Detection of Colletotrichum spp. Resistant to Benomyl by Using Molecular Techniques

Colletotrichum species is known as the major causal pathogen of red pepper anthracnose in Korea and various groups of fungicides are registered for the management of the disease. However, the consistent use of fungicides has resulted in the development of resistance in many red pepper-growing areas of Korea. Effective management of the occurrence of fungicide resistance depends on constant monitoring and early detection. Thus, in this study, various methods such as agar dilution method (ADM), gene sequencing, allele-specific polymerase chain reaction (PCR), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for the detection of benzimidazole resistance among 24 isolates of Colletotrichum acutatum s. lat. and Colletotrichum gloeosporioides s. lat. The result of the ADM showed that C. gloeosporioides s. lat. was classified into sensitive and resistant isolates to benomyl while C. acutatum s. lat. was insensitive at ≥1 µg/ml of benomyl. The sequence analysis of the β-tubulin gene showed the presence of a single nucleotide mutation at the 198th amino acid position of five isolates (16CACY14, 16CAYY19, 15HN5, 15KJ1, and 16CAYY7) of C. gloeosporioides s. lat. Allele-specific PCR and PCR-RFLP were used to detect point mutation at 198th amino acid position and this was done within a day unlike ADM which usually takes more than one week and thus saving time and resources that are essential in the fungicide resistance management in the field. Therefore, the molecular techniques established in this study can warrant early detection of benzimidazole fungicide resistance for the adoption of management strategies that can prevent yield losses among farmers.