Poster: Diseases of Plants: Disease Detection & Diagnosis
Detection of Erysiphe necator fungicide-resistant alleles in leaf and air samples using novel molecular diagnostic techniques
J. Yamagata (1), B. Warneke (2), T. Neill (3), W. Mahaffee (4), L. Miles (5), A. Schilder (6) (1) CSU Monterey Bay, U.S.A.; (2) Oregon State University, U.S.A.; (3) USDA ARS, U.S.A.; (4) USDA ARS, U.S.A.; (5) Hartnell College, U.S.A.; (6) Michigan State U
Erysiphe necator (En) the causal agent of grapevine powdery mildew is managed using fungicides such as quinone outside inhibitors (QoIs). Unfortunately, En is known to develop resistance to QoIs by a single nucleotide polymorphism, resulting in an amino acid substitution (G143A) in cytochrome b. To monitor this allele, a TaqMan assay was developed and validated against powdery mildew DNA from strawberry, blueberry, snap pea, squash, Gerbera daisy, rose, and oak plants. Single spored DNA isolates (n = 103) were used to verify the accuracy of the TaqMan assay by comparing it to previous trifloxystrobin germination tests. In-vitro tests of single spored isolates identified 20% as the necessary proportion of the mutant allele in a mixed DNA sample to test positive for resistance. Leaf and air samples from Oregon (n = 130) were utilized in field validations. Digital droplet PCR was also utilized to quantify the allele distribution in a mixed DNA sample and lowered the detection limit the mutant allele for positive resistance to <5% of the total E. necator DNA present. This study resulted in novel allele-specific TaqMan and Digital droplet PCR assays that detect G143A in En. This assay could be used to significantly reduce the labor required in dealing with leaf and air samples of En in the laboratory and could be used to more closely monitor resistance development for a standard management program of grapevine powdery mildew.