Research Project: Integrative Genomic and Biological Approaches to Detect and Manage Emerging Foreign Fungal and Oomycete Plant Pathogens

Location: Foreign Disease-Weed Science Research

2025 Annual Report

Objectives
Objective 1: Generate and utilize genomic and proteomic sequence information of emerging and threatening fungal plant pathogens to develop diagnostic assays. (NP303, C1, PS1A, PS1B) Sub-objective 1.A: Develop accurate and rapid means for the identification and detection of foreign fungal plant pathogens. Subobjective 1.B: Determine origins and distribution routes of foreign and recently introduced plant pathogens. Sub-objective 1.C: Evaluate chemical control methods for emerging fungal plant pathogens. Objective 2. 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 2.A: Diagnostic tools and resources for downy mildew pathogens of cereals and grasses. Sub-objective 2.B: Apply previously validated isothermal RPA-lateral flow assays for rapid point of care detection of Phytophthora ramorum in nursery irrigation water (Non-hypothesis research). Sub-objective 2.C: A molecular assay can be developed to detect and identify downy mildew species. Objective 3: Characterize pathogen biology and epidemiology of emerging and invasive oomycete plant pathogens. (NP303, C2, PS2A, PS2C) Sub-objective 3.A: Characterize the pathogen biology of emerging and invasive Phytophthoras. Sub-objective 3.B: Characterize the pathogen biology of emerging and invasive downy mildews. Sub-objective 3.C: Determine risk potential posed by exotic or hybrid oomycete pathogen species to U.S. agricultural plant species. Sub-objective 3.D: Determine the diversity and virulence of foreign Phytophthora infestans genotypes. Objective 4: Identify factors involved in fungal pathogen infection and virulence. (NP303, C2, PS2A, PS2C) Sub-objective 4.A: Determine environmental factors affecting sporulation of fungal plant pathogens. Sub-objective 4.B: Evaluate chemical control methods for emerging fungal plant pathogens. Sub-objective 4.C: Identify genes and proteins contributing to virulence in fungal plant pathogens. Objective 5: Screen germplasm and identify resistance genes to emerging and threatening fungal plant pathogens. (NP303, C2, PS2B, C3, PS3A) Sub-objective 5.A: Screen germplasm for resistance to foreign fungal plant pathogens. Sub-objective 5.B: Identify accession-specific resistance genes for emerging and threatening fungal plant pathogens. Sub-objective 5.C: Identify new races of fungal pathogens that can be controlled by existing resistance genes.

Approach
Genomic sequence information will be generated from host plants and foreign fungal plant pathogens. Bioinformatic analyses will be conducted to identify resistance genes, pathogenicity factors, markers for evaluating pathogen populations, and unique targets for DNA-based diagnostic assay development. Pathogen proteins or isoforms will be identified and used to generate antibodies to develop immunodiagnostic assays. Fungicides will be evaluated for their efficacy in controlling emerging fungal plant pathogens. Controlled environment growth chambers will be used to determine the effect of temperature and humidity on the sporulation of fungal plant pathogens. Containment greenhouses will be utilized to screen plant germplasm with foreign fungal plant pathogens to identify sources of host resistance.

Progress Report
Under Objective 1, Sub-objective 1.A, a rapid isothermal diagnostic assay to detect the boxwood blight fungal pathogens Calonectria pseudonaviculata and C. henricotiae was successfully validated on a high-throughput fluorescent cycler using analytical DNA from multiple isolates of the boxwood blight pathogens to ensure specificity. Analytical and diagnostic sensitivity tests were completed, and specificity was confirmed to be limited exclusively to the target species. The research results were presented in a national webinar to diagnosticians from laboratories in the U.S. National Plant Diagnostic Network (NPDN), who responded with interest in participating in a ring test of the assay. This new diagnostic capability directly supports the USDA-ARS mission by enabling earlier, more accurate detection of an economically important ornamental disease. Once deployed at NPDN labs, the rapid assay would enhance national diagnostic capacity by replacing slower microscopic methods with a faster tool that offers higher sensitivity, improved accuracy, and greater throughput—strengthening stakeholder response to boxwood blight threats, both domestic and foreign. Under Objective 2, Sub-objective 2.B, research continued to determine the lower limit of detection of a diagnostic assay targeting the Sudden Oak Death pathogen Phytophthora ramorum in nursery irrigation water. The assay successfully detected as few as 10 spores captured from water using a filtration-based method. The total time of spore capture, extraction and assay was approximately one hour, using an automated instrument to determine positive or negative outcomes. A standardized assay workflow was drafted to support adoption by diagnostic labs. The technology is being prepared for transfer to diagnostic labs in states where regulations require testing irrigation water from nurseries with infected plants for potential pathogen contamination. If adopted, this assay will significantly enhance regulatory detection capacity by replacing the current USDA Animal and Plant Health Inspection Service (APHIS) “Bottle of Bait” protocol, which requires up to 10 days for confirmation, provides regulators and plant health labs with faster, more effective tools to protect U.S. nurseries. Under Objective 3, Sub-objective 3.A, we continued to characterize the biology and epidemiology of the emerging oomycete plant pathogen Peronospora sparsa, the causal agent of rose downy mildew. Oospores were tested for viability and changes in morphology; they remained viable in all samples and their appearance changed in later samples. Samples taken in March, when placed on rose petals in vitro, germinated and infected the rose tissue, demonstrating that oospores could overwinter and serve as inoculum the following season. Under 3.B, to establish graminaceous downy mildews (GDM) pathogen host range, wild and cultivated sugarcane have been propagated along with wild and domesticated maize. Under 3.C, we initiated the evaluation of P. sparsa oospores that had been stored at different temperatures to determine survival over different temperatures and time intervals. Preliminary results indicate that the changes in morphology observed in field plots are also observed in culture. Under Objective 4, Sub-Objective 4.A, we studied the effects of temperature and humidity on boxwood blight disease caused by two species of Calonectria across six boxwood cultivars. Comparing the two pathogens, we found little difference in disease severity at any given temperature, although both caused more severe symptoms at 24 C. Critically, cultivar-based differences in susceptibility were only apparent at the higher temperature, while lower temperatures showed no significant variation among cultivars. We also examined the influence of humidity on sporulation and disease severity. Four cultivars were inoculated with both boxwood blight pathogens maintained in humidity chambers set at 40, 60, 80 and 100% relative humidity. Sporulation was observed infrequently at 80% relative humidity and commonly at 100% relative humidity, indicating a strong environmental threshold for pathogen reproduction. These findings enhance understanding of disease dynamics under field- relevant conditions and inform predictive models for disease outbreaks. Under Sub-objective 4.C, we advanced research on peanut smut, a foreign disease caused by the fungal pathogen Thecaphora frezzii, by refining inoculation methods for cultivated peanuts, using culture material from compatible mating types. This approach will enable more consistent disease development in controlled environments, particularly when spore availability for inoculation is limited. We also finished the assembly of a reference-quality genome sequence for T. frezzii, which is now being used to assess the pathogen’s genetic diversity of the pathogen. Analysis revealed multiple mating types, suggesting several independent introductions into cultivated systems and offering valuable insight into its potential spread. These advances provide critical insight into T. frezzii biology and epidemiology, equipping breeders, farmers, and regulators with improved tools for screening and disease management to strengthen biosecurity and preparedness against foreign peanut smut threats. As part of ongoing research on Coniothyrium glycines, we also used genomics, transcriptomics and metabolomics to confirm that the pathogen produces a light-activated phytotoxin. This is the first evidence regarding how C. glycines may infect its host, and this finding will support efforts to identify resistant germplasm. As part of ongoing research on soybean rust, transcriptomes generated from resting spores, germinating spores, and appressoria of the pathogen Phakopsora pachyrhizi were annotated. This dataset captures critical stages of infection and provides a foundation for identifying genes involved in pathogenicity. Downstream analyses are now underway to predict putative effector proteins, which are key molecular tools pathogens use to manipulate host defenses. These findings will support the discovery of virulence-associated proteins, informing development of resistant soybean cultivars and guiding future disease management strategies. Under Objective 5, Sub-objective 5.A, we developed a new protocol for inoculating plants with the peanut smut pathogen Thecaphora frezzii. We are currently evaluating the efficacy of this method on multiple wild peanut relatives grown under high containment conditions. Also under 5.A, advanced soybean breeding lines developed by ARS scientists at Stoneville, Mississippi were screened for resistance to soybean rust. Resistant lines will move into field trials to evaluate performance under natural infection conditions and undergo yield testing identify top candidates for released. Under 5.B, mapping data for the soybean rust resistance gene Rpp7, along with the genome of the susceptible soybean cultivar ‘Williams 82’ were used to identify candidate Rpp7 genes in the resistant accession PI 605823. A loss-of-resistance phenotype was confirmed through virus-induced gene silencing using partial sequences of the candidate genes. Additionally, a bacterial artificial chromosome spanning the Rpp7 locus was fully sequenced. Gene expression of all candidate genes has been completed and will guide identification of the Rpp7 candidate gene responsible for resistance. This work advances the identification of Rpp7 and supports the development of soybean cultivars with durable resistance to soybean rust, strengthening disease management for growers. Under 5.C, a reference genome for Hemileia vastatrix, the causal agent of coffee leaf rust, was completed, including a small nucleotide polymorphism (SNP)-based phylogeny of race-typed isolates. In planta RNA sequencing is underway to support annotation of candidate effector genes. Preliminary findings suggest that the pathogen has spread globally in a largely clonal pattern. To support research of foreign cereal rusts, 56 samples of rust fungi affecting wheat, barley, oats and rye were received from international collaborators in Spain, Canada, Bhutan, Azerbaijan, and the United Kingdom under an international USDA APHIS permit. A new Material Transfer Agreement was signed between ARS and the National Plant Protection Center in Bhutan to enable in-depth analysis of cereal rust samples collected across that country, where emerging pathogen variants have recently been reported. Viable rust pathogen material was harvested and increased on wheat, barley and/or oat seedlings. Preliminary virulence tests were completed on all recovered samples. Subsamples of rust spores and ethanol-killed infected wheat leaves from all samples were shipped to ARS researchers at the Cereal Disease Lab in St. Paul, Minnesota, for genotyping and wheat differential analysis.

Accomplishments
1. Spinach downy mildew traced to infected seed, threatening U.S. crop health. Spinach downy mildew, caused by Peronospora effusa, is a major threat to global spinach production. Although rare in the Northeastern U.S. before 2014, recent outbreaks raised concerns about contaminated seed as a possible source. In a world where clean seed is essential to healthy crops, ARS researchers in Frederick, Maryland and Salinas, California, investigated whether the pathogen’s durable oospores were present and viable on commercial seed. Examining over 40,000 seeds from growers and suppliers, they found infestations on up to 7% of seeds, with viability reaching 57% in one lot. Some infected seeds produced diseased seedlings, confirming seedborne transmission. This discovery reveals an overlooked pathway for disease spread and advances understanding of pathogen epidemiology. With U.S. spinach valued at over $400 million annually, the findings help seed companies, diagnostic labs, and American farmers adopt better screening and sanitation practices to prevent outbreaks and protect livelihoods.

2. ARS releases rust- and rot-resistant soybean germplasm for breeders and growers. The long-term success of U.S. soybean farming—an industry valued at over $60 billion annually—relies on access to disease-resistant, high-yielding varieties that reduce input costs and protect crop performance. Soybean rust, a devastating fungal disease, remains a persistent late-season threat in the southeastern U.S., and most commercial lines remain susceptible. To address this, ARS scientists in Frederick, Maryland; Stoneville, Mississippi; Urbana, Illinois; and West Lafayette, Indiana, in partnership with the Paraguayan Institute for Agricultural Technology, developed and released five new soybean germplasm lines with unique resistance to soybean rust and Phytophthora root and stem rot. These lines demonstrated strong yields under disease pressure and introduce novel resistance genes now available through USDA’s Germplasm Resources Information Network. By expanding the genetic diversity of resistance sources and making them accessible to breeders, this research enables more efficient development of improved cultivars. These lines are already being adopted into breeding pipelines, offering producers a practical path to reduce disease losses and chemical inputs while strengthening the long-term resilience of U.S. soybean production.

3. Toxin discovery in deadly foreign soybean pathogen reveals pathway for disease prevention. Safeguarding U.S. soybean production requires anticipating threats from foreign pathogens and closing knowledge gaps before disease outbreaks occur. Red leaf blotch (RLB), caused by the foreign Select Agent fungus Coniothyrium glycines, is a deadly foliar disease currently restricted to Africa that threatens the $57.5 billion U.S. soybean industry, with potential yield losses exceeding 50%. Despite decades of research, how this pathogen infects its host remained unknown. To address this critical gap, ARS scientists in Maryland, Illinois, and Mississippi—together with partners at the International Institute for Tropical Agriculture in Kenya—analyzed the pathogen’s genome, gene expression, and metabolite production. They discovered that C. glycines produces a light-activated phytotoxin with near-universal plant toxicity, providing the first evidence of how it causes disease on the molecular level. This breakthrough explains why traditional resistance breeding has failed and offers a clear target for developing resistant soybean varieties. These findings equip breeders and plant health officials with essential tools to reduce the risk of future outbreaks, strengthen biosecurity, and protect American farmers and global food security.

4. ARS advances pathogen identification with precision DNA tool to strengthen agricultural biodefense. Fast, accurate identification of plant pathogens is essential to protect crops, ecosystems, and farmers’ livelihoods. Oomycetes—a group of fungus-like organisms that includes Phytophthora and Pythium—cause some of the world’s most destructive plant diseases. Traditional DNA markers used to identify them are often too short to distinguish closely related species. To address this, ARS scientists in Frederick, Maryland developed a long-read DNA method using Oxford Nanopore sequencing (a portable technology that reads long strands of DNA in real time) to target the full-length cox1/spacer/cox2 region of the oomycete genome. The method generated high-quality DNA sequences from 184 species across 23 genera, identified only oomycetes in environmental samples, and reliably distinguished both known and difficult-to-detect species. It also revealed new species and a novel genus affecting major crops like corn, sugarcane, citrus, and turfgrass, with broader relevance to potato, soybean, and tomato. This tool enables earlier, more accurate identification of crop pathogens—accelerating response and strengthening America’s defense against emerging biological threats to agriculture.

5. Validated rapid diagnostic assay enables early detection of Sudden Oak Death pathogen. Protecting U.S. agriculture and natural resources requires fast, science-based responses to emerging plant threats. Phytophthora ramorum, the federally regulated cause of Sudden Oak Death, continues to spread through nurseries in California and Oregon, triggering quarantines and underscoring the need for rapid detection. To address this, ARS scientists in Frederick, Maryland and Salinas, California—working with academic and industry partners—validated a rapid isothermal Recombinase Polymerase Amplification (RPA) diagnostic assay for P. ramorum. The test delivers results in under an hour and showed high sensitivity and specificity, avoiding false positives from over 100 related pathogens. It passed a rigorous blind Test Performance Study across five independent National Plant Diagnostic Network (NPDN) laboratories, confirming its reliability under operational conditions. The assay is being marketed and is currently being evaluated by diagnosticians in NPDN plant health clinics and USDA Animal and Plant Health Inspection Service for adoption into programs for regulatory confirmation of the pathogen. This research delivered a field-ready diagnostic tool that improves plant disease response, reduces economic losses for nurseries, and advances USDA-ARS’s goal of safeguarding U.S. agriculture through science-based solutions.

6. ARS identifies key source of resistance to guide U.S. wheat breeding for wheat blast defense. Wheat is one of the world’s most important food crops, providing nearly 20% of human calories. A secure U.S. wheat supply depends on varieties that can resist emerging diseases and protect American farmers’ livelihoods. Wheat blast—a fast-spreading, highly destructive fungal disease—has caused major losses in South America and has since spread to Asia and Africa, prompting concern about a potential U.S. outbreak. To help prepare wheat growers for wheat blast, ARS scientists in Maryland, Nebraska, and Kansas evaluated entries from the USDA Hard Winter Wheat Regional Performance Nurseries using a virulent wheat blast isolate. All potentially resistant lines shared a 2NS chromosomal segment, a piece of DNA from a wild wheat relative known to carry disease-fighting genes, confirming it as a key source of wheat blast resistance. This discovery improves our understanding of wheat blast defense and offers breeders a clear genetic target for building resilience into Great Plains wheat. Findings were shared with public and private breeding networks through nursery reports and professional meetings to help reduce future disease losses and protect U.S. food security.

7. DNA-based identification using herbarium specimens strengthens accurate identification of downy mildew pathogens in cereal crops. Protecting staple food crops like corn, sugarcane, and millets requires accurate identification of the pathogens that threaten them, but many downy mildew species are difficult to distinguish due to limited DNA information—especially from historic reference samples preserved in herbarium collections. ARS scientists addressed this gap using an oomycete-specific target enrichment high-throughput DNA sequencing approach to extract and sequence hundreds of genes from 58 herbarium specimens, including 17 that define the pathogens’ identities, and constructed a phylogenetic family tree to resolve species boundaries and correct widespread misidentifications. Their work validated 13 species, revealed 12 previously unnamed lineages, and confirmed the distinct identity of high-risk pathogens such as Peronosclerospora philippinensis, a destructive downy mildew of corn responsible for major yield losses in Asia. These results give plant health officials improved information for early, accurate identification of emerging pathogens—supporting American farmers, defending against biological threats, and helping safeguard the nation’s food supply.

8. New genomic resource fuels peanut smut diagnostics and surveillance to protect U.S. agriculture. A secure agricultural system relies on early detection and monitoring of foreign pathogens that threaten key crops. Peanut smut, caused by Thecaphora frezzii, has severely impacted Argentina’s peanut production and poses a risk to the U.S. peanut industry, which was valued at approximately $1.7 billion in 2024. Yet without high-quality genome data, efforts to develop diagnostic tools and monitor the pathogen’s spread have been hindered. To close this gap, ARS researchers in Frederick, Maryland and Stillwater, Oklahoma, with Argentinian collaborators, developed a method to extract high-quality DNA from T. frezzii spores and completed the first nearly complete, phased genome for the pathogen. A phased genome captures both chromosome sets, helping scientists track genetic variation that could influence virulence—or how aggressively the pathogen infects plants and overcomes resistance. At 38.8 megabases, it’s the largest smut fungus genome sequenced to date. This reference genome is now being used to develop diagnostics, guide breeding, and support surveillance, and is already informing cross-border prevention and preparedness to protect American farmers and the peanut supply chain.

9. New water- and soilborne pathogens identified to enhance early detection and safeguard U.S. agriculture. Protecting crop health requires the swift detection of harmful pathogens in irrigation water and soils. Meeting this critical need is complicated by the emergence of poorly characterized species within groups like the water mold Phytopythium, which causes root and crown rot on citrus, ornamentals, peppers, tomatoes, and other valuable crops. To address this risk, ARS researchers in Frederick, Maryland, examined the appearance and DNA of samples from U.S. irrigation water and Vietnamese soil, identifying two new species of Phytopythium. These species closely match global genetic records, suggesting they are more widespread than previously known and may contribute to future outbreaks of foreign and invasive pathogens. The results improve diagnostics, support international surveillance, and help U.S. growers and regulators respond faster to disease threats. This research equips ARS partners with better tools to detect and manage risk early, helping protect America's farmers, food supply, and agricultural economy.

Review Publications
Wang, N., Weckler, P.R., Zhang, Y., Errabelly, A., Payton, M.E., Baldessari, J., Chamberlin, K.D., Rodriguez, A., Koch Bach, R.A., Maestro, M., Bennett, R. 2025. Evaluation of a line-scan X-ray system for phenotyping peanut smut resistance using faux-infected pods. Peanut Science. 52(1):89-96. https://doi.org/10.3146/0095-3679-52.1-PS-1629.
Li, X., Weiland, J.E., Ohkura, M., Luster, D.G., Daughtrey, M.L., Gouker, F.E., Chen, G., Kong, P., and Hong, C. 2024. Cultivars and production environments shape shoot endophyte profiles of boxwood with different blight resistance. Phytofrontiers 4:602-615. https://doi.org/10.1094/PHYTOFR-03-24-0023-R
Martin, F.N., Batuman, O., Luster, D.G., Miles, T., Rivera, Y., Sharma, P., Geiser, D., Cardwell, K. 2025. How controls improve diagnostic assay performance: Hitchhiker's guide to diagnostic assay controls. PhytoFrontiers. 5(2):127-137. https://doi.org/10.1094/PHYTOFR-10-24-0118-FI.
Neugebauer, K., Davenport, B., Harmon, C., Byrne, J., Miles, L., Snover-Clift, K., Rooney-Latham, S., Martin, F.N., Luster, D.G., Miles, T. 2025. Validation of a rapid, high throughput isothermal recombinase polymerase amplification screening assay for Phytophthora ramorum. PhytoFrontiers. 5(2):220-228. https://doi.org/10.1094/PHYTOFR-10-24-0114-FI.
Stupar, R.M., Locke, A.M., Allen, D.K., Stacey, M.G., Ma, J., Weiss, J., Nelson, R., Hudson, M.E., Joshi, T., Li, Z., Song, Q., Jedlicka, J., Macintosh, G.C., Grant, D., Parrott, W.A., Clemente, T.E., Graham, M.A., O'Rourke, J.A., Stacey, G., An, Y., Aponte-Rivera, J., Bhattacharyya, M.K., Baxter, I., Bilyeu, K.D., Campbell, J.D., Cannon, S.B., Clough, S.J., Mcgrinn, M., Curtin, S.J., Diers, B.W., Dorrance, A.E., Gillman, J.D., Graef, G.L., Hancock, N., Hudson, K.A., Hyten, D.L., Kachroo, A., Koebernick, J., Libault, M., Lorenz, A.J., Mahan, A.L., Massman, J.M., Meksem, K., Okamuro, J.K., Pedley, K.F., Rainey, K.M., Scaboo, A.M., Schmutz, J., Song, B., Steinbrenner, A.D., Stewart-Brown, B.D., Toth, K., Wang, D., Weaver, L., Zhang, B. 2024. Soybean genomics research community strategic plan: a vision for 2024-2028. The Plant Genome. https://doi.org/10.1002/tpg2.20516.
Greatens, N., Murithi, H., Coyne, D., Clough, S.J., Sulyok, M., Okunowo, W., Abbas, H.K., Shier, W., Koch Bach, R.A. 2025. Production of the light-activated elsinochrome phytotoxin in the soybean pathogen Coniothyrium glycines hints at virulence factor. PLOS ONE. https://doi.org/10.1371/journal.pone.0321896.
Rocha, V., Ferreira, E., Castanho, F., Kuwahara, M., Godoy, C., Meyer, M., Pedley, K.F., Voegele, R., Grigoriev, I., Lipzen, A., Barry, K., Loehrer, M., Schaffrath, U., Sirven, C., Duplessis, S., Guimarães, F. 2025. Analysis of the genetic diversity of the soybean rust pathogen Phakopsora pachyrhizi reveals two major evolutionary lineages. Fungal Genetics and Biology. 179. Article 103990. https://doi.org/10.1016/j.fgb.2025.103990.
Pedley, K.F., Boehm Jr, J.D., Bai, G., St Amand, P.C., Peterson, G.L., Vinyard, B.T. 2025. Evaluation of wheat blast resistance in the USDA hard winter wheat (Triticum aestivum L.) Northern and Southern Regional Performance Nurseries. Plant Disease. https://doi.org/10.1094/PDIS-09-24-1941-RE.