Molecular detection tools for the powdery mildew genus Podosphaera

Powdery mildews are caused by obligate biotrophic fungi that pose a significant threat to a broad range of economically important crops across a diverse range of plant families. However, conventional morphological characterization and PCR-based identification using the internal transcribed spacer (ITS) region often lack the resolution necessary to differentiate closely related species within Erysiphaceae, limiting the precision of current diagnostic approaches. In this study, we developed species-specific TaqMan qPCR assays targeting the nuclear gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to detect Podosphaera shepherdiae (North American strawberry powdery mildew) and P. leucotricha (apple powdery mildew) in environmental samples. To evaluate assay specificity, powdery mildew specimens representing multiple genera were collected between 2020 and 2024 from diverse hosts across Michigan, California, New York, Oregon, and Washington (USA) and were sequenced. The qPCR assays exhibited high specificity and sensitivity, with detection limits of 10 pg of DNA and 100 gene copies for P. shepherdiae, and 5 pg of DNA and 10 gene copies for P. leucotricha, with no cross-reactivity with other collected powdery mildew species. Application of the P. shepherdiae assay to a Fragaria x ananassa 'Annapolis' infection time-course confirmed consistent detection at both early (5 dpi) and later stages of infection. In silico TaqMan qPCR assays for additional powdery mildew taxa were developed using GAPDH sequences obtained from a broad panel of genera within the family Erysiphaceae. Additionally, we document four novel host-pathogen associations that are being reported globally for the first time. These assays provide a robust molecular toolset for early detection and monitoring of powdery mildew fungi, supporting improved management, enabling accurate species identification, and advancing species-specific diagnostics within the Erysiphaceae.