Rapid visual detection of benzimidazole resistance in Botrytis cinerea by recombinase polymerase amplification...

Benzimidazole resistance in Botrytis cinerea is related to point mutations in the target β-tubulin gene (TUB2). Three mutations (E198A, E198K, E198V) at codon 198 account for most of the resistant strains. A rapid on-site diagnostic assay would be useful to detect the presence and monitor further spread of this resistance mechanism.

A recombinase polymerase amplification combined with lateral flow detection (RPA-LFD) method was established for the rapid detection of methyl benzimidazole carbamate (MBC) resistance in B. cinerea. Based on the three mutations at TUB2 codon 198, three sets of RPA-LFD primers were designed, and each of these primer sets was able to specifically amplify the DNA containing its corresponding mutation; no amplification was detected with other mutated or wild-type DNA. The assay was optimized for specificity and sensitivity and was shown to detect the presence of 2 × 10² copies μl^-1 of target DNA per reaction within 10 min. DNA from eight other common fungal species of small fruit did not yield a signal. The system worked well over a wide range of temperatures from 25 to 45°C. Crude DNA obtained from boiled mycelium and conidia of symptomatic fruit could be used as templates, which simplified the assay process.

This study developed a novel assay based on RPA-LFD for the rapid and equipment-free detection of MBC-resistant isolates. In combination with a simple DNA extraction method, the assay could detect B. cinerea MBC-resistant isolates even without specialized equipment within 30 min. Considering its specificity, stability and simplicity, the RPA-LFD assay could be a promising tool for rapid on-site diagnosis of fungicide-resistant isolates. © 2021 Society of Chemical Industry.