2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The most critical problem facing the U.S. hop industry is the need for hop varieties that provide superior brewing quality and that contain resistance to two serious diseases, powdery and downy mildew. Although downy mildew has been a problem in U.S. hop producing regions for many years, powdery mildew disease was first identified in the Pacific Northwest growing regions of USA in 1997. Downy mildew breeding has not progressed significantly over the past half century due to the highly variable nature of disease expression in the hop plant. The increased costs associated with controlling these two diseases threaten the economic viability of the entire U.S. hop industry. Presently, the only way to control powdery and downy mildew is by growing of resistant varieties or applying expensive fungicides as many as eight times per season. The costs of these applications can easily exceed the gross receipts for the crop. When powdery mildew first emerged in U.S. hop production fields, only a few resistant varieties were in production and we had no understanding of genetic mechanisms that would contribute to disease resistance. Furthermore, we know even less about the epidemiology of, and genetic mechanisms for resistance to, downy mildew in hop. Without significant and rapid progress in developing economic means to control the disease, U.S. hop production cannot remain economic and competitive in global hop markets, farm profitability will severely decline, and reliance of brewers on foreign hop imports will increase. At the same time, it also is important to identify specific compounds in hop cones that contribute superior flavor and to develop superior varieties that produce enhanced levels of these compounds for commercial production. The objectives of this research are:. 1)Identify, sequence, map and characterize hop genes that confer specific phenotypic functions of economic importance.. 2)Characterize genetic diversity of hop accessions maintained in the USDA hop germplasm collection.. 3)Identify and develop hop germplasm possessing new and unique traits that have economic impact on the American hop industry. To solve these problems, both cultivated and wild hop germplasm are being evaluated for their potential use in development of new varieties, traditional plant breeding is being used to incorporate traits that provide disease resistance and superior brewing characteristics into new germplasm, and molecular markers are being developed to decrease the length of time required to develop new varieties. This research addresses objectives of National Programs #301, Plant, Microbial and Insect Germplasm, Conservation and Development-Germplasm Enhancement and Manipulation, and National Program #303, Plant Disease Host Plant Resistance to Disease. This work is relevant to U.S. breweries, hop marketing companies and hop producers and critical to maintain U.S. leadership in hop production and export. 2.List the milestones (indicators of progress) from your Project Plan. Year 1 (FY2003) Make Genomic Libraries Estimate Genetic diversity/ Publish results Make year 2003 Crosses Year 2 (FY2004) Run micro-satellites/Take field data-1st yr Collect second year data estimating heritability of specific traits Year 3 (FY2005) Finish sequencing genes/ analyze data for Objective 1 Finish micro-satellites/ 2nd year data collection Collect 1st year data and release superior germplasm from year 2001 crosses Year 4 (FY2006) Collect 2nd year data for Objective 2. Collect 2nd year data for Objective 3 Year 5 (FY2007) Release superior germplasm from year 2003 crosses 4a.What was the single most significant accomplishment this past year? Molecular Markers for Hop Downy Mildew Resistance Identified: USDA-ARS Forage Seed and Cereal Research Unit discovered highly significant correlations between five AFLP molecular markers and resistance to hop downy mildew in the USDA-ARS hop germplasm collection. Traditional breeding methods have resulted in limited genetic gains for hop downy mildew resistance. We utilized AFLP analysis on 100 hop accessions and scored these same accessions for downy mildew resistance over a two-year period in field plots inoculated with downy mildew. This discovery was important because all previous downy mildew scoring methods for identifying resistance involved inaccurate, laborious and expensive field trials. These molecular markers and methods, coupled with the identified genetic materials, will allow more rapid and inexpensive development of downy mildew resistant varieties and result in less need for fungicide applications, reduced input costs for producers, and enhance environmental protection. 4b.List other significant accomplishments, if any. Potential Molecular Markers for Hop Powdery Mildew Resistance Identified: The USDA-ARS Forage Seed and Cereal Research Unit and Horticulture Research Unit in Corvallis, OR identified two molecular markers for hop powdery mildew resistance acting as potential promoter regions involved in transcription under plant stress response. Hop powdery mildew is a recent invasive pathogen that is causing serious quality and production losses and a quick genetic solution involving germplasm development is required to reduce or eliminate the need for numerous fungicide applications. Differential display was used to identify unique AFLP bands presumably associated with resistance to powdery mildew and then isolated, cloned and sequenced 11 of the resulting markers. The results of this study are significant as they provide both molecular markers for selection and also address basic genetic questions on plant response to hop powdery mildew infection. Markers that prove to be linked to powdery mildew resistance will speed up and improve accuracy of selection for powdery mildew resistance and reduce the costs associated with germplasm development. The Role of Leaf Wetness and Temperature in Hop Powdery Mildew Infection: The USDA-ARS Forage Seed and Cereal Research Unit discovered that leaf wetness greatly reduces the severity of infection of hop by its powdery mildew pathogen. The environmental conditions that impact the occurrence and spread of the hop powdery mildew are poorly understood, limiting the development of accurate risk assessment systems. Controlled environment studies investigated the effect of varying durations of leaf wetness and temperature on severity of powdery mildew infection of hop, revealing that short durations of leaf wetness are sufficient to reduce infection. This finding is significant because it suggests that surface wetness, and not necessarily rain fall as currently thought, is associated with reduced disease severity in certain environments and climates. Knowledge of this previously un-quantified environmental factor should lead to better prediction of powdery mildew diseases since leaf wetness is not considered in current risk assessment models, and result in less need for fungicide applications, reduced input costs for producers, and enhanced environmental protection. Long-Term Retention of Insensitivity to Fungicides in Downy Mildew Populations: The USDA-ARS Forage Seed and Cereal Research Unit in collaboration with Washington State University discovered that insensitivity to certain fungicides has persisted in the downy mildew pathogen populations for nearly 20 years in the near absence of their use. Hop downy mildew management relies heavily on chemical inputs, but indiscriminate use of fungicides has resulted in the rapid development of pathogen resistance. Laboratory evaluations of hop downy mildew isolates from the Pacific Northwest measured pathogen sensitivity to certain fungicides, revealing that insensitive phenotypes are present at a high frequency within the population. This discovery will allow for the elimination of chemical controls to which the pathogen remains insensitive, and result in optimized fungicide use, reduced input costs for producers, and enhanced environmental protection. 4c.List any significant activities that support special target populations. None 4d.Progress report. None 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. AFLP Fingerprinting and estimation of genetic diversity of the USDA-ARS germplasm pool has allowed the identification of specific genetically-diverse male and female parental pairs for heterotic crosses. Through the process of identifying genetically diverse parents, we determined that a significant genetic bottleneck exists in the hop varieties grown in the USA. We analyzed the AFLP fingerprints of 54 recently collected wild North American hop accessions and determined their relationship to advanced lines present in the USDA-ARS germplasm collection. Use of the genetic diversity information will allow for better decisions regarding choice of parents for future germplasm development work. Differential display analysis was used to identify several molecular markers for PM resistance. Additional work using AFLP analysis among seven near-isogenic hop accessions (bred for the presence of the seven different R-genes for powdery mildew) revealed 24 different molecular markers. Too date, several of these markers have been determined as coding for promoter regions involved in transcription regulation during plant stresses such as response to disease infection. In work focused upon downy mildew resistance, we identified five AFLP molecular markers that are significantly linked to resistance to downy mildew in hop. This work represents the first time a relationship between specific molecular markers and downy mildew resistance in hop has been demonstrated. All of the work identifying molecular markers for disease resistance will enable breeders to utilized marker assisted selection for disease resistance—speeding up germplasm development and reducing costs associated with breeding and for the grower. We released the hop varieties ‘Newport’, ‘Santiam’, ‘Sterling’, ‘Horizon’, ‘Bittergold’ and ‘Teamaker’. Newport is a replacement for the old USDA variety called ‘Galena’ and it possesses superior levels of bittering acid, higher yields and, more importantly, resistance to both powdery and downy mildews (Galena is susceptible to both diseases). Sterling is a replacement for the German variety, ‘Perle’. Superior, higher-yielding, disease resistant public varieties enables growers to reduce costs for production and ultimately allows brewers to produce a less expensive, more competitive product. The hop powdery mildew resistance genes from three different sources of resistance were incorporated into single genetic background via traditional breeding methods. This germplasm development step will hopefully result in long-term resistance as the pathogen must overcome three resistance genes rather than one. In collaboration with Dr. Elizabeth Seignor (Located in Huell, Germany) we successfully inserted the chitinase gene isolated and identified by our lab in 1999. This gene is thought to provide broad-based resistance to fungal pathogens and is being used as a model system for future genetic modification work. An additional gene from hops was identified and cloned (beta-glucanase) but has not yet been inserted into hop lines. With the recent hiring of the USDA-ARS hop pathologist we have now discovered that leaf wetness greatly reduces the severity of infection of hop by its powdery mildew pathogen. This information greatly enhances our understanding of the powdery mildew pathogen and will be used to further develop a hop powdery mildew infection model. Finally, in collaboration with Washington State University we have discovered that insensitivity to certain fungicides has persisted in downy mildew pathogen populations for nearly 20 years in the near absence of their use—underscoring the importance of judicious use of fungicides and the need for multi-tactic strategies for long-term management of the disease. . 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? 1) Hop research program tours were presented to representatives of Anheuser Busch and Miller Brewing Companies, Hop Growers of America and individual hop producers that included discussions about the integrated USDA-ARS greenhouse, field, chemistry analysis and molecular biology research approach in Oregon. 2) Participated in field tours for the Washington Hop Commission and Oregon Hop Commission and presented a lecture on hop production to students at Oregon State University in the Introductory Brewing Science course. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Gave oral or poster presentations to the following organizations: -Hop Research Council (January, 2005 and August, 2005) -Oregon Hop Research Council (July 2005) -Washington Hop Commission Scientific Meeting (December, 2004) Review Publications Hummer, K.E., Henning, J.A. 2005. Proceedings of the first international humulus symposium. Acta Horticulturae.668. Leuven, Belgium:ISHS. p.262. Bassil, N.V., Gilmore, B.S., Oliphant, J.M., Henning, J.A., Hummer, K.E. 2005. Genbank-derived microsatellite markers in hops. Acta Horticulturae. 668:47-52. Townsend, M.S., Henning, J.A. 2005. Potential Heteroitc groups in hop as determined by aflp analysis. Crop Science. v.45. p. 1901-1907. Danilova, T.V., Henning, J.A. 2005. Sex molecular markers reveal differences of y-chromosome molecular structure among hops of european and american origin. Acta Horticulturae. (ISHS) 668:85-92.