Location: Foreign Disease-Weed Science Research
2022 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
New information increased our understanding of the genus Phytopythium, a group of foreign plant pathogenic oomycetes. Using a collection of 781 samples collected from Vietnam in 2018, approximately 150 isolates were identified at the individual species level. Although at the time of collection the samples were thought to be isolates of Phytophthora based on their physical appearance, many of the sampled oomycetes were identified as Phytopythium, a relatively newly described genus of plant pathogens that causes root rots on many different crops. We characterized Phytopythium isolated from citrus by morphology, determined their optimal growth temperatures and conducted pathogenicity tests on citrus fruit. The two most frequently isolated species, P. helicoides and P. vexans, have both been recently described in the literature to cause root rots and gummosis on citrus in Tunisia, Thailand, and China. By improving our understanding of the conditions under which these pathogens thrive, we hope to be able to identify U.S. production areas at highest risk of incursion of P. helicoides and P. vexans. Methods for risk assessment of hybrid Phytophthoras were refined. To date, 30 isolates of Phytophthora spp. have been screened for their sensitivity to mefenoxam and fluopicolide, oomycete-specific fungicides to utilize the fungicides as a selective factor in confirming successful zoospore fusion. Two P. cactorum and four P. nicotianae isolates have been identified as resistant to mefenoxam, and one P. plurovora isolate has been identified as resistant to fluopicolide. In planned hybridization experiments, Phytophthora spp. with differential sensitivity will be combined in equal amounts and environmental parameters will be adjusted. Any individual that successfully grows on dual-fungicide media will be used in downstream hybridization confirmation analyses. We completed the development and validation of an assay to detect hybrids using parental isolates and known hybrids. Development and refinement of current methods for filtration and concentration of Phytophthora ramorum propagules in spiked nursery water samples continued during the winter of 2021 into spring 2022. Antibodies reactive against P. ramorum were tested with water samples containing infectious propagules of P. ramorum, and immunoassay diagnostic protocols for nursery water samples were refined. In preparation for the Winter 2022 sampling season, an open retention pond was again constructed using existing facilities, capturing runoff from an adjacent plot infested with Phytophthora ramorum from research activity. The retention pond was set up in Dec 2021 and infested with P. ramorum-inoculated leaves in bags with copious irrigation, draining into the mock retention pond; new inoculum was added monthly. 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, and mid-June 2022, with a break due to the pandemic. Filtration and sampling were conducted for diagnostic assays on multiple instrument platforms. Flocculation with aluminum sulfate plus ferric 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. The combination of ferric and aluminum salt flocculates successfully recovered P. ramorum zoospores from retention pond water. Preliminary trials applying a recombinase polymerase diagnostic assay (RPA) specific for P. ramorum were also successful, and further RPA trials will continue into 2023. Monitoring activities included biweekly conference calls and frequent emails, as well as sharing of research results. Planned travel in April and May 2022 was cancelled due to USDA COVID-19 travel restrictions. Research findings were presented virtually to university, industry, state and federal government stakeholders in March 2022.
This is the final report for the Project 8064-22000-045-000D. Over the course of the project, 13 peer-reviewed scientific publications were written on oomycete pathogens. A Trichoderma species was discovered and patented for use to reduce levels of the sudden oak death pathogen in infested nursery soil, aiding in remediation of infested nurseries and allow such nurseries to resume interstate trade. Rapid water sampling methods were developed for capture and assay of P. ramorum propagules in nursery water sources using ELISA with monoclonal antibodies or immunofluorescence with polyclonal antibodies, improving rapid identification of the pathogen in nursery irrigation water and aiding in remediation of infested nurseries. Epidemiological parameters of Phytophthora ramorum were evaluated to understand the dynamics of forest and nursery epidemics; significantly larger numbers of spores were produced during dark periods compared with light periods, a phenomenon not previously reported for P. ramorum. To further our understanding of downy mildews, serious plant pathogens whose life cycle and epidemiology are poorly understood, oospores of Impatiens downy mildew (P. destructor) were produced in detached stem culture & induced to germinate for the first time; mating behavior was discovered to be homothallic. Seedlings could be systemically infected via roots using sporangia or oospores. These findings helped confirm that impatiens downy mildew can overwinter in soil and oospores can lead to fresh infections; these findings were directed to Extension scientists. A downy mildew “Citizen Science project” connected research activities to the general public.
Accomplishments
Review Publications
Crouch, J.A., Davis, W.J., Shishkoff, N., Castroagudin, V.L., Martin, F.N., Michelmore, R., Thines, M. 2022. Peronosporaceae species causing downy mildew diseases of Poaceae, including nomenclature revisions and diagnostic resources. Fungal Systematics and Evolution. 9(1):43-86. https://doi.org/10.3114/fuse.2022.09.05.
Barlow, W.B., Pfeufer, E. 2021. Small scale fungicide delivery equipment to manage early blight in three types of tomato. Plant Health Progress. 23(1):45-48. https://doi.org/10.1094/PHP-06-21-0091-RS.
