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United States Department of Agriculture

Agricultural Research Service

Research Project: Reducing the Impact of Diseases on Hop Production

Location: Forage Seed and Cereal Research

2016 Annual Report

1a. Objectives (from AD-416):
Objective 1: Identify new genetic markers for selecting resistant hop germplasm to downy and powdery mildews. Objective 2: Develop new disease resistant germplasm for public release. Objective 3: Construct optimized integrated management approaches for powdery mildew susceptible cultivars. Sub-objective 3.A: Describe the ontology of crown bud development, susceptibility of crown buds to powdery mildew in different developmental stages, and dynamics of flag shoot emergence. Sub-objective 3.B: Develop a PCR assay to rapidly identify mating type in P. macularis; determine prevalence of mating types among isolates of P. macularis in the Pacific Northwest. Objective 4: Develop and apply genotyping approaches to assess the diversity, geneticdifferentiation, and sexual recombination in the downy mildew and powdery mildew pathogens. Sub-objective 4.A: Identify and develop simple sequence repeat markers in Pseudoperonospora humuli and elucidate the degree of diversity, selfing, and population differentiation within and among population at multiple hierarchical scales. Sub-objective 4.B: Identify polymorphic loci among isolates of Podosphaera macularis and characterize the genetic diversity, population structure, and relatedness of the population in the Pacific Northwest U.S. to other populations of the pathogen in the world.

1b. Approach (from AD-416):
Objective 1: Identify molecular markers associated with plant resistance to P. humuli and P. macularis. Progeny developed from crosses of resistant and susceptible parents will be screened by pathogen challenge, and single nucleotide polymorphic (SNP) marker identification and genotyping-by-sequencing will be performed. Objective 2: Development of multiple pathogen resistant germplasm or varieties. Progeny of crosses of parental material that possess relative resistance to powdery and downy mildews will be successively challenged with each disease to identify germplasm with enhanced resistance to both. Resistant germplasm that possess excellent agronomic and brewing characteristics will be released. Objective 3: Construct optimized integrated management approaches for powdery mildew susceptible cultivars. Hypothesis 3.A: Successful perennation of the powdery mildew fungus occurs via infection of juvenile crown buds and such crown buds develop asynchronously. Management factors that reduce late season severity of powdery mildew will reduce overwintering survival of the pathogen. Crown bud development phenology will be determined and plants will be challenged with powdery mildew at selected stages. Treatment at different stages will be evaluated. Hypothesis 3.B: The absence of the ascigerious stage of Podosphaera macularis in the Pacific Northwestern U.S. is due to the absence of one of requisite mating types of the fungus. PCR amplification of conserved regions in MAT1-1 and MAT1-2 loci of P. macularis will be optimized and pathogen isolates collected from a variety of cultivars and hop yards in the Pacific Northwest will be tested to determine the frequency of each mating type. Objective 4: Develop and apply genotyping approaches. Hypothesis 4.A: P. humuli is heterothallic, possessing a high degree of genetic diversity in the Pacific Northwestern U.S, and the population is structured at the scale of individual fields. P. humuli isolates will be obtained from three hop yards in western Oregon. After suitable SSR loci and primers are developed from pyrosequences, genotyping will be performed by capillary sequencing of 7 to 10 SSR loci per isolate. Hypothesis 4.B: The population of Podosphaera macularis in the Pacific Northwestern U.S. exhibits a low degree of genetic diversity or structure based on geography or cultivar host. Multiple nuclear loci will be identified, PCR-amplified, and sequenced from Pacific Northwest, northeastern U.S., and European isolates. Population genetic parameters will be calculated and differentiation among geographic populations will be estimated.

3. Progress Report:
In collaboration with Oregon State University (OSU), progress was made in identifying molecular markers linked to expression of plant resistance to both powdery mildew and downy mildew (Objective 1). New molecular markers were developed from the recently completed USDA-OSU hop genome ( and utilized to develop new genetic maps for mapping populations. New markers linked to powdery mildew resistance were identified with all significantly linked markers found on the same chromosome. First stage validation of molecular markers for use in selecting powdery mildew resistant lines was initiated. Previously identified molecular markers linked to expression of resistance to downy mildew were double validated upon a second set of 150 hop varieties and found to be consistent with identifying offspring or parents possessing resistance. In collaboration with Oregon hop growers and Washington State University (WSU), studies continued on evaluating resistance to both powdery and downy mildew pathogens in crosses designed for dual resistance (Objective 2). Approximately 60 different offspring were selected, harvested and chemically characterized for their brewing potential based upon possession of resistance to both pathogens, high yield potential and other highly desirable morphological traits during the 2015 growing season. These same lines were evaluated for disease resistance during spring and early summer 2016 with most showing continued resistance under normal field production practices in off-station field trials. Several families possess superior resistance levels than others and the parents of these crosses were noted for future use. In collaboration with scientists from Oregon State University and Wye Hops Ltd. (Canterbury, United Kingdom) studies were completed identifying the regions of the hop genome controlling the expression of “dwarfing” trait in hop. One chromosome contained all the major regions determining whether an offspring was short versus tall as well as the regions determining the extent of dwarfism. These chromosomal regions were investigated at the genomic level to see what proteins were present and if these proteins were potentially responsible for the dwarfing trait. Several proteins were identified with one protein standing out as being responsible for dwarfism in other plant species. Validation of a set of five molecular markers for use in breeding programs will be implemented during 2016. In collaboration with scientists from Oregon State University, studies were completed on the identification of Y-specific chromosome regions as well as regions of the X and Y chromosome that pair and recombine. Proteins expressed from genes located on these regions were identified and the expression of these genes across male and female plants housed in the USDA-ARS hop germplasm collection were determined. Some genes showed dramatic differences in expression between sexes. In addition, a large set of male-specific molecular markers were identified that will enable simple molecular assays used to screen out male offspring from breeding nurseries. In collaboration with Washington State University, progress was made on understanding how the powdery mildew fungus overwinters (Objective 3, sub-objective 3A). Results of infection studies demonstrate that bud infection, leading to pathogen perennation, may occur over an extended period of time, but is most likely during juvenile stages of bud development. Emergence of the pathogen during the ensuing season was linked to host growth. Analysis of historical data collected since 2000 identified that prior to overwintering of the pathogen, disease levels in the previous season, certain cultural practices, and winter temperature were associated with the occurrence and frequency of survival of the pathogen. Field experiments were designed and implemented (in collaboration with Washington State University and cooperating producers) to quantify the association of disease control measures on ability of the pathogen to overwinter. Results from these experiments have been inconclusive given the low level of overwintering of the pathogen. In collaboration with Oregon State University scientists, studies were continued determine how the frequency and location of overwintering of the powdery mildew pathogen influences disease development at the landscape level. We discovered that in Oregon, landscape-level outbreaks of powdery mildew are associated with survival of the pathogen in less than 5% of hop yards in April and May, and subsequently spread from this small number of yards. A preliminary statistical model has been developed that predicts disease spread based on proximity to other fields, variety susceptibility, and wind variables associated with pathogen dispersal. The model will be validated with data collected in 2017, and could become an important component of a disease hazard system that predicts long distance dispersal of the fungus. Studies were largely completed that describe the mating system of the hop downy mildew pathogen (Objective 4, sub-objective 4A). New collaborations with scientists in Israel and Germany were begun to expand the scope of this work. The results indicate the pathogen does not require two different strains to produce its survival spores. These survival spores are readily produced in artificial and natural conditions, and appear to have a dormancy period that is not easily disrupted. This has important implications for understanding the diversity of the pathogen as well as disease management considerations. Studies were planned and initiated to quantify genetic variability of the downy mildew pathogen at multiple spatial scales. Large collections of isolates of the downy mildew pathogen were genetically fingerprinted and the data analysis was mostly completed. The results indicate that there is a low level of genetic variation in the pathogen, although differences at the field and plant-level do exist. This finding supports the finding that the pathogen can produce its survival spores individually (selfing). This research was extended through a collaboration with scientists at North Carolina State University that are sequencing the genome of the pathogen and the related downy mildew pathogen on cucurbits to identify unique genes in each organism and develop diagnostic DNA tests. RNA genetic data was obtained for the powdery mildew fungus to aid in development of genetic fingerprinting methods for this organism. Data analysis is underway, as per the project plan (Objective 4, sub-objective 4B). In response to emergence of strains of the powdery mildew fungus that can attack the previously resistant variety Cascade, studies were begun to understand pathogenic diversity of the emerging strain(s), assess the reaction of critical germplasm, and clarify the distribution and incidence of disease on now susceptible cultivars in the Pacific Northwest. This research has: clarified that Cascade adapted isolates are widespread in Washington State; quantified the susceptibility of Cascade under field conditions; demonstrated that Cascade adapted isolates are distinct from other virulent strains that overcome commonly deployed powdery mildew resistance genes; discovered that virulent strains are specifically adapted to Cascade; found that the resistance in Cascade appears to be based on multiple rather than a single resistance gene; and identified multiple sources of resistance to these strains in publically available germplasm. This work has provided the epidemiological scaffolding for growers and breeding programs to respond appropriately to this new threat, both in the near and long-term.

4. Accomplishments

5. Significant Activities that Support Special Target Populations:
ARS scientists in Corvallis, Oregon, shared research results and best practices for integrated pest management with small shareholder farmers in Indiana and Wisconsin at conferences and field day events in these states.

Review Publications
Scott, J.B., Gent, D.H., Hay, F.S., Pethybridge, S.J. 2015. Estimation of pyrethrum flower number using digital imagery. HortTechnology. 25(5):617-624.

Probst, C., Nelson, M.E., Grove, G.G., Twomey, M.C., Gent, D.H. 2016. Hop powdery mildew control through alteration of spring pruning practices. Plant Disease. 100(8):1599-1605. doi: 10.1094/PDIS-10-15-1127-RE.

Diaz-Lara, A., Gent, D.H., Martin, R.R. 2016. Identification of extrachromosomal circular DNA in hop via rolling circle amplification. Cytogenetics and Genome Research. 148(2-3):237-240. doi: 10.1159/000445849.

Wolfenbarger, S.N., Quesada-Ocampo, L.M., Gent, D.H. 2016. Powdery mildew caused by Podosphaera macularis on hop (Humulus lupulus) in North Carolina. Plant Disease. 100(6):1245. doi: 10.1094/PDIS-12-15-1525-PDN.

Woods, J., Gent, D.H. 2016. Susceptibility of hop cultivars to downy mildew: associations with chemical characteristics and region of origin. Plant Health Progress. 17:42-48.

Wolfenbarger, S.N., Massie, S.T., Ocamb, C., Eck, E.B., Grove, G.G., Nelson, M.E., Probst, C., Twomey, M.C., Gent, D.H. 2016. Distribution and characterization of Podosphaera macularis virulent on hop cultivars possessing R6-based resistance to powdery mildew. Plant Disease. 100(6):1212-1221. doi: 10.1094/PDIS-12-15-1449-RE.

Howard, E., Whittock, S.P., Jakse, J., Carling, J., Matthews, P.D., Probasco, G., Henning, J.A., Darby, P., Cerenak, A., Javornik, B., Kilian, A., Koutoulis, A. 2011. High-throughput genotyping of hop (Humulus lupulus L.) utilising diversity arrays technology (DArT). Theoretical and Applied Genetics. 122(7):1265-1280.

Henning, J.A., Gent, D.H., Twomey, M.C., Townsend, M.S., Pitra, N.J., Matthews, P.D. 2015. Genotyping-by-sequencing of a bi-parental mapping population segregating for downy mildew resistance in hop (Humulus lupulus L.). Euphytica. 208(3):545-559. doi: 10.1007/s10681-015-1600-3.

Henning, J.A., Coggins, J., Peterson, M. 2015. Simple SNP-based minimal marker genotyping for (Humulus lupulus L.) identification and variety validation. BMC Research Notes. 8(1):1-12. doi: 10.1186/s13104-015-1492-2.

Hay, F., Gent, D.H., Pilkington, S., Pearce, T., Scott, J.B., Pethybridge, S.J. 2015. Changes in distribution and frequency of fungi associated with a foliar disease complex of pyrethrum in Australia. Plant Disease. 99:1227-1235.

Summers, C., Gulliford, C., Lillis, J.A., Carlson, M., Cadle Davidson, L.E., Gent, D.H., Smart, C. 2015. Identification of genetic variation between obligate plant pathogens Psuedoperonospora cubensis and P. humuli using RNA sequencing and genotyping-by-sequencing. PLoS One. doi: 10.1371/journal.pone.0143665.

Hill, S.T., Coggins, J., Liston, A., Hendrix, D., Henning, J.A. 2016. Genomics of the hop psuedo-autosomal regions. Euphytica. 209:171-179. doi: 10.1007/s10681-016-1655-9.

Twomey, M.C., Stone, J.K., Gent, D.H. 2016. Black wilt of hop (Humulus lupulus) caused by Diplodia seriata in New York State. Plant Disease. 100(4):861. doi: 10.1094/PDIS-10-15-1140-PDN.

Gent, D.H., Probst, C., Nelson, M.E., Grove, G.G., Massie, S.T., Twomey, M.C. 2016. Interaction of basal foliage removal and late season fungicide applications in management of Hop powdery mildew. Plant Disease. 100:1153-1160.

Woods, J.L., James, D.G., Lee, J.C., Gent, D.H. 2011. Evaluation of airborne methyl salicylate for improved conservation biological control of two-spotted spider mite and hop aphid in Oregon hop yards. Experimental and Applied Acarology. 55(4):401-416.

Withers, S., Gongora-Castillo, E., Gent, D.H., Thomas, A., Ojiambo, P., Quesada-Ocampo, L. 2016. Using next-generation sequencing to develop molecular diagnostics for Pseudoperonospora cubensis, the cucurbit downy mildew pathogen. Phytopathology. doi: 10.1094/PHYTO-10-15-0260-FI.

Last Modified: 10/17/2017
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