Quantitative Sequencing as a High-Throughput Tool for Monitoring QoI Fungicide Resistance in Colletotrichum scovillei Causing Pepper Anthracnose

Anthracnose caused by Colletotrichum scovillei is a major constraint on red pepper (Capsicum annuum) production in South Korea. Intensive use of quinone outside inhibitor (QoI) fungicides has led to widespread resistance, mainly associated with the G143A mutation in the cytochrome b gene. Rapid and reliable resistance monitoring is required to support sustainable disease management. This study evaluated quantitative sequencing (QS) as a molecular tool for detecting and quantifying QoI resistance allele frequencies. Genomic DNA and spore suspensions of pyraclostrobin-sensitive and -resistant isolates were mixed at known ratios to generate pseudo-populations. QS accurately reflected the expected G143A allele frequencies, showing nearly perfect linear correlations (R² > 0.99) for both genomic DNA and spore pools. The applicability of QS was further confirmed using artificially inoculated pepper fruits, where allele frequencies determined from lesion-derived DNA were consistent with inoculum ratios. Field surveys conducted from 2020 to 2023 revealed that the frequency of pyraclostrobin-resistant isolates increased from 72.0% to 91.6%. Resistance frequencies obtained by QS were strongly correlated with those determined by the conventional mass agar dilution method (R² = 0.82-0.97), validating QS as a high-throughput monitoring tool. These results demonstrate that QS provides a robust, sensitive, and scalable approach for monitoring fungicide resistance in C. scovillei populations and can be implemented to guide fungicide use strategies and delay further resistance development.