A General Framework for Spatio-Temporal Modeling of Epidemics With Multiple Epicenters: Application to an Aerially Dispersed Plant Pathogen

The spread dynamics of long-distance-dispersed pathogens are influenced by the dispersal characteristics of a pathogen, anisotropy due to multiple factors, and the presence of multiple sources of inoculum. In this research, we developed a flexible class of phenomenological spatio-temporal models that extend a modeling framework used in plant pathology applications to account for the presence of multiple sources and anisotropy of biological species that can govern disease gradients and spatial spread in time. We use the cucurbit downy mildew pathosystem (caused by Pseudoperonospora cubensis) to formulate a data-driven procedure based on the 2008 to 2010 historical occurrence of the disease in the U.S. available from standardized sentinel plots deployed as part of the Cucurbit Downy Mildew ipmPIPE program. This pathosystem is characterized by annual recolonization and extinction cycles, generating annual disease invasions at the continental scale. This data-driven procedure is amenable to fitting models of disease spread from one or multiple sources of primary inoculum and can be specified to provide estimates of the parameters by regression methods conditional on a function that can accommodate anisotropy in disease occurrence data. Applying this modeling framework to the cucurbit downy mildew data sets, we found a small but consistent reduction in temporal prediction errors by incorporating anisotropy in disease spread. Further, we did not find evidence of an annually occurring, alternative source of P. cubensis in northern latitudes. However, we found a signal indicating an alternative inoculum source on the western edge of the Gulf of Mexico. This modeling framework is tractable for estimating the generalized location and velocity of a disease front from sparsely sampled data with minimal data acquisition costs. These attributes make this framework applicable and useful for a broad range of ecological data sets where multiple sources of disease may exist and whose subsequent spread is directional.

Temporal Dynamics and Severity of Cucurbit Downy Mildew Epidemics as Affected by Chemical Control and Cucurbit Host Type

Cucurbit downy mildew caused by the oomycete Pseudoperonospora cubensis is an important disease that affects members of Cucurbitaceae family globally. However, temporal dynamics of the disease have not been characterized at the field scale to understand how control strategies influence disease epidemics. Disease severity was assessed visually on cucumber and summer squash treated with weekly alternation of chlorothalonil with either cymoxanil, fluopicolide or propamocarb, during the 2018 spring season and 2019 and 2020 fall seasons in North Carolina, and the 2018 and 2020 fall seasons in South Carolina. Disease onset was observed around mid-June during the spring season and early September during the fall season, followed by a rapid increase in severity until mid-July in the spring season and late September or mid-October in the fall season, typical of polycyclic epidemics. The Gompertz, logistic and monomolecular growth models were fitted to disease severity using linear regression and parameter estimates used to compare the effects of fungicide treatment and cucurbit host type on disease progress. The Gompertz and logistic models were more appropriate than the monomolecular model in describing temporal dynamics of cucurbit downy mildew, with the Gompertz model providing the best description for 34 of the 44 epidemics examined. Fungicide treatment and host type significantly (P < 0.0001) affected standardized area under disease progress curve (sAUDPC), final disease severity (Final DS) and weighted mean absolute rates of disease progress (ρ), with these variables, in most cases, being significantly (P < 0.05) lower in fungicide treated plots than in untreated control plots. Except in a few cases, sAUDPC, Final DS and ρ were lower in cases where chlorothalonil was alternated with fluopicolide or propamocarb than in cases where chlorothalonil alternated with cymoxanil or when chlorothalonil was applied alone. These results characterized the temporal progress of cucurbit downy mildew and provided an improved understanding of the dynamics of the disease at the field level. Parameters of disease progress obtained from this study could serve as inputs in simulation studies to assess the efficacy of fungicide alternation in managing fungicide resistance in this pathosystem.

Efficacy of Fungicides for Pseudoperonospora cubensis Determined using Bioassays over Multiple Years in the Mid-Atlantic and Northeastern United States

In the United States, fungicides are the primary management option for cucumber growers to protect their crops from Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew. Pathogen resistance to some fungicides can quickly develop with the repeated applications needed to protect yield. In order to determine fungicide efficacy and monitor it over time, bioassays were conducted from 2016-2019 in Delaware, Maryland, Pennsylvania, and New York. Potted cucumber plants were either sprayed with fungicides or not treated, placed next to field-grown plants with cucurbit downy mildew for up to two days, then kept in a greenhouse until symptoms developed. Severity of symptoms or number of lesions on leaves was recorded 6-14 days after exposure started and used to determine fungicide efficacy. Quadris (azoxystrobin) was ineffective in seven of the nine bioassays, while Revus (mandipropamid) was ineffective in six of seven bioassays. Forum (dimethomorph) and Presidio (fluopicolide) were ineffective in three of eight and four of nine bioassays, respectively. The most effective fungicides were Bravo (chlorothalonil), Zing! (zoxamide + chlorothalonil), and Orondis (oxathiapiprolin), all of which consistently suppressed disease severity more than 90% when compared with the untreated control. Previcur Flex (propamocarb hydrochloride) and Ranman (cyazofamid) were also effective in every bioassay.

Detection of airborne sporangia of Pseudoperonospora cubensis and P. humuli in Michigan using Burkard spore traps coupled to qPCR

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 U.S., infecting historically resistant cucumber cultivars and requiring the adoption of an intensive fungicide program. The pathogen cannot overwinter in Michigan fields but due to an influx of airborne sporangia cucurbit downy mildew 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 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 below 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 two 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 approximately 5 -10 days before cucurbit downy mildew 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.

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.

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 US 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 qPCR assay was developed based on species- and clade-specific nuclear genomic markers. The assay detected as few as 10 sporangia or 100 fg/ml of DNA for both clades and was validated in the field by deploying roto-rod 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.

Diagnostic Guide for Cucurbit Downy Mildew

Cucurbit downy mildew caused by the oomycete Pseudoperonospora cubensis is the most devastating foliar disease on cultivated cucurbitaceous crops. Failure of host resistance in cucumber and previously effective fungicides has occurred in the last few years in the United States and Europe, making accurate and early diagnosis critical for timely disease management. The objective of this diagnostic guide is to describe the current taxonomy, host, geographic range, symptoms, and signs as well as effective techniques for pathogen identification, evaluation, isolation, and storage for P. cubensis.

Disease Control Attributes of Oxathiapiprolin Fungicides for Management of Cucurbit Downy Mildew

Oxathiapiprolin, a novel oomycete fungicide recently registered by DuPont, was reported to have high intrinsic activity against cucurbit downy mildew (Pseudoperonospora cubensis). The goal of this study was to characterize disease control attributes of oxathiapiprolin-based fungicides critical to effective management of cucurbit downy mildew. In growth chamber and greenhouse studies, oxathiapiprolin-based fungicides were compared with mandipropamid, mefenoxam + mancozeb, fluopicolide + propamocarb, cymoxanil + mancozeb, and ametoctradin + dimethomorph products for pre- and postinfection activity, local systemic movement, and protection of new growth produced after fungicide application. In preventive application, oxathiapiprolin-based fungicides significantly (P < 0.0001) inhibited downy mildew development, with the highest level of disease observed being 0.4% compared with 86.7% observed for mandipropamid. When applied postinfection, oxathiapiprolin-based fungicides significantly (P < 0.0001) suppressed disease development, but disease control was reduced relative to that observed for preventive application. There was a significant effect of formulation on the postinfection activity of oxathiapiprolin, whereby the oil dispersion (OD) formulation was more inhibitory than the water-dispersible granule formulation (0.001 ≤ P ≤ 0.049). Disease severity on the outer half leaf portion protected from spray deposition during fungicide application was lower for oxathiapiprolin-based fungicides (1.6 to 6.6%) than observed for fluopicolide + propamocarb (10.9 to 23.7%), mefenoxam + mancozeb (40.3 to 51.4%), and the nontreated controls (83.3 to 84.9%), which indicates significant acropetal movement within the leaf. Postinfection applications of oxathiapiprolin-based formulations had the greatest effect on lesion growth and sporangia production compared with the other fungicides in the experiment. When applied preventively to rapidly growing plants in a greenhouse, oxathiapiprolin-based fungicides consistently protected new growth that was not present at the time of application, with the OD formulation reducing disease severity by >75% relative to nontreated plants. The practical implications of these observations are discussed.