Location: Forage Seed and Cereal Research2018 Annual Report
1a. Objectives (from AD-416):
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)
1b. Approach (from AD-416):
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.
3. Progress Report:
This report documents progress for project 2072-21000-051-00D, which started in March 2018 and continues research from project 2072-21000-046-00D, “Reducing the Impact of Diseases on Hop Production." Significant progress was made in support of Objective 1. Thirty-two (32) new crosses were performed using popular cultivars crossed with male germplasm possessing strong resistance to fungal pathogens. Seeds were germinated, and seedlings subsequently inoculated with powdery mildew spores. Susceptible offspring were culled and the remaining lines cut-back. Remaining offspring were inoculated with downy mildew spores and infection allowed to progress. Unfortunately, this inoculation failed due to regionally high temperatures. Seedlings were transplanted into 18.9-liter pots for grow out to select for sex (Fall 2018) and downy mildew resistance (Spring 2019). New research was initiated, focused on identifying molecular markers linked to resistance to fungal pathogens. This project utilizes a bi-parental mapping population designed to access resistance to powdery mildew as well as downy mildew. Genotyping-by-sequencing is to be used to identify single-nucleotide polymorphism (SNP) markers for both quantitative trait locus (QTL) analysis as well as genome-wide scans. A cross between Comet (resistant to powdery mildew, susceptible to downy mildew) and USDA 64035M (resistant to downy mildew, susceptible to powdery mildew) was performed, seeds collected, cold scarified and germinated. The healthiest 315+ seedlings were selected and cloned. The first round of inoculations with the powdery mildew pathogen were begun. Research to identify molecular markers associated with virulence of the powdery mildew fungus began (Objective 3). An initial assessment of population genetic differentiation based on pathogen race was conducted using standard methods for RNA sequencing (Illumina short-insert libraries) using RNA obtained from the pathogen growing on the leaf surface. Across numerous isolates of the fungus, ARS researchers did not identify evidence of genetic differentiation associated solely with pathogen race. Efforts are underway to increase inoculum of multiple strains, isolate specialized infection structures (haustoria) within infected plants, and deeply sequence RNA from these structures using different sequencing techniques that can yield longer stretches of RNA sequence data. In support of Sub-objective 4A, substantial progress was made in characterizing the aggressiveness, fitness, and race of the isolates of the powdery mildew pathogen that attack the cultivar, Cascade, one of the most widely planted cultivars in the U.S., and the most commonly used in craft beer. In a collection of the isolates of the pathogen made in the western U.S., nearly all isolates were able to infect Cascade in laboratory inoculations. However, the greatest number of colonies, most conidia produced, and the shortest latent period 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. There was no evidence of a known major R-gene in Cascade, as seven isolates of P. macularis with contrasting virulence all infected Cascade. Amongst 158 isolates obtained from hop yards planted to Cascade, only two (1.3%) were able to infect the cultivar, Nugget, which possesses the resistance factor termed R6, indicating that isolates of P. macularis virulent on Nugget are largely distinct from those adapted to Cascade. Further, race characterization indicated Cascade-adapted isolates of P. macularis were able to overcome R-genes Rb, R3, and R5, but not other known R-genes. Therefore, multiple R-genes and other sources of partial resistance are expected to provide resistance to Cascade-adapted strains of the fungus. In Sub-objective 4B, two growth chamber experiments were completed to identify and quantify the impact of supraoptimal temperature on host susceptibility to and development of powdery mildew on the cultivar, Cascade. ARS researchers discovered that the prior exposure of Cascade plants to elevated temperature reduces susceptibility to powdery mildew disproportionately as compared to other, susceptible cultivars. Furthermore, the amount of disease that develops following inoculation and incubation at constant, elevated temperatures is also significantly less on Cascade versus other susceptible cultivars. Follow up growth chamber and field studies are planned to more fully characterize the apparent heat-induced resistance to powdery mildew in the cultivar, Cascade, and resolve how this is unique from other susceptible cultivars. This work will provide the foundation for revision of a powdery mildew risk index for Cascade and related cultivars. In Sub-objective 4C, good progress was made in characterizing publically available male germplasm for its reaction to multiple strains of the powdery mildew fungus. Strains of the fungus virulent on cultivars possessing the resistance factor termed R6 and the cultivar Cascade have become widespread in the Pacific Northwestern U.S., rendering most cultivars grown susceptible to the disease at some level. A total of 136 male accessions of hop contained in the U.S. Department of Agriculture collection were obtained, propagated. Screening began to identify potential sources of resistance to extant races of the pathogen.
1. Completion of hop draft genome with gene annotations. Hop breeding is hindered due to a lack of information on gene families, promoters and transcription factors associated with traits of economic importance. Previously published genomes for hop were incomplete and missing up to 33% of the estimated hop genome along with the genes found in these missing regions. ARS scientists in Corvallis, Oregon, along with collaborators at Oregon State University, Hopsteiner Inc., and Pacific Bioscience Inc., completed sequencing and assembly of the complete (2.8 Gigabase) genome using single-molecule long-read sequencing data coupled with advanced genome assemble techniques. Simultaneous gene expression experiments were performed, and the location and plausible function of genes were added to the genome. Scientists now have the capability of determining and identifying which genes or gene families are expressed during pathogen attack, hop cone formation and plant growth, with new gene expression studies currently underway. These new research abilities enable hop breeders to more quickly develop improved cultivars that address industry needs.
Gent, D.H., Claassen, B.J., Twomey, M.C., Wolfenbarger, S.N., Woods, J.L. 2018. Susceptibility of Hop crown buds to powdery mildew and its relation to perennation of Podosphaera macularis. Plant Disease. 102(7):1316-1325.
Gent, D.H., Mahaffee, W.F., Turechek, W., Ocamb, C.M., Twomey, M.C., Woods, J.L., Probst, C. 2018. Risk factors for bud perennation of Podosphaera macularis on hop. Phytopathology. https://doi.org/10.1094/PHYTO-04-18-0127-R.