Antifungal action of menthol and menthone against Botrytis cinerea: Physiological responses associated with membrane integrity, mitochondrial activity, and oxidative stress, and lipid nanoparticle formulation for its application.

Botrytis cinerea causes gray mold and is considered the second most important fungal phytopathogen. Natural compounds such as menthol and menthone have emerged as sustainable tools for reducing fungal infections and mitigating the adverse effects of synthetic fungicides. Nevertheless, there is limited information about the physiological mechanisms of menthol and menthone on B. cinerea. This study focused on elucidating the response of B. cinerea to the exposure to menthol and menthone in reducing fungal growth and exploring their use in lipid nanoparticles. The results showed that both compounds significantly reduced mycelial growth by ∼ 100%. Otherwise, menthol reduced spore germination by 71-100%, whereas menthone achieved only 28-37% inhibition. Both compounds reduced the dry weight of fungal cultures by more than 80% and increased pH and electrical conductivity, reflecting dysfunction of cell membranes. Besides, the compounds enhanced the release of cellular material that absorbs at OD260nm and soluble proteins. An increased cell membrane damage and reduced esterase activity, mitochondrial activity, and cell membrane potential were observed. Otherwise, both compounds significantly increase oxidative stress and the production of hydrogen peroxide and free hydroxyl radicals. The encapsulation of menthol and menthone in solid lipid nanoparticles (SLNs) demonstrated greater efficiency in reducing spore germination than in reducing mycelial growth. These results support the idea that menthol and menthone can act as sustainable antifungal agents and that their encapsulation in SLNs can be an important formulation tool to advance their application, but more studies are required to optimize and achieve more effective control.