A Lateral Flow-Recombinase Polymerase Amplification Method for <i>Colletotrichum gloeosporioides</i> Detection

The greater yam (<i>Dioscorea alata</i>), a widely cultivated and nutritious food crop, suffers from widespread yield reduction due to anthracnose caused by <i>Colletotrichum gloeosporioides</i>. Latent infection often occurs before anthracnose phenotypes can be detected, making early prevention difficult and causing significant harm to agricultural production. Through comparative genomic analysis of 60 genomes of 38 species from the <i>Colletotrichum</i> genus, this study identified 17 orthologous gene groups (orthogroups) that were shared by all investigated <i>C. gloeosporioides</i> strains but absent from all other <i>Colletotrichum</i> species. Four of the 17 <i>C. gloeosporioides</i>-specific orthogroups were used as molecular markers for PCR primer designation and <i>C. gloeosporioides</i> detection. All of them can specifically detect <i>C. gloeosporioides</i> out of microbes within and beyond the <i>Colletotrichum</i> genus with different sensitivities. To establish a rapid, portable, and operable anthracnose diagnostic method suitable for field use, specific recombinase polymerase amplification (RPA) primer probe combinations were designed, and a lateral flow (LF)-RPA detection kit for <i>C. gloeosporioides</i> was developed, with the sensitivity reaching the picogram (pg) level. In conclusion, this study identified <i>C. gloeosporioides</i>-specific molecular markers and developed an efficient method for <i>C. gloeosporioides</i> detection, which can be applied to the prevention and control of yam anthracnose as well as anthracnose caused by <i>C. gloeosporioides</i> in other crops. The strategy adopted by this study also serves as a reference for the identification of molecular markers and diagnosis of other plant pathogens.