Rapid and simple detection of anilinopyrimidine resistance in Botrytis cinerea by combining recombinase polymerase amplification with the CRISPR/Cas12a assay.

Abstract

Anilinopyrimidine (AP) fungicides have been widely adopted to control Botrytis cinerea since the 1990s. As a high-risk pathogen for the development of fungicide resistance, B. cinerea developed resistance to AP fungicides soon after their application. To ensure the proper use of these fungicides, it is necessary to establish a rapid and simple method for resistance detection. Our previous study demonstrated that the E407K mutation in Bcmdl1 was the major mutation conferring AP resistance in China. Based on the combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a nucleic acid detection assay (RPA/Cas12a detection assay), a simple method for the rapid detection of AP resistance was established by specifically identifying this resistance-related mutation. The new detection assay could precisely identify the E407K mutants from other mutants and wild-type isolates within 50 minutes, relying solely on a water/metal bath and a UV flashlight. Moreover, this assay could detect genomic DNA at concentration as low as 1.8 × 106 fg/μL, which is comparable with conventional PCR, indicating its high sensitivity. High specificity among different species were also observed in this assay. Above all, this assay was compatible with a two-minute DNA extraction method, implying its feasibility for field application. In conclusion, the RPA/Cas12a detection assay developed in this study is rapid and simple, making it an ideal method for AP resistance detection in local agencies and other points of care. Instant information on resistance monitoring can provide important guidance on resistance management.