Location: Forage Seed and Cereal Research Unit
2023 Annual Report
Objectives
The goal of this project is to maintain and enhance the competitiveness of the U.S. hop industry through development of publicly available genetic resources, tools, and knowledge-based pest management systems. This will be accomplished through interdisciplinary research that addresses high priority documented stakeholder needs. Over the next 5 years, the specific objectives to be accomplished are:
Objective 1: Develop and release new hop cultivars and germplasm possessing superior disease resistance, yield, and brewing characteristics. (Henning)
Objective 2: Identify, characterize, and validate molecular markers associated with qualitative and quantitative resistance to important foliar diseases. (Henning)
Objective 3: Identify molecular markers associated with virulence of Podosphaera macularis and use the information to rapidly determine pathogen races. (Gent)
Objective 4: Quantify the aggressiveness, fitness, and race of Podosphaera macularis isolates able to overcome partial host resistance and identify new sources of resistance to diverse strains of the pathogen in public germplasm. (Gent)
Sub-objective 4A: Characterize the aggressiveness, fitness, and race of Podosphaera macularis virulent on the cultivar Cascade. (Gent)
Sub-objective 4B: Identify and quantify the impact of supraoptimal temperature on host susceptibility to and development of powdery mildew on the cultivar Cascade. (Gent)
Sub-objective 4C: Characterize publicly available male germplasm for its reaction to multiple strains of Podosphaera macularis. (Gent)
Approach
Objective 1 Research Goal: Develop multiple pathogen resistant germplasm or cultivars. Controlled crosses of cultivars two cultivars will be made using resistant males. Progeny will be screened for disease resistance and phenotypic traits including hop aroma. Selected offspring will be advanced for further evaluation.
Objective 2 Research Goal: Identify molecular markers associated with plant resistance to P. humuli and P. macularis. Genetic maps and genome-wide surveys for marker association will be conducted using a bi-parental mapping population derived from a powdery mildew resistant female line and a downy mildew resistant male line.
Objective 3 Hypothesis: Markers associated with pathogenic variation in P. macularis can be identified. Isolates of P. macularis from Pacific NW will be collected and race-validated using differential host panels. RNA will be collected from P. macularis isolates and subsequently sequenced using next gen sequencing. SNP markers will be identified from this data. SNPs will be used to fingerprint different isolates and a set of unique markers for each isolate identified.
Sub-objective 4A Hypothesis: Strains of P. macularis virulent on Cascade are specifically adapted to this cultivar. Controlled environment experiments will be conducted to determine the aggressiveness and fitness of isolates of P. macularis originating from Cascade to provide fundamental information to guide breeding efforts and disease risk assessment. These races will also be characterized using a differential set of cultivars possessing different resistance genes.
Sub-objective 4B Hypothesis: Partial resistance to powdery mildew in the Cascade is modulated by brief exposure to supra-optimal temperature. An extensive set of controlled environment studies will be conducted to define the environmental conditions that moderate infection risk on the Cascade to derive rules for adapting the HOPS powdery mildew risk index to Cascade and similar cultivars.
Sub-objective 4C Research Goal: Characterize resistance of USDA males to multiple strains of powdery mildew. A set of 150 individuals –resistant to downy mildew--will be tested for their resistance to multiple races of P. macularis. Resistance to three different isolates-each with unique virulence genes—will be sequentially scored across all male lines. Remaining resistant individuals will be further evaluated to determine the nature of resistance.
Progress Report
This is the final report for project 2072-21000-051-000D, Breeding, Genomics, and Integrated Pest Management to Enhance Sustainability of U.S. Hop Production and Competitiveness in Global Markets, which will be replaced by new project 2072-21000-061-000D, Development of Superior Hops and Resilient Hop Production Systems. For additional information, see the new project report.
In support of Objective 1, research concluded on collection of data on field grown selections from crosses between Cascade and Centennial and USDA male accessions. In addition, data from other crosses made simultaneously were also collected for the third and last year of evaluation. While offspring from crosses made with Cascade and Centennial did not yield well, some of the Cascade offspring exhibited good resistance to powdery and downy mildew. Other crosses made between USDA male accessions and other hop cultivars and USDA female germplasm lines performed significantly better and exhibited higher yields. Approximately 15 offspring from crosses made for this objective will be cloned and then advanced into large multi-plot evaluations for continued observations on yield, disease resistance and other agronomic characters. These advanced selections will also be evaluated for brewing quality for the first time as larger quantities of harvested hop cone material will be available from replicated plots.
In support of Objective 2, research was completed by the submission and/or publication of two manuscripts covering the identification of molecular markers controlling the expression of resistance to either downy mildew or powdery mildew. Research on resistance to downy mildew demonstrated that this trait was most likely under the control of multiple genes each with independent segregation. The number of markers associated with resistance ranged from three to five depending upon the phenotypic trait evaluated (qualitative scoring of disease severity versus percent leaf area infected). These results suggest that selection for resistance to downy mildew will require multiple rounds of phenotypic selection to increase the accumulation of favorable genes controlling resistance. Research on resistance to powdery mildew yielded much clearer results with the identification of one 5 Mb region on chromosome 6 as the primary region controlling resistance. Analysis of the putative genes within this region identified 140 genes with 27 plant resistance-like genes (R-genes) found in nine clusters. Six sulfur-rich proteins genes with homology to patatins, thionins, agglutinin were identified in two clusters. Two glucan-endo-1,3-beta-glucosidase genes were identified bordering different R-gene clusters. Finally, putative up-regulators of transcription and stress-response genes were identified. The combination of R-gene clusters, sulfur-rich proteins, endo-1,3-beta-glucosidase and stress-response genes may be responsible for resistance to powdery mildew in the cultivar Comet.
In support of Objective 3, we developed a rapid molecular assay to differentiate two of the dominant pathogenic races of the hop powdery mildew fungus present in the western United States. Expressed genes were sequenced in 46 isolates of the pathogen and were used to identify genetic regions unique to each race. From this data, we developed a simple PCR-based differential assay. We tested the assay against 100 isolates of the fungus from the Unites States and Europe. The assay had perfect discrimination of virulence among isolates of the pathogen originating from the western United States but failed to predict virulence in three isolates collected from Europe. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed. Indeed, the assay has already been used in other settings to understand movement of the pathogen on planting material, predict which hop varieties may be at risk of powdery mildew when the disease is first detected on a farm, and speed routine diagnostics.
In support of Sub-objective 4A, we completed studies that demonstrated that certain strains of the hop powdery mildew fungus are specifically adapted to overcome the resistance found in the popular hop cultivar Cascade. We found that nearly all isolates of the fungus tested were able to infect Cascade under laboratory conditions. However, the greatest number of colonies, the most prolific reproduction of the fungus, and the shortest generation time of the pathogen was only observed with isolates derived originally from Cascade, as compared to other isolates derived from other cultivars. Further, the enhanced aggressiveness of these isolates was only manifested on Cascade and not six other susceptible cultivars, further indicating a specific adaptation to Cascade by the isolates. Cascade virulence was distinct from the virulence that enables the pathogen to infect cultivars possessing the known resistance factor termed R6. Race characterization indicated Cascade-adapted isolates of the fungus could overcome three named resistance genes in hop, but not other known resistance genes. Therefore, multiple sources of host resistance are expected to provide resistance to Cascade-adapted strains of the fungus. Given the plasticity of the powdery mildew fungus, breeding strategies for powdery mildew need to consider the potential for adaptation to both qualitative and partial resistance in the host.
In support of Sub-objective 4B, given that disease resistance in the cultivar Cascade has been overcome by a host-adapted strain of the fungus, there is an urgent need for growers to manage the disease to minimize crop damage. However, these management efforts may not be efficient because of the lack of historical experience with the disease on this cultivar and lack of basic information on disease response to the environment. We completed growth chamber experiments that characterized the response of Cascade to constant, transient, and diurnal cycles of supraoptimal temperatures, and how this cultivar differs in its responses from others. We identified specific temperature thresholds and durations that are permissive for powdery mildew on certain susceptible cultivars, but are suppressive to the disease on Cascade. Based on this information, we developed a powdery mildew risk index to estimate disease hazard on Cascade. A beta version of the web app and an automated email notification system was developed with university collaborators and revised based on usability testing with stakeholders. The web app is available at: http://uspest.org/risk/hpm_app. Field validation of the risk index over years found that growers could safely eliminate one to two fungicide applications per year as compared to their standard practices without compromising disease control.
In support of Sub-objective 4C, we systematically evaluated 136 accessions of male hop lines contained in the USDA collection for their powdery mildew reaction. Iterative inoculations with three isolates of hop powdery mildew fungus with varying race identified 23 (16.9%) accessions with apparent resistance to all known races of the pathogen present in the Pacific Northwest. Of these 23 accessions, 12 were resistant when inoculated with three additional isolates obtained from Europe that possess novel virulences. The nature of resistance in these individuals is unclear, but does not appear to be based on known R genes. Identification of possible novel sources of resistance to powdery mildew will be useful to hop breeding programs in the western United States and elsewhere. We have already made crosses using a subset of these males to introduce the apparently novel resistance into germplasm intended for future release to the public.
Accomplishments
1. Chromosome-scale assembly of the hop genome. The hop genome is complex in comparison to other crop species and previous attempts to assemble the hop genome resulted in large numbers of individual fragments. Placing these fragments into chromosome scale assemblies with genes, promoters and other expression factors is vital for genetic studies along with molecular breeding. ARS scientists in Corvallis, Oregon, and university collaborators utilized a combination of sequencing technologies to finally assemble the first complete chromosome-scale genome for hop. Approximately 25,000 unique genes and expression factors were identified, aligned to the genome and made available to the public at http://hopbase.cqls.oregonstate.edu as a genome browser. Stakeholder benefits range from brewery scientists evaluating the presence of genes thought only to be present in yeast, hop scientists identifying genes associated with traits of economic importance, and hemp scientists evaluating co-evolution.
2. Release of new USDA-ARS hop cultivar "Vista". New high yielding hop cultivars with improved traits are needed to advance the U.S. hop industry. ARS scientists in Corvallis, Oregon, and collaborators from Oregon State University, released the high yielding, disease-tolerant aroma line named “USDA Vista”. In 2022 this cultivar was planted on approximately 50 acres across the Pacific Northwest with additional acres planted during 2023. The release of this new cultivar provides brewers with a new hop cultivar with a unique flavor profile that excels in India pale ale (IPA) and pale ale styles but has also found niche use in fruity sour beers. Benefits for U.S. producers are the production of a high-yielding new cultivar that is seedless and picks cleanly. Additionally, the cultivar is harvested later than other cultivars and allows a grower to add acreage of production without eliminating other hop lines currently in their inventory.
Review Publications
Havill, J.S., Wiseman, M.S., Henning, J.A., Gent, D.H., Muehlbauer, G.J. 2022. Registration of seven powdery mildew-resistant wild hop germplasm lines. Journal of Plant Registrations. 17(1):171-179. https://doi.org/10.1002/plr2.20255.
Olatoye, O.M., Wiseman, M., Gent, D.H., Henning, J.A., Altendorf, K.R. 2022. Genetic characterization of downy mildew resistance from the hop (Humulus lupulus L.) line USDA 64035M. Crop Science. 63(3):1082-1091. https://doi.org/10.1002/csc2.20880.
Laurie, R.W., Richardson, B.J., Ross, C.J., Gent, D.H. 2022. Yard age, cultivar susceptibility, and spring pruning practices as risk factors for overwintering of Podosphaera macularis on hop. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-10-22-0112-R.
Gent, D.H., Block, M., Massie, S.T., Phillips, C.L., Richardson, B.J., Shellhammer, T.H., Trippe, K.M., Wiseman, M.S. 2023. Nitrogen and sulfur fertility practices: Influences on hop chemistry, aroma, and nitrate accumulation. Journal of the American Society of Brewing Chemists. https://doi.org/10.1080/03610470.2023.2204412.
Havill, J.S., Richardson, B.J., Rohwer, C.L., Gent, D.H., Henning, J.A., Muehlbauer, G.J. 2023. Identification of quantitative trait loci involved in R1-mediated resistance to powdery mildew and sex determination in hop (Humulus lupulus L.). Journal of Theoretical and Applied Genetics. 136. Article 154. https://doi.org/10.1007/s00122-023-04399-7.
