Comparison of Next Generation Sequencing and qPCR Methodologies for Quantification of Fungicide Resistance in Mixed Samples of Colletotrichum siamense

Measuring the frequency of fungicide-resistance alleles in pathogen populations is critical for understanding resistance spread and severity. TaqMan qPCR is a standard method for allele quantification, while next-generation sequencing (NGS) technologies offer an alternative for high-resolution detection. As studies exploring NGS for this purpose are limited, the objective of this study was to compare the accuracy and feasibility of Illumina NGS amplicon sequencing with a previously validated TaqMan qPCR assay to quantify resistance alleles to thiophanate-methyl fungicide in a set of mixed Colletotrichum siamense DNA samples. For validation, a set with known frequencies of resistance to thiophanate-methyl was used as a control. Both the NGS- and qPCR-based methods were highly precise with an R2 of 0.99 and 0.97, respectively, for the C. siamense DNA samples with known resistance frequencies. However, with the samples of unknown resistance frequencies that were collected from detached blueberry fruit assays, the NGS method demonstrated more variability than the TaqMan method (p < 0.001). This variability was likely associated with low target DNA concentration, and the presence of host and non-target fungal DNA, which reduced PCR efficiency. This study highlighted strengths and weaknesses of both technologies and important assay design considerations. It was concluded that NGS-based methodologies may be preferential for large sample sizes and for detecting multiple or novel mutations within a region, while the qPCR assay can be more efficient for rapid quantification of known alleles.