Grape powdery mildew and downy mildew, caused by Erysiphe necator and Plasmopara viticola, respectively, are considered as major threats to grape production worldwide. To manage these pathogens, grape growers rely heavily on various contact and systemic fungicides. However, repeated fungicide applications have led to widespread resistance in E. necator and P. viticola populations, affecting disease management strategies. This dissertation explores the prevalence and geographic distribution of fungicide resistance in Plasmopara viticola populations across vineyards in the eastern United States and Canada. It also focuses on developing rapid detection tools for identifying QoI (Quinone oxide inhibitors, Fungicide Resistance Action Committee (FRAC) 11) resistance in E. necator and CAA (Carboxylic Acid Amides, FRAC 40) resistance in P. viticola populations. Additionally, this research explores the impact of sublethal doses of powdery mildew-specific fungicides (cyflufenamid, flutianil, metrafenone, and quinoxyfen) on E. necator conidial germination and assesses associated changes in gene expression.The first part of this research involves development of rapid detection tool for QoI fungicide resistance in E. necator. The G143A mutation in the cytochrome b gene is commonly associated with QoI resistance in E. necator populations in the field. A peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA-LAMP) assay was developed to detect the G143A mutation, achieving 100% specificity and sensitivity in distinguishing QoI-sensitive and resistant isolates. The assay demonstrated rapid detection capabilities, completing tests in under 30 minutes. The PNA-LNA-LAMP assay showed 73.3% concordance with the TaqMan probe-based assay (previously developed assay) when both G-143 and A-143 alleles were present in a sample. Validation across three laboratories demonstrated 94.4% accuracy in one laboratory and 100% in the other two. This diagnostic tool is faster and more cost-effective than the TaqMan assay, as it does not require expensive equipment, making the PNA-LNA-LAMP assay a valuable tool for diagnostic laboratories to detect QoI resistance in E. necator. In the second part, a spore germination assay assessed the effects of sub-lethal doses of specialized fungicides—cyflufenamid, flutianil, metrafenone, quinoxyfen, and trifloxystrobin—on E. necator. While flutianil, quinoxyfen and trifloxystrobin effectively inhibited conidial germination, cyflufenamid and metrafenone showed no inhibitory effects, even at high concentrations (100 ppm). Transcriptomic analyses of flutianil, quinoxyfen and trifloxystrobin revealed that all of these fungicides induced distinct gene expression profiles in E. necator. Flutianil, quinoxyfen, and trifloxystrobin treatments upregulated genes involved in respiration, including pathways of the electron transport chain, glycolysis, and the Krebs cycle. Specific genes such as Cytochrome P450 monooxygenase, Maltose permease MAL31, and NADP-dependent oxidoreductase RED1 were upregulated. Exposure to quinoxyfen resulted in decreased expression of protein kinase genes, indicating disruption in signal transduction. In contrast, flutianil treatment led to increased expression of sugar transporters and chitinase, possibly indicating increased energy requirements and cell wall remodeling. Additionally, overexpression of multidrug transporters (MFS transporters and ABC drug transporters) in quinoxyfen and flutianil treatments indicated an elevated risk of developing fungicide resistance. The final section addresses fungicide resistance in P. viticola populations, with a focus on QoI, CAA, Quinone inside inhibitors (QiIs, FRAC 21), and Quinone inside and outside inhibitor, stigmatellin binding mode (QioSI, FRAC 45) fungicides. A total of 658 samples were collected from vineyards across the eastern United States and Canada. Sequencing of CesA3 and Cytb genes, along with ITS1 region analysis was conducted. Three prevalent clades: aestivalis, vinifera, and riparia were identified. QoI resistance, driven by the A-143 genotype, was widespread among aestivalis and vinifera clades. The G143A mutation was not specific to any particular clade. The G1105S mutation, linked to CAA resistance, was predominantly detected in P. viticola clade aestivalis clade samples from Georgia, New York, and Ontario. Mixed-genotype samples (containing both G-1105 and S-1105 alleles) were identified in P. viticola clades vinifera and riparia clades from Michigan, New York, and Wisconsin. No mutations associated with QiI and QioSI resistance were detected in the eastern United States and Canada. A TaqMan probe-based assay developed for detecting the G1105S mutation associated with CAA fungicide resistance demonstrated high sensitivity and specificity, effectively distinguishing resistant genotypes in both leaf and air samples. These findings are valuable for implementing effective fungicide resistance management strategies in grape cultivation.
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