Cucurbit downy mildew (CDM) caused by the oomycete obligate pathogen, Pseudoperonospora cubensis, incites foliar blighting of several cucurbit genera worldwide. In 2004, the pathogen re-emerged in the U.S. infecting historically resistant cucumber cultivars and requiring the adoption of an intensive fungicide program. Due to an influx of aerially dispersed sporangia from overwinter sources, CDM occurs annually in cucumber growing regions of northern U.S. The genetic monitoring of incoming P. cubensis populations is essential for growers to make informed decisions regarding CDM management strategies. However, the scale and resolution of genetic studies of downy mildews (Peronosporaceae) remains limited due to the logistical constraints involved in the genotyping of these species (e.g. obtaining DNA of sufficient quantity and quality). To gain an evolutionary and ecological perspective of P. cubensis, we describe a targeted enrichment (TE) protocol able to genotype environmental samples of Pseudoperonospora spp. using less than 50 ng of DNA for library preparation. Using the TE protocol, we were able to enrich 736 target genes across 101 samples and identified 2,978 high quality SNP variants. This SNPs resolved the population structure of P. cubensis in Michigan and detected significant (AMOVA, P=0.01) genetic differentiation among the P. cubensis populations from squash (clade I) and cucumber (clade II). No evidence of location-based differentiation was detected within the P. cubensis (clade II) subpopulation of Michigan. Timely alerts of an influx of airborne inoculum of two distinct host-adapted clades of P. cubensis can assist Michigan growers in assessing the need to initiate fungicide sprays. However, the inability to distinguish between the morphologically identical sporangia of P. humuli and P. cubensis has been a significant shortcoming. Using spore traps and qPCR assays, an improved methodology for the aerial monitoring of each Pseudoperonospora taxa was identified. A highly specific qPCR assay differentiated Pseudoperonospora humuli, the causal agent of downy mildew on hop, and the two host-adapted clades of P. cubensis (clade I and II) on spore trap samples. After two years of monitoring, P. cubensis clade II DNA was detected in spore trap samples >2 days before CDM symptoms were first observed in commercial cucumber fields (August), while P. humuli DNA was only detected early in the growing season (May and June). P. cubensis clade I DNA was not detected in air samples before or after the disease onset in cucumber fields. Additionally, the probability for P. cubensis detection in Burkard spore trap samples was higher compared to impaction spore trap samples with approximately the same number of sporangia, suggesting that the efficiency of recovery of sporangia by Burkard spore traps exceeds the recovery of impaction spore traps. The methodology described in this study to monitor the airborne concentrations of Pseudoperonospora spp. sporangia could be used as part of a CDM risk advisory system to time fungicide applications that protect cucurbit crops in Michigan.
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