Rapid On-Site Detection of Colletotrichum gloeosporioides Using EASY DNA Extraction (EZ-D) Method Combined with...

Anthracnose, caused by Colletotrichum gloeosporioides, is a globally distributed phytopathogenic disease with a broad host range, posing a serious threat to the healthy growth of forest trees, including Cunninghamia lanceolata. To enable rapid and accurate on-site detection of this pathogen, this study developed a comprehensive field-deployable detection method. The approach integrates the EZ-D method (EASY DNA extraction) for rapid nucleic acid extraction with recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system. A specific target gene, designated Cglo6922, was identified for the detection of C. gloeosporioides. The entire detection process can be completed within approximately 25 min, comprising a 10-min isothermal RPA at 39 °C followed by a 15-min Cas12a cleavage reaction. Specificity evaluation showed that the method successfully detected two C. gloeosporioides isolates derived from different hosts, while no cross-reactivity was observed against a panel of 32 other isolates, including ten Colletotrichum species, eight Phytophthora species, six Pythium species, seven Fusarium species, and one Botryosphaeria dothidea isolate, demonstrating robust species-level specificity. Sensitivity testing revealed that the method achieved a limit of detection (LOD) of 10 pg/μL of genomic DNA for C. gloeosporioides. Furthermore, by incorporating the EZ-D rapid extraction method (requiring only one minute for DNA extraction at a cost of approximately $0.03 USD per sample), target nucleic acid was successfully extracted from artificially inoculated Cunninghamia lanceolata branch samples and proved compatible with the RPA-CRISPR/Cas12a detection system. In conclusion, this study establishes a novel field-deployable detection method for C. gloeosporioides that is rapid, cost-effective, highly specific, and highly sensitive, providing a powerful tool for point-of-care testing (POCT) of this disease.