Grapevine black-foot disease is a destructive trunk disease with a complex pathogen composition that often involves mixed and latent infections, making timely field diagnosis challenging. To improve rapid field detection, we developed a rapid, sensitive, and low instrument-dependent nucleic acid assay. The assay integrates recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a for the detection of Ilyonectria and Dactylonectria, two genera associated with grapevine black-foot disease. Conserved regions of the histone H3 and β-tubulin genes were selected for the design of specific RPA primers and corresponding CRISPR RNAs (crRNAs) for Ilyonectria and Dactylonectria, respectively. A workflow integrating RPA, Cas12a-mediated recognition, and lateral flow assay (LFA)-based visualization was established. The reaction conditions were optimized to enhance amplification efficiency and Cas12a recognition stability. Specificity was evaluated using DNA from target and non-target fungi, and sensitivity was determined using serially diluted templates. Under optimized conditions, the assay detected Ilyonectria DNA at concentrations as low as 3.6 ng/μL within 1 h at 39 °C. For Dactylonectria, the detection limit reached 80 fg/μL within 50 min at 41 °C. No cross-reactivity was observed. The LFA strips exhibited positive and negative bands within minutes, enabling rapid visual interpretation. This RPA-CRISPR/Cas12a-LFA system provides a rapid, visually interpretable approach for detecting selected grapevine black-foot disease-associated species in China. The workflow reduces the requirement for specialized thermocycling and fluorescence detection equipment during amplification and readout, following DNA extraction.
