Abstract
Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew, causes severe annual losses in cucurbit production. Management relies on frequent fungicide applications, with oxathiapiprolin (OXTP; FRAC 49) being a cornerstone of current programs. Because OXTP is classified as high risk for resistance development, monitoring for resistance alleles is critical. Although OXTP resistance mutations have been reported in other oomycetes, none had previously been documented in P. cubensis. In this study, we reconstructed the P. cubensis oxysterol-binding protein (OSBP) gene, the target of OXTP, and identified the N772I resistance mutation by Sanger sequencing. We then developed a novel qPCR assay for high-throughput detection of resistance alleles. Screening more than 500 isolates revealed that the N772I resistance mutation is strongly associated with clade 2 isolates, which typically infect cucumber and cantaloupe and are also resistant to QoI and CAA fungicides. In contrast, clade 1 isolates primarily infecting squash, pumpkin, and watermelon remained largely sensitive. These findings provide the first evidence of OXTP resistance mutations in P. cubensis, demonstrate clade-specific occurrence of resistance mutations, and provide a diagnostic tool for rapid screening. Integration of this assay into biosurveillance systems will enable in-season monitoring and support more effective fungicide programs for cucurbit downy mildew.