Development of a qPCR assay for ready-to-use in-field detection and quantification of resistance to SBI-class III fungicides in Botrytis cinerea

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 all over the word. The fungus is recognised as a high-risk pathogen for the development of fungicide resistance, also due to intensive usage of fungicides with single-site mode of action, promoting the emergence of resistant mutants in fungal populations. Resistance development has been observed for several fungicides, leading to reduced effectiveness against grey mould and then a continuous monitoring and the adoption of appropriate anti-resistance strategies need to 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 both mycelium and conidia of wild-type strains, normally sensitive to SBI-III, and several mutants of the fungus showing high resistance (HydR3+) because 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 protocols of DNA extraction and rapid methods for sample preparation were tested and compared to minimize field time and improve the efficiency of the assay. The qPCR assay proved to be sensitive (even 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 the pathogen populations reducing time and labour requirements over traditional laboratory detection methods, which is useful for managing the evolving resistance in crop protection.