Development of a rapid visual detection method for walnut anthracnose based on RPA-LFD

Walnut anthracnose is caused by pathogenic fungi of the genus <i>Colletotrichum</i>. Current detection approaches primarily depend on single-pathogen assays, which frequently fail to identify complex <i>Colletotrichum</i> populations in field environments, often resulting in a high rate of false negatives. To overcome this constraint, we developed a broad-spectrum detection method using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD), enabling rapid detection of <i>Colletotrichum</i> species. Targeting the conserved internal transcribed spacer (ITS) region, we designed specific primers and probes, selecting the optimal set through systematic screening. The assay specifically identified <i>Colletotrichum</i> species without cross-reacting with other walnut-associated fungi. The optimized RPA-LFD detection system exhibited a 10 min reaction time at 39°C and showed distinct sensitivity thresholds, detecting <i>C</i>. <i>fioriniae</i>, <i>C</i>. <i>gloeosporioides</i>, <i>C</i>. <i>godetiae</i>, <i>C</i>. <i>karsti</i> and <i>C</i>. <i>nymphaeae</i> at 10 pg/μL, while achieving 100 pg/μL for <i>C</i>. <i>fructicola</i> and <i>C</i>. <i>siamense</i>. This RPA-LFD system is simple to operate, with high sensitivity and specificity. It enables visual result interpretation and shows broad application potential in walnut anthracnose management.