Location: Forage Seed and Cereal Research Unit
2020 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, researchers at Corvallis, Oregon, completed greenhouse screening of all offspring from crosses made for developing superior cultivars possessing disease resistance. Disease resistant selections were subsequently grown out in a 2.4-m “short-trellis” to identify females and screen for hop cone shape and preliminary cone aroma. One additional round of selection for disease resistance was performed in March. Selections from these steps were transplanted out into the experimental hopyard under 1.2-m spacing in April, 2020. This nursery was scored for disease resistance and vigor during spring/Summer 2020. A number of full-sibling families with powdery and downy mildew resistance had no visible infections of either disease and appear to be quite vigorous.
In support of Objective 2, researchers produced multiple clones of the offspring and parents from the cross between ‘Comet’ x ‘USDA 64035M’. Replicate lines of these samples were subsequently established in the greenhouse and the first and second round of scoring for powdery mildew was completed. Inoculation for first round of downy mildew resistance was initiated during Spring 2020 (once environmental conditions became optimum for screening—typically late Winter early Spring) as this requires cool, humid conditions for optimum expression. Plant leaf-tissue from each line was harvested for DNA extraction prior to initiating inoculation with fungal pathogens. DNA from approximately 300 different genotypes was obtained and subsequently sequenced to identify molecular markers. Over 1.2 million molecular markers were identified with a subset of approximately 50,000 highly informative markers chosen for use in identifying genomic regions linked to disease resistance. Preliminary genome wide association studies along with preliminary quantitative trait locus (QTL) analyses identified eight regions associated with resistance to powdery mildew that contain tandem putative disease resistance genes on one chromosome along with two other minor-associated regions on different chromosomes. These results were expected as resistance to powdery mildew in ‘Comet’ follows quantitative inheritance patterns (multi-genic) rather than qualitative (single gene).
In support of Objective 3, researchers completed development of a simple, rapid, diagnostic assay that can differentiate between two widely prevalent pathogenic races of the hop powdery mildew fungus. Current methods for race determination in the fungus are slow, costly, and labor intensive. Based on genetic variants identified in expressed genes in the fungus, the scientists developed a DNA-based test (quantitative real-time PCR) that can reduce the time required for determining pathogen race from 21 days or longer to hours. The assay was tested with a large collection of powdery mildew isolates from across the United States and Europe. The assay had perfect discrimination of two races of the fungus among isolates originating from the western United States, but was population-specific and failed to predict race in 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.
In support of Sub-objective 4A, the cultivar ‘Cascade’ has been grown in the U.S. hop industry for decades and historically was not seriously affected by powdery mildew due to its partial resistance to the disease. This changed near 2012 when severe powdery mildew was reported on Cascade plants in Washington State. Studies were completed that characterized the aggressiveness, fitness, and race of the isolates of the powdery mildew pathogen that attack this cultivar. This research demonstrates that isolates of the fungus that cause severe disease on Cascade are specifically adapted to this cultivar. The scope of this research was expanded to describe whether the powdery mildew fungus develops local adaptation for increased aggressiveness on cultivars that do not possess known resistance when these cultivars are widely planted in the landscape. The sum of this research has important implications for mitigating disease risk.
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 cultivar and lack of basic information on disease response to the environment. Researchers 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. They 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, they 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 and web app are underway.
In support of Sub-objective 4C, powdery mildew screening of 136 male accessions of hop in the USDA germplasm collection was completed, and resistance to extant strains of the powdery mildew fungus was identified in a subset of 12 plants. Researchers expanded the scope of this objective and established a collaborative research project with the University of Minnesota to characterize disease resistance in wild hop plants collected across North America. A subset of 23 individuals with putatively novel resistance were sequentially challenged with different strains of the fungus. New forms of resistance appear to be present in this germplasm, which will provide breeders with new genetic resources for managing this destructive disease.
Accomplishments
1. Assembly and annotation of pseudo-chromosomes of the hop genome. The hop genome is one of the most intractable genomes within the plant kingdom with extremely high levels of DNA diversity and repetition making it one of the more difficult genomes to work with. Researchers at Corvallis, Oregon, utilized previously reported long-read sequences along with optical sequencing methods to assemble the presumed 10 chromosomes of hop. This genome assembly was used to identify genome regions (and the putative genes present within) on single chromosomes linked with disease resistance as well as plant height. The development of this draft genome covering the 10 chromosomes will allow hop scientists to better ascertain genic interactions both locally and distally on different chromosomes and further scientific understanding of the complexities of flavor components in hop that are of interest to brewers.
2. Diagnostic assays for predicting pathogenic race of the hop powdery mildew fungus. Powdery mildew of hop is one of the most damaging diseases affecting hop crops worldwide. Multiple pathogenic races of the fungus occur, which complicates management because of uncertainty of what races are present, and therefore what varieties of hop may be at risk of disease. To solve this problem, researchers at Corvallis, Oregon, and Oregon State University collaborators, developed a diagnostic DNA test that can confirm pathogen race within hours, which is critical for implementing timely control measures. This is significant because the assay permits growers and others to make precise management decisions when pathogen race is known with certainty. The assay has practical applications in hop breeding, epidemiological studies, and other settings where rapid confirmation of pathogen race is needed.
Review Publications
Gent, D.H., Claassen, B.J., Gadoury, D.M., Grunwald, N.J., Knaus, B.J., Radisek, S., Weldon, W., Wiseman, M.S., Wolfenbarger, S.N. 2020. Population diversity and structure of Podosphaera macularis in the Pacific Northwestern U.S. and other populations. Phytopathology. 110(5):1106-1116. https://doi.org/10.1094/PHYTO-12-19-0448-R.
Gent, D.H., Block, M., Claassen, B. 2020. High levels of insensitivity to phosphonate fungicides in Pseudoperonospora humuli. Plant Disease. 104(5):1400-1406. https://doi.org/10.1094/PDIS-10-19-2067-RE.
Padgitt-Cobb, L., Kingan, S.B., Henning, J.A. 2019. Genomic analysis of powdery mildew resistance in a hop (Humulus lupulus L.) bi-parental population segregating for “R6-locus”. Euphytica. 216:10. https://doi.org/10.1007/s10681-019-2543-x.
Eriksen, R.L., Rutto, L.K., Dombrowski, J.E., Henning, J.A. 2020. Photosynthetic activity of six hop (Humulus lupulus L.) cultivars under different temperature treatments. HortScience. 55(4):403-409. https://doi.org/10.21273/HORTSCI14580-19.
Purayannur, S., Miles, T.D., Gent, D.H., Pigg, S., Quesada-Ocampo, L.M. 2020. Hop downy mildew caused by Pseudoperonospora humuli: A diagnostic guide. Plant Health Progress. 21(3):173-179. https://doi.org/10.1094/PHP-10-19-0072-DG.
Purauannur, S., Cano, L.M., Bowman, M.J., Childs, K.L., Gent, D.H., Quesada-Ocampo, L.M. 2020. The effector repertoire of the hop downy mildew pathogen, Pseudoperonospora humuli. Frontiers in Genetics. 11. https://doi.org/10.3389/fgene.2020.00910.
