Mechanism Of Action Of Carvacrol On Tea Leaf Spot Caused By Lactodiplodia Theobroma Caca

Tea leaf spot caused by L.theobromae is a new disease,induced the loss in the yield and quality of tea leaves in Guizhou Province.In order to effectively prevent and control tea leaf spot,finding out safe and efficient agents,and studying the action mechanism of agents on pathogenic fungus are of great significance for the prevention and treatment of tea leaf spot.This paper focuses on the screening of the antifungal activity of carvacrol（car）and thymol（thy）on L.theobromae,and initially observed the effect on the external morphology of L.theobromae.The binding mode of car and thy to ctDNA and the effects of transcriptome methods on the DNA transcriptional fragments of L.theobromae were studied.The main research contents are as follows:1.Screening the antifungal activity of monoterpenoid in vitro.The results showed that most of the compounds showed higher antifungal activity,including compounds thymol(EC₅₀=46.11μg/m L),carvacrol(EC₅₀=50.70μg/m L),3-methyl-4-isopropylphenol(EC₅₀=55.17μg/m L),2,3,5-trimethylphenol(EC₅₀=119.19μg/m L),eugenol(EC₅₀=193.00μg/m L)and menthol(EC₅₀=460.55μg/m L).Among them,car and thy exhibited higher antifungal activity.2.The morphological effects of the treatment of L.theobromae by car and thy were further studied through morphology.The results show that L.theobromae was treated with car and thy EC₅₀for 24 h,the hyphae were fewer,shorter,more deformed,and slightly protuberant.The surface of some hyphae were observed by scanning electron microscope to be swollen,with protuberances and invaginations.After treating L.theobromae with EC₉₀ for 24 h,the surface deformation of mycelia was more obvious,some hyphae branches decreased,short and thick swelling and collapsed appeared under light microscope.Under the observation of scanning electron microscope,the thickness of hyphae was not uniform,and some hyphae surface had protrusion,fold,collapse and even dry deformation.3.The binding mode and mechanism of car and thy to calf thymus DNA（ctDNA）were studied by multi-spectral method.（1）With the increase of drug concentration and absorption light intensity measured by ultraviolet-visible spectrum,there is a hyperchromic effect,which indicates that the drug interacts with ctDNA and interacts by groove binding.（2）Fluorescence spectroscopy was used to study the quenching mechanism of the reaction system by fluorescence experiment.The binding constants of car and ctDNA were calculated to be 3.88×10⁶ M,and those of thy and ctDNA were1.04×10¹⁰ M.The K_q values of the two small molecules were both greater than the maximum collision quenching rate constant K_dif of the quenching agent for biological macromolecules,and the main force type was determined to be the groove bind.After the two small molecules were combined with single-stranded ctDNA respectively,the binding constants of car and thy to single-stranded ctDNA were significantly smaller than those to double-stranded ctDNA.It was further verified that car and thy could bind to ctDNA grooves.The competitive rates of car against DAPI and EB were 17.55%and 5.01%respectively,and that of thy against DAPI and EB were 13.43%and 5.44%respectively,indicating that car and thy replaced the DAPI bound by ctDNA,and further confirmed the groove binding mode between car and thy and ctDNA.（3）Through circular dichroism,it was observed that there was no significant change in CD signal value after car and thy reacted with ctDNA.At the same time,after DAPI groove binder and classical intercalator EB reacted with ctDNA,the CD signal value did not change significantly with the increase of DAPI-ctDNA system,and the CD signal decreased regularly with the increase of EB-ctDNA system.Two small molecules was similar to that of DAPI.The interference of grooves and electrostatic binding on their CD spectra was relatively small.The groove binding between car and thy and ctDNA also showed similar results.4.Transcriptome sequencing was used to further study the treatment of L.theobromae with different concentrations and different times of car and thy.The data analysis showed that the gene fragment size of L.theobromae was the largest in the range of 1,000～2,000 bp after the compounds treatment.The fragments that affect the DNA transcription of car and thy are mainly concentrated in the up regulated gene fragments with a fragment interval of 1,000～2,000 bp.