Location: Forage Seed and Cereal Research2014 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:
Phenotyping of downy mildew resistance in the population, resulting from ‘Teamaker’ x USDA 21422M was completed, as was phenotyping of resistance to powdery mildew in the population resulting from ‘Newport’ x USDA 21110M. DNA from samples from both mapping populations were submitted for sequencing and genotyping to identify molecular markers associated with disease resistance. ARS scientists in Corvallis, Oregon, also identified and phenotyped downy mildew resistance in diverse genotypes, including hop varieties representing world hop growing regions as well as male accessions housed in the USDA-ARS hop collection. DNA from these populations was submitted for sequencing and genotyping. In support of Objective 1, we initiated a project to sequence the hop genome using de novo sequencing of two genotypes, ‘Teamaker’ and male ‘21422M’. Initial attempts to assemble the genome have resulted in a partial map covering approximately 2/3 of the total hop genome. An additional round of sequencing is underway to gain better coverage of the genome. Crosses designed to combine resistance to powdery and downy mildews were germinated and inoculated with both diseases and selections made in the greenhouse during 2013. Further validation of disease resistance under field conditions (grown as potted plants) as well as identification of female lines for variety development was also completed. Approximately 650 female selections were cloned and transplanted into breeding nursery for agronomic and chemical assessment, which is in progress. Studies were launched to clarify overwintering of the powdery mildew fungus as proposed in the project plan. Preliminary findings indicate that bud infection leading to pathogen perennation may occur over an extended period of time and that emergence of the pathogen during the ensuing season is linked to host growth. Experiments describing the phenology of crown bud development were initiated. Field experiments were designed and implemented in Washington with University collaborators and cooperating producers to quantify the association of disease control measures in the current season on subsequent success of pathogen overwintering. A total of 317 isolates of the hop powdery mildew fungus, Podosphaera macularis, were collected in the Pacific northwestern U.S. and an additional 56 isolates were obtained from the eastern U.S. and Europe. Mating experiments were conducted with isolates from the Pacific Northwestern U.S. and from other populations, which demonstrated that P. macularis has two mating types (heterothallism) and that development and maturation of the overwintering structures proceeds similarly to other powdery mildew fungi. Viability and infectivity of overwintering spores of the fungus (ascospores) were confirmed for the first time. A shotgun sequence of the pathogen genome was obtained and assembled, allowing identification of the MAT1-1 mating type gene. Genome sequencing of another isolate of the fungus is planned to identify the full length of the MAT1-2 mating type gene. New collaborations with university researchers at Cornell and North Carolina State University were initiated to identify genetic markers suitable for population genetic studies in the hop and cucurbit downy mildew pathogens. Isolates of the hop pathogen were collected from the Pacific northwestern and northeastern U.S. and purified in culture. DNA and RNA were provided to collaborators for sequencing and genotyping. Over 150 isolates of the pathogen have been collected and preserved for later genotyping once suitable genetic markers are identified. Isolates of the powdery mildew fungus continue to be collected, increased, and preserved to support future population genetic diversity analyses. Genome sequencing, as noted above, was conducted to accelerate identification of genetic markers appropriate for measuring differentiation of populations of the fungus.
Pethybridge, S.J., Gent, D.H., Hingston, L., Frost, P. 2014. Quantifying the effects of uniconazole on growth and yield of pyrethrum in Australia. New Zealand Journal of Crop and Horticultural Science. 42(1):50-59.