Occurrence and distribution of Meloidogyne spp. in fields rotated with sweetpotato and host range of a North Carolina population of Meloidogyne enterolobii.

Root-knot nematodes (RKN, Meloidogyne spp.) are some of the most economically important and common plant parasitic nematodes in North Carolina (NC) cropping systems. Soil samples collected from fields planted with crops rotated with sweetpotato [Ipomoea batatas (L.) Lam.] in 39 NC counties in 2015-2018 were processed at the NC Nematode Assay Laboratory. The occurrence of second-stage juvenile (J2) RKN populations was examined based on collection year, month, county, and previous planted crop. The highest number of RKN positive samples originated from Cumberland (53%), Sampson (48%), and Johnston (48%) counties. The highest average RKN population density was detected in Sampson (147 J2/500 cm3 soil) and Nash (135 J2/500 cm3 soil) counties, while Wayne (7 J2/500 cm3 soil) and Greene (11 J2/500 cm3 soil) counties had the lowest average RKN population density. Meloidogyne enterolobii is a new invasive species that is impacting sweetpotato growers of NC. The host status of a NC population of M. enterolobii, the guava-root knot nematode, was determined by examining eggs per gram of fresh root (ER) and the final nematode egg population divided by the initial population egg count (reproductive factor, RF) in greenhouse experiments. This included eighteen vegetable, field, cover crops and weed species. The tomato 'Rutgers' was used as a susceptible control. Cabbage 'Stonehead', pepper 'Red bull', and watermelon 'Charleston gray' and 'Fascination' were hosts and had similar mean ER values to the positive control, ranging from 64 to 18,717. Among field crops, cotton, soybean 'P5018RX', and tobacco were hosts with ER values that ranged from 185 to 706. Members of the Poaceae family such as sweet corn (Zea mays) and sudangrass (Sorghum x drummondii) were non-hosts to M. enterolobii and the mean ER values ranged from 1.85 to 7. The peanut 'Tifguard' and winter wheat (Triticum aestivum) also had lower ER values than the vegetable hosts. Growers should consider planting less susceptible or non-hosts such as peanut, sudangrass, sweet corn, and winter wheat in 2-3 year crop rotations to lower populations of this invasive nematode.

Open Access and Reproducibility in Plant Pathology Research: Guidelines and Best Practices.

The landscape of scientific publishing is experiencing a transformative shift toward open access, a paradigm that mandates the availability of research outputs such as data, code, materials, and publications. Open access provides increased reproducibility and allows for reuse of these resources. This article provides guidance for best publishing practices of scientific research, data, and associated resources, including code, in The American Phytopathological Society journals. Key areas such as diagnostic assays, experimental design, data sharing, and code deposition are explored in detail. This guidance aligns with that observed by other leading journals. We hope the information assembled in this paper will raise awareness of best practices and enable greater appraisal of the true effects of biological phenomena in plant pathology.

Effector Repertoire of the Sweetpotato Black Rot Fungal Pathogen Ceratocystis fimbriata.

In 2015, sweetpotato producers in the United States experienced one of the worst outbreaks of black rot recorded in history, with up to 60% losses reported in the field and packing houses and at shipping ports. Host resistance remains the ideal management tool to decrease crop losses. Lack of knowledge of Ceratocystis fimbriata biology represents a critical barrier for the deployment of resistance to black rot in sweetpotato. In this study, we scanned the recent near chromosomal-level assembly for putative secreted effectors in the sweetpotato C. fimbriata isolate AS236 using a custom fungal effector annotation pipeline. We identified a set of 188 putative effectors on the basis of secretion signal and in silico prediction in EffectorP. We conducted a deep RNA time-course sequencing experiment to determine whether C. fimbriata modulates effectors in planta and to define a candidate list of effectors expressed during infection. We examined the expression profile of two C. fimbriata isolates, a pre-epidemic (1990s) isolate and a post-epidemic (2015) isolate. Our in planta expression profiling revealed clusters of co-expressed secreted effector candidates. Based on fold-change differences of putative effectors in both isolates and over the course of infection, we suggested prioritization of 31 effectors for functional characterization. Among this set, we identified several effectors that provide evidence for a marked biotrophic phase in C. fimbriata during infection of sweetpotato storage roots. Our study revealed a catalog of effector proteins that provide insight into C. fimbriata infection mechanisms and represent a core catalog to implement effector-assisted breeding in sweetpotato. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Long-read sequencing genome assembly of Ceratocystis fimbriata enables development of molecular diagnostics for sweetpotato black rot.

Ceratocystis fimbriata is a destructive fungal pathogen of sweetpotato (Ipomoea batatas (L.) Lam.) that leads to losses at all stages of sweetpotato production. Accurate detection of C. fimbriata would allow for more efficient deployment of management tactics in sweetpotato production. To develop a diagnostic assay, a hybrid genome assembly of C. fimbriata isolate AS236 was generated. The resulting 31.7 MB assembly was near-chromosome level, with 18 contigs, 6,481 predicted genes, and a BUSCO completion score of 98.4% when compared to the fungi-specific lineage database. Additional Illumina DNA reads from C. manginecans, C. platani, and a second C. fimbriata isolate (C1421) were then mapped to the assembled genome using BOWTIE2 and counted using HTSeq, which identified 148 genes present only within C. fimbriata as molecular diagnostic candidates; 6 single-copy and 35 highly multi-copy (>40 BLAST hits), as determined through a self-BLAST-P alignment. Primers for PCR were designed in the 200 bp flanking region of the first exon for each candidate, and the candidates were validated against a diverse DNA panel containing Ceratocystis species, sweetpotato pathogens, and plants. After validation, two diagnostic candidates amplified only C. fimbriata DNA and were considered to be highly specific to the species. These genetic markers will serve as valuable diagnostic tools with multiple applications including the detection of C. fimbriata in seed, soil, and wash water in sweetpotato production.

Sensitivity of Meloidogyne incognita, Fusarium oxysporum f.sp. niveum, and Stagonosporopsis citrulli to succinate dehydrogenase inhibitors used for control of watermelon diseases.

Watermelon is affected by diseases such as Fusarium wilt, gummy stem blight, and root-knot nematode. Succinate dehydrogenase inhibitors (SDHIs) with potential fungicide and nematicide activity provide the opportunity to control multiple diseases with one compound. In this study, we aimed to determine the sensitivity of Meloidogyne incognita race 4 (MI4), Fusarium oxysporum f.sp. niveum (FON), and Stagonosporopsis citrulli (SCIT), to existing SDHIs: benzovindiflupyr, fluopyram, cyclobutrifluram, and pydiflumetofen. All SDHIs had fungicidal activity against 19 SCIT isolates in mycelial growth assays, but isolates were most sensitive to pydiflumetofen (median EC50 = 0.41 µg/ml). Most of 50 FON isolates tested were sensitive to cyclobutrifluram for mycelial growth (median EC50 = 4.04 µg/ml) and conidial germination (median EC50 = 0.2 µg/ml) assays but were not sensitive to fluopyram. MI4 was most sensitive to cyclobutrifluram for egg hatch (mean EC50 = 0.0019 µg/ml) and J2 motility (mean EC50 = 1.16 µg/ml) assays but was not sensitive to pydiflumetofen. Significant positive correlations between the sensitivity of SCIT (mycelial growth) and FON (mycelial growth and conidial germination) for cyclobutrifluram and benzovindiflupyr (SCIT r=0.88, FON r =0.7, P< 0.0001) and cyclobutrifluram and pydiflumetofen (SCIT r=0.83, FON r =0.67 and 0.77, P< 0.0001) indicate a potential for cross-resistance between these SDHIs for these fungal pathogens. Overall, results suggest that cyclobutrifluram may be used for managing root-knot nematode, whereas it should be used judiciously for Fusarium wilt of watermelon and gummy stem blight due to the existence of insensitive isolates to the fungicide.

Effect of Cucurbit Host, Production Region, and Season on the Population Structure of Pseudoperonospora cubensis in Florida.

Pseudoperonospora cubensis, the causal agent of Cucurbit downy mildew (CDM), is one of the most important diseases affecting cucurbit production in the United States. This disease is especially damaging to Florida production areas, as the state is a top producer of many cucurbit species. In addition, winter production in central and south Florida likely serves as a likely source of P. cubensis inoculum for spring and summer cucurbit production throughout the eastern United States, where CDM is unable to overwinter in the absence of a living host. Over 2 years (2017 and 2018) and four seasons (spring 2017, spring 2018, fall 2017, and fall 2018), 274 P. cubensis isolates were collected from cucurbit hosts at production sites in south, central, and north Florida. The isolates were analyzed with 10 simple sequence repeat (SSR) markers to establish population structure and genetic diversity and further assigned to a clade based on a qPCR assay. Results of population structure and genetic diversity analyses differentiated isolates based on cucurbit host and clade (1 or 2). Of the isolates assigned to clade by qPCR, butternut squash, watermelon, and zucchini were dominated by clade 1 isolates, whereas cucumber isolates were split 34 and 59% between clades 1 and 2, respectively. Clade assignments agreed with isolate clustering observed within discriminant analysis of principal components (DAPC) based on SSR markers, although watermelon isolates formed a group distinct from the other clade 1 isolates. For seasonal collections from cucumber at each location, isolates were typically skewed to one clade or the other and varied across locations and seasons within each year of the study. This variable population structure of cucumber isolates could have consequences for regional disease management. This is the first study to characterize P. cubensis populations in Florida and evaluate the effect of cucurbit host and clade-type on isolate diversity and population structure, with implications for CDM management in Florida and other United States cucurbit production areas.

Phytophthora capsici, 100 Years Later: Research Mile Markers from 1922 to 2022.

In 1922, Phytophthora capsici was described by Leon Hatching Leonian as a new pathogen infecting pepper (Capsicum annuum), with disease symptoms of root rot, stem and fruit blight, seed rot, and plant wilting and death. Extensive research has been conducted on P. capsici over the last 100 years. This review succinctly describes the salient mile markers of research on P. capsici with current perspectives on the pathogen's distribution, economic importance, epidemiology, genetics and genomics, fungicide resistance, host susceptibility, pathogenicity mechanisms, and management.

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.

Phytophthora capsici Populations Are Structured by Host, Geography, and Fluopicolide Sensitivity.

Phytophthora capsici epidemics are propelled by warm temperatures and wet conditions. With temperatures and inland flooding in many locations worldwide expected to rise as a result of global climate change, understanding of population structure can help to inform management of P. capsici in the field and prevent devastating epidemics. Thus, we investigated the effect of host crop, geographical origin, fungicide sensitivity, and mating type on shaping the population structure of P. capsici in the eastern United States. Our fungicide in vitro assays identified the emergence of insensitive isolates for fluopicolide and mefenoxam. A set of 12 microsatellite markers proved informative to assign 157 P. capsici isolates to five distinct genetic clusters. Implementation of Bayesian structure, population differentiation, genetic diversity statistics, and index of association analysis, allowed us to identify population structure by host with some correspondence with genetic clusters for cucumber and squash isolates. We found weak population structure by state for geographically close isolates. In this study, we discovered that North Carolina populations stratify by fluopicolide sensitivity with insensitive isolates experiencing nonrandom mating. Our findings highlight the need for careful monitoring of local field populations, improved selection of relevant isolates for breeding efforts, and hypervigilant surveillance of resistance to different fungicides.

Duration of Downy Mildew Control Achieved with Fungicides on Cucumber Under Florida Field Conditions.

Cucurbit production in Florida is impacted by downy mildew on a yearly basis. Cucurbit downy mildew (CDM), caused by Pseudoperonospora cubensis, is one of the most devastating cucurbit diseases and can lead to complete yield loss. Nearly continuous production of cucurbits occurs temporally throughout Florida, which puts extensive pressure on the pathogen population to select for individuals that are resistant to fungicides in use labeled for CDM. Loss of efficacy as a result of fungicide resistance developing is becoming a major concern for Florida cucurbit growers who rely on these products to manage CDM. This study was established to evaluate the field activity of 11 utilized fungicides by determining their duration of activity when applied at various intervals for the management of CDM in cucumber under Florida field conditions. By comparing levels of percent CDM control and area under the disease progress curve values, the fungicide's duration of field activity was established. Field activities were <1 week for dimethomorph and fluopicolide; 1 week for cymoxanil; 1 to 2 weeks for chlorothalonil and mancozeb; 2 weeks for ethaboxam; 1 to 3 weeks for propamocarb, cyazofamid, and ametoctradin + dimethomorph; and 2 to 4 weeks for oxathiapiprolin and fluazinam. Knowledge of duration of field activity can potentially improve the development of CDM management programs and slow the resistance selection.

The hop downy mildew pathogen Pseudoperonospora humuli.

Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew, one of the most devastating diseases of cultivated hop, Humulus lupulus. Downy mildew occurs in all production areas of the crop in the Northern Hemisphere and Argentina. The pathogen overwinters in hop crowns and roots, and causes considerable crop loss. Downy mildew is managed by sanitation practices, planting of resistant cultivars, and fungicide applications. However, the scarcity of sources of host resistance and fungicide resistance in pathogen populations complicates disease management. This review summarizes the current knowledge on the symptoms of the disease, life cycle, virulence factors, and management of hop downy mildew, including various forecasting systems available in the world. Additionally, recent developments in genomics and effector discovery, and the future prospects of using such resources in successful disease management are also discussed. TAXONOMY: Class: Oomycota; Order: Peronosporales; Family: Peronosporaceae; Genus: Pseudoperonospora; Species: Pseudoperonospora humuli. DISEASE SYMPTOMS: The disease is characterized by systemically infected chlorotic shoots called "spikes". Leaf symptoms and signs include angular chlorotic lesions and profuse sporulation on the abaxial side of the leaf. Under severe disease pressure, dark brown discolouration or lesions are observed on cones. Infected crowns have brown to black streaks when cut open. Cultivars highly susceptible to crown rot may die at this phase of the disease cycle without producing shoots. However, foliar symptoms may not be present on plants with systemically infected root systems. INFECTION PROCESS: Pathogen mycelium overwinters in buds and crowns, and emerges on infected shoots in spring. Profuse sporulation occurs on infected tissues and sporangia are released and dispersed by air currents. Under favourable conditions, sporangia germinate and produce biflagellate zoospores that infect healthy tissue, thus perpetuating the infection cycle. Though oospores are produced in infected tissues, their role in the infection cycle is not defined. CONTROL: Downy mildew on hop is managed by a combination of sanitation practices and timely fungicide applications. Forecasting systems are used to time fungicide applications for successful management of the disease. USEFUL WEBSITES: https://content.ces.ncsu.edu/hop-downy-mildew (North Carolina State University disease factsheet), https://www.canr.msu.edu/resources/michigan-hop-management-guide (Michigan Hop Management Guide), http://uspest.org/risk/models (Oregon State University Integrated Plant Protection Center degree-day model for hop downy mildew), https://www.usahops.org/cabinet/data/Field-Guide.pdf (Field Guide for Integrated Pest Management in Hops).

A Comprehensive Characterization of Ecological and Epidemiological Factors Driving Perennation of Podosphaera macularis Chasmothecia on Hop (Humulus lupulus).

Hop powdery mildew, caused by the ascomycete fungus Podosphaera macularis, is a consistent threat to sustainable hop production. The pathogen utilizes two reproductive strategies for overwintering and perennation: (i) asexual vegetative hyphae on dormant buds that emerge the following season as infected shoots; and (ii) sexual ascocarps (chasmothecia), which are discharged during spring rain events. We demonstrate that P. macularis chasmothecia, in the absence of any asexual P. macularis growth forms, are a viable overwintering source capable of causing early season infection two to three orders of magnitude greater than that reported for perennation via asexual growth. Two epidemiological models were defined that describe (i) temperature-driven maturation of P. macularis chasmothecia; and (ii) ascosporic discharge in response to duration of leaf wetness and prevailing temperatures. P. macularis ascospores were confirmed to be infectious at temperatures ranging from 5 to 20°C. The organism's chasmothecia were also found to adhere tightly to the host tissue on which they formed, suggesting that these structures likely overwinter wherever hop tissue senesces within a hop yard. These observations suggest that existing early season disease management practices are especially crucial to controlling hop powdery mildew in the presence of P. macularis chasmothecia. Furthermore, these insights provide a baseline for the validation of weather-driven models describing maturation and release of P. macularis ascospores, models that can eventually be incorporated into hop disease management programs.

Fantastic Downy Mildew Pathogens and How to Find Them: Advances in Detection and Diagnostics.

Downy mildews affect important crops and cause severe losses in production worldwide. Accurate identification and monitoring of these plant pathogens, especially at early stages of the disease, is fundamental in achieving effective disease control. The rapid development of molecular methods for diagnosis has provided more specific, fast, reliable, sensitive, and portable alternatives for plant pathogen detection and quantification than traditional approaches. In this review, we provide information on the use of molecular markers, serological techniques, and nucleic acid amplification technologies for downy mildew diagnosis, highlighting the benefits and disadvantages of the technologies and target selection. We emphasize the importance of incorporating information on pathogen variability in virulence and fungicide resistance for disease management and how the development and application of diagnostic assays based on standard and promising technologies, including high-throughput sequencing and genomics, are revolutionizing the development of species-specific assays suitable for in-field diagnosis. Our review provides an overview of molecular detection technologies and a practical guide for selecting the best approaches for diagnosis.

A Multiplex TaqMan qPCR Assay for Detection and Quantification of Clade 1 and Clade 2 Isolates of Pseudoperonospora cubensis and Pseudoperonospora humuli.

The ability to detect and quantify aerially dispersed plant pathogens is essential for developing effective disease control measures and epidemiological models that optimize the timing for control. There is an acute need for managing the downy mildew pathogens infecting cucurbits and hop incited by members of the genus Pseudoperonospora (Pseudoperonospora cubensis clade 1 and 2 isolates and Pseudoperonospora humuli, respectively). A highly specific multiplex TaqMan quantitative polymerase chain reaction (PCR) assay targeting unique sequences in the pathogens' mitochondrial genomes was developed that enables detection of all three taxa in a single multiplexed amplification. An internal control included in the reaction evaluated whether results were influenced by PCR inhibitors that can make it through the DNA extraction process. Reliable quantification of inoculum as low as three sporangia in a sample was observed. The multiplexed assay was tested with DNA extracted from purified sporangia, infected plant tissue, and environmental samples collected on impaction spore traps samplers. The ability to accurately detect and simultaneously quantify all three pathogens in a single multiplexed amplification should improve management options for controlling the diseases they cause.

Effects of Water Temperature, Inoculum Concentration and Age, and Sanitizers on Infection of Ceratocystis fimbriata, Causal Agent of Black Rot in Sweetpotato.

Black rot, caused by Ceratocystis fimbriata, is a devastating postharvest disease of sweetpotato that recently re-emerged in 2014. Although the disease is known to develop in storage and during export to overseas markets, little is known as to how pathogen dispersal occurs. This study was designed to investigate dump tank water as a means of dispersal through four different types of water treatments: inoculum concentration (0, 5, 5 × 101, 5 × 102, and 5 × 103 spores/ml), inoculum age (0, 24, 48, 96, and 144 h), water temperature (10°C, 23°C, 35°C, and 45°C), and presence of a water sanitizer (DryTec, SaniDate, FruitGard, and Selectrocide). Wounded and nonwounded sweetpotato storage roots were soaked in each water treatment for 20 min, stored at 29°C for a 14-day period, and rated for disease incidence every other day. Disease was observed in sweetpotato storage roots in all water treatments tested, except in the negative controls. Disease incidence decreased with both inoculum concentration and inoculum age, yet values of 16.26% and up to 50% were observed for roots exposed to 5 spores/ml and 144-h water treatments, respectively. Sanitizer products that contained a form of chlorine as the active ingredient significantly reduced disease incidence in storage roots when compared with control roots and roots exposed to a hydrogen-peroxide based product. Finally, no significant differences in final incidence were detected in wounded sweetpotato storage roots exposed to water treatments of any temperature, but a significant reduction in disease progression was observed in the 45°C treatment. These findings indicate that if packing line dump tanks are improperly managed, they can aid C. fimbriata dispersal through the build-up of inoculum as infected roots are unknowingly washed after storage. Chlorine-based sanitizers can reduce infection when applied after root washing and not in the presence of high organic matter typically found in dump tanks.

The Effector Repertoire of the Hop Downy Mildew Pathogen Pseudoperonospora humuli.

Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew (DM), one of the most destructive diseases of cultivated hop that can lead to 100% crop loss in susceptible cultivars. We used the published genome of P. humuli to predict the secretome and effectorome and analyze the transcriptome variation among diverse isolates and during infection of hop leaves. Mining the predicted coding genes of the sequenced isolate OR502AA of P. humuli revealed a secretome of 1,250 genes. We identified 296 RXLR and RXLR-like effector-encoding genes in the secretome. Among the predicted RXLRs, there were several WY-motif-containing effectors that lacked canonical RXLR domains. Transcriptome analysis of sporangia from 12 different isolates collected from various hop cultivars revealed 754 secreted proteins and 201 RXLR effectors that showed transcript evidence across all isolates with reads per kilobase million (RPKM) values > 0. RNA-seq analysis of OR502AA-infected hop leaf samples at different time points after infection revealed highly expressed effectors that may play a relevant role in pathogenicity. Quantitative RT-PCR analysis confirmed the differential expression of selected effectors. We identified a set of P. humuli core effectors that showed transcript evidence in all tested isolates and elevated expression during infection. These effectors are ideal candidates for functional analysis and effector-assisted breeding to develop DM resistant hop cultivars.

Population Structure of Pythium ultimum from Greenhouse Floral Crops in Michigan.

Pythium ultimum causes seedling damping-off and root and crown rot in greenhouse ornamental plants. To understand the population dynamics and assess population structure of P. ultimum in Michigan floriculture crops, simple sequence repeats (SSRs) were developed using the previously published P. ultimum predicted transcriptome. A total of 166 isolates sampled from 2011 to 2013 from five, one, and three greenhouses in Kalamazoo, Kent, and Wayne Counties, respectively, were analyzed using six polymorphic and fluorescently labeled SSR markers. The average unbiased Simpson's index (λu, 0.95), evenness (E5, 0.56), and recovery of 12 major clones out of the 65 multilocus genotypes obtained, suggests that P. ultimum is not a recent introduction into Michigan greenhouses. Analyses revealed a clonal population, with limited differentiation among seasons, hosts, and counties sampled. Results also indicated the presence of common genotypes among years, suggesting that sanitation measures should be enhanced to eradicate resident P. ultimum populations. Finally, the presence of common genotypes among counties suggests that there is an exchange of infected plant material among greenhouse facilities, or that there is a common source of inoculum coming to the region. Continued monitoring of pathogen populations will enhance our understanding of population dynamics of P. ultimum in Michigan and facilitate improvement of control strategies.

Advances in Diagnostics of Downy Mildews: Lessons Learned from Other Oomycetes and Future Challenges.

Downy mildews are plant pathogens that damage crop quality and yield worldwide. Among the most severe and notorious crop epidemics of downy mildew occurred on grapes in the mid-1880s, which almost destroyed the wine industry in France. Since then, there have been multiple outbreaks on sorghum and millet in Africa, tobacco in Europe, and recent widespread epidemics on lettuce, basil, cucurbits, and spinach throughout North America. In the mid-1970s, loss of corn to downy mildew in the Philippines was estimated at US$23 million. Today, crops that are susceptible to downy mildews are worth at least $7.5 billion of the United States' economy. Although downy mildews cause devastating economic losses in the United States and globally, this pathogen group remains understudied because they are difficult to culture and accurately identify. Early detection of downy mildews in the environment is critical to establish pathogen presence and identity, determine fungicide resistance, and understand how pathogen populations disperse. Knowing when and where pathogens emerge is also important for identifying critical control points to restrict movement and to contain populations. Reducing the spread of pathogens also decreases the likelihood of sexual recombination events and discourages the emergence of novel virulent strains. A major challenge in detecting downy mildews is that they are obligate pathogens and thus cannot be cultured in artificial media to identify and maintain specimens. However, advances in molecular detection techniques hold promise for rapid and in some cases, relatively inexpensive diagnosis. In this article, we discuss recent advances in diagnostic tools that can be used to detect downy mildews. First, we briefly describe downy mildew taxonomy and genetic loci used for detection. Next, we review issues encountered when identifying loci and compare various traditional and novel platforms for diagnostics. We discuss diagnosis of downy mildew traits and issues to consider when detecting this group of organisms in different environments. We conclude with challenges and future directions for successful downy mildew detection.

Resurgence of cucurbit downy mildew in the United States: Insights from comparative genomic analysis of Pseudoperonospora cubensis.

Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew (CDM), is known to exhibit host specialization. The virulence of different isolates of the pathogen can be classified into pathotypes based on their compatibility with a differential set composed of specific cucurbit host types. However, the genetic basis of host specialization within P. cubensis is not yet known. Total genomic DNA extracted from nine isolates of P. cubensis collected from 2008 to 2013 from diverse cucurbit host types (Cucumis sativus, C. melo var. reticulatus, Cucurbita maxima, C. moschata, C. pepo, and Citrullus lanatus) in the United States were subjected to whole-genome sequencing. Comparative analysis of these nine genomes confirmed the presence of two distinct evolutionary lineages (lineages I and II) of P. cubensis. Many fixed polymorphisms separated lineage I comprising isolates from Cucurbita pepo, C. moschata, and Citrullus lanatus from lineage II comprising isolates from Cucumis spp. and Cucurbita maxima. Phenotypic characterization showed that lineage II isolates were of the A1 mating type and belonged to pathotypes 1 and 3 that were not known to be present in the United States prior to the resurgence of CDM in 2004. The association of lineage II isolates with the new pathotypes and a lack of genetic diversity among these isolates suggest that lineage II of P. cubensis is associated with the resurgence of CDM on cucumber in the United States.

Analysis of microsatellites from the transcriptome of downy mildew pathogens and their application for characterization of Pseudoperonospora populations.

Downy mildew pathogens affect several economically important crops worldwide but, due to their obligate nature, few genetic resources are available for genomic and population analyses. Draft genomes for emergent downy mildew pathogens such as the oomycete Pseudoperonospora cubensis, causal agent of cucurbit downy mildew, have been published and can be used to perform comparative genomic analysis and develop tools such as microsatellites to characterize pathogen population structure. We used bioinformatics to identify 2,738 microsatellites in the P. cubensis predicted transcriptome and evaluate them for transferability to the hop downy mildew pathogen, Pseudoperonospora humuli, since no draft genome is available for this species. We also compared the microsatellite repertoire of P. cubensis to that of the model organism Hyaloperonospora arabidopsidis, which causes downy mildew in Arabidopsis. Although trends in frequency of motif-type were similar, the percentage of SSRs identified from P. cubensis transcripts differed significantly from H. arabidopsidis. The majority of a subset of microsatellites selected for laboratory validation (92%) produced a product in P. cubensis isolates, and 83 microsatellites demonstrated transferability to P. humuli. Eleven microsatellites were found to be polymorphic and consistently amplified in P. cubensis isolates. Analysis of Pseudoperonospora isolates from diverse hosts and locations revealed higher diversity in P. cubensis compared to P. humuli isolates. These microsatellites will be useful in efforts to better understand relationships within Pseudoperonospora species and P. cubensis on a population level.