Location: Foreign Disease-Weed Science Research
2021 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. Project 8044-22000-045-00D
Under Objective 1, Sub-objective 1.A.: 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. 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.1.: Approximately 200 tropical Phytophthora isolates are in varying states of revival from storage from the 781-isolate, 2018 collection from Vietnam (assembled by a former Foreign Disease-Weed Science Research Unit ARS scientist). Vietnam has been suggested as one potential center of origin of P. ramorum and numerous highly diverse, naturally occurring populations have been shown to be present. Vietnam is the likely origination point for P. ramorum, a pathogen that has severely disrupted natural ecosystems in the Western U.S. and resulted in significant trade complications in ornamental crop nurseries, both domestic and international. To assess the risk other new Phytophthora pose from the same region, 55 isolates have been fully revived and/or re-purified so far. DNA extractions from these individuals are being completed in order to classify them within Phytophthora. A workflow for identifying tropical Phytophthora to clade (group of species), species, putative new species, or determining hybrid status has been designed with input from collaborators. Briefly, this entails identification to clade (species group) using Internal Transcribed Spacers (ITS) sequence, identification at the sub-clade level using sequence of 3-4 housekeeping genes, hypothetical assignment to species, new species, or hybrid based on those genetic sequences, then confirmation or refutation of the hypothesis using next-generation sequencing of an additional two genes. Developing and documenting this workflow has the potential to change the way researchers evaluate highly diverse pathogen populations from potential centers of origin.
Under Objective 3, Sub-objective 3.B.2.: Phytophthora citricola, P. cryptogea, and P. cactorum isolates have been acquired and grown, with DNA extracted and evaluated for suitability in the hybrid detection protocol. P. cactorum in particular has been shown to be a parent to two different, economically influential Phytophthora hybrids, while P. cryptogea has also been shown to readily hybridize with closely related Phytophthora. P. citricola is an important pathogen causing disease in citrus, woody forest species, and avocado, among others. Methods are being tested to induce sporulation in these isolates so fusion experiments can begin. High resolution melt qPCR was chosen as the hybrid identification method. Briefly, this method has been shown to discriminate among parent and hybrid species by evaluating critical genetic sites that are known to be intermixed when certain Phytophthora parents hybridize. Recently published protocols are available for P. cactorum detection, while new protocols will be developed for other parent lines. Bio-Rad high resolution melt qPCR software and adapters have been purchased and fitted to existing equipment. Team members have software licenses and are developing proficiencies. Development of these protocols will broaden the utility of the high resolution melt workflow to more end-users.
B. This is a progress report for Project 8044-22000-045-03S; (Non-Assistance Cooperative Agreement No. 58-8044-8-003; April 4, 2021 to April 4 2026. “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, California (NORS-DUC). Work on this project continued at NORS-DUC during winter 2020 into to spring 2021 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 2020-21 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 December 2020 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. 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. Water was sampled every two weeks January- mid March and mid-May- mid June 2021, 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, ferric sulfate, and bentonite clay were also tested for rapid recovery of P. ramorum propagules. Monitoring activities included weekly conference calls and frequent emails, as well as sharing of research results. Research findings were presented to university, industry, state and federal government NORS-DUC stakeholders in March 2021 during the virtual NORS-DUC Steering Committee Meeting.
Accomplishments
1. Rapid detection of Sudden Oak Death in nursery irrigation water. Phytophthora ramorum, causal agent of sudden oak death, continues to threaten U.S. forest ecosystems and the nursery industry. Rapid methods are needed for recovery and detection of P. ramorum propagules from water sources, as current methods can take 8-10 days to complete. Working at the USDA-funded National Ornamental Research Site at Dominican University of California (NORS-DUC), we are developing methods for rapid recovery and detection of P. ramorum from nursery irrigation water sources. Results with spiked samples taken from a mock nursery irrigation pond at NORS-DUC indicate that the pathogen can be detected in 24 h or less in nursery water samples, significantly reducing the time for diagnostic labs to report results to regulatory agencies. Reducing the time frame from sampling to detection will allow regulatory agencies to quickly identify infested nursery sites and decrease the risk of shipping P. ramorum -infected material out of quarantine zones.
2. Plant pathogens found in non-host plants. The movement of plants is considered to be the main avenue for introduction of invasive plant pathogens. Regulatory agencies try to reduce the risk of introductions by conducting import inspections, but limited personnel and the high volumes of imported plants make this job difficult. Port inspectors are trained to identify known plant pathogen-host associations, but in some cases plant pathogens may infect plants that are not known hosts without showing disease symptoms. We surveyed Rhododendron plants in native stands in the Eastern U.S. and cultured microbes from the native plants, using molecular sequencing methods to identify the microbes. Of the identified fungal microbes cultured from rhododendron plants, approximately 22% were reported to be pathogens of other host plants. Applying standard plant pathology methods, we found that two of the fungi that we recovered were pathogenic on other host plants, further demonstrating that plant pathogens living within non-host plants could escape detection and be transported to new locations where their host is present. This study highlights that plant import inspection methods may not be adequate to detect the importation and spread of new invasive plant pathogenic species.
