Location: Forage Seed and Cereal Research Unit
2024 Annual Report
Objectives
Objective 1: Breed new hop varieties possessing superior traits including disease resistance, yield, and brewing characteristics.
Sub-objective 1.A: Combine multiple sources of resistance to powdery mildew disease into new germplasm.
Sub-objective 1.B: Evaluate, identify, and promote superior advanced female hop germplasm from the former Washington State University breeding program for potential public variety release.
Sub-objective 1.C: Generate new variation via crossing, make selections, and advance selected material.
Objective 2: Characterize hop germplasm for tolerance to priority traits such as water-use-efficiency and pest and disease resistance including systematic evaluation of powdery mildew resistance in new germplasm.
Sub-objective 2.A: Assess germplasm for water use efficiency characteristics and abiotic stress resiliency.
Sub-objective 2.B: Evaluate male germplasm for resistance to powdery mildew.
Objective 3: Identify, characterize, and validate molecular markers associated with qualitative and quantitative traits of economic importance in hop such as novel powdery mildew resistance.
Sub-objective 3.A: Identify molecular markers for WUE using bi-parental mating designs, genotyping-by-sequencing (GBS) and quantitative trait loci (QTL) analyses.
Sub-objective 3.B: Identify and validate QTL in the hop cultivar Comet for quantitative resistance to powdery and downy mildews in field environments.
Sub-objective 3.C: Develop a multi-parent population to identify novel sources of powdery mildew resistance and create and utilize a consensus genetic map.
Objective 4: Evaluate management practices that improve crop productivity and crop health using computational analyses to identify optimal control policies for powdery mildew disease at the landscape level and abiotic stress physiology and management.
Sub-objective 4.A: Assess reduced late-season irrigation as a cultural management strategy to improve hop cone quality.
Sub-objective 4.B: Identify determinants and model pesticide use intensity at the field level based on characteristics of growers’ production systems.
Approach
Objective 1 Research include 1) develop new disease resistant varieties possessing unique, desirable brewing qualities, and 2) evaluate germplasm from Washington State University (WSU) for the same. Germplasm with different and complementary resistance will be crossed to produce offspring to be screened for disease resistance. Resistant offspring will be field-evaluated for resistance, agronomics, aroma and brewing chemistry. Germplasm evaluation from WSU includes screening for the same metrics. Possible challenges exist with 1) ensuring successful controlled pollination; addressed using greenhouses for crosses if necessary, and 2) inoculation for disease resistance screening; alleviated by multiple inoculation attempts.
Objective 2 posits 1) hop varieties with higher water-use efficiency (WUE) can be identified, and 2) newly acquired male hop germplasm exhibits varying levels of powdery mildew resistance. Experiments to test hypotheses include 1) evaluating four hop varieties over two growth seasons near Prosser, WA for WUE, along with agronomic, physiological assessments, and brewing quality metrics, and 2) employing an elimination screening procedure with diverse isolates of the powdery mildew fungus. Contingencies include alternative strategies such as broadening hop variety evaluations and additional germplasm sources for Hypothesis 1, while Hypothesis 2 may involve additional genetic analyses or functional investigations.
Objective 3 posits 1) WUE under drought conditions is under polygenic control, 2) quantitative trait loci (QTL) for resistance to downy and powdery mildew are consistent between greenhouse and field environments and 3) resistance to powdery mildew in male hops is heritable and novel. Bi-parental crossing schemes will be used to develop multiple populations. Populations will be phenotyped for WUE in the greenhouse or disease resistance in the field or via high-throughput systems. Germplasm will be genotyped using molecular methods. QTL statistical analyses will be performed to identify significant QTL. Contingencies for addressing problems range from use of increased water for WUE, use of spatial adjustment of phenotypic data for differences environmental conditions, and use of larger population sizes and/or increased depth of DNA sequencing.
Objective 4 involves 1) developing late-season irrigation guidelines to improve hop quality, and 2) hypothesizes that production efficiency and pesticide use levels vary among farms and are explained by pathogen, host, and environment. Field studies will compare regular irrigation to reduced late-season irrigation for improving hop quality. Soil water will be monitored to maintain deficit water conditions. Yield and brewing quality metrics will be quantified. In 4.B data from industry cooperators or developed in-house will be used to identify determinants of pesticide use on commercial farms. Machine learning will be used to identify factors predicting pesticide use. Contingencies include increasing biological replicates to resolve irrigation effects and developing additional weather or biological variables to refine models if results do not describe pesticide use intensity.
Progress Report
For Sub-objective 1.A, ARS scientists in Corvallis, Oregon, successfully crossed Cascade x 19058M, germinated seedlings in the greenhouse and inoculated with powdery mildew and made selections for resistant lines. Genetic segregation for resistance was observed among offspring with most offspring showing resistance or partial resistance. Nevertheless, multiple offspring were highly-susceptible and these lines were culled. Out of an initial seedling population of approximately 1,800 offspring, 300 highly-resistant lines were selected. These have been transplanted into one1-gallon pots and placed out in the field for further selection. ARS scientists will be selecting female lines using molecular markers prior to planting into off-station nurseries.
For Sub-objective 1B, ARS scientists evaluated 18 selections from the former Washington State University (WSU) breeding program in 7-hill plots in Washington for a second year. One genotype, originating from the USDA-ARS, 2001006-084A, was selected and expanded in a one1-acre plot starting in 2024. W1108-333, a WSU line, was evaluated for a second year on-farm in two2-acre plots in Idaho and Washington. Both genotypes were evaluated for downy mildew and powdery mildew resistance under controlled environment conditions in preparation for variety release. Rhizomes of W1108-333 and 2001006-084A were also planted in Oregon for evaluation. These clones were planted at the off-station research plots located outside of Mount Angel, Oregon. A third genotype from the WSU program, W1121-059, was eliminated due to high levels of susceptibility to powdery mildew.
For Sub-objective 1.C, W2022 crosses were germinated, inoculated with hop powdery mildew spores in the greenhouse, and susceptible individuals were culled. Approximately 2,000 seedlings were sexed by molecular marker and propagated into four replications. Two replications were established in pots on- farm in Oregon and were inoculated and rated for downy mildew resistance. Two replications were planted in Washington under a short trellis, and females were evaluated for disease incidence, cone morphology, brewing chemistry, visual estimates of yield, and aroma. 130 offspring were selected, cloned, and subjected to additional disease screening in the greenhouse. After disease screening, 30 selections were transplanted into 7-hill plots in Washington in two blocks with commercial checks.
Under Sub-objective 2.A, field plots in Washington were established in 2022. Three irrigation regimes (50% ET, 100% ET, and 150% ET) were incorporated across three hop varieties (Cascade, Citra, and Mosaic) in a randomized complete block design with six replications. In 2024, hop varieties were subjected to irrigation regimes, which began on June 10th and will continue until the end of the season. The irrigation regimes are based on accumulated evapotranspiration (ET) over a three-to-four-day period. To verify irrigation regimes, soil moisture was monitored bi-weekly on half of the plots. To assess physiological response to treatments, ARS scientists measured stomatal conductance, photosynthesis, carbon assimilation, and respiration every two to three weeks using a photosynthetic measurement system. Using measurements of carbon assimilation and transpiration, they are able to calculate intrinsic water use efficiency. Crop water productivity will be calculated from the total hop cone yield, and the total water applied at the end of the season from the plots subjected to the 100% ET irrigation regime. The crop low-water stress resiliency level will be determined based on the total hop cone yield and physiological measurements made throughout the season.
For Sub-objective 2.B, ARS scientists conducted follow up evaluation of 13 hop accessions that, in Year 1, were identified as resistant to pathogenic races of hop powdery mildew fungus found in the western United States. They conducted controlled inoculations under quarantine conditions with three isolates of the fungus that possess novel virulences not known to occur in the United States. From these inoculations, they identified seven males that were entirely resistant. In 2024, ARS scientists obtained another new isolate of the fungus with a novel virulence not known to occur in the United States. Inoculations are underway with this new isolate to identify if the powdery mildew resistance in the USDA hop germplasm collection is indeed novel.
For Sub-objective 3.A, ARS scientists successfully germinated seedlings from a cross between drought tolerant parent, ‘Cascade’, and presumed drought-susceptible parent, ‘19058M’, in the greenhouse. A set of 400 offspring were selected for powdery mildew resistance from the original population. Genotyping-by-sequencing of all samples were completed July 2024. Two hundred offspring from this population were randomly selected for cloning to be replicated into a randomized complete block design. Materials and instruments to run the drought experiment in the greenhouse have been purchased.
For Sub-objective 3.B, a Comet x 64035M bi-parental population was maintained in a randomized complete block design with two blocks in Washington for field evaluation of powdery mildew resistance to validate marker discovery. Spreader rows with the susceptible cultivar Zeus were planted every two rows and inoculated with the pathogen that causes hop powdery mildew. Disease incidence and severity were low in 2023. Infected Zeus plants were planted amongst existing Zeus plants to try and promote disease. Conditions appear more conducive to disease development in 2024, and plots are being maintained and monitored for rating opportunities.
For Sub-objective 3.C, ARS scientists completed powdery mildew evaluations of four populations of hop generated by crossing four male hop lines with putative novel powdery mildew resistance to a common female parent. Scientists conducted phenotyping using two approaches. First, they conducted traditional greenhouse evaluations with disease enumeration by human raters who counted the number of pathogen colonies that formed per leaf. Second, leaf disks from each individual were inoculated and maintained under controlled conditions in a laboratory and subsequently imaged using the Blackbird automated image capture system. Each image was processed and assessed using a machine learning algorithm to estimate the percentage of the leaf area covered with mycelium or sporulation structures of the hop powdery mildew fungus. Tissue samples from all offspring and parents were submitted for genotyping-by-sequencing for use in genetic map construction.
For Sub-objective 4.A, ARS scientists conducted an experiment in collaboration with a Washington hop grower during 2022-2023 to evaluate the impact of late-season drought on hops. In this study, mature Cascade hops were subjected to drought conditions for 15 days preceding harvest. Physiological responses to drought were assessed by measuring stomatal conductance and stem water potential. To evaluate the crop's physical response to drought, scientists measured dry matter, hop cone yield, and hop quality through chemical analysis. This investigation revealed practical insights. Treatments resulted in a 9% reduction in hop cone yield, equating to a loss of approximately 177 pounds per acre. Their results also showed that late-season drought didn’t affect alpha and beta acids levels, which are responsible for giving beer its bitterness and aroma. Additionally, ARS scientists observed a trend where monoterpene flavor compounds, such as myrcene, decreased when subjected to drought, while sesquiterpenes, such as humulene, increased under the same conditions.
For Sub-objective 4.B, ARS scientists analyzed two data sets to identify variables that are predictive of pesticide use intensity in commercial hop yards. In the first data set, they drew upon data covering incidence of hop plants with powdery mildew from yards in Oregon during 2014 to 2017 and associated metadata on grower cultural practices, cultivar susceptibility to powdery mildew, and pesticide application records. ARS scientists applied multiple machine learning methods in either a predictive (associational) or causal inference framework. One model identified growers’ spring pruning thoroughness, cultivar susceptibility to strains of hop powdery mildew, network centrality of yards during May-June/June-July time transitions, and the initial strain of the fungus detected as important variables determining the number of pesticides applied by growers and the associated costs they incurred. Exposure-response function models fit after covariate weighting indicated both the number of pesticides applied and their costs scaled linearly with the seasonal mean incidence of plants with powdery mildew. Biological and production factors collectively influence the incidence of powdery mildew, which directly affects the number and cost of pesticide applications. ARS scientists identified several potential strategies for reducing pesticide use and costs for management of powdery mildew on hop. They initiated a follow-up study on when growers may use various types of fungicides and herbicides for powdery mildew management and factors that predict when growers may switch from non-synthetic fungicides to more potent and expensive synthetic fungicides. They then analyzed a census data set of pesticide use on a powdery-mildew-susceptible cultivar and quantified variation in the seasonal use of powdery mildew fungicides by producers in Oregon, Idaho, and Washington, and how pesticide use has changed over time. ARS scientists found broad variance in the average number of fungicide applications made by producers and identified producers that represent the extremes of the distribution. Further, they identified 30 hop yards for field surveys to collect disease, production, and pesticide use data, and validate the findings through grower interviews.
Accomplishments
1. The Y-chromosome of male hop plants contains genes of interest for hop traits of economic importance. Currently, the most complete genome assemblies are only available for female hop cultivars, the sex of the crop used in beer brewing. These cultivars do not possess the Y-chromosome found in males, which may contain genes of importance for beer brewing and are therefore important for breeding new female cultivars. ARS researchers in Corvallis, Oregon, along with scientists at Oregon State University, sequenced and assembled two male hop genomes. While a large portion of the Y-chromosome contains non-coding regions with unknown function, genes presumed to be involved in expression of sex and flowering have been identified as well as genes for other traits of economic importance, including disease resistance and hop flavoring compounds. The completion of these two male genomes also completes a triune of mother-father-son genomes and as such provides vital information on genetic recombination in hops. This new genomic information and genome assemblies, available to the public at hopbase.org, will provide hop scientists with advanced selection tools that place hop in the same categories as other crop species regarding use of molecular tools for plant breeding.
2. Impact of late season drought on hop cone yield and quality in the Yakima Valley. The Yakima Valley, located in Washington State, is the highest hop-producing region in the United States, accounting for over 70 percent of the country’s hop production. This important production is threatened by changes in water availability driven by climate change. To investigate the impact of drought, ARS researchers in Corvallis, Oregon, conducted an experiment in a grower’s field where mature Cascade hops were subjected to 15 days without irrigation during the final 15 days before harvest. The study revealed that a loss of irrigation during this critical period significantly affected the plants’ ability to assimilate carbon through photosynthesis, as evidenced by reduced stomatal conductance in plants subjected to drought (78 percent drop in stomatal conductance). The late-season drought resulted in a nine percent reduction in hop cone yield, equating to a loss of approximately 199 kg per hectare.
3. A new molecular marker in hop enables early selection of male and female plants. Hop is a dioecious species with male and female plants. Females are used in production, and males are used exclusively for breeding. Traditional methods involve selecting males and females using phenotypic assessments during the first year of the breeding cycle. ARS researchers in Corvallis, Oregon, identified a new molecular marker using genome-wide association studies (GWAS) and developed it into a PCR allele competitive extension (PACE) assay that can be utilized in-house to screen large populations rapidly (around two hours for a 96-well plate), cheaply (0.15 cents per sample), and with 96.2 percent accuracy. The marker enabled a 97 percent all-female seedling yard in 2023, and a 25 percent increase in the ratio of female:male seedlings evaluated in the same field space as 2022.
Review Publications
Grunwald, N.J., Bock, C.H., Chang, J.H., Alves De Souza, A., Del Ponte, E.M., du Toit, L.J., Dorrance, A.E., Dung, J., Gent, D.H., Goss, E.M., Lowe-Power, T., Madden, L.V., Martin, F.N., McDowell, J., Naegele, R.P., Potnis, N., Quesada-Ocampo, L.M., Sundin, G.W., Thiessen, L., Vinatzer, B.A., Zeng, Q. 2024. Open access and reproducibility in plant pathology research: Guidelines and best practices. Phytopathology. 114(5):910-916. https://doi.org/10.1094/PHYTO-12-23-0483-IA.
Richardson, B.J., Gent, D.H. 2024. Suppression of hop downy mildew as influenced by the timing of selected fungicides. Plant Health Progress. https://doi.org/10.1094/PHP-10-23-0086-BR.
Garcia-Figuera, S., Lowder, S.R., Lubell, M.N., Mahaffee, W.F., McRoberts, N., Gent, D.H. 2024. Free-riding in plant health: A social-ecological systems approach to collective action. Annual Review of Phytopathology. 62:4.1-4.28. https://doi.org/10.1146/annurev-phyto-121423-041950.
Gent, D.H., Adair, N.L., Hatlen, R., Miles, T.D., Richardson, B.J., Rivedal, H.M., Ross, C., Wiseman, M.S. 2024. Detection of Podosphaera macularis in air samples by quantitative PCR. Plant Disease. https://doi.org/10.1094/PDIS-04-24-0894-RE.