Location: Foreign Disease-Weed Science Research
2020 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
Under Objective 1, Sub-objective 1.A.1: we identified extracellular protein candidates to serve as antigens for generation of antibodies to use 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 have been tested for specificity and sensitivity in our laboratory, exhibiting specificity for P. ramorum and moderate sensitivity in detecting propagules of the pathogen in leaf and filtered irrigation water samples. The Animal and Plant Health Inspection Service (APHIS) staff have not expressed interest in moving forward with formal validation of the antibody-based assays at this time.
Under Objective 3: Sub-objective 3.B: we evaluated the methods for identifying Phytophthora hybrids using molecular techniques from twenty one peer-reviewed publications and chose the most likely ones to be useful for us in creating polyploidy zoospores.
This is a progress report for Project 8044-22000-045-03S; (Non-Assistance Cooperative Agreement 58-8044-8-003; July 1 2018- June 30, 2021); “Immunoassay for Phytophthora ramorum in Nursery Water Samples”, with Dominican University of California, at the National Ornamental Research Site at Dominican University of California, in San Rafael CA (NORS-DUC).
Work on this project continued at NORS-DUC during the winter 2019 into to spring 2020 under the objectives 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. In preparation for the winter 2020 sampling season, an open retention pond was again constructed by NORS-DUC staff using existing facilities, capturing runoff from an adjacent plot infested with Phytophthora ramorum from research activity. The retention pond was set up in Dec 2019 by NORS-DUC staff and 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 again generate P. ramorum propagules and flush them into the retention pond, simulating an infested nursery situation. Water was sampled every two weeks January- mid March and mid-May- mid June 2020, with a break during the Dominican University closure due to the pandemic. Filtration and sampling were conducted for diagnostic assays on multiple instrument platforms. Flocculation with aluminum sulfate was also tested for rapid recovery of P. ramorum propagules. Baiting of the retention pond was conducted using mesh bags containing rhododendron leaves, plated on semi-selective media for identification of Phytophthora spp. in the pond. Monitoring activities included weekly conference calls and frequent emails, as well as sharing of research results. In addition, meetings and site visits were held with the Cooperator at Dominican University in February and March 2020 (planned travel in April and May 2020 was cancelled due to USDA COVID-19 travel restrictions). Research findings were presented to university, industry, state and federal government NORS-DUC stakeholders in March 2020.
Accomplishments
