Location: Forage Seed and Cereal Research2015 Annual Report
1a. Objectives (from AD-416):
Objective 1: Identify new genetic markers for selecting resistant hop germplasm to downy and powdery mildews. Objective 2: Develop new disease resistant germplasm for public release. Objective 3: Construct optimized integrated management approaches for powdery mildew susceptible cultivars. Sub-objective 3.A: Describe the ontology of crown bud development, susceptibility of crown buds to powdery mildew in different developmental stages, and dynamics of flag shoot emergence. Sub-objective 3.B: Develop a PCR assay to rapidly identify mating type in P. macularis; determine prevalence of mating types among isolates of P. macularis in the Pacific Northwest. Objective 4: Develop and apply genotyping approaches to assess the diversity, geneticdifferentiation, and sexual recombination in the downy mildew and powdery mildew pathogens. Sub-objective 4.A: Identify and develop simple sequence repeat markers in Pseudoperonospora humuli and elucidate the degree of diversity, selfing, and population differentiation within and among population at multiple hierarchical scales. Sub-objective 4.B: Identify polymorphic loci among isolates of Podosphaera macularis and characterize the genetic diversity, population structure, and relatedness of the population in the Pacific Northwest U.S. to other populations of the pathogen in the world.
1b. Approach (from AD-416):
Objective 1: Identify molecular markers associated with plant resistance to P. humuli and P. macularis. Progeny developed from crosses of resistant and susceptible parents will be screened by pathogen challenge, and single nucleotide polymorphic (SNP) marker identification and genotyping-by-sequencing will be performed. Objective 2: Development of multiple pathogen resistant germplasm or varieties. Progeny of crosses of parental material that possess relative resistance to powdery and downy mildews will be successively challenged with each disease to identify germplasm with enhanced resistance to both. Resistant germplasm that possess excellent agronomic and brewing characteristics will be released. Objective 3: Construct optimized integrated management approaches for powdery mildew susceptible cultivars. Hypothesis 3.A: Successful perennation of the powdery mildew fungus occurs via infection of juvenile crown buds and such crown buds develop asynchronously. Management factors that reduce late season severity of powdery mildew will reduce overwintering survival of the pathogen. Crown bud development phenology will be determined and plants will be challenged with powdery mildew at selected stages. Treatment at different stages will be evaluated. Hypothesis 3.B: The absence of the ascigerious stage of Podosphaera macularis in the Pacific Northwestern U.S. is due to the absence of one of requisite mating types of the fungus. PCR amplification of conserved regions in MAT1-1 and MAT1-2 loci of P. macularis will be optimized and pathogen isolates collected from a variety of cultivars and hop yards in the Pacific Northwest will be tested to determine the frequency of each mating type. Objective 4: Develop and apply genotyping approaches. Hypothesis 4.A: P. humuli is heterothallic, possessing a high degree of genetic diversity in the Pacific Northwestern U.S, and the population is structured at the scale of individual fields. P. humuli isolates will be obtained from three hop yards in western Oregon. After suitable SSR loci and primers are developed from pyrosequences, genotyping will be performed by capillary sequencing of 7 to 10 SSR loci per isolate. Hypothesis 4.B: The population of Podosphaera macularis in the Pacific Northwestern U.S. exhibits a low degree of genetic diversity or structure based on geography or cultivar host. Multiple nuclear loci will be identified, PCR-amplified, and sequenced from Pacific Northwest, northeastern U.S., and European isolates. Population genetic parameters will be calculated and differentiation among geographic populations will be estimated.
3. Progress Report:
Disease response and genetic characterization of downy mildew resistance in the population resulting from ‘Teamaker’ x USDA 21422M was completed, as was the disease response and genetic characterization of resistance to powdery mildew in the population resulting from ‘Newport’ x USDA 21110M as proposed in Objective 1. USDA-ARS scientists identified and made publically available genetic markers linked to resistance to downy mildew in hop. USDA-ARS scientists also characterized disease resistance to downy mildew in a diverse collection of 116 hop cultivars housed in the USDA-ARS germplasm collection. These same cultivars were sequenced and genetic markers related to disease reaction were identified. In support of Objective 2, approximately 650 female selections from crosses designed to combine resistance to powdery and downy mildews were transplanted to on-farm plots for further evaluation under normal growing conditions. First year validation of disease resistance as well as plant vigor under field conditions (grown as single plants per plot) was completed. In addition, new crosses were made to address the recent emergence of a strain of powdery mildew into the Pacific Northwestern U.S. that is capable of infecting formerly resistant cultivars. Seed from these crosses were germinated, grown out in a greenhouse, and inoculated with multiple strains of the powdery mildew and downy mildew pathogens. Disease susceptible offspring were culled from this breeding nursery and the resulting offspring (approximately 2400) were evaluated in a breeding nursery. Further selection in this nursery for disease resistance, plant vigor and whether or not they are female or male plants is ongoing. Current DNA fingerprinting methods for hop utilize outdated technology that is both time consuming and expensive. Studies were completed to identify a minimum set of genetic markers needed to DNA fingerprint hop cultivars. USDA-ARS scientists devised a quick, inexpensive technique to help identify unknown hop cultivars using modern technologies. In collaboration with Washington State University, progress was made on understanding how the powdery mildew fungus overwinters (Objective 1, sub-objective 3A). Results of infection studies suggest that bud infection, leading to pathogen perennation, may occur over an extended period of time, but is most likely during juvenile stages of bud development. Emergence of the pathogen during the ensuing season was linked to host growth. Analysis of historical data collected since 2000 identified that prior to overwintering of the pathogen, certain cultural practices such as pruning method, and winter temperature were associated with the occurrence and frequency of survival of the pathogen. Field experiments were designed and implemented (in collaboration with Washington State University and cooperating producers) to quantify the association of disease control measures on ability of the pathogen to overwinter. Results from these experiments have been inconclusive given the low level of overwintering of the pathogen. Further studies are underway to link late season disease severity to the likelihood of pathogen survival. In collaboration with Oregon State University, studies were initiated to determine how the frequency and location of overwintering of the powdery mildew pathogen influences disease development at the landscape level. We discovered that in Oregon, landscape-level outbreaks of powdery mildew affecting two-thirds of hop yards were associated with survival of the pathogen in less than 5% of hop yards in April and May. Efforts are underway to model risk factors for regional disease spread and development, laying the foundation for an area-wide management approach for the disease. A three-year study, in collaboration with Washington State University, to quantify the prevalence and incidence of powdery mildew on cultivars possessing a resistance factor called R6, which formerly provided high levels of resistance to powdery mildew, was completed. The distribution of powdery mildew on these cultivars was quantified, and found to be widespread and increasing in severity. Differences in susceptibility among cultivars possessing R6 also were identified. Characterization of the pathogen found two resistance genes remain effective against the newly emerged strains of the fungus, and this information was used to inform new crosses made during 2015. Screening of USDA germplasm was initiated to identify new sources of resistance to virulent strains of the powdery mildew pathogen; several promising sources have been identified. Studies were conducted to describe the mating system of the hop downy mildew pathogen. Results of this work indicates that most or all strains of the pathogen are capable of producing survival spores in large numbers. However, the survival spores appear to have a conditioning requirement before being infective. This finding advances our understanding of pathogen survival and may have implications for disease management. Isolates of the powdery mildew fungus continue to be collected, increased, and preserved to support future population genetic diversity analyses in support of project plant objectives. Progress was made in developing methods to extract DNA and RNA of sufficient quality for sequencing for both the powdery mildew and downy mildew pathogens. A study with scientists at Cornell University was conducted to compare the fidelity and suitability of RNA and DNA-based methods for discovery of genetic markers in the downy mildew pathogen. Based on this work, a strategy for assessing genetic diversity of the downy mildew pathogen has been established. Simple assays to identify and differentiate the hop downy mildew pathogen from the closely related cucurbit downy mildew pathogen were developed and made publically available. Identification of genes underlying host range of the cucurbit and hop downy mildew pathogens is underway with collaborating scientists at the North Carolina State University.
Henning, J.A., Gent, D.H., Twomey, M., Townsend, M.S., Pitra, N., Matthews, P. 2015. Precision QTL mapping of downy mildew resistance in Hop (Humulus lupulus L.). Euphytica. 202(3):487-498.