A Single Tube RPA/Cas12a-Based Diagnostic Assay for Early, Rapid, and Efficient Detection of <i>Botrytis cinerea</i> in Sweet Cherry

The fungus <i>Botrytis cinerea</i> is a devastating plant pathogen that causes gray mold rot in many fruits and vegetables, leading to substantial losses globally. The pathogen induces a broad range of symptoms, making it challenging to differentiate from other pathogens, especially when plants exhibit no symptoms during visual inspection. Therefore, an efficient early warning and rapid detection system is crucial for surveillance and monitoring plant health, as well as for quantitative pathogen risk assessment to reduce microbial risks. In this study, we developed a single-tube detection method for <i>B. cinerea</i> based on RPA/Cas12a (Bc-RPA/Cas12a), which can be performed under field conditions and visualized using mini-UV light and ordinary mobile phone light within 45 min at a constant temperature. The Bc-RPA/Cas12a assay can detect <i>B. cinerea</i> without cross-reactivity with other pathogens and without compromising sensitivity. The universality of the Bc-RPA/Cas12a assay was demonstrated through its adaptability across different geographical regions of China. Furthermore, field simulation assays revealed that fruits inoculated with the target pathogen showed fluorescence, whereas fruits inoculated with nontargeted pathogens and double-distilled water did not exhibit fluorescence. The findings suggest that this assay can be applied directly in field conditions for rapid and accurate detection of <i>B. cinerea</i> without the need for specialized equipment.