Identification and detection of chili anthracnose using three new species-specific PCR primers

Three new species-specific primer sets were designed to detect C. truncatum, C. gloeosporioides species complex, and C. scovillei, which were reported as the cause of chili anthracnose disease. These new primers were designed based on the sequence data of the nuclear ribosomal DNA (rDNA) region to amplify and identify short size target DNA (<200 bp). The short size target DNA is more efficient for DNA hybridization, and will improve the sensitivity and specificity of DNA assay. The amplicon size obtained for C. gloeosporioides species complex, C. truncatum, and C. scovillei, was 66, 112, and 168 bp, respectively. Each primer set could identify its specific species of Colletotrichum as confirmed by the absence of PCR product with DNA of two other Colletotrichum species and DNA from different fungal genera, indicating high specificity. The sensitivity test was employed, and the results showed that these new primer sets could strongly amplify DNA at the concentration in femto- to picogram levels. Furthermore, these primer sets can be used to amplify and identify Colletotrichum species DNA directly from infected seeds and fruit tissue. The short PCR products obtained by the new primers have the potential to improve the sensitivity in DNA biosensors applications.