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Research Project: Integrated Disease Management of Exotic and Emerging Plant Diseases of Horticultural Crops

Location: Horticultural Crops Research

2020 Annual Report

1a. Objectives (from AD-416):
Objective 1: Describe the pathogen biology and disease epidemiology of exotic and emerging plant pathogens affecting horticultural crops. Sub-objective 1A: Comparative genomics of Phytophthora pathogens. Sub-objective 1B: Population genomics and evolution of Phytophthora pathogens. Sub-objective 1C: Characterize the fungal, oomycete and bacterial microbiome associated with horticultural crops. Sub-objective 1D: Disease surveys of small fruits in the Pacific Northwest. Objective 2: Develop improved integrated disease management of pathogens of horticultural crops. Sub-objective 2A: Integrate disease risk forecasters with models for air turbulence to predict pathogen dispersal and spatially explicit disease risk. Sub-objective 2B: Develop methods to monitor presence of fungicide resistance in pathogen inoculum. Sub-objective 2C: Optimize fungicide selection and application timing to manage powdery mildew on grape berries. Sub-objective 2D: Identify inoculum sources of Botrytis cinerea in caneberry fields and evaluate methods to reduce overwintering populations. Sub-objective 2E: Develop and evaluate alternative control measures for management of diseases that reduce fruit yield or quality.

1b. Approach (from AD-416):
The long-term goal of this project is to develop the knowledge and tools needed to respond to plant disease epidemics using approaches that are economically and environmentally sustainable, with emphasis on increasing our ability to respond to exotic, emerging, and re-emerging pathogens. This will be accomplished through trans-disciplinary approaches that increase our knowledge of pathogen genetics, biology, and disease epidemiology and incorporates this information into decision support aids for horticultural crops. The biology of exotic, emerging, and re-emerging plant pathogens is either poorly understood or inadequate to enable economic and environmentally sustainable management. We will develop and test methods for monitoring pathogen dispersion and describe the genomes, evolutionary history, population structure, genetics, epidemiology, and ecology of these pathogens. This knowledge will then be used in Objective 2 to develop decision support tools for producers of horticultural crops. Once there is a more detailed understanding of pathogen ecology, this knowledge will be translated into disease management strategies that are continually optimized and/or improved to address changing climate, market and regulatory pressures. We will develop and improve disease management strategies for select pathogens affecting horticultural crops. The development and improvement of integrated disease management strategies for endemic pathogens will also improve our ability to respond to changing climatic conditions while enhancing the economic and sustainable production of horticultural crops.

3. Progress Report:
Under Sub-objective 1A, comparative genomic analyses of Phytophthora pathogens were conducted. ARS researchers compared genomes of the plant pathogen, Phytophthora infestans, and documented that populations at the center of origin in Mexico are predominantly diploid. This is significant because Phytophthora infestans clonal lineages that are dominant around the world are predominantly triploid. This work has important implications for understanding emergence of plant pathogens in agriculture. An expansion and divergence of Argonaute genes in the Oomycete genus Phytophthora were documented. This knowledge provides novel insights into the silencing machinery available in oomycetes. The whole genomes of the raspberry and strawberry pathogens Phytophthora rubi and P. fragariae were sequenced and characterized. This work provides a novel tool for effector-mediated plant breeding. For Sub-objective 1B, a population genomic study was conducted characterizing Phytophthora plurivora in Oregon nurseries on rhododendron. Overall, presence of one dominant clonal lineage was detected with a broad range of sensitivity to the fungicide mefenoxam and phosphorous acid. These findings provide the first perspective into P. plurivora population structure and phenotypic plasticity in Pacific Northwest nurseries. A population genetic analysis on a new emergence of the sudden oak death pathogen in the U.S. Midwest was shown to be caused by the NA2 clonal lineage that was previously only detected on the U.S. West Coast. Prior documented lineages obtained from P. ramorum-infected plants in Eastern receiving states have only harbored the NA1 clonal lineage. This result documents that we are seeing continued migration of the sudden oak death pathogen in the nursery industry. The population dynamics of Phytophthora rubi, the causal agent of Phytophthora root rot in raspberry, were studied. P. rubi is found in the Western United States, where most of the fresh and processed raspberries are produced. Overall, populations of P. rubi show low genetic diversity across the Western United States. These findings provide new insights into the evolution and structure of populations of the raspberry pathogen P. rubi, indicating that human activity might be involved in moving the pathogen among regions and fields. To address Sub-objective 1C, the composition of the fungal and oomycete microbiome of rhododendron roots under varying growth conditions, nurseries, and cultivars was evaluated. Few oomycetes were found and fungal communities were dominated by neutral and beneficial types. Nurseries that grew plants in containers and in-field had a significantly higher diversity of fungi than those that only grew plants in containers. Microbiome composition differed significantly among growth conditions and nurseries, but not among cultivars. This suggests that, among these cultivars of rhododendron, environment is important in structuring the root microbiome, but cultivar is not. A new technique for visualizing microbiomes was developed and implemented in the R package metacoder. The technique provides a tree-based visualization that is color coded based on significant differences among taxons and communities characterized using metabarcoding. This Heat-tree tool provides much deeper insights into microbiome data than previously available with stacked barcharts or piecharts. An unknown Phytophthora species was discovered in the central Peruvian Andes on blighted foliage of the native South American plant species Urera lacineata. Urera is a genus of native flowering shrubs in the nettle family Urticaceae. This new taxon Phytophthora urerae sp. nov. was formally described based on extensive morphological analysis, and phylogenetic analysis. This pathogen is a close relative of the potato late blight pathogen and provides novel insights into host adaptation and evolution of the potato late blight pathogen. A second year microbiome sampling of red raspberry flowers and berries in commercial fields in northern Washington was completed. Botrytis was isolated at various time points throughout the season and at harvest, and about 40% of berry samples were infected with the pathogen. Initially, most sequences obtained from samples were plant DNA; thus, we are optimizing technologies to block the amplification of plant DNA. This method should result in more comprehensive information on the yeasts, fungi, and bacteria present on red raspberry flowers and berries over the growing season. To address Sub-objective 1D, disease surveys of small fruits in the Pacific Northwest were repeated. Over 600 Botrytis isolates from commercial fields were tested for resistance to five commonly used fungicides. Approximately 60% of the isolates were resistant to boscalid, a Fungicide Resistance Action Committee (FRAC) group 7 fungicide that previously provided excellent control of the disease. Alarmingly, 37% of the boscalid-resistant isolates also were resistant to cyprodinil (FRAC group 9) and fenhexamid (FRAC group 17). Stakeholders were informed of the multifungicide-resistant Botrytis at meetings. Many growers switched from boscalid to fluopyram (FRAC group 7) and reduced the incidence of in-field gray mold. Monitoring for fungicide resistance has helped stakeholders develop management plans for gray mold in light of emergence of multifungicide-resistant Botrytis. Multifungicide-resistant Botrytis are persistent in conventionally managed fields and leave growers with fewer options for effective chemical control of gray mold. Aerial crown gall of blueberry attributed to Agrobacterium tumefaciens was shown to occur in 50 to 99% of mature blueberry plants in machine harvested fields. Infected plants had significantly reduced yields due to fewer and smaller berries on galled stems. A novel tissue culture isolation method was developed in order demonstrate Koch’s postulates and collect isolates to improve diagnostics and disease management. After 4 months in tissue culture, galls formed on the base of the plantlets and a novel Agrobacterium-like pathogen was isolated from each of the galls. We reproduced the disease symptoms on blueberry inoculated with the novel pathogen. Results from preliminary assays indicate that the bacterial pathogen is related to a recently described Rhododendron stem gall pathogen in Europe. Under Sub-objective 2A, models for describing particle flow over sparse canopies were parameterized and compared to field data. This work identified the need to better describe canopy heterogeneity in the models. These models were used to simulate impact of nearby obstacles on spore sampler detection from different scenarios for pathogen overwintering or inoculum dispersion from external sources. In Sub-objective 2B, swabbing of worker gloves after conducting routine canopy maintenance was shown to be as effective as using spore samplers for monitoring airborne inoculum at 20% of the cost. Swabbing gloves was also shown to be more sensitive and cost effective than classical visual disease scouting with the added benefit of identifying the presence of fungicide resistance markers for FRAC groups 3 and 11 fungicides. FRAC uses a number and letter system to group active ingredients will similar modes of action. An improved bioassay for determining the fungicide resistance phenotype for E. necator was shown to be suitable for examining 7 different FRAC groups. The assay can examine 14 different fungicide x concentrations for less than $100/isolate, which is significantly less than the traditional leaf bioassay. Leaf bioassays cost more than $400 to examine a single fungicide chemistry. For Sub-objective 2C, numerous fungicides were shown to be mobile by translaminar, translocation, or vapor redistribution. These data were used to design and test a disease management program that targeted mobile fungicide applications to specific crop growth stages. The experiments showed that excellent disease management was achieved with reduced fungicide and economic inputs. In addition, results and recommendation were presented through several outreach events and popular press outlets. Sub-objective 2D focuses on identifying and managing overwintering inoculum sources of Botrytis spp. in a caneberry field. Progress was delayed due to insufficient growth of many plants in the field site due to problems with the drip irrigation system, which have been corrected. For Sub-objective 2E, developing and evaluating alternative control measures for diseases that reduce fruit yield or quality, we found that specific aluminum salts are toxic to Botrytis at 1 to 10 parts per million concentrations. We are currently evaluating if aluminum salts are phytotoxic to blackberry plants. Aureobasidium pullulans are commercially available yeast-like fungi used for biological control of gray mold. The strains of A. pullulans in the commercial products are sensitive to and incompatible with fungicides used in conventional management of gray mold on small fruits. We isolated Aureobasidium from conventionally-managed caneberry fields and tested isolates for multifungicide resistance. We are currently testing the fitness of multifungicide-resistant isolates of Aureobasidium on caneberries and their potential for gray mold control under field conditions.

4. Accomplishments
1. Discovery of the NA2 clonal lineage in the Midwestern United States. The pathogen, Phytophthora ramorum, causing sudden oak death, is a threat to agronomic and natural ecosystems. The pathogen causes severe losses due to containment and management costs. ARS researchers at Corvallis, Oregon, demonstrated that a new emergence of P. ramorum in the Midwest was caused by the NA2 clonal lineage and related to the lineage from the West Coast. This research documents that there is continued movement of sudden oak death associated with interstate movement of nursery plants.

2. Phenological-timed fungicide applications for improved disease management of grape powdery mildew. Grape powdery mildew is difficult and expensive to control in the western United States and accounts for 78% of the pesticide use in grapes. In addition, fungicide resistance has been an increasing problem, making management more difficult. ARS researchers at Corvallis, Oregon, demonstrated that numerous fungicides were mobile through at least one of three different mechanisms tested. These data were then used to design disease management programs that targeted mobile fungicides to critical crop growth stages. Applying micronized sulfur every 14 days all season and timing mobile fungicides to late bloom through BB-sized berries resulted in excellent disease management. This method reduces fungicide costs to >$90/acre and uses only two non-sulfur applications per season. These data will aid in developing improved fungicide resistance and mitigation strategies.

5. Record of Any Impact of Maximized Teleworking Requirement:
Maximized telework significantly reduced our ability to meet objectives for the parent project, as some existing projects were shut-down and new experiments and projects could not be started during the pandemic. Inability to purchase specific reagents and inability to complete sequencing projects from outside facilities has hampered research progress. Limitations for staffing density in laboratories and field sites continues to detrimentally impact laboratory and time-sensitive field research. On the positive side, the maximized telework for our laboratory has allowed time and space to reorganize and move our research laboratory into another room, and to update, enter, and edit data in our extensive culture collection database. Maximized telework also significantly reduced the ability to meet objectives for several extramurally funded projects. Genomic and transcriptomic sequencing of E. necator was halted due to the inability to extract nucleic acids from collected samples due to restrictions on number of persons in the lab and inability to get sequencing done from outside contractors. Processing of ascospore, conidia, and chasmothecia samples was halted, although collection continued, because of restriction on number of personnel in the lab while laminar flow hood is in use. Field collection became more expensive because of the requirement of a single person per vehicle. This resulted in one individual needing 3 or more days to collect samples instead 2 people requiring one day. Due to the restriction on overnight travel, collection of E. necator isolates from California was not possible and there is currently limited participation from cooperating consultants and growers struggling to deal with COVID-19 restrictions in their area. In lieu of lab work, numerous safety and standard operating procedure (SOP) documents were prepared or updated/refined and manuscripts were prepared.

Review Publications
Diao, Y., Larsen, M.M., Kamvar, Z.N., Zhang, C., Wang, W., Li, D., Peng, Q., Knaus, B.J., Foster, Z.S., Grunwald, N.J., Liu, X. 2019. Genetic differentiation and clonal expansion of Chinese Botrytis cinerea populations from tomato and other crops in China. Phytopathology. 110(2):428-439.
Warneke, B., Thiessen, L., Mahaffee, W.F. 2020. Effect of fungicide mobility and application timing on the management of grape powdery mildew. Plant Disease. 104(3):1167-1174.
Kozhar, O., Larsen, M.M., Grunwald, N.J., Peever, T.L. 2020. Botrytis cinerea populations infecting small fruit hosts in the Pacific Northwest are highly structured on a local scale. Applied and Environmental Microbiology. 86(9):e02908-19.
Grunwald, N.J., Kamvar, Z.N., Everhart, S.E., Tabima, J.F., Knaus, B.J. 2019. Population genetics and genomics in R. [online]. Available:
Grunwald, N.J., Kamvar, Z.N., Foster, Z.S. 2019. Reproducible science in R. [online]. Available:
Foster, Z.S., Grunwald, N.J. 2019. Analysis of microbiome community data in R. [online]. Available:
Taylor, A.S., Knaus, B.J., Grunwald, N.J., Burgess, T. 2019. Population genetic structure and cryptic species of Plasmopara viticola in Australia. Phytopathology. 109(11):1975-1983.
Dung, J., Knaus, B.J., Fellows, H., Grunwald, N.J., Vining, K. 2019. Genetic diversity of Verticillium dahliae isolates from mint detected with genotyping by sequencing. Phytopathology. 109(11):1966-1974.
Franic, I., Prospero, S., Hartmann, M., Allan, E., Auger-Rozenberg, M., Grunwald, N.J., Kenis, M., Roques, A., Schneider, S., Sniezko, R., Williams, W., Eschen, R. 2019. Are traded forest tree seeds a potential source of nonnative pests? Ecological Applications. 29(7).
Press, C.M., Fieland, V.J., Creswell, T., Bonkowski, J., Miles, L., Grunwald, N.J. 2020. First report of the NA2 clonal lineage of Phytophthora ramorum in Indiana causing ramorum leaf blight on Rhododendron. Plant Disease. 104(6):1875.
Weisberg, A., Davis, E.W., Tabima, J., Belcher, M.S., Miller, M., Kuo, C., Lai, E., Loper, J.E., Grunwald, N.J., Putnam, M.L., Chang, J.H. 2020. Unexpected conservation and global transmission of agrobacterial virulence plasmids. Science. 368(6495).
Weiland, G.E., Scagel, C.F., Grunwald, N.J., Davis, E.A., Beck, B.R., Foster, Z.S., Fieland, V.J. 2020. Soilborne Phytophthora and Pythium diversity from rhododendron in propagation, container, and field production systems of the Pacific Northwest. Plant Disease. 104(6):1841-1850.
Foster, Z.S., Weiland, G.E., Scagel, C.F., Grunwald, N.J. 2020. The composition of the fungal and oomycete microbiome of Rhododendron roots under varying growth conditions, nurseries, and cultivars. Phytobiomes Journal. 4(2):156-164.
Miles, T.D., Neill, T.M., Colle, M., Warneke, B., Robinson, G., Stergiopoulos, I., Mahaffee, W.F. 2020. Allele-specific detection methods for QoI fungicide resistant Erysiphe necator in vineyards. Plant Disease.
Klein, J.M., Stockwell, V.O., Minsavage, G.V., Vallad, G.E., Goss, E.M., Jones, J.B. 2020. Improved deferred antagonism technique for detecting antibiosis. Letters in Applied Microbiology.