Functional Analysis Of Bcmix17 And Bcpos5 Genes And Rapid Detection Of Anilinopyrimidine Fungicide Resistance In Botrytis Cinerea

Gray mold caused by Botrytis cinerea,one of the top ten plant diseases in the world,leads to hundreds of million dollars economic losses every year.Chemical application is still the main method to control gray mold.Anilinopyrimidine fungicides(APs),one of the common fungicide groups,have specific effects on many ascomycetes including B.cinerea.However,the occurrence of fungicide resistance has seriously affected its control efficacy.At present,the resistance mechanism of APs is not clear.This research focused on two aspects:one is to analyze the function of genes related to the AP resistance in B.cinerea;The other is to develop a rapid detection method for AP resistance based on RPA/Cas12a methods.This study laid a foundation for clarifying the resistance mechanisms of APs and provided a simple method for the detection of field resistant strains.The main research results are as follows:(1)The function of Bcmix17 and Bcpos5 genes related to AP resistance was analyzed.In previous study,it was found that point mutations in nine mitochondrial genes of B.cinerea such as Bcmix17 and Bcpos5 can cause resistance to APs using whole genome sequencing and reverse genetics.This study focused on Bcmix17 and Bcpos5,the corresponding knockout and complemented transformants of Bcmix17and Bcpos5 were obtained respectively from the resistant parental isolate HBTom-400by PEG mediated genetic transformation.The phenotypic assay showed that Bcpos5knockout transformants had no significant differences with the parental isolate and complemented transformants in terms of the resistance to cyprodinil,sensitivity to exogenous stresses,sporulation,conidial germination,acid production and virulence,except for the significantly decreased mycelium growth.These results indicated that Bcpos5 was not directly involved in the AP resistance.Compared with the parental isolate and the complemented transformants,the Bcmix17 knockout transformants showed no significant differences in sensitivity to exogenous stress,but the mycelium growth,resistance to cyprodinil,acid production,and virulence was significantly decreased,and no conidia and sclerotia were produced.These results indicated that Bcmix17 was not involved in the stress responses to osmotic stress,oxidative stress,and the cell wall/membrane integrity,but involved in the regulation of vegetative growth,acid production and virulence,as well as the resistance to APs in B.cinerea.(2)Based on the mutation E407K in Bcmdl1 gene of resistant isolates,a method was developed by using RPA/Cas12a reactions to detect AP resistance in B.cinerea.In the previous study,the E407K mutation in Bcmdl1 was proved to be involved in resistance to APs using QTL-seq and genetic transformation,and this mutation could be detected in more than 60%of tested resistant field isolates.In this study,two pairs of RPA primers were designed by introducing one mismatch and forming two PAM sites.The results showed that both RPA primers could amplify the target sequence.Then four cr RNAs targeting the E407K mutation were designed near two PAM sites for Cas12a cleavage reaction,and the results showed that only cr RNA2 could specifically identify the resistant isolates containing the E407K mutation.The reaction system was optimized by adjusting the concentration of Cas12a,the ratio of Cas12a to cr RNA,the concentration of FQ-reporter,and the reaction time.The optimized system could detect the resistant isolates with the limitation of 1.8×10~4 fg/μL g DNA,which was 10 times lower than the conventional PCR(1.8×10~3 fg/μL).The specificity tests showed that this system could specifically identify AP resistant isolates containing E407K mutation from the negative controls.Finally,the detection method was further simplified by using rude DNA from the diseased host in the field which was inoculated with B.cinerea,and the simplified RPA/Cas12a system could accurately detect the resistant isolates within 55 min,without the limitation of geographical conditions and complex instruments.In summary,this study analyzed the functions of Bcmix17 and Bcpos5 genes related to the resistance to APs,and developed a simple,accurate and efficient method for AP resistance detection.It is not only important for further understanding the AP resistance mechanisms,but also facilitate the detection of AP resistance and the monitoring of occurrence of resistant population of B.cinerea in fields.