LD50 ≥ 2000 mg/kg) toward chitin synthase I (CHS I), a key enzyme in chitin biosynthesis. The oil exhibited strong inhibitory activity, with MIC values of 0.156 µL/mL against P. expansum and 0.312 µL/mL against P. brevicompactum, along with significant morphological and physiological alterations. Computational analyses indicated that trans-β-caryophyllene oxide and α-humulene form stable interactions at both the active and an allosteric site of CHS I, supporting a putative dual inhibitory mechanism. These findings highlight clove essential oil as a promising ecological alternative to synthetic fungicides and underscore the value of computational approaches for elucidating antifungal mechanisms in understudied species.