Development of species-specific primers and highly sensitive duplex ddPCR assay for the identification and detection of chili anthracnose

Chili anthracnose, caused by Colletotrichum species is a major disease of chili leading to severe economic loss worldwide. Here, two new species-specific primer pairs were designed based on the actin gene sequence and used for the detection of Colletotrichum truncatum (Ct) and Colletotrichum scovillei (Cs), the main causal agents of chili anthracnose. The primers amplified short sequences of 96 and 126 bp for Ct and Cs, respectively. These two species-specific primer pairs were applied in a droplet digital PCR (ddPCR) for duplex detections of Ct and Cs. Highly specific and sensitive assays for the detection of Ct and Cs in infected chili fruit and seeds were presented. The absence of PCR product for template DNA from other Colletotrichum species and different fungal genera was used to inform about the specificity of these primers for Ct and Cs. The sensitivity of duplex ddPCR was 10,000-fold lower than the conventional PCR.