Location: Foreign Disease-Weed Science Research
2019 Annual Report
Objectives
Objective 1: Develop immuno-diagnostic assays for detection of Phytophthora ramorum and other emerging Phytophthora species in host plants as well as nursery irrigation and aquatic environmental samples. [NP303, C1, PS1]
Subobjective 1A: Develop a molecular detection assay for Phytophthora ramorum based on cell wall-specific proteins.
Objective 2: Develop formulations of antagonistic microorganisms including Trichoderma spp. that can be used as a management tool to reduce soil and leaf populations of Phytophthora ramorum and other emerging Phytophthora species. [NP303, C2, PS2]
Subobjective 2A. Investigate the use of biocontrol agents against selected Phytophthora species, seek to enhance their effectiveness, and identify new biocontrol agents.
Objective 3: Characterize the biology and epidemiology of emerging plant diseases caused by oomycetes such as Plasmopara obducens (Impatiens downy mildew) as the basis for improved disease management strategies. [NP303, C2, PS2C]
Subobjective 3A: Determine key characteristics of epidemiology of emerging oomycete plant pathogens.
Subobjective 3B: Determine the risk potential posed by exotic oomycete species to US agricultural plant species.
Approach
Using specialized containment facilities, we will obtain data in key research areas to assist in detecting and managing emerging oomycete pathogens including Phytophthora ramorum, P. kernoviae, and Plasmopara obducens. For pathogen detection, our approach is to develop enzyme-linked immunosorbent and lateral flow device immunoassays for detection of P. ramorum in plant products and nursery irrigation and runoff samples. We will also continue to develop formulations of antagonistic microorganisms including Trichoderma sp. that can be used as a management tool to reduce soil and leaf populations of Phytophthora ramorum and other emerging Phytophthora species, and will seek to identify new biocontrol agents. To elucidate key characteristics of epidemiology of emerging oomycete pathogens, we will use a variety of experimental approaches in specialized laboratory and greenhouse facilities. We will determine the nature of systemic infection of Impatiens sp. by Plasmopara obducens, and whether the pathogen is seed-borne. We will also define parameters for sporulation and survival of P. ramorum on a key host plant species, and improve Phytophthora kernoviae detection in soil using plant leaves as bait. Understanding key features of biology, epidemiology, and detection will contribute to development of improved management practices and recommendations. Our results will also provide a scientific basis for development of nursery industry best management practices aimed at minimizing disease outbreaks and enhancing interstate commerce.
Progress Report
A. Our objectives fall under National Program 303 Component 1: Etiology, Identification, Genomics and Systematics; Problem Statement 1: Diagnostics, Etiology, Genomics and Systematics of Plant Disease and Associated Microbes and Component 2: Biology, Ecology, and Genetics of Plant Pathogens and Plant-Associated Microbes; Problem Statement 2C: Ecology and Epidemiology of Plant Diseases. Under Objective 1, Sub-objective 1A of our project plan, we applied a proteomic approach to identify proteins secreted by the Phytophthora ramorum, and identified extracellular proteins which will serve as antigens for generation of antibodies. The antibodies will be used in the development of immunoassays to detect P. ramorum in nursery irrigation and surface water samples. To date, rabbit polyclonal and mouse monoclonal antibodies have been generated and are in testing in our laboratory for specificity and sensitivity. Antibodies were tested against 14 domestic and foreign isolates of P. ramorum and three other closely related Phytophthora species to date, exhibiting specificity for P. ramorum and moderate sensitivity in detecting propagules of the pathogen. Under Objective 3, Subobjective 3A, we determined the temperature range at which isolates of foreign pathogen Phytophthora kernoviae from 3 different geographic regions (United Kingdom, New Zealand, and Chile) can infect its host, Rhododendron ponticum. Pathogenicity expressed in terms of lesion development on inoculated, detached leaves occurred consistently at 20 and 21°C, was limited at 22 °C, and did not occur at 23-25°C. In replicated experiments, isolates from New Zealand showed significantly lower average lesion sizes over the range of temperatures 20-25°C compared with isolates from the United Kingdom and Chile. Also, under Objective 3, Subobjective 3A, significant progress was made in understanding the life cycle of Impatiens downy mildew (IDM). Downy mildew is a disease that infects Impatiens plants, a crop with a wholesale value of more than $130 million. Beginning in 2009, outbreaks were reported in New York and Florida; by 2013, IDM was identified in 37 states. Previously our lab had shown that sporangia of the pathogen could infect Impatiens roots and cause systemic infection that eventually travelled up the stem and into leaves, where airborne spores were produced. We have now shown that the same thing will happen if roots are inoculated with cold-conditioned oospores. This provides further evidence that oospores are the overwintering stage in the disease cycle, and therefore, that if soil is infested with the pathogen the host should not be planted the next year.
B. This is the final report for Project 8044-22000-045-02N; (Non-Funded Cooperative Agreement #58-8044-8-001N) which terminated Jan 31, 2019; “Immunoassay for Phytophthora ramorum in Nursery Water Samples”, with Dominican University of California, at the National Ornamental Research Site at Dominican University of California (NORS-DUC), in San Rafael, California.
The specific objectives of this research were to develop or refine current methods for filtration and concentration of Phytophthora ramorum propagules in spiked nursery water samples; test antibodies reactive against P. ramorum with water samples containing infectious propagules of P. ramorum, and refine immunoassay diagnostic protocols for nursery water samples. Progress was made on all objectives, which fall under National Program 303, Plant Diseases. A mock nursery irrigation retention pond was constructed in November 2017 by NORS-DUC staff capturing runoff from an adjacent plot infested with P. ramorum-inoculated leaves in bags with copious irrigation, draining into the mock retention pond. New inoculum was added monthly by NORS-DUC staff. The objective was to generate P. ramorum propagules and flush them into the new retention pond. Water from the runoff pond spiked with P. ramorum propagules was then used to test microfiltration protocols by the Principal Investigator and NORS-DUC staff during visits throughout the winter of 2017-18. Laboratory trials were conducted at NORS-DUC to trap zoospores from 1.0 L batches of retention pond water spiked with zoospores. Samples were pre-filtered through mesh macro-filters to clarify the sample and remove silt and debris, followed by filtration through membrane filters. After an overnight incubation to encourage zoospore encystment and germination, filters were extracted in commercial buffer bags using buffers compatible with immunoassay protocols, and extracts were then plated for colorimetric immunoassays. Bait bag sampling by NORS-DUC staff in the mock retention pond was positive for P. ramorum in January and February 2018 with other Phytophthora spp. also present. Immunoassays using our anti- P. ramorum polyclonal antibody on filter extracts from spiked zoospore samples were positive in samples from January through March 2018, indicating successful recovery of P. ramorum propagules on filters. Monitoring activities included weekly conference calls and frequent emails, as well as sharing of research results. In addition, meetings were held when the Principal Investigator visited the Cooperator on site at Dominican University in December 2017, January, February and June 2018. Research findings were presented to university, industry, state and federal government NORS-DUC stakeholders in March 2018.
C. This is a progress report for Project 8044-22000-045-03S; (Non-Assistance Cooperative Agreement #58-8044-8-003; July 1 2018- June 30 2019); Immunoassay for Phytophthora ramorum in Nursery Water Samples, with Dominican University of California, at the National Ornamental Research Site at Dominican University of California (NORS-DUC), in San Rafael, California.
This project continued work at NORS-DUC on the same objectives as NFCA #58-8044-8-001N, under this NACA. The specific objectives of this research were to develop or refine current methods for filtration and concentration of Phytophthora ramorum propagules in spiked nursery water samples; test antibodies reactive against P. ramorum with water samples containing infectious propagules of P. ramorum, and refine immunoassay diagnostic protocols for nursery water samples.
Progress was made on all objectives, which fall under National Program 303, Plant Diseases. A mock nursery irrigation retention pond was constructed in December 2018 by NORS-DUC staff, capturing runoff from an adjacent plot infested with P. ramorum-inoculated leaves in bags with copious irrigation, draining into the mock retention pond. New inoculum was added monthly by NORS-DUC staff. The objective was to generate P. ramorum propagules and flush them into the new retention pond. Water from the runoff pond spiked with P. ramorum propagules was then used to test microfiltration protocols by the PI and NORS-DUC staff during visits throughout the winter of 2018-19. Laboratory trials were conducted at NORS-DUC to trap zoospores from 1.0 L batches of retention pond water. Filters were extracted in commercial buffer extraction bags for colorimetric immunoassays. Bait bag sampling by NORS-DUC staff in the mock retention pond was positive for P. ramorum in January through April 2019. Immunoassays using our anti- P. ramorum polyclonal antibody on filter extracts from samples were positive, indicating successful recovery and detection of P. ramorum propagules on filters.
Monitoring activities included weekly conference calls and frequent emails, as well as sharing of research results. In addition, meetings were held when the Principal Investigator visited the Cooperator on site at Dominican University in February 2019 (planned travel in January 2019 was cancelled due to the government shutdown). Research findings were presented to university, industry, state and federal government NORS-DUC stakeholders, in March, 2019, and will be presented as a poster at the 2019 Sudden Oak Death 7 Meetings in San Francisco, California, in June 2019.
Accomplishments
Review Publications
Widmer, T.L. 2019. Compatibility of Trichoderma asperellum isolates to selected soil fungicides. Crop Protection. 120:91-96. https://doi.org/10.1016/j.cropro.2019.02.017.
Shishkoff, N. 2019. The oospore stage of Plasmopara obducens. Mycologia. 111:632-646. https://doi.org/10.1080/00275514.2019.1601986.
Tooley, P.W., Browning, M.E. 2019. The effect of moisture on Phytophthora ramorum zoospore infection of Rhododendron ‘Cunningham’s White’ and Viburnum tinus. Plant Disease. https://doi.org/10.1094/PDIS-09-18-1681-RE.
Kandel, S.L., Mou, B., Shishkoff, N., Shi, A., Subbarao, K.V., Klosterman, S.J. 2019. Spinach downy mildew: Advances in our understanding of the disease cycle and prospects for disease management. Plant Disease. 103:791-803. https://doi.org/10.1094/PDIS-10-18-1720-FE.
