Location: Horticultural Crops ResearchTitle: Allele-specific detection methods for QoI fungicide resistant Erysiphe necator in vineyards
|MILES, TIM - Michigan State University|
|COLLE, MARIVI - Michigan State University|
|WARNEKE, BRENT - Oregon State University|
|ROBINSON, GUY - University Of California, Davis|
|STERGIOPOULOS, IOANNIS - University Of California, Davis|
|Mahaffee, Walter - Walt|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/17/2020
Publication Date: 7/1/2020
Publication URL: https://handle.nal.usda.gov/10113/7071190
Citation: Miles, T.D., Neill, T.M., Colle, M., Warneke, B., Robinson, G., Stergiopoulos, I., Mahaffee, W.F. 2020. Allele-specific detection methods for QoI fungicide resistant Erysiphe necator in vineyards. Plant Disease. https://doi.org/10.1094/PDIS-11-19-2395-RE.
Interpretive Summary: During the 2015 grape growing season in Oregon, powdery mildew epidemics in some vineyards were not responding to standard fungicide programs. Isolates of Erysiphe necator, the fungus which causes powdery mildew were collected from these vineyards, did not respond to quinone outside inhibitor (QoI) fungicides in bioassays. Sequencing of the cytochrome b gene indicated that there was a single genetic mutation associated with all resistance isolates. In order to develop rapid diagnostics to identify QoI resistant E. necator, nucleic acid amplification methods were developed that were specific and sensitive for the mutation. The suitability of these methods for practical monitoring during the season was demonstrated, thus, these methods should be useful to inform grower's fungicide selection decisions and reduce ineffective pesticide usage in grape growing regions.
Technical Abstract: Grapevine powdery mildew (GPM), caused by the fungal pathogen Erysiphe necator (En), is one of the most devastating diseases on grapes worldwide and management requires numerous prophylactic fungicide applications. GPM control failures in 2015 lead to the exploration of the possibility of quinone outside inhibitor (QoI) fungicide resistance present in the Oregon En population. Sequencing of the cytochrome b (cytß) gene from isolates detected only the single nucleotide polymorphism (G143A) associated with the resistance phenotype in conidia germination assays on fungicide amended agar. To enable rapid detection of the QoI resistance in E. necator, TaqMan quantitative PCR (qPCR) and digital droplet PCR (ddPCR) assays were developed with allele-specific TaqMan probes that target the G143A alleles. Primers and probes were specific for the G143A polymorphism in En based on sequencing of product and results of other Erysiphales species and environmental samples. Both assays were sensitive to single conidia and did not detect heteroplasia in individual isolates. These assays were used to characterize leaf and air samples from vineyards in Oregon and indicated the QoI the G143A polymorphism begin developing in 2013. The qPCR and ddPCR assays developed in this study present a useful tool for characterizing QoI fungicide resistance of E. necator from a variety of sample types, even when multiple genotypes are present. These identification tools can be implemented for both leaf and air samples and potentially inform grower's fungicide selection decisions and reduce ineffective pesticide usage in grape growing regions.