Development of a qPCR assay for ready-to-use in-field detection and quantification of resistance to SBI-class III fungicides in Botrytis cinerea using a portable real-time-PCR thermocycler

Botrytis cinerea Pers. is the fungal pathogen causing grey mould, one of the major diseases affecting yield and quality on numerous fruit and vegetable crops worldwide. The fungus is recognised as a high-risk plant pathogen for developing fungicide resistance, also due to intensive usage of fungicides with a single-site mode of action. For several fungicides, resistance development led to reduced effectiveness of grey mould control, and then continuous monitoring and the adoption of appropriate anti-resistance strategies should be implemented in disease management. In this study, a real-time quantitative polymerase chain reaction (qPCR) assay was developed for rapid in-field detection and quantification of B. cinerea isolates resistant to fenhexamid, a sterol biosynthesis inhibitor (SBI-III), using a Franklin® Three9 portable thermocycler for real-time PCR (Biomeme, Inc., Philadelphia, PA, USA). DNA was extracted from mycelium and conidia of wild-type strains, usually sensitive to SBI-III, and several mutants of the fungus showing high resistance (HydR3+), carrying different point mutations in the erg27 gene responsible for several amino acid changes at position 412 of the encoded 3-keto reductase enzyme (F412I, F412V, F412S, and F412C). Different DNA extraction protocols and rapid sample preparation methods were tested and compared. The qPCR assay proved to be sensitive (up to 10-50 pg of target DNA) and specific for quantitative detection of field resistance to SBI-III fungicides in B. cinerea. It provides a novel tool for fast and accurate in-field monitoring of plant pathogen populations, reducing time and labour requirements compared to traditional laboratory detection methods.