Bio-preservative potential of marjoram and fennel essential oil nano-emulsions against toxigenic fungi in citrus: integrating in-vitro, in-vivo, and in-silico approaches

Citrus fruits, known for their vibrant flavours and health benefits, are susceptible to fungal attacks, particularly from toxigenic Penicillium fungi, which pose a significant pre- and post-harvest hazard. However, aromatic oils and their nanoparticles may effectively address this issue. Marjoram and fennel oils, alongside their nanoparticles, were extracted, and their aromatic constituents and antimicrobial activities were evaluated. A simulated medium with fungal spores was used to assess anti-toxigenic activity, and a simulated infection experiment was conducted with orange and lemon fruits. The capacity and mechanisms of aromatic constituents were analysed through molecular docking assays targeting enzymes involved in fungal growth and mycotoxin production. The nanoparticles exhibited good stability (89.17%-92.41%) and compact formulation (density of 0.92-0.96 g/mL). Results demonstrated substantial effectiveness of nano-emulsions against toxigenic fungi, with major aromatic compounds identified as terpinene-4-ol (18%) and gamma-terpinene (11%) in marjoram and estragole (38%) and anethole (29%) in fennel oil. Diffusion assays revealed significant anti-pathogen effects (8.33-11 mm) and antifungal activity (33.33 +/- 2.88-89.33 +/- 1.15 mm) of marjoram and fennel nano-emulsions. Results regarding simulated infected fruit reflect spoilage delay without impacting fruit quality or sensory. The interactions between oil or nano-emulsions and fungal enzymes showed strong binding-free energy values, with significant docking scores (-6.6 to -7.0 kcal/mol) for aromatic constituents. In conclusion, aromatic antifungals offer a promising strategy for controlling Penicillium, enhancing the safety and quality of oranges and lemons, with oil nanoparticles improving antifungal efficacy by significantly reducing mycelium weight and spore germination.