Clade-Specific Monitoring of Airborne Pseudoperonospora spp. Sporangia Using Mitochondrial DNA Markers for Disease Management of Cucurbit Downy Mildew.

Management of cucurbit downy mildew (CDM) caused by Pseudoperonospora cubensis, relies on an intensive fungicide program. In Michigan, CDM occurs annually due to an influx of airborne sporangia and timely alerts of airborne inoculum can assist growers in assessing the need to initiate fungicide sprays. This research aimed to improve the specific detection of airborne P. cubensis sporangia by adapting quantitative real-time polymerase chain reaction (qPCR) assays to distinguish among P. cubensis clades I and II and P. humuli in spore trap samples from commercial production sites and research plots. We also evaluated the suitability of impaction spore traps compared with Burkard traps for detection of airborne sporangia. A multiplex qPCR assay improved the specificity of P. cubensis clade II detection accelerating the assessment of field spore trap samples. After 2 years of monitoring, P. cubensis clade II DNA was detected in spore trap samples before CDM symptoms were first observed in cucumber fields (July and August), while P. cubensis clade I DNA was not detected in air samples before or after the disease onset. In some commercial cucumber fields, P. humuli DNA was detected throughout the growing season. The Burkard spore trap appeared to be better suited for recovery of sporangia at low concentrations than the impaction spore trap. This improved methodology for the monitoring of airborne Pseudoperonospora spp. sporangia could be used as part of a CDM risk advisory system to time fungicide applications that protect cucurbit crops in Michigan.

Application of Target Enrichment Sequencing for Population Genetic Analyses of the Obligate Plant Pathogens Pseudoperonospora cubensis and P. humuli in Michigan.

Technological advances in genome sequencing have improved our ability to catalog genomic variation and have led to an expansion of the scope and scale of genetic studies over the past decade. Yet, for agronomically important plant pathogens such as the downy mildews (Peronosporaceae), the scale of genetic studies remains limited. This is, in part, due to the difficulties associated with maintaining obligate pathogens and 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 downy mildews, adaptable methods for the genotyping of their populations are required. Here, we describe a targeted enrichment (TE) protocol to genotype isolates from two Pseudoperonospora species (P. cubensis and P. humuli), using less than 50 ng of mixed pathogen and plant DNA for library preparation. We were able to enrich 830 target genes across 128 samples and identified 2,514 high-quality single nucleotide polymorphism (SNP) variants. Using these SNPs, we detected significant genetic differentiation (analysis of molecular variance [AMOVA], P = 0.01) between P. cubensis subpopulations from Cucurbita moschata (clade I) and Cucumis sativus (clade II) in the state of Michigan. No evidence of location-based differentiation was detected within the P. cubensis (clade II) subpopulation in Michigan. However, a significant effect of location on the genetic variation of the P. humuli subpopulation was detected in the state (AMOVA, P = 0.01). Mantel tests found evidence that the genetic distance among P. humuli samples was associated with the physical distance of the hop yards from which the samples were collected (P = 0.005). The differences in the distribution of genetic variation of the Michigan P. humuli and P. cubensis subpopulations suggest differences in the dispersal of these two species. The TE protocol described here provides an additional tool for genotyping obligate biotrophic plant pathogens and the execution of new genetic studies.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Detection of Airborne Sporangia of Pseudoperonospora cubensis and P. humuli in Michigan Using Burkard Spore Traps Coupled to Quantitative PCR.

Cucurbit downy mildew (CDM), caused by the oomycete pathogen Pseudoperonospora cubensis, is a devastating foliar disease on cucumber resulting in reduced yields. In 2004, the pathogen re-emerged in the United States, infecting historically resistant cucumber cultivars and requiring the adoption of an intensive fungicide program. The pathogen cannot overwinter in Michigan fields but because of an influx of airborne sporangia CDM occurs annually. In Michigan, spore traps are used to monitor the presence of airborne P. cubensis sporangia in cucumber growing regions to guide the initiation of a fungicide program. However, Pseudoperonospora humuli sporangia, the causal agent of downy mildew on hop, are morphologically indistinguishable from P. cubensis sporangia. This morphological similarity reduces the ability to accurately detect P. cubensis from spore trap samples when examined with the aid of light microscopy. To improve P. cubensis detection, we adapted a qPCR-based assay to allow the differentiation between P. cubensis and P. humuli on Burkard spore trap samples collected in the field. Specifically, we evaluated the specificity and sensitivity of P. cubensis detection on Burkard spore trap tapes using a morphological-based and quantitative-PCR (qPCR)-based identification assay and determined whether sporangia of P. cubensis and P. humuli on Burkard samples could be distinguished using qPCR. We found that the qPCR assay was able to detect a single sporangium of each species on spore trap samples collected in the field with Cq values <35.5. The qPCR assay also allowed the detection of P. cubensis and P. humuli in samples containing sporangia from both species. However, the number of sporangia quantified using light microscopy explained only 54 and 10% of the variation in the Cq values of P. cubensis and P. humuli, respectively, suggesting a limited capacity of the qPCR assay for the absolute quantification of sporangia in field samples. After 2 years of monitoring using Burkard spore traps coupled with the qPCR in cucumber fields, P. humuli sporangia were detected more frequently than P. cubensis early in the growing season (May and June). P. cubensis sporangia were detected ∼5 to 10 days before CDM symptoms were first observed in cucumber fields during both years. This research describes an improved sporangial detection system that is key for the monitoring and management of P. cubensis in Michigan.