Screening Of Chemical Fungicides And Potential Drug Resistance Mechanism Of Poplar Leaf Blight

Poplar is a tree species with wide distribution and strong adaptability.Not only can it prevent wind and sand,protect water and soil,but its wood can also be used as an excellent raw material for making wood boards and pulp,with high ecological and economic value.Poplar is also a tree species with frequent pests and diseases.Among them,poplar leaf blight,as a leaf disease,seriously affects the growth of poplar and the various benefits of poplar.Based on genomics and transcriptomics research on the changes in physiological response indicators and transcriptome data of the pathogenic Alternaria alternata（AaNEFU1）that causes poplar leaf blight after being treated with chemical fungicide stress,this paper seeks to obtain A.alternata drug resistance-related genes include ABC transporter,MFS transporter,cell membrane pigment P450 family genes,cell wall formation and degradation-related genes,stress resistance and antioxidant-related genes,and drug resistance-related pathways include ergosterol biosynthesis and melanin biosynthesis pathways.The experimental results are as follows:1.Isolation,identification and genome sequencing of pathogenic fungiIn this paper,the pathogen of poplar leaf blight was isolated and purified.It was preliminarily identified as A.alternata and named AaNEFU1.The genome of AaNEFU1 was sequenced and analyzed,and the genome integrity of AaNEFU1 was 98.4%.Theβ-tubulin,Alt a1,endo PG and gapdh sequences of A.alternata were used to further cluster analysis,and it was found that it was closely related to Zhejiang strain A.alternata Z7.2.Sensitivity test of fungicides against A.alternata（AaNEFU1）The drug sensitivity experiment of AaNEFU1 was carried out by using 12 chemical fungicides.Finally,three fungicides with significant antibacterial effect on AaNEFU1 were screened out,which were three fungicides with different action mechanisms.According to the EC₅₀ from small to large,they were 25（g/L）fludioxonil suspension concentrate(EC₅₀=0.28μg/m L)of phenylpyrrole PPs,450（g/L）prochloraz water emulsion(EC₅₀=1.35μg/m L)of demethylation inhibitors（Demethylation Inhbithors）DMIs,and 80%mancozeb wettable powder(EC₅₀=15.49μg/m L)of multi-sites.3.The pathogenicity,spore germination rate and physiological indexes of A.alternata（AaNEFU1）under fungicide stress were measured and analyzed.The A.alternata treated with the three fungicides were inoculated into poplar leaves for pathogenicity determination.By measuring the size of poplar leaf blight lesions,their pathogenicity was ranked from large to small as CK group>mancozeb treatment group>fludioxonil treatment group>prochloraz treatment group.Using three fungicides to treat Alternaria spores,it was found that the spore germination rate of all treatment groups increased rapidly within 2-6hpi,and the spore germination rate increased slowly within 6hpi-16hpi.The order of spore germination rate at 16hpi was CK group>mancozeb treatment group>fludioxonil treatment group>prochloraz treatment group.The physiological indexes of A.alternata treated with EC₃₀,EC₅₀ and EC₇₀concentrations of fludioxonil,mancozeb and prochloraz were measured and analyzed.The results showed that under the stress of fungicide concentration from small to large,the soluble protein content of the pathogen decreased,while the tyrosinase activity,melanin content and MDA content increased,CAT and SOD activity increased first and then decreased.From the perspective of fungicide category,compared with CK group,the soluble protein content increased most significantly after prochloraz treatment;the melanin content increased most significantly after treatment with fludioxonil and prochloraz;the tyrosinase activity increased most significantly after treatment with fludioxonil and prochloraz;the physiological indexes of antioxidant enzyme system after fludioxonil treatment were more intense than those after other fungicides.4.Transcriptome-based analysis of the potential resistance mechanism of A.alternata（AaNEFU1）Transcriptome sequencing was performed on Alternaria alternata treated with EC₅₀concentrations of three fungicides with different mechanisms of action.The results showed that there were 2939 DEGs（1141 up-regulated and 1798 down-regulated）between CK and fludioxonil.There were 744 DEGs（159 up-regulated and 585 down-regulated）between CK and mancozeb.There were 1028 DEGs（312 up-regulated and 716 down-regulated）between CK and prochloraz.The experimental results showed that more DEGs were involved in the response of Alternaria to fludioxonil treatment.Most of the DEGs after prochloraz treatment were the same as those after fludioxonil treatment,but the expression differences of DEGs in the same part were lower than those in the fludioxonil treatment group.After mancozeb treatment,most DEGs and fludioxonil and prochloraz had different expressions,indicating that compared with other fungicides,the genes of A.alternata after mancozeb treatment had unique responses.GO enrichment analysis of DEGs of A.alternata showed that ribosomal biosynthesis was involved after fludioxonil treatment.Mancozeb treatment involves lipid and carbohydrate metabolism processes;prochloraz treatment involves carbohydrate metabolism process.KEGG enrichment analysis of Alternaria DEGs showed that ribosomal biogenesis was involved after fludioxonil treatment.Prochloraz treatment involves ribosome biogenesis;mancozeb treatment involves pyruvate metabolism.The collinearity trend analysis showed that the up-regulated response trend of A.alternata genes after fludioxonil treatment was the strongest,followed by prochloraz,and the up-regulated response after mancozeb treatment was the most unique,which was consistent with the results of DEGs analysis,GO and KEGG database annotation analysis of DEGs.Based on the KEGG annotation of DEGs of A.alternata after treatment with three fungicides,31 multidrug resistance（MDR）genes were found.Among them,there were 25 MFS transporter genes,belonging to DHA1 family and DHA2 family,and 6 ABC transporter genes,belonging to subfamily B,subfamily C and subfamily G,respectively.According to the KEGG database annotation and previous research results,a total of 85 up-regulated DEGs related to fungal resistance were identified and divided into 5 categories.There were 14 DEGs of ABC transporter,29 DEGs of MFS transporter,6 DEGs of cytochrome P450 family genes,11 DEGs of cell wall formation and degradation related genes,and 17 DEGs of stress resistance and antioxidant related genes.Analysis of DEGs-based drug resistance-related pathways found that in the ergosterol synthesis route and the entire melanin production pathway,the up-regulated gene response after fludioxonil treatment was the strongest.