Development of an RPA-CRISPR/Cas12a-Based Rapid Diagnosis Strip for the Tangerine Pathotype of Alternaria alternata

Citrus alternaria brown spot, caused by the tangerine pathotype of <i>Alternaria alternata</i>, is one of the most severe fungal diseases affecting citrus crops. Currently, there is a critical need for rapid and visual detection techniques to identify the tangerine pathotype of <i>A. alternata</i>. In this study, a novel detection system was developed by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology, targeting the <i>ACTT3</i> gene specific to the tangerine pathotype of <i>A. alternata</i>. Through optimization of reaction time and component concentrations, the assay demonstrated a detection sensitivity of 1 pg μL^-1 within 40 min at a constant temperature of 37 °C. The results can be visually interpreted using nucleic acid test strips, offering advantages in specificity, sensitivity, and speed. This system has been successfully validated for the rapid detection of the pathogen within plant tissues, including leaves and fruits, providing an efficient and practical solution for real-time field detection of the tangerine pathotype of <i>A. alternata</i>.