The Development of a Fungicide Tolerance Phenotyping Assay Utilizing Digital Microscopy with the Obligate Biotroph, Erysiphe necator

Grape powdery mildew (Erysiphe necator) poses a significant challenge to vineyards that is primarily managed through fungicide applications. However, the emergence of fungicide-tolerant populations necessitates the development of rapid phenotyping methods. Traditional approaches for obligate biotrophs, such as live leaf disk assays, are effective but labor-intensive and prone to error which hinders throughput and accuracy. Using high-resolution automated digital microscopy, a novel phenotyping method capable of rapidly and objectively assessing E. necator germination was developed for strobilurin (QoI) and quinoxyfen fungicides. Notably, a multiclass image classification model based on conidia shape metrics can accurately distinguish conidia germination status while effectively eliminating image artifacts (accuracy >95%). All isolates with the CYTB QoI resistance allele (A143) were discernable from wild-type isolates (G143) across four unique QoI fungicides. Additionally, isolates exhibiting reduced sensitivity to quinoxyfen in live leaf disk assays were correlated with higher germination rates in the described imaging-based in vitro assay. However, sterol demethylation inhibitor (DMI) tolerance could not be accurately phenotyped. Compared to traditional live leaf disk assays or manually counted germination assays, the described imaging-based in vitro method offers notable advantages in terms of time and cost-effectiveness but is not suitable for all chemistries. This development in phenotyping technology not only enhances our ability to better characterize E. necator but also opens avenues for further research into quinoxyfen fungicide tolerance mechanisms.