Development Of Detection Methods Of Fungicide Resistance And Exploration Of Nucleic Acid Fungicides For Monilinia Fructicola

Peach brown rot,caused by Monilinia spp.,is a significant peach tree disease.This disease can occur both pre-and post-harvest,causing severe damage to peach yield and quality,resulting in substantial economic losses.Chemical fungicides such as benzimidazoles（MBCs）and sterol demethylation inhibitors（DMIs）are commonly used in production to control peach brown rot.However,with the extensive use of fungicides,Monilinia fructicola strains resistant to MBC or DMI have emerged around the world.The research findings indicate that MBC fungicide-resistant strains of M.fructicola commonly exhibit the E198A mutation in theirβ-tubulin（Mf Tub2）gene.Additionally,DMI fungicide-resistant strains are frequently found to have a 65bp insertion element named"Mona"upstream of their Mf CYP51 gene,which is believed to be associated with overexpression of the Mf CYP51 gene.In this study,based on Recombinase polymerase amplification（RPA）and CRISPR/Cas12a technologies,two RPA/Cas12a detection methods were developed for the detection of resistance to DMI or MBC fungicides in M.fructicola,targeting the E198A mutation and the"Mona"element,respectively.And a method was proposed to reduce the dependence of Cas12a on PAM sites by modifying RPA primers.The main results are as follows:（1）Four RPA primers and two cr RNAs were designed to detect the resistance of DMI fungicides.After screening,it was found that the RPA primer pair M-DMI-F2/M-DMI-R1could successfully amplify the target sequences of M.fructicola;DMI-cr RNA1 could guide Cas12a protein to quickly target the target sequence in resistant strains and quickly cleave FQ-reporter to produce a large number of fluorescence signals within 20 min.After optimization,the optimal RPA reaction time was 15 min,and the optimal CRISPR/Cas12a reaction system was 10μL reaction system,including 50 n M Lb Cas12a,50 n M cr RNA,6000 n M FQ-reporter,1μL LRPA reaction product,1×NEBuffer 2.1,filled with 10μL reaction system by RNase-Free H₂O.（2）Six RPA primers and four cr RNAs were designed to detect MBC fungicide resistance.After screening,it was found that the RPA primer pair M-MBC-F2/M-MBC-R2（PAM）could successfully amplify the target sequences of M.fructicola,and PAM sites were added to the amplification products of target sequences without PAM sites.M-MBC-tcr RNA2 could guide Cas12a protein to quickly target the target sequences in resistant strains,and successfully distinguish the E198A mutation,and quickly cut FQ-reporter to produce a large number of fluorescence signals within 40 min.After optimization,the optimal RPA reaction time was 20 min,and the optimal CRISPR/Cas12a reaction system was obtained,specifically 10μL reaction system,including 150 n M Lb Cas12a,600 n M cr RNA,4000 n M FQ-reporter,1μL LRPA reaction product,2.5×Reaction Buffer,10μL reaction system filled by RNase-Free H₂O.The two RPA/Cas12a detection methods developed only requiring a simple constant temperature device to obtain the detection results within 1 h,and could be visualized by UV flashlight at 365 nm wavelength or ordinary white flashlight irradiation.It is simple,rapid and highly specific,and could detect3×10¹ fg target DNA from one microliter sample DNA.This study investigated whether M.fructicola can be controlled by RNAi-based spray-induced gene silencing（SIGS）technology.The main results are as follows:（3）By co-culturing the spores of M.fructicola with Fluorescein-12-UTP-labeled ds RNA,it was observed under a fluorescence microscope that there was no fluorescence signal in M.fructicola,indicating that ds RNAs in the environment were not effectively absorbed by M.fructicola.（4）The Mf Tub2 gene was selected because it could affect the growth of M.fructicola after silencing.According to the sequence which could cause the Mf Tub2 silencing,ds RNA was synthesized and co-cultured with M.fructicola.It was found that the Mf Tub2 silencing was not induced because the growth of M.fructicola was not affected by adding corresponding ds RNAIn summary,ds RNA in the environment could not be effectively absorbed by M.fructicola,and could not induce its target gene silencing,indicating that the potential of SIGS to control M.fructicola was limited.