Identification Of Colletotrichum Spieces On Tea Plants And The Antifungal Effect Of Atractylodes Oil On Colletotrichum Species

Tea brown blight disease caused by Colletotrichum species,seriously damages tea leaves and leads to quality and production decrease.The study on the accurate identification and control of Colletotrichum species can effectively prevent the occurrence and development of tea brown blight diseases.At the same time,chemical fungiicides are mainly used in the prevention and control of Colletotrichum spieces.Although chemical pesticides have good control effects,they may bring great security risks to the environment,it is still necessary to select safer and more efficient control agents.In this paper,the main Colletotrichum species on tea plants in Chongqing area were investigated,isolated and identified,and the control effects of atractylodes oil,chlorothalonil,carbendazim and methyl thiazim against different Colletotrichum species were compared and studied.The main antifungi components and mechanism of atractylodes oil and the differences of resistance mechanism of five Colletotrichum species were systematically studied by gas chromatography-mass spectrometry tandem and transcriptome analysis.The main conclusions are as follows:1.The Colletotrichum species in Chongqing was isolated and identified through morphological identification combined with multi-gene analysis（including ITS,GAPDH,TUB2,CAL,ACT,and CHS）,five species of Colletotrichum were identified and recorded,including four known species（C.gloeosporioides,C.camelliae,C.fioriniae,and C.karstii）and the newly discovered（C.chongqingense）.C.chongqingense exhibited significant morphological differences compared to the others,and the pathogenicity of ten strains of C.chongqingense was evaluated through injective inoculation,and the results demonstrated an incidence rate of 41.7%to 61.7%after 5 d of inoculation,with CB8-4 being the most virulent and CB12-3 being the least virulent.2.The effects of the essential oil,chlorothalonil,carbendazim,and methyl thiocarmine on C.camelliae,C.chongqingense,C.karstii,C.gloeosporioides,and C.fioriniae were assessed using the growth rate method.The EC₅₀ of atractylodes oil against the five Colletotrichum species were 0.108,0.085,0.089,0.165,and 0.205 mg·mL^-1,respectively,the EC₅₀ of chlorothalonil were 11.474,14.134,17.812,27.074,and 55.674μg·mL^-1,respectively,the carbendazim were 0.055,0.078,0.076,0.066,and 2.381μg·mL^-1,respectively,and methyl thiocarmine were 0.129,0.202,0.288,and 3.323μg·mL^-1,respectively.Except for carbendazim,the efficacy order of the other three treatments against the five Colletotrichum species was C.camelliae>C.karstii>C.gloeosporioides>C.fioriniae.The vapar phase method was used to investigate the antifungal effect of atractylodes oil against the five Colletotrichum species.Under the conditions of EC₅₀ and EC₈₀ concentrations,the antifungal rate of atractylodes oil against the five Colletotrichum species was lower than 20%.3.The main constituents of atractylodes oil were identified by gas chromatography and mass spectrometry analyses.Among them,diethyl phthalate,curcuma furalene,and cis-5-vinyl-5-methyl-4-(1-methylvinyl-2-（1-methyl-methyl-methyl）-cyclohexanone were found to have the highest content ratio,accounting for 77.35%of the total peak area.Diethyl phthalate,the most abundant component with a peak area ratio of 62.10%,and is presumed to be the main agent in atractylodes oil;The antifungi effects of atractylodes oil was systematic investigated.The effects of atractylodes oil on the morphology of Colletotrichum species were analyzed by scanning electron microscopy.Following treatment,the colony surface gradually became rough,the color faded,and the mycelium clustered.Notably,spores development was significantly affected,with spores appearing concave and rough and displaying local irregular enlargement,elongation or shortening at both ends.The permeability of the protoplasmic membrane and cell wall of the four types of Colletotrichum mycelia changed after treatment with atractylodes oil,and within12 h of treatment,the A260,alkaline phosphatase（AKP）,and total protein（TP）levels were significantly elevated.Additionally,the mycelium membrane lipid peroxidation was enhanced,and the protoplasmic membrane structure was destroyed,causes the contents of CAT,SOD,and malondialdehyde（MDA）increased in the mycelium of all five types of Colletotrichum within 12 h of treatment,and these levels continued to increase after 24 h of treatment.4.The drug resistance mechanism of Colletotrichum species was investigated using transcriptome analysis.GO enrichment analysis demonstrated that the most significant gene enrichment was related to REDOX enzyme activity and membrane structure and function.KEGG pathway enrichment analysis revealed that ABC transporter and tryptophan metabolic pathways were the most significantly enriched.Differential gene expression analysis indicated that the top three up-regulated genes were involved in ABC transporter,MFS transporter,and cytochrome P450 functions.Furthermore,gene-GCG54＿00000018,which is related to the ABC transporter,was the most significantly up-regulated gene,with a fold change of 4729.22,and gene-GCG54＿00013384,which is associated with the function of MFS transporter,was the most significantly down-regulated gene,with a fold change of 804.85.Five genes（gene-GCG54＿00000018,gene-GCG54＿00010091,gene-GCG54＿00011590,gene-GCG54＿00013384,gene-GCG54＿00008127）that related to drug resistance of fungal wree selected for verification,the result shows that the expression of those five genes in Colletotrichum species were quite different,which indicating that the mechanism of drug resistance of four fungi may not be consistent.