Studies On Bioactivity Of Eugenol, A Natural Compound, Against Botrytis Cinerea

The grey mold disease caused by Botrytis cinerea which causes severe damage in many fruits,vegetables and ornamental crops in pre- and post-harvest is very destructive on crops all over the world.Nowadays,control of B.cinerea is mainly reliant on multiple applications of chemical fungicides such as benzimidazoles, dicarboximides,diethofuncarband and the sterol biosynthesis inhibitors etc.However, frequent application of these fungicides has resulted in the emergence of resistant pathogen strains in several countries.Therefore,there is a worldwide trend to explore new alternatives to synthetic fungicides in order to comply with food safety standards.Eugenol,the essential component from clove oil,has been reported to inhibit the growth of several pathogenic fungi,however,there is no report on the effect of eugenol on B.cinerea and its mechanism of action.In this paper,antifungal activity of eugenol against Botrytis cinerea and its mechanism of action were studied in vitro,in order to evaluate its potential as a fungicide for use in control of B.cinerea.And the results were described as the following:(1)Antifungal activity of eugenol was determined by mycelial growth rate method in vitro against 10 pathogenic fungi.It was found that mycelial growth of B.cinerea was the most sensitive to eugenol with EC₅₀value of 38.62μg·mL^-1.(2)The effectiveness of eugenol treatment on mycelium growth of B.cinerea was concentration-dependent,and not influenced by pH.Further study showed that eugenol could reduce the number of sclerotium,and inhibit its germination and sporulation,but had no effect on spores germination of B.cinerea.(3)Light and SEM, TEM observation on pathogen hyphae,exposed to eugenol,revealed considerable morphological alterations in hyphae such as vacuolation and hyphal shrivelling.(4) Hyphae of B.cinerea treated with eugenol showed strong propidium iodide(PI) fluorescence in the cytosol viewed with a fluorescence microscope,indicating eugenol-mediated damage of cytoplastmic membrane.But no PI staining was observed on spore treated with eugenol,which revealed that spore membrane were resistant to the given concentration of eugenol.In addition,concentration of potassium ion and cellular materials in the medium increased after the treatment with eugenol by atomic absorption spectrophotometry and ultraviolet spectrophotometry, which indicated that permeability of hyphal membrane was interrupted and cellular materials were released.(5)It was observed that increasing of Ca²⁺concentration occurred early in eugenol-treated mycelia,and subsequently H₂O₂ and MDA was over-produced.In conclusion,our results strongly supported the notion that the antifungal activity of eugenol was due to the occurrence of a number of processes,including initial Ca²⁺increasing and subsequent H₂O₂ production,followed by lipid peroxidation and disruption of both plasma and inner membranes.