Dismantling of Necrotroph Alternaria alternata by Cellular Intervention of Peppermint oil Nanoemulsion (PNE)

Abstract

Alternaria alternata, a common necrotrophic fungal pathogen, poses a significant threat to various crops, causing substantial yield losses and quality deterioration. In the present study, we explore the potential fungicidal properties of Peppermint Oil Nanoemulsion (PNE) against A. alternata and investigate its impact on the fungal phenotype. Our previous study synthesized the PNE using a nanoemulsion approach, optimizing its formulation for enhanced stability and efficacy. The present study extended the assessment of a multidisciplinary approach to comprehensively analyze the fungicidal efficacy of PNE against A. alternata. Notably, in a liquid growth medium, 0.5% of PNE could reduce A. alternata's biomass by 96%. PNE-treated mycelia were stained with a nitro-blue tetrazolium (NBT) dye to assess ROS accumulation during oxidative stress induced by PNE. A higher degree of ROS generative potential of PNE has appeared in 72 h treated mycelia. PNE-treated mycelium showed cell wall alterations, with red fluorescence peaking at 0.5%, indicating a dose-dependent effect compared to the untreated control. Consequently, PNE treatment led to a significant early hour increase in electrical conductivity (EC), extended to 306.03-353.33 μS/cm compared to 277.67-280.33 μS/cm untreated control. Scanning Electron Microscopy (SEM) analysis of A. alternata reflects the osmotic imbalance and structural damage in mycelia as the obvious cause of fungal inhibition. In addition, a phenotype microarray analysis of PNE-treated A. alternata mycelia revealed a significant phenotypic loss in 37 out of 708 substrates, potentially impacting metabolic pathways essential for fungi's functional processes. The study found that downregulation of genes like Cre A, NmrA, SOD, IMP, EfP, and Erg, which are linked to A. alternata's stress coping mechanisms, leads to alterations in survival and adaptation. Additionally, understanding the phenotypic changes induced by PNE contributes to our knowledge of the mode of action of this nanoemulsion against A. alternata. In conclusion, this study provides a comprehensive analysis of the fungicidal and phenotypic effects of PNE, offering a promising avenue for sustainable fungal control. The implications of our research extend to the development of novel, natural fungicidal agents for agricultural applications.