Hop Downy Mildew Research Articles

Bibliographic Analysis of Scientific Research on Downy Mildew (Pseudoperonospora humuli) in Hop (Humulus lupulus L.)

This study focused on downy mildew in hop caused by the pathogen Pseudoperonospora humuli. A systematic literature review was conducted using bibliometric analysis to explore trends in publishing, prominent research themes, and where research is being conducted on hop downy mildew. The databases Scopus, Web of Science, and ScienceDirect were used to identify publications spanning from 1928 to 2023. The analysis yielded 54 publications, with the most cited studies primarily focusing on disease management and host resistance. Additionally, these studies explored the genetic and pathogenic relationship between P. cubensis and P. humuli. A word co-occurrence map revealed that the main themes addressed in the publications included “hop”, “disease”, “downy”, “humuli”, “mildew”, and “Pseudoperonospora”. Notably, there was a particular emphasis on subtopics such as disease management, the disease reaction of hop cultivars, and the influence of weather factors on hop downy mildew. Notably, there was limited knowledge about the disease in regions with tropical climates. This study provides valuable information that can support and guide future research endeavors concerning downy mildew in hop cultivation.

Suppression of Hop Downy Mildew as Influenced by the Timing of Selected Fungicides

Downy mildew of hop is a disease that is controlled by the implementation of cultural practices along with the application of fungicides, as there are few cultivars with high levels of resistance to this disease. We conducted studies in western Oregon to elucidate the effect of using synthetic fungicides of various modes of action early versus late in disease management programs when rotated with a copper-based fungicide. When analyzed over the 4 years of this study, there were independent effects from both the specific fungicide mode of action and application timing on disease control. The use of cymoxanil + copper hydroxide or mandipropamid in a program reduced the area under the disease progress curve by 66.3 or 59.7 units (17.7 or 19.6%), respectively, as compared with phosphorous acid. Using synthetic fungicides in the first two applications in a program provided better disease control than using the same fungicides in the third and fourth applications, reducing the area under the disease progress curve by 47.8 units (14.4%). By simply applying modes of action found to be more efficacious earlier in the growing season, the severity of downy mildew was reduced as much as 32% without increasing the total number of applications made.

Impact of Infection Timing and Autumnal Fungicide Applications on Perennation of Pseudoperonospora humuli and Severity of Hop Downy Mildew.

Pseudoperonospora humuli, causal agent of hop downy mildew, is known to survive winter as systemic mycelium in the crown and developing buds of hop (Humulus lupulus). Field studies were conducted over three growing seasons to quantify the association of infection timing to overwintering of P. humuli and development of downy mildew. Cohorts of potted plants were inoculated sequentially from early summer to autumn, overwintered, and then evaluated for symptoms of systemic downy mildew in emerging shoots. Shoots with systemic P. humuli developed after inoculation at any time in the previous year, with the most severe disease typically resulting from inoculation in August. Independent of the timing of inoculation, diseased shoots emerged coincident with the emergence of healthy shoots, beginning as early as late February and continuing through late May to early June. Surface crown buds on inoculated plants exhibited internal necrosis associated with P. humuli at rates ranging from 0.3 to 1.2%, whereas P. humuli was detected by PCR on 7.8 to 17.0% of asymptomatic buds depending on the timing of inoculation and year. Four experiments were conducted to quantify the impact of foliar fungicides applied in autumn on downy mildew the following spring. There was a small reduction of disease in only one study. Together, these studies indicate that infection by P. humuli that leads to overwintering can occur over a broad period of time, but delaying infection until autumn tends to reduce disease levels in the following year. However, in established plantings, postharvest application of foliar fungicides appeared to have little impact on severity of downy mildew in the ensuring year.

Optimizing Molecular Detection for the Hop Downy Mildew Pathogen Pseudoperonospora humuli in Plant Tissue

Downy mildew-free hop plantlets and rhizomes are essential to limit the introduction of this destructive pathogen, Pseudoperonospora humuli, into hopyards. The objective of this research was to determine which DNA-based diagnostic tools are optimal for P. humuli detection in plant tissue. Quantitative real-time PCR (qPCR) assays with TaqMan probes for nuclear (c125015.3e1) and mitochondrial (orf359) DNA loci were developed and tested side by side. A recombinase polymerase amplification (RPA) assay was designed based on the orf359 DNA locus. The mitochondrial qPCR assay had a 10-fold lower limit of detection (100 fg of genomic DNA) and was 60% more effective in detecting P. humuli in asymptomatic stems than the nuclear-based assay. Both qPCR assays had linear standard curves (R2 > 0.99) but lacked the quantitative precision to differentiate leaf infections beyond 1 day postinoculation. A wide range of Cq values (≥4.9) in standardized tests was observed among isolates, suggesting that the number of mitochondria and nuclear DNA targets can vary. The absence of P. humuli DNA in symptomatic rhizomes was explained, in part, by the detection of Phytophthora DNA. However, the Phytophthora-specific atp9-nad9 assay cross-reacted with P. humuli, leading to false positive amplification. Sensitivity in the RPA assay was reduced by crude plant DNA extract. Improvements to the objectivity of calling positive amplifications and determining the onset of amplification from RPA fluorescence data were realized by applying the first and second derivatives, respectively. The orf359 qPCR assay is specific and sensitive, making it well suited for P.humuli diagnostics in plant tissue.

Fungicide Efficacy Against Pseudoperonospora humuli and Point Mutations Linked to Carboxylic Acid Amide Resistance in Michigan.

Hops have expanded as a niche crop in Michigan and other production areas in the eastern United States, but growers in these regions face annual downy mildew outbreaks incited by Pseudoperonospora humuli, exacerbated by frequent rainfall and high relative humidity. We evaluated the efficacy of foliar- and drench-applied fungicides against downy mildew and examined Michigan isolates for point mutations linked to carboxylic acid amide (CAA) resistance. Disease severity and density were assessed weekly in 2016 and 2017 in nontrellised research hop yards in Michigan. Area under the disease progress curve values for disease severity were significantly lower for plants treated with oxathiapiprolin, ametoctradin/dimethomorph, fluopicolide, cyazofamid, or mandipropamid (90.6 to 100% control) compared with those treated with fosetyl-Al (64.3 to 93.0% control) at both locations for both years. Drench treatments of fluopicolide and oxathiapiprolin/mefenoxam reduced disease density and severity at both locations but were only moderately effective (76.4 to 91.5% control). To assess CAA resistance, the cellulose synthase CesA3 gene was aligned using reference downy mildew species and primers designed to amplify the 1105 and 1109 amino acids. Point mutations conferring CAA resistance were not detected at these loci for sporangia from 42 symptomatic shoots collected from 11 commercial hop yards. These efficacy results for hop downy mildew are needed to guide disease recommendations in this expanding Michigan industry. The absence of resistant genotypes indicates that Michigan growers can continue to utilize CAA-containing commercial fungicides as part of an overall downy mildew management program.

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.TaxonomyClass: Oomycota; Order: Peronosporales; Family: Peronosporaceae; Genus: Pseudoperonospora; Species: Pseudoperonospora humuli.Disease symptomsThe 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 processPathogen 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.ControlDowny 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).

Pseudoperonospora humuli might be an introduced species in Central Europe with low genetic diversity but high distribution potential

The downy mildew pathogen Pseudoperonospora humuli is a major disease of cultivated and wild hop (Humulus lupulus) in many hop-growing areas of the world. Despite its frequent occurence and economic impact, so far very little is known with respect to its population structure, which would be a prerequisite for understanding population dynamics and for successful resistance breeding efforts. We collected a set of 70 P. humuli samples in five European countries (Austria, the Czech Republic, France, Germany, Italy) around the Alps, a high mountain range and a known major hurdle for dispersal, and examined them with microsatellite markers. For this purpose we developed a set of new markers and used them along with markers published for P. humuli by other authors. While the 11 microsatellites that were reported as polymorphic for North America did not show variation for the European samples, the five markers newly established in this study were variable and could be used to investigate the population structure of hop downy mildew in Europe. The overall genetic variation found in European populations was low, with only few heterozygous loci detected. Overall, 20 multilocus genotypes were observed, which clustered into 2 or 4 groups, depending on clustering criteria. We observed a random distribution of genotypes on the surveyed area in line with predominant selfing and long range dispersal. Thus, our investigations suggest that Pseudoperonospora humuli forms a large, potentially panmictic population with little local adaptation and frequent long-distance exchanges. This suggests that a wide array of Pseudoperonospora humuli strains should be used in any resistance breeding programs. In addition, the low degree of variation in Europe in contrast to North America suggests that, in line with its report in Europe as late as the 1930‘s, hop downy mildew might be an introduced species in Europe. Whether the introduction occurred from North America or from East Asia needs to be clarified in future studies.

High Levels of Insensitivity to Phosphonate Fungicides in Pseudoperonospora humuli.

Phosphonate (phosphite; HPO3-2) is fungicidal against oomycetes and certain other organisms. The Fungicide Resistance Action Committee has deemed phosphonate to be at low risk of resistance development, and reduced sensitivity to phosphonate has been reported only occasionally in plant pathogens. Reduced sensitivity to the fungicide fosetyl-Al was documented in the hop downy mildew pathogen, Pseudoperonospora humuli, in the early 2000s, but disease caused by insensitive isolates could still be managed commercially if the fungicide rate was doubled from 2.24 to 4.48 kg/ha. In this research, we document the occurrence of isolates of P. humuli in Oregon that possess even higher levels of insensitivity to fosetyl-Al and other phosphonate fungicides. The median estimated effective concentration required to reduce infection by 50% (EC50) for isolates collected from two farms reporting disease control failures was 2.7% (vol/vol) phosphonate (range = 1.6 to 164.2), which was 1.6 times (range = 0.9 to 96.0) the maximum labeled rate of the phosphonate fungicide utilized. In contrast, the median EC50 for isolates obtained from experimental plots that have received only a single application of a phosphonate fungicide was 0.6% (vol/vol) phosphonate (range = 0.11 to 2.3) or 0.3 times the maximum allowable rate. Sensitivity of isolates to a phosphorous acid fungicide, fosetyl-Al, and a plant nutrient product containing an unspecified level of phosphorous acid were linearly related. Insensitivity to the maximum allowable rate of a phosphorous acid fungicide was widespread within and among hop farms in Oregon. Among 54 isolates assayed for phosphonate insensitivity, 96% had EC50 values that exceeded the maximum allow rate of the fungicide used in the assays. Field studies conducted in 2 years further demonstrated that a phosphorous fungicide, a nutrient product containing phosphorous acid, and fosetyl-Al failed to provide commercially acceptable suppression of downy mildew when applied at the maximum allowable rates and even double these rates, whereas fungicides with different modes of action provided 91% or greater disease control. The whole of this research indicates that P. humuli has been selected to tolerate fosetyl-Al and other phosphonate fungicides at rates four times greater than those used earlier to obtain satisfactory suppression of downy mildew. This finding has implications for management of the disease not only in Oregon but also, in other production regions should insensitive isolates be introduced on infected planting material.

Genome Sequencing and Transcriptome Analysis of the Hop Downy Mildew Pathogen Pseudoperonospora humuli Reveal Species-Specific Genes for Molecular Detection.

Pseudoperonospora humuli is an obligate oomycete pathogen of hop (Humulus lupulus) that causes downy mildew, an important disease in most production regions in the Northern Hemisphere. The pathogen can cause a systemic infection in hop, overwinter in the root system, and infect propagation material. Substantial yield loss may occur owing to P. humuli infection of strobiles (seed cones), shoots, and cone-bearing branches. Fungicide application and cultural practices are the primary methods to manage hop downy mildew. However, effective, sustainable, and cost-effective management of downy mildew can be improved by developing early detection systems to inform on disease risk and timely fungicide application. However, no species-specific diagnostic assays or genomic resources are available for P. humuli. The genome of the P. humuli OR502AA isolate was partially sequenced using Illumina technology and assembled with ABySS. The assembly had a minimum scaffold length of 500 bp and an N50 (median scaffold length of the assembled genome) of 19.2 kbp. A total number of 18,656 genes were identified using MAKER standard gene predictions. Additionally, transcriptome assemblies were generated using RNA-seq and Trinity for seven additional P. humuli isolates. Bioinformatics analyses of next generation sequencing reads of P. humuli and P. cubensis (a closely related sister species) identified 242 candidate species-specific P. humuli genes that could be used as diagnostic molecular markers. These candidate genes were validated using polymerase chain reaction against a diverse collection of isolates from P. humuli, P. cubensis, and other oomycetes. Overall, four diagnostic markers were found to be uniquely present in P. humuli. These candidate markers identified through comparative genomics can be used for pathogen diagnostics in propagation material, such as rhizomes and vegetative cuttings, or adapted for biosurveillance of airborne sporangia, an important source of inoculum in hop downy mildew epidemics.

Investigating Phenylamide Insensitivity in Wisconsin Populations of Pseudoperonospora humuli

Hop downy mildew caused by the oomycete Pseudoperonospora humuli is primarily managed with fungicides because commercial varietal resistance is unavailable. Mefenoxam, a phenylamide-class fungicide, is highly effective for systemic disease control but is at high risk for the development of pathogen insensitivity. Due to the recent expansion of hop production in Wisconsin, it was unknown if P. humuli could be managed with mefenoxam because insensitivity has been documented in other regions. During 2015 to 2017, isolates of P. humuli were collected from commercial yards throughout Wisconsin. Isolates were screened for insensitivity using a leaf disk assay with fungicide-amended water agar. Insensitivity was determined using a single discriminatory dose of 25 μg/ml of mefenoxam, and isolates were considered insensitive if they exhibited at least 50% sporulation on amended media relative to nonamended water agar. Over all years, nearly 48% of all isolates were insensitive to mefenoxam. The incidence of insensitivity varied between individual hop yards, with most yards exhibiting a mixture of sensitive and insensitive isolates. At this time, mefenoxam-insensitive populations do not appear to be predominant in Wisconsin. Growers should monitor the effectiveness of mefenoxam applications in their hop yards and be responsive to changes in crop response.

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.

Susceptibility of Hop Cultivars to Downy Mildew: Associations with Chemical Characteristics and Region of Origin

Hop downy mildew (caused by Pseudoperonospora humuli) is a yield-limiting disease in many hop-production regions of the world. In this research, 110 cultivars that are or were widely grown in the United States, Europe, or Australasia were evaluated in western Oregon over three years for their reaction to the shoot infection phase of downy mildew and vigor. There was a large range of downy mildew susceptibility and vigor amongst commercial cultivars, with some cultivars possessing a very high level of resistance. Overall, however, disease resistance and vigor were significantly greater in cultivars originating from Europe than those originating from the United States, Japan, and Australia/New Zealand. Amongst a subset of 79 cultivars, vigor was negatively correlated with levels, in cones, of cohumulone, a chemical constitute of bittering acids typically found in germplasm derived from North America. The generally poor vigor observed in cultivars derived outside of Europe likely is indicative of a lack of tolerance to the crown infection phase of the disease. Thus, the best sources of downy mildew resistance seems to be found in cultivars from the United Kingdom and continental Europe, and such cultivars are typically lower yielding and lack distinctive aroma and flavor characteristics presently desired by craft brewers. Introgression of downy mildew resistance into North American germplasm with high yield and desirable brewing characteristics is needed. Accepted for publication 3 March 2016. Published 7 March 2016.

Pseudoperonospora cubensis and P. humuli detection using species-specific probes and high definition melt curve analysis

Real-time PCR assays using locked nucleic acid (LNA) probes and high resolution melt (HRM) analysis were developed for molecular differentiation of Pseudoperonospora cubensis and P. humuli, causal agents of cucurbit and hop downy mildew, respectively. The assays were based on a previously identified single nucleotide polymorphism (SNP) in the cytochrome oxidase subunit II (cox2) gene that differentiates the two species. Sequencing of the same region from 15 P. cubensis isolates collected in New York State for the current study confirmed that all isolates shared the conserved SNP. LNA probes were specific and sensitive, detecting as few as 10 sporangia for both species and as little as 1 fg P. cubensis total DNA and 10 fg P. humuli total DNA. The LNA assay detected both pathogens from air sampled using spore traps placed in vegetable fields and a hop yard during the summers of 2013 and 2014 and correctly diagnosed symptomatic leaf tissue. High resolution melt analysis (HRM) correctly identified all tested isolates as well as those isolates from symptomatic plants collected in the field. The LNA and HRM assays correctly identified both organisms when tested independently in a second laboratory. The results confirm that the LNA and HRM assays developed can provide reliable identification of both species despite the high molecular similarity of the cox2 gene.

Pre- and Postinfection Activity of Fungicides in Control of Hop Downy Mildew.

Optimum timing and use of fungicides for disease control are improved by an understanding of the characteristics of fungicide physical mode of action. Greenhouse and field experiments were conducted to quantify and model the duration of pre- and postinfection activity of fungicides most commonly used for control of hop downy mildew (caused by Pseudoperonospora humuli). In greenhouse experiments, control of downy mildew on leaves was similar among fungicides tested when applied preventatively but varied depending on both the fungicide and the timing of application postinfection. Disease control decreased as applications of copper were made later after inoculation. In contrast, cymoxanil, trifloxystrobin, and dimethomorph reduced disease with similar efficacy when applied 48 h after inoculation compared with preventative applications of these fungicides. When fungicides were applied 72 h after inoculation, only dimethomorph reduced the sporulating leaf area similarly to preinoculation application timing. Adaxial chlorosis, necrosis, and water soaking of inoculated leaves, indicative of infection by P. humuli, were more severe when plants were treated with cymoxanil, trifloxystrobin, and dimethomorph 48 to 72 h after inoculation, even though sporulation was suppressed. Trifloxystrobin and dimethomorph applied 72 h after inoculation suppressed formation of sporangia on sporangiophores as compared with all other treatments. In field studies, dimethomorph, fosetyl-Al, and trifloxystrobin suppressed development of shoots with systemic downy mildew to the greatest extent when applied near the timing of inoculation, although the duration of preventative and postinfection activity varied among the fungicides. There was a small reduction in efficacy of disease control when fosetyl-Al was applied 6 to 7 days after inoculation as compared with protective applications. Trifloxystrobin had 4 to 5 days of preinfection activity and limited postinfection activity. Dimethomorph had the longest duration of protective activity. Percent disease control was reduced progressively with increasing time between inoculation and application of dimethomorph. These findings provide guidance to the use of fungicides when applications are timed with forecasted or post hoc disease hazard warnings, as well as guidance on tank-mixes of fungicides that may be suitable both for resistance management considerations and extending intervals between applications.

Precision QTL mapping of downy mildew resistance in hop (Humulus lupulus L.)

Hop downy mildew (DM) is an obligate parasite causing severe losses in hop if not controlled. Resistance to this pathogen is a primary goal for hop breeding programs. The objective of this study was to identify QTLs linked to DM resistance. Next-genera- tion-sequencing was performed on a mapping popu- lation segregating for DM resistance levels. Cloned plants were grown in a RCBD with three replicates under three environments: greenhouse (GH), field plots in Oregon (OR), Corvallis field plots in Wash- ington (WA), Yakima). The linkage map of 3,341 SNP markers was determined with a four-stage process using Rqtl, TMAP, Joinmap v 4.0 and MERGEMAP. QTL analysis was performed using JMP Genomics and TASSEL 5.0. SNP markers were distributed across 11 linkage groups (LGs) with an average distance between markers of 0.2 cM and total distance of 745.9 cM. QTLs for all three environments were identified using multiple interval mapping. Overall heritability across the three environments varied from h 2 = 0.38 (GH) to 0.57 (OR). A total of 22 QTLs across 8 LG were identified for DM resistance: 5 identified from OR field data, 12 using WA data and five from GH DM data. No epistasis was observed. This study points out the complexity of genetic control of DM resistance in hop and identifies several markers that can be potentially be used to select for DM resistance in hop. It also provides the first linkage map suitable for genome sequencing due to the high density of SNP markers.

Forecasting and Management of Hop Downy Mildew.

Downy mildew of hop (Humulus lupulus), caused by Pseudoperonospora humuli, is managed in the Pacific Northwestern United States by regular application of fungicides. A degree-day model that forecasts the first emergence of shoots systemically infected with P. humuli (termed basal spikes) and a risk index for secondary spread of the disease were evaluated over four seasons in western Oregon. In surveys conducted in 34 hop yards, the predicted first spike emergence occurred on average 11.6 days (median 12 days) after spike emergence using a simple average degree-day model (base temperature 6.5°C) developed for Washington State. Predictions based on a single sine model (base temperature 6°C) provided on average 4.9 days (median -0.5 days) of advanced warning before the first spike emerged. Downy mildew severity in a previous season was negatively correlated with the degree-day emergence date of spikes the following year (r = -0.39). In experimental plots, disease severity was significantly greater where fungicide applications were timed using a risk index compared to routine fungicide applications in 2005 and 2007, but statistically similar between these treatments in 2006 and 2008. However, in 2006, 2007, and 2008, treatments initiated using a degree-day threshold resulted in an area under the disease progress curve similar to or smaller than in treatments with routine fungicide applications. Model-aided treatments required four fewer fungicide applications compared to routine fungicide applications. These studies indicate that downy mildew can be managed effectively with fewer fungicide applications than currently made by hop growers in this region if fungicide applications are timed to coincide with the predicted emergence of basal spikes and subsequent disease risk forecasts.

PCR detection of Pseudoperonospora humuli in air samples from hop yards

Downy mildew of hop, caused by Pseudoperonospora humuli, is an important disease in most regions of hop production and is managed largely with regular fungicide applications. A PCR assay specific to P. humuli and the related organism P. cubensis was developed and used to monitor airborne inoculum in hop yards to initiate fungicide applications. The PCR amplified as little as 1 fg of genomic DNA of P. humuli, and yielded an amplicon in 70% of reactions when DNA was extracted from single sporangia. In the presence of 25 mg of soil, an amplicon was amplified in 90% of reactions when DNA was extracted from 10 or more sporangia. During nine location‐years of validation, PCR detection of the pathogen in air samples occurred no later than 8 days after the appearance of trace levels of disease signs and/or detection of airborne spores in a volumetric spore sampler. Inoculum was detected on average 4·5 days before (range −8 to 14 days) the first appearance of basal spikes in six commercial yards, or 1·3 days after (range −5 to 1 days) sporangia were detected in a volumetric spore sampler in experimental plots. In commercial yards, use of PCR to initiate the first fungicide application led to enhanced disease control or a reduction in fungicide use in four of six yards compared to growers’ standard practices. These results indicate that the efficiency and efficacy of hop downy mildew management can be improved when control measures are timed according to first detection of inoculum.

Hop Downy Mildew Caused by Pseudoperonospora humuli in Argentina.

In Argentina, hop downy mildew disease caused by Pseudoperonospora humuli first appeared in Alto Valle of Rio Negro and Neuquen (1957), Mar del Plata (1962), and El Bolson (1963) (1). The disease occurred in the hop (Humulus lupulus L.) planting areas of El Bolson (Rio Negro) and Lago Pueblo (Chubut) in 2002 and 2003. Surveys were conducted in 30 commercial hop fields from December 2002 to March 2003 to estimate disease incidence and susceptibility of cultivars planted in these fields. Hop fields were divided into five sections and 100 plants were randomly selected and assessed for the presence of disease. Symptoms that were observed in early spring included dark brown rootstocks and primary basal spikes (stunted plants with pale and curled leaves), which are characteristic of systemic infection. Later in the season, secondary infections were characterized by dark purple-to-black lesions on leaves, flowers, cones, and lateral and terminal spikes. Plant symptoms and fungal morphological markers (dichotomously branched sporangiophores; ellipsoid and papillate sporangia) agreed with hop downy mildew disease and the fungus P. humuli. Yield loss was estimated as the reduction in yield compared with the 2001-2002 season observed from five hop growers. On December 10, 70% of the hop fields had greater than 50% disease incidence and seven fields reached 100% incidence. The reduction in cone yield varied between 20 and 34% in fields without a rootstock fungicide treatment. One field with a rootstock fungicide treatment (mefenoxam, copper oxiclorure, phosphorous acid, copper sulfate, and fosetyl-Al) and regular fungicide applications had a 30% increase in cone yield compared with 2001-2002. Systemically infected plants were recorded for hop cvs. Bullion, Cascade, CEZ, GS-19, Hallertauer Mfr., Nugget, Spalt, and Traful. Previously, Cascade was rated as a resistant cultivar to the root systemic infection (1). To our knowledge, this is the first record of a hop downy mildew outbreak in Argentina during the last 30 years. Reference: (1) L. Leskovar. El Lúpulo: Su Cultivo y Procesamiento. Hemisferio Sur. Buenos Aires, 1978.

PCR detection of Pseudoperonospora humuli and Podosphaera macularis in Humulus lupulus

Hop downy mildew (<I>Pseudoperonospora humuli</I>) and hop powdery mildew (<I>Podosphaera macularis</I>) are the most important pathogens of hop (<I>Humulus lupulus</I>). The early detection and identification of these pathogens are often made difficult by symptomless or combined infection with another pathogens. Molecular analysis of internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Therefore, specific PCR assays were developed to detect the pathogens <I>Pseudoperonospora humuli</I> and <I>Podosphaera</I> <I>macularis</I> in naturally infected hop plants. The specific PCR primer combinations P1 + P2 and S1 + S2 amplified specific fragments from <I>Pseudoperonospora humuli</I> and <I>Podosphaera macularis</I>, respectively, and did not cross-react with hop DNA nor with DNA from other fungi. PCR primer combinations R1 + R2 and R3 + R4 could be used in multiplex PCR detection of <I>Pseudoperonospora humuli</I>, <I>Podosphaera macularis</I>, <I>Verticillium albo-atrum</I> and <I>Fusarium sambucinum</I>. Phylogenetic relationships were inferred for 42 species of the <I>Erysiphales</I> from nuclear rDNA (ITS1, 5.8S, ITS2). The molecular characterisation and phylogenetic analyses confirmed the species identification of hop powdery mildew. The PCR assays used in this study proved to be accurate and sensitive for detection, identification, classification and disease-monitoring of the major hop pathogens.

Sensitivity of Pseudoperonospora humuli (the Causal Agent of Hop Downy Mildew) from Oregon, Idaho, and Washington to Fosetyl-Al (Aliette)

Failures of fosetyl-Al (Aliette) to control hop (Humulus lupulus) downy mildew, caused by Pseudoperonospora humuli, have recently occurred in northern Idaho and Oregon. To determine if resistance of the pathogen to the fungicide has developed, leaf disk assays were conducted to compare sensitivity of P. humuli isolates from the different U.S. hop-growing regions to isolates from a research yard where exposure to fosetyl-Al had not occurred for at least 10 years. Dose response curves of transformed data were linear. The fosetyl-Al concentration effective against 50% of the P. humuli isolates (ED50) from each location was estimated from the linearized data. The ED50 values indicate that fosetyl-Al was about one-third as effective against P. humuli isolates from commercial hop yards in northern Idaho and Oregon and about one-half as effective against isolates from southern Idaho compared to isolates from the research yard. Commercial yards in Washington were similar to the research yard. Accepted for publication 3 August 2004. Published 11 August 2004.