Antifungal Activity Of CBT-diols Against Botrytis Cinerea

Gray mold,caused by Botrytis cinerea,is one of major fungal diseases on crops.Botanical fungicides are preferred over chemical fungicides to control gray mold since they are less toxic to the environment and are resistant to fungicide.Cembratrien?CBT?-diols are carbocyclic diterpenes found in abundance in Nicotiana,especially in tobacco inflorescence,and possess a wide range of biological activities.In order to develop the application value of CBT-diols in biological control of gray mold,the antifungal effect of CBT-diols on B.cinerea was evaluated in vitro and in vivo,and the antifungal mechanism was investigated by studying the effects of CBT-diols on the morphology and structure of B.cinerea mycelium,the main components and related enzyme activities of cell wall and cell membrane,cell membrane permeability and oxidative stress.In addition,combination of CBT-diols and different agents on the growth of B.cinerea mycelium was also studied.The main results are as follows:1.The antifungal activity of CBT-diols against B.cinerea was studied by mycelium growth rate test,spore germination test,and the live inoculation test in vivo and in vitro.Results showed that the effects of two isomers of CBT-diols??-and?-CBT-diols?were similar but non-synergistic.CBT-diols show inhibition on the mycelium growth of wild and multi-resistant strains of B.cinerea with the EC₅₀0 ranging from 9.67?g/mL to 16.38?g/mL,and the EC₅₀0 values were significantly lower than that for other registered botanical fungicides such as osthole and eugenol.The inhibition rate of CBT-diols against B.cinerea in different fruits was 71.90?84.29%at concentration of 200?g/mL.2.The cellular structure of B.cinerea treated with CBT-diols was observed by SEM,TEM,crystal violet-congo red staining,and PI 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.CBT-diols could increase the activity of chitinase,resulting in an accumulation of D-GlcNAc,which is a precursor for chitin biosynthesis.CBT-diols caused significant increase in conductivity and cellular contents in 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 and increased the malondialdehyde concentration that were evidence of CBT-diols damaged B.cinerea membrane and induced membrane lipid peroxidation.With deepening research,it has been found that the damage of B.cinerea membrane system was mainly associated with the oxidative stress reaction induced by CBT-diols,with the increase of ROS levels,antioxidant enzymes CAT and POD activities,and decreasing SOD activity and H₂O₂ content.3.The toxicity of CBT-diols with four chemical fungicides and seven plant extracts against B.cinerea was studied by the co-toxicity method.Results showed that there were no synergistic effect between CBT-diols and the selected agents.In order to develop the application value of CBT-diols as a fungicide in controlling gray mold,the further research needs to study the synergistic effect between CBT-diols and other biologicals or low-toxic chemical fungicides in-depth,and develop the field efficacy of CBT-diols and their compounds on the basis of the antifungal mechanism.