The Resistance Molecular Detection And The Research Of Sensitive-Relative Genes Of Phenamacril In Fusarium Graminearum

Wheat scab caused by Fusarium graminearum is an epidemic disease,which causes severe wheat yield loss.In addition,the mycotoxin produced by Fusarium graminearum could contaminate wheat,which seriously threated the health of humans and animals.Phenamacril is a new fungicide developed by the National Southern Pesticide Creation Center in Jiangsu Province to control wheat scab.Previous studies have found that Fusarium graminearum is easy to develop resistance to phenamacril in indoor induction conditions,and that the mutation of myosin-5 amino acid in Fusarium graminearum cause the resistance of Fusarium graminearum to phenamacril.In order to better monitor the resistance of Fusarium graminearum to phenamacril in the field,further explain the reason why the myosin-5 amino acid point mutation causes the resistance of Fusarium graminearum to phenamacril,this paper performs in the following three aspects:First,a rapid diagnostic technique for detecting the resistance of Fusarium graminearum to phenamacril was developed using PCR-RFLP technology.The results showed that the differentially mutated strains A135T,S217L and E420K with different resistance levels were distinguished from the wild-type sensitive strain PH-1 by Kpnl,TasI and Dral digestion.After establishing the above detection method,we tested 48 strains of phenamacril-resistant strains by PCR-RFLP technique and traditional mycelial growth rate assay,then found this method has high accuracy and PCR-RFLP is more time-saving and labor-saving than growth rate measurement.PCR-RFLP technology not only provides a more concise and convenient way to monitor the resistance of wheat scab to phenamacril,but also provides a scientific basis for future field resistance control.Secondly,based on the sensitive strain PH-1 of Fusarium graminearum,the mutant strain of A135T,S217L,S418R and E420K fixed-point mutation on myosin-5 and a hyphamycin-labeled PH-1 control strain were constructed respectively.Then,four amino acid point mutation resistant strains and a hygromycin-labeled control strain PH-1-HPH were used for transcriptome sequencing,and a detailed transcriptome report was obtained.The results showed that compared with the control strain,some genes up-regulated or down-regulated expressed in the four site-directed mutant strains.The GO function classification and enrichment analysis of the above differentially expressed genes showed that the DEGs GO categories of the four Myosin-5 different amino acid point mutants focus on the molecular function,cellular component and the biological process compared with the control group.The KEGG biological pathway classification and enrichment analysis of the above genes showed that the pathway of four Myosin-5 different amino acid point mutant DEGs was mainly concentrated in metabolic pathways,biosynthesis of secondary metabolites and other pathways compared with the control group.Further analysis showed that compared with the control strain,among the four amino acid point mutants,there were 14 genes up-regulated together and 24 genes down-regulated together,so we speculate that the above genes may interact with Myosin-5 and participate the regulation of the susceptibility of Fusarium graminearum to phenamacril.This result provides some clues to look for the genes related to the resistance of Fusarium graminearum to the resistance to phenamacril,and provides a basis for further elucidating the mechanism of resistance of Fusarium graminearum to phenamacril.Thirdly,according to the transcriptome data,we deleted the gene FgDbf2 and FgMetl in four amino acid point mutants to study the drug sensitivity and gene function of phenamacril.The results showed that the knockdown of FgDbf2 gene decreased the EC50 significantly in the high-resistance strain YP-1.The study of gene function found that FgDbf2 had an effect on the growth,reproduction,stress and pathogenicity of the strain.The knockdown of FgMet1 had no significant effect on the susceptibility of phenamacril and had no effect on reproduction,stress and pathogenicity,but it could slow the growth rate of the strain.