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

Walnut anthracnose is caused by pathogenic fungi of the genus Colletotrichum. Current detection approaches primarily depend on single-pathogen assays, which frequently fail to identify complex Colletotrichum 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 Colletotrichum 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 Colletotrichum 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 C. fioriniae, C. gloeosporioides, C. godetiae, C. karsti and C. nymphaeae at 10 pg/μL, while achieving 100 pg/μL for C. fructicola and C. siamense. 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.