In vitro and in vivo antifungal activity and preliminary mechanism of cembratrien-diols against Botrytis cinerea

Gray mold, caused by Botrytis cinerea, is one of the major fungal diseases in crops. Botanical fungicides are preferred over chemical fungicides to control gray mold since they are less toxic to the environment and could reverse the resistance to chemical fungicides in pathogens. Cembratrien (CBT)-diols are carbocyclic diterpenes found in abundance in Nicotiana, especially in tobacco inflorescence, and possess a wide range of biological activities. In this study, the antifungal activities of two isomers of CBT-diols (alpha- and beta-CBT-diols) against B. cinerea were evaluated in vitro and in vivo. In addition, the antifungal mechanism was studied on cytoderm, membrane system, and oxidative stress. Results showed that CBT-diols could inhibit wild and multi-resistant strains of B. cinerea, and the half-maximal effective concentration was within 9.67-16.38 mu g/mL. The effects of aand beta-CBT-diols were similar but non-synergistic. The inhibition rate of CBT-diols (200 mu g/mL) against B. cinerea in different fruits was between 71.9 and 84.2 %. The cellular structure of B. cinerea treated with CBT-diols was observed by scanning electron microscopy, transmission electron microscopy, Crystal violet-Congo red staining, and propidium iodide staining. Results showed that there was almost no change in cytoderm integrity; however, a significant damage to the membrane system integrity was observed, which led to disintegration of the organelles. Cembratrien-diols could increase the activity of chitinase, resulting in an accumulation of N-acetyl-D-glucosamine, which is a precursor for chitin biosynthesis. Cembratrien-diols caused significant increase in conductivity and cellular contents in the culture medium by increasing the membrane permeability of B. cinerea. Furthermore, CBT-diols decreased the levels of essential membrane components such as ergosterol and linoleic acid, but increased the malondialdehyde concentration, which indicated that CBT-diols damaged B. cinerea membrane and induced membrane lipid peroxidation. With growing research, it has been found that the damage to B. cinerea membrane system is mainly associated with the oxidative stress reaction induced by CBT-diols, due to the increase in reactive oxygen species levels and activities of catalase and peroxidase, and the decrease in superoxide dismutase activity and hydrogen peroxide content. The results provide a scientific foundation for the application of CBT-diols as an alternative biological agent against B. cinerea, which may ultimately promote organic fruits and vegetable crops production.