Carboxylic Acid Amide but Not Quinone Outside Inhibitor Fungicide Resistance Mutations Show Clade-Specific Occurrence in Pseudoperonospora cubensis Causing Downy Mildew in Commercial and Wild Cucurbits.

Since its reemergence in 2004, Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew (CDM), has experienced significant changes in fungicide sensitivity. Presently, frequent fungicide applications are required to control the disease in cucumber due to the loss of host resistance. Carboxylic acid amides (CAA) and quinone outside inhibitors (QoI) are two fungicide groups used to control foliar diseases in cucurbits, including CDM. Resistance to these fungicides is associated with single nucleotide polymorphism (SNP) mutations. In this study, we used population analyses to determine the occurrence of fungicide resistance mutations to CAA and QoI fungicides in host-adapted clade 1 and clade 2 P. cubensis isolates. Our results revealed that CAA-resistant genotypes occurred more prominently in clade 2 isolates, with more sensitive genotypes observed in clade 1 isolates, while QoI resistance was widespread across isolates from both clades. We also determined that wild cucurbits can serve as reservoirs for P. cubensis isolates containing fungicide resistance alleles. Finally, we report that the G1105W substitution associated with CAA resistance was more prominent within clade 2 P. cubensis isolates while the G1105V resistance substitution and sensitivity genotypes were more prominent in clade 1 isolates. Our findings of clade-specific occurrence of fungicide resistance mutations highlight the importance of understanding the population dynamics of P. cubensis clades by crop and region to design effective fungicide programs and establish accurate baseline sensitivity to active ingredients in P. cubensis populations.

Clade-Specific Biosurveillance of Pseudoperonospora cubensis Using Spore Traps for Precision Disease Management of Cucurbit Downy Mildew.

Pseudoperonospora cubensis is an obligate oomycete and cause of cucurbit downy mildew (CDM), the most destructive foliar disease affecting cucurbit hosts. Annual epidemics develop throughout the United States as windborne sporangia travel great distances and survive prolonged exposure to solar radiation. Recent genomic evidence suggests that P. cubensis isolates display host adaptation based on their respective clade. Early detection is key for fungicide application timing, and identification of the host-adapted clade provides information on the risk of infection for specific cucurbit crops. In this study, a multiplex quantitative PCR assay was developed based on species- and clade-specific nuclear genomic markers. The assay detected as few as 10 sporangia or DNA at 100 fg/ml for both clades and was validated in the field by deploying rotorod spore samplers in cucurbit sentinel plots located at two research stations in North Carolina. Using this assay, sporangia DNA was detected in spore trap sampling rods before signs of P. cubensis or CDM symptoms were observed in the sentinel plots. Both clade 1 and clade 2 DNA were detected in late-season cucumber and watermelon plots but only clade 2 DNA was detected in the early-season cucumber plots. These results will significantly improve disease management of CDM by monitoring inoculum levels to determine the cucurbit crops at risk of infection throughout each growing season.

Population Analyses Reveal Two Host-Adapted Clades of Pseudoperonospora cubensis, the Causal Agent of Cucurbit Downy Mildew, on Commercial and Wild Cucurbits.

Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew, is an airborne, obligate oomycete pathogen that re-emerged in 2004 and causes foliar disease and yield losses in all major cucurbit crops in the United States. Approximately 60 species in the family Cucurbitaceae have been reported as hosts of P. cubensis. Commercial hosts including cucumber, cantaloupe, pumpkin, squash, and watermelon are grown in North Carolina and many host species occur in the wild as weeds. Little is known about the contribution of wild cucurbits to the yearly epidemic; thus, this study aimed to determine the role of commercial and wild cucurbits in the structuring of P. cubensis populations in North Carolina, a region with high pathogen diversity. Ten microsatellite markers were used to analyze 385 isolates from six commercial and four wild cucurbits from three locations representing different growing regions across North Carolina. Population analyses revealed that wild and commercial cucurbits are hosts of P. cubensis in the United States, that host is the main factor structuring P. cubensis populations, and that P. cubensis has two distinct, host-adapted clades at the cucurbit species level, with clade 1 showing random mating and evidence of recombination and clade 2 showing nonrandom mating and no evidence of recombination. Our findings have implications for disease management because clade-specific factors such as host susceptibility and inoculum availability of each clade by region may influence P. cubensis outbreaks in different commercial cucurbits, timing of fungicide applications, and phenotyping for breeding efforts.