A Microplate-Based Assay to Determine DMI Fungicide Sensitivity of Cercospora beticola

Cercospora beticola, the causal agent of Cercospora leaf spot (CLS) in sugar beet, is a major pathogen that significantly impacts crop productivity. The emergence and spread of resistance to demethylation inhibitor (DMI) fungicides further complicate effective disease management. Current methods for monitoring DMI sensitivity, such as radial growth and molecular assays, are limited by low throughput or binary detection of known resistance alleles, respectively. In this study, we developed and validated a high-throughput microplate-based assay to determine DMI fungicide sensitivity in C. beticola using optical density (OD) measurements. The assay was optimized for media type, inoculum preparation, wavelength, fungicide concentration gradients, and incubation time. Relative effective fungicide concentrations that inhibit 50% of growth (EC 50 ) were determined to provide quantitative assessment of fungicide sensitivity to tetraconazole, prothioconazole, difenoconazole, and mefentrifluconazole. The assay demonstrated consistency across two microplate brands and strong correlation with CbCyp51 haplotypes associated with DMI resistance. Our results show that the microplate assay is an efficient tool for monitoring quantitative fungicide sensitivity in C. beticola, offering improved throughput compared to the conventional Petri plate method.