Rapid visual detection technologies for Botrytis cinerea resistance to QoI and SDHI fungicides based on RPA-CRISPR/Cas12a

Grape gray mold caused by Botrytis cinerea is one of the most important fungal diseases affecting grape production and postharvest quality. The extensive use of fungicides has led to the emergence of resistance in B. cinerea, particularly to quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHI), which compromises the efficacy of disease control. Therefore, detection techniques for fungicide resistance in B. cinerea are needed to provide scientific guidance for disease management. Based on recombinase polymerase amplification (RPA)-CRISPR/Cas12a technology, this study designed specific RPA primer pairs and crRNA targeting resistance-related mutation sites in fungicide target genes cytb and SdhB, incorporated single-stranded fluorescent reporter molecules, and optimized key reaction components. This study achieved rapid fluorescence-based resistance detection within 1 h, with detection sensitivities of 450 and 430 pg/µL for the two fungicide genes.