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Research Project: Breeding, Genomics, and Integrated Pest Management to Enhance Sustainability of U.S. Hop Production and Competitiveness in Global Markets

Location: Forage Seed and Cereal Research Unit

2019 Annual Report

Objectives
The goal of this project is to maintain and enhance the competitiveness of the U.S. hop industry through development of publically 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 publically 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
In support of Objective 1, research continued on development and release of new hop cultivars and germplasm possessing superior disease resistance, yield, and brewing characteristics. Unusually warm dry weather during 2018 delayed selection for seedlings possessing downy mildew resistance. As a result, the seedlings selected for resistance to powdery mildew were over-wintered outside of greenhouse settings for natural downy mildew inoculant as well as artificially induced inoculation during Spring of 2019. Selection of downy mildew resistant seedlings, as well as culling of powdery mildew susceptible lines that escaped selection routines during 2018, were completed. The disease resistant seedlings that remained were potted up into 18.9-liter pots and transplanted to a low-trellis nursery for identification of female lines as well as lines possessing vigor and good hop cone aroma. Additional progress was made on previous project objectives that resulted in advanced selections from previous crosses. Two USDA-ARS experimental hop cultivars were selected for expansion into commercial sized nurseries (0.8 Hectares) in multiple states. The first line, USDA 2000010-008, represents an “aroma-hop” line that possesses high yield potential and is harvested two weeks earlier than most other hop cultivars. The greatest benefit this line offers to stakeholders is the expansion of the harvest window beyond the normal harvest period, ultimately increasing total harvestable crop without investment into additional harvesting equipment. The second line, USDA 2006009-074, is a seedless, high-yielding, disease-tolerant hop cultivar with excellent aroma and brewing characteristics. It offers high yields in excellent aroma line specifically for India Pale Ale (IPA) beers. Related to Objective 2, research continued on the Identification, characterization, and validation of molecular markers associated with qualitative and quantitative resistance to important foliar diseases. The 288 offspring from the cross, ‘Comet’ x USDA 64037M, were cloned and experimental evaluation of resistance levels to powdery mildew of these offspring is ongoing during 2019. Preliminary data shows that of the 288 offspring evaluated, 261 had some level of powdery mildew with 27 lines exhibiting no colonies. In addition, DNA from each of these lines was extracted and purified for identification of molecular markers for this study. Inoculation studies for downy mildew resistance will commence once environmental conditions become optimum for screening, typically late Winter early Spring 2020, as it requires cool, humid conditions for optimum expression. In support of Objective 3, we continued research to identify molecular markers associated with virulence of the powdery mildew fungus as described in the project plan. Genetic variants in the expressed RNA between pathogenic races of the pathogen were identified through an iterative bioinformatics process. From the candidate genetic variants, molecular diagnostic assays were tested to identify genetic variants that consistently differentiate races of the pathogen. Through this process, we have tentatively identified a gene with a single nucleotide polymorphism that consistently differs between isolates of the fungus that can infect plants with the resistance gene termed R6 (an important source of resistance to powdery mildew) and those that cannot. A rapid diagnostic assay (a quantitative polymerase chain reaction (PCR)) is in the late stages of development. Once developed, this assay will provide growers, researchers, and quarantine officials with a quick, simple, and reliable means to determine pathogen race without the need for laborious traditional inoculation tests. In support of Sub-objective 4A, studies were largely completed that characterized the aggressiveness, fitness, and race of the isolates of the powdery mildew pathogen that attack the cultivar Cascade, one of the most widely planted hop cultivars in the U.S. and the most commonly used cultivar in craft beer. On the whole, the research indicates that isolates of the powdery mildew fungus that cause severe disease on Cascade are specifically adapted to this cultivar. While these isolates may infect other cultivars, elevated levels of disease are observed only on Cascade as compared to other, extant isolates. Further, R-genes and R-gene combinations that appear effective against this race of the pathogen have been identified. Future research to identify genetic determinants of adaptation to the cultivar Cascade are in the planning stages. Given the importance of understanding the factors that determine the development of powdery mildew in formerly resistant cultivars, we planned and conducted new growth chamber experiments to characterize disease responses due to brief periods of unfavorable, high temperature. This work supports Sub-objective 4B. We discovered that even brief exposure to high temperature in the early stages of the infection process substantially reduces disease on Cascade plants as compared to other susceptible cultivars. This suggests that disease risk indexes can be revised to better reflect conditions favorable for or suppressive to powdery mildew on Cascade. Follow up growth chamber and field studies are planned to more fully characterize the apparent heat-induced resistance to powdery mildew in Cascade. Initial work with university collaborators has started to develop a mobile device web application for a powdery mildew risk index. Related to Sub-objective 4C, powdery mildew screening of 136 male accessions of hop in the USDA germplasm collection was completed. A subset of 12 plants were identified that have apparent resistance to the prevalent races of the fungus in the U.S. and Europe. Additional screening of other male germplasm in the USDA collection is in the initial planning stages.

Accomplishments
1. Development of new genetic maps for multiple traits using new complete draft hop genome. Much of the hop genome remains unexplored for genes and expression factors controlling traits of interest in hop production. ARS scientists located in Corvallis, Oregon, developed three new genetic maps for studies on short-trellis growth habit, powdery mildew resistance and downy mildew resistance. Using the recently completed and fully annotated joint USDA-Oregon State University developed hop genome (HOPBASE.ORG), scientists were able to identify putative resistance genes responsible for powdery mildew resistance found in cultivar USDA ‘Newport’; the putative gibberellin-insensitive genes responsible for dwarfing in hop; and many of the putative quantitatively expressed genes thought to be responsible for resistance to downy mildew. The anticipated products from these studies are the development and identification of molecular markers linked to genes responsible for the expression of these traits.

2. Origin and spread of the hop powdery mildew fungus. Powdery mildew is one of the most important diseases of hop in the U.S., costing the industry an estimated 15 percent of crop value annually. The powdery mildew fungus was introduced into the western U.S. during the mid-1990s, although the source of the original introduction is unknown. Scientists at Corvallis, Oregon, identified genetic variants and reproductive types (mating types) in a worldwide collection of the pathogen. The pathogen population in the western U.S. consistently grouped with isolates from Europe; genetic fingerprints and mating type of populations on cultivated plants in western and eastern U.S. were similar, but differed from those found on isolates on wild plants. The genetic and phenotypic patterns suggest a European origin of the pathogen in the western U.S., followed by spread of the pathogen from the western U.S. to re-emergent production regions in the eastern U.S. This finding has important policy implications for limiting future spread of the pathogen in association with planting material.

3. Prediction of disease spread at the landscape level. Epidemics of airborne plant diseases result because of dispersal, but predicting dispersal can be exceedingly difficult. Scientists in Corvallis, Oregon, and university collaborators developed a statistical model to predict spread of the hop powdery mildew fungus between hop yards in the landscape. The model accounted for 34 percent to 90 percent of the observed variation in disease incidence at the field level, depending on the year and season. Insights from the model indicate that pathogen dispersal is dominated by relatively localized dispersal events (less than 2 kilometers) among the network of fields, being mostly restricted to the same or adjacent farms. The model was used to identify characteristics of individual hop yards that are highly important in disease spread. The model can inform both within-season disease risk and also growers’ strategic decisions on how to reduce the overall risk of disease development and spread.

U.S. DEPARTMENT OF AGRICULTURE

Forage Seed and Cereal Research Unit: Corvallis, OR