PCR Based Assays for the Specific Detection of Colletotrichum species Causing Apple Bitter Rot in Korea

Colletotrichum species of the C. gloeosporioides species complex are the major pathogens of bitter rot of apples in Korea. To ensure early and quick detection of these species, species-specific primers based on the single nucleotide polymorphisms (SNPs) in the nucleotide sequences of the β-tubulin gene were designed and used in PCR assays. Primers were designed by making the SNP the 3‘ end and introducing a single nucleotide artificial mismatch at the penultimate base to the SNP. Primer pairs Cae-Bt462F/CgSc-Bt691R, Cfr-Bt492F/CgSc-Bt691R, Cgl-591F/CgSc-Bt691R and Csi- Bt567F/CgSc-Bt691R successfully amplified specific PCR fragments from C. aenigma, C. fructicola, C. gloeosporioides and C. siamense respectively in pure cultures and inoculated apple fruits. Each primer pair only amplified fragments from their respective species. The sensitivity assay to determine the lowest detectable DNA concentration of the primers showed that the primers could detect as low as 10 to 100 pg of purified DNA. A duplex PCR assay was conducted with primer pairs Cfr-Bt492F/CgSc-Bt691R and Csi-Bt567F/CgSc-Bt691R for the detection of C. fructicola and C. siamense, the two most isolated species from bitter rot infected apple in Korea. The primers could specifically detect each species from one reaction. This PCR-based protocol could be used for quick detection of Colletotrichum gloeosporioides species complex causing apple bitter rot in Korea and could save the cost of bitter rot diagnosis. KCI Citation Count: 0