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ARS Home Northeast Area Frederick, Maryland Foreign Disease-Weed Science Research Research





- Shishkoff, Nina
- Luster, Douglas - Doug
- Puig, Alina
- Crouch, Jo Anne
- 2023
- 2022
- Publications
- Plant Diseases (303)

Research Project: Diagnostics and Biological Approaches for Management of Emerging Oomycete Plant Pathogens **Location:
                            Foreign Disease-Weed Science Research** 2023 Annual Report **Objectives**
                                

Objective 1. Develop genomic sequence resources and broad range nucleic acid and antibody-based diagnostics for novel emerging and invasive oomycete plant pathogens. (NP303, C1, PS1A, PS1B)
Sub-objective 1.A: Online barcode sequence resources will help researchers understand downy mildews (non-hypothesis driven research).
Sub-objective 1.B: Apply isothermal RPA-lateral flow assays for rapid point of care detection of Phytophthora ramorum in nursery irrigation water. (non-hypothesis driven).
Sub-objective 1.C: Development of a molecular diagnostic assay to detect Hyaloperonospora brassicae from plant material.
Objective 2: Characterize pathogen biology and epidemiology of emerging and invasive oomycete plant pathogens. (NP303, C2, PS2A, PS2C).
Sub-objective 2.A: Characterize the pathogen biology of emerging and invasive Phytophthora.
Sub-objective 2.B:  Characterize the pathogen biology of emerging and invasive downy mildews.
Sub-objective 2.C: Determine risks posed by exotic or hybrid oomycete pathogens to U.S. agriculture. **Approach**
                                

This project will discover and utilize new knowledge to develop a low-cost and efficient detection assay for the invasive oomycete Phytopthora ramorum in nursery irrigation water and inoculate plants to confirm the pathogen’s host range. Newly introduced Phytophthora species also  risk hybridizing with native Phytophthoras to create pathogens of unknown host range and pathogenicity; we propose to study the risk of hybrid development and evaluate isolates from a center of Phytophthora diversity. We will also better identify downy mildews and understand their life cycles. Hyaloperonospora brassicae, the crucifer downy mildew, is newly delimited with an unclear host range. Peronospora sparsa, a newly re-emerging disease of roses and Plasmopara destructor, invasive downy mildew of Impatiens, are poorly understood. This work capitalizes on the existing Biological Safety Level 3 Plant Pathogen Containment Facility at Fort Detrick, Maryland, required to study foreign plant pathogens and regulated plant pathogens limited to specific geographical regions within the U.S. The characterization of foreign or reemerging pathogens enables the development of robust detection methods, provides the critical information required for risk assessments and control measures, and allows for the screening of potential hosts to restrict spread. **Progress Report**
                                

Objective 1. Research continued on genomic resources and diagnostics for novel emerging and invasive oomycete plant pathogens. For downy mildews, which are serious pathogens of plants but poorly understood, a comprehensive website was created in collaboration with USDA-APHIS to support accurate identification and disease diagnoses. This freely accessible website contains barcode sequence resources, species descriptions and images to help stakeholders understand the currently known geographic range, host range and taxonomy. 
 
Sudden oak death, caused by Phytophthora ramorum, is a serious disease of forest trees. When the pathogen infects nursery plants, it can be shipped across the country, spreading the disease to new nurseries and potentially, streams and forests. Quick and inexpensive methods of detection of the pathogen in nurseries is essential to protect the nursery and forestry industries. Development and refinement of current methods to concentrate Phytophthora ramorum propagules in spiked nursery water samples continued during the winter of 2022 into spring 2023 in a plot of land simulating an infested nursery during the winter rainy season. An open retention pond captured runoff from an adjacent plot infested with Phytophthora ramorum; new inoculum was added monthly. 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 from January through mid-May 2023 for diagnostic assays. Flocculation with aluminum sulfate plus ferric sulfate salts was tested for rapid recovery and concentration of P. ramorum propagules and found to successfully recover P. ramorum zoospores from retention pond water. Experiments to test a recombinase polymerase diagnostic assay (RPA) specific for P. ramorum were successful in detecting P. ramorum in flocculates and filtrates, and further RPA trials will continue in 2023 using a newly validated commercial P. ramorum RPA diagnostic kit. Research findings were presented virtually to university, industry, state and federal government stakeholders in March 2023.

For research of Brassica downy mildews, six samples infested with Hyaloperonospora spp. were contributed by collaborators or collected, with DNA extracted, amplified, and sequenced. Of seven DNA regions targeted for sequencing, only two of the markers reliably amplified among most samples, despite using multiple different methods from the literature for each gene region. This suggests the potential for high genetic variability within the understudied oomycete genus Hyaloperonospora. 

Objective 2.  Research continued to characterize pathogen biology and epidemiology of emerging and invasive water molds.  Downy mildews are serious pathogens of many important crops worldwide, yet their life cycles, taxonomy and host ranges are poorly understood.  Of the brassica downy mildew (Hyaloperonospora spp.) cultures collected last year, six genomic DNA extracts are stored in -20C. Hyaloperonospora oospores were identified in fresh Cardamine concatenata tissue and were documented in publication. H. brassicae was identified on a minority of brassica samples (2 of 6). Research was continued to understand the spread of impatiens downy mildew (Plasmopara) within plants and in different accessions bred for greater resistance to the disease.  Research was initiated on rose downy mildew (Peronospora), which has been increasingly reported on Knock-out roses, the most important category of landscape roses.  Methods were developed to culture the pathogen. Research was also initiated to determine if spinach downy mildew could be spread on seed, particularly in organic spinach production, where growers have few options for control.  

Phytopythium, a group of warm-climate plant pathogens, is relatively unstudied and its impact on U.S. agriculture with climate warming is unknown. Of the isolates in our collection made from Vietnam, approximately 60% were surprisingly identified as Phytopythium spp., though the original target had been Phytophthora spp. All but one isolate from agricultural areas were Phytopythium spp. Using clade-level information, analyses are being applied to the oomycete diversity present in river water, uncultivated forest soils, and agricultural soils. In addition, due to the large number of Phytopythium spp. present, evaluations are underway to evaluate the pathogenicity, virulence, and microbiological attributes of Phytopythium associated with citrus production. 

Another potentially serious threat to U.S. agriculture is the development of hybrid oomycete pathogens, and research continued to determine the frequency of hybrid events and what environmental conditions might increase that risk. In the Vietnam collection, one putative new hybrid Phytophthora was identified within this objective, for which we have DNA sequences for the gene regions ITS, LSU, cox2, cox1, rps10, and ypt1. These regions suggest the hybrid is in clade 7 of Phytophthora, but it is not similar to known species or hybrids.

Black pod rot, the most significant disease affecting cacao, is caused by several species of Phytophthora. Key pathogen isolates are undergoing molecular characterization and effector sequencing to understand how these pathogens overcome host plant resistance. **Accomplishments**
                                

1. Seedborne spores of the spinach downy mildew pathogen can initiate disease. The U.S. is the second largest producer of spinach, a crop valued at $496 million dollars a year in 2021. Spinach is a high value crop for large agribusiness as well as for small organic growers. However, spinach downy mildew, caused by the water mold Peronospora effusa, is a major limiting factor for spinach production. ARS researchers in Salinas, California, and Frederick, Maryland, set out to solve the mystery of how some outbreaks were initiated in spring, particularly in places with no history of the disease. The research showed that a type of P. effusa spore obtained from spinach leaves gave rise to downy mildew disease on spinach when mixed with seeds at planting. They further demonstrated that commercial sources of organic spinach seed contained the spores. When the infested seeds were germinated, a few of the new seedlings were infected with the downy mildew pathogen. These results are useful to spinach growers by demonstrating new sources of outbreaks, which are costly to control with pesticides and extremely difficult to control in organic systems. 2. Assessing new and emerging water mold causes of plant diseases in subtropical areas. Safeguarding U.S. agriculture and landscapes requires rapid insight into new plant disease risks. To improve U.S. preparedness for plant disease threats, ARS researchers in Frederick, Maryland, collected, identified, and tested water molds collected from countries outside the U.S., including those from citrus production areas. New variants of the ‘plant killer’ group, Phytophthora, were the initial target of the collection. These 'plant killers' average 6% losses to Florida citrus, reducing crop value by $26 million. Surprisingly, most of the water molds common to citrus were in a new group, Phytopythium. To understand their impact on citrus, the new foreign water molds were compared to citrus-damaging water molds from the U.S. in greenhouse trials. The foreign water molds caused up to twice as much root rot as the well-known, domestic-origin water molds. While the new water molds undergo more risk tests, Florida citrus soils are being surveyed to connect U.S. water mold presence and abundance to levels of citrus root rot. 3. Genes associated with resistance to cacao frosty pod rot disease. A sustainable strategy for reducing the amount of crops lost to diseases is to develop disease resistant plants through breeding. Up to 40% of the global harvest of cacao is lost to diseases. Frosty pod disease, caused by the fungus Moniliophthora roreri, causes losses of up to 90% in areas. ARS scientists at Frederick, Maryland, Beltsville, Maryland, and Miami, Florida analyzed gene expression by the fungus and found that cacao plants that are resistant to frosty pod disease expressed more genes responsible for defense and cellular communication compared with plants that are highly susceptible to the disease, even when the plants were not infected by the fungus. Ten of the genes that were more highly expressed in resistant plants were located within parts of the fungus’s genome that are known to be associated with resistance to frosty pod. These findings are important to scientists because it helps understand how resistant plants respond to infection and these genes could be targets for future cacao improvement. **Review Publications**
                            *Pfeufer, E., Harrison, L.K. 2022. High incidence of Hyaloperonospora cardamines-laciniatae on Cardamine concatenata in Maryland, USA. New Disease Reports. 46:e12143. https://doi.org/10.1002/ndr2.12143.*
                                *Pfeufer, E., Barlow, W.B., Pearce, R. 2023. Green yield as a potential predictor of cured burley tobacco yield in a Phytophthora nicotianae-infested field over four seasons. Plant Health Progress. https://doi.org/10.1094/PHP-09-22-0085-RS.*
                                *Puig, A.S., Cohen, S.P., Koch Bach, R.A., Arciniegas Leal, A., Gutierrez, O.A., Bailey, B.A. 2023. Comparative RNA-seq analysis of resistant and susceptible Theobroma cacao fruits in response to infection with frosty pod pathogen (Moniliophthora roreri). PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-09-22-0101-R.*
                                *Salgado-Salazar, C., Catstroagudin, V., Leblanc, N.R., Daughtrey, M., Hausbeck, M., Palmer, C., Shishkoff, N., Warfield, C.Y., Crouch, J. 2023. The impatiens downy mildew epidemic in the U.S. is caused by new, introgressed lineages of Plasmopara destructor with prominent genotypic heterogenicity and high evolutionary potential. Plant Disease. https://doi.org/10.1094/PDIS-08-22-1872-RE.*


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