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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Research Project #432679

Research Project: Identification, Biology, Epidemiology, and Control of Foreign and Emerging Fungal Plant Pathogens

Location: Foreign Disease-Weed Science Research

2021 Annual Report

Objective 1: Generate and utilize genomic, transcriptomic, and proteomic sequence information of foreign fungal plant pathogens to develop diagnostic assays. [NP303, C1, PS1] Sub-objective 1.A - Develop accurate and rapid means for identification and detection of foreign fungal plant pathogens. Objective 2: Determine the effects of temperature, moisture and their interactions on the germination, growth, and survival of foreign fungal plant pathogens and development of disease. [NP303, C2, PS2A] Sub-objective 2.A - Determine the effects of temperature and moisture on infection and development of disease. Sub-objective 2.B - Determine the effects of temperature and moisture on the survival of foreign fungal plant pathogens. Objective 3: Utilize genomic and transcriptomic sequence information to identify and characterize genes and proteins required for infection and pathogenicity of foreign fungal plant pathogens. [NP303, C2, PS2B] Sub-objective 3.A - Identify secreted proteins from foreign fungal plant pathogens. Objective 4: Screen germplasm and identify resistance genes to foreign fungal plant pathogens. [NP303, C3, PS3A] Sub-objective 4.A. Screen germplasm for resistance to foreign fungal plant pathogens. Sub-objective 4.B. Identify genes and pathways involved in resistance to foreign fungal plant pathogens.

Genomic sequence information will be generated from foreign fungal plant pathogens and bioinformatic analyses will be conducted to identify genes and proteins. The genomic sequence data will be mined to identify unique target sequences to develop rapid DNA-based diagnostic assays. Unique pathogen proteins or isoforms will be identified and used to generate antibodies to develop immunodiagnostic assays. Secreted proteins from fungal plant pathogens that contribute to pathogenicity will be identified using assays to detect secreted proteins and/or interactions between host- and pathogen-derived proteins. Temperature-controlled growth chambers will be used to determine effects of low temperatures and durations on pathogen survival. Additionally, the effects of moisture levels, chemical sterilants, endophytes, and antagonistic biocontrol organisms on plant pathogen survival will be assessed. Germplasm will be inoculated with foreign fungal plant pathogens and screened for resistance.

Progress Report
Boxwood blight: Boxwood blight, caused by two species of Calonectria, is a serious threat to the U.S. boxwood industry. Effective management strategies require the development of detection methods for the pathogen and control measures to reduce the spread of diseased stock. Under Objective 1: We generated rabbit polyclonal antibodies against two new protein targets for use in the development of immunoassays to detect Calonectria pseudonaviculata and C. henricotiae in boxwood leaf and environmental samples. Antibodies were tested for specificity with boxwood (Buxus sempivirens cv. Justin Brouwer) leaves inoculated with isolates of the two pathogen species. Extraction protocols were refined, using various buffers, sample heating and centrifugation. Antibodies reacted strongly in capture enzyme-linked immunosorbent(ELISA) assays with extracts from inoculated boxwood leaves and showed no reaction against uninoculated control leaf extracts or extracts from leaves expressing Buxus fungal endophytes. The antibodies are currently undergoing additional testing for specificity with leaves inoculated with Pseudonectria and Fusarium spp. boxwood pathogens. Boxwood diagnostic samples are being received for testing from diagnosticians at Bartlett Tree Experts, Cornell University, Rutgers University and the University of Maryland. Under Objective 2: Research on the efficacy of biological control agents to reduce survival of the boxwood blight pathogen in fallen leaf litter was completed and data was analyzed with the help of an ARS statistician in Beltsville, Maryland, and the results were used to produce posters for two online meetings. To aid in the diagnosis of boxwood blight, photographs of characteristic symptoms of Volutella blight were taken and morphology of the three Volutella pathogens on boxwood were morphologically characterized for a diagnostic guide. Red leaf blotch of soybeans: Coniothyrium glycines, the causal agent of Red Leaf Blotch of soybeans, is known to cause serious soybean loses in southern Africa and produces special melanized structures called “sclerotia” that are important for survival and spread of the pathogen. Under Objective 2: Additional growth media and environmental conditions were evaluated further to determine optimal cultural conditions for C. glycines hyphal growth and the production of sclerotia on solid and liquid media. Soybean rust: Soybean rust, caused by the pathogen Phakopsora pachyrhizi, is an aggressive disease of soybean affecting production in all major growing areas of the world. Identifying natural sources of resistance and developing cultivars with durable host plant resistance is the preferred means of managing the disease. Under Objective 4: The resistance gene Rpp1b maps to an overlapping region of the soybean locus where Rpp1 is located, but it confers a distinctly different type of resistance. To identify the Rpp1b gene, we sequenced overlapping bacterial artificial chromosomes (BACs) spanning the Rpp1 locus obtained from a BAC library constructed from a soybean line harboring Rpp1b. The initial set of virus-induced gene silencing (VIGS) constructs designed to assess the function of candidate Rpp1b genes did not generate the expected loss of resistance phenotype when used in Rpp1b-resistance plants inoculated with an avirulent isolate of P. pachyrhizi. New VIGS vectors are being designed against other candidate resistance genes at this locus. Also under this objective, we completed the screening of soybean breeding lines created by ARS scientists at Stoneville, Mississippi, for rust resistance. The soybean lines evaluated were found to contain the resistance genes Rpp1, Rpp1b, Rpp2, Rpp3, or Rpp4. Wheat blast: Wheat blast, caused by the fungal pathogen Magnaporthe oryzae Triticum (MoT) pathotype, is an emerging seed borne disease of wheat. Although the pathogen was restricted to South America for nearly 30 years, in 2016 MoT was discovered in Bangladesh and during the 2017 – 2018 rainy season it was found in Zambia. The pathogen is expected to spread to other areas in Asia and Africa underlining the concern that it may be imported to other parts of the world including the U.S. Identifying resistant wheat germplasm that can be deployed is an essential component of the USDA strategy to combat wheat blast. Under Objective 4: The evaluation of the 2020 Northern Regional Performance Nurseries (NRPN) and Southern Regional Performance Nurseries (SRPN) was completed with six entries identified as resistant to wheat blast. Initial testing of the 2020 USDA Uniform Eastern Soft Red Wheat nurseries (UESR) and Southern Soft Red Wheat nurseries (USSR) was completed with nine entries identified as potentially resistant to wheat blast. In addition, under an agreement with Kansas State University and the Association of Oilseeds and Wheat Producers (ANAPO), potentially resistant lines identified at Ft. Detrick are sent for confirmational field study testing in Bolivia. In all the studies conducted thus far, wheat germplasm identified as resistant to MoT possessed the translocation of the 2NS chromosomal arm from Aegilops ventricosa to the 2AS arm of bread wheat. In cooperation with ARS scientists at the ARS Center for Grain and Animal Health Research Center in Manhattan, Kansas, and collaborators at the Univ. of Nottingham, United Kingdom, we are evaluating 110 wheat/Amblyopyrum muticum and wheat/Triticum urartu chromosome introgressions as well as 126 hexaploid wheat/Aegilops tauschii crosses as a possible new source if blast resistance from wild wheat relatives. To date, 35 of the wheat/Amblyopyrum muticum lines have been tested and no resistance has been observed. We anticipate completing these evaluations by the end of the 2021 reporting period. Wheat stem rust: Rust diseases, caused by species of Puccinia, are among the most important causes of yield loss in wheat in the U.S. and worldwide. Global surveillance of cereal rusts with the goal of identifying new races of Puccinia spp. as they emerge, is an important mitigation measure in order to ensure the timely deployment of resistance as races spread between cereal production areas. Under Objective 4, we received samples of various wheat rust species under Animal and Plant Health Inspection Service, Plant Health Quarantine (APHIS PPQ) permit from Kenya, Spain and Ethiopia. We inoculated fungal material from all viable samples onto wheat seedlings, increased and shipped spores from 117 samples to the ARS Cereal Disease Laboratory, St Paul, Minnesota, for genotyping and wheat line resistance screens.

1. Resistance to soybean rust. Soybean is the most important legume grown in the United States used for food, feed, and fuel. Soybean rust is a devastating disease caused by the fungal pathogen Phakopsora pachyrhizi that occurs on soybeans worldwide. While fungicides are available that control the disease, the pathogen has developed tolerance to some fungicides. Identifying potential sources of resistance to soybean rust is an important first step in developing soybean cultivars for growers to use to manage disease losses. To assess for resistance to soybean rust, 53 soybean genotypes from ARS scientists in Stoneville, Mississippi, were infected with 16 different isolates of the fungal pathogen collected throughout the world inside the ARS Biosafety Level 3 Plant Pathogen Containment Facility at Ft. Detrick, Maryland. The previously identified rust resistance gene, Rpp1, was found in 18 of the soybean lines tested. Two soybean lines were found to contain two soybean resistance genes, Rpp2 and Rpp4, while another soybean line possessed the resistance genes Rpp1b and Rpp4. Three soybean lines were identified as having possible new sources of resistance to soybean rust. The resistance identified in these soybean lines could be used as sources to develop high yielding soybean lines with broader resistance to soybean rust.

2. Development of training manual and virtual workshops. An ARS scientist at Frederick, Maryland, developed a training manual for quantitating teliospores of the fungal pathogen, Tilletia controversa Kuhn (TCK), that causes Dwarf bunt in grain samples and conducted virtual workshops on the Dwarf bunt testing procedure that was attended by individuals from USDA, universities, U.S. Wheat Associates, Wheat Marketing Center, Cargill, and several private testing laboratories. The Federal Grain Inspection Service (FGIS) adopted the training manual as the official USDA protocol.

3. New diagnostic guide for diseases of boxwood, pachysandra and sweetbox. Boxwood, pachysandra and sweetbox plants (all in the boxwood plant family) are threatened worldwide by the boxwood blight pathogen. Accurate diagnosis of boxwood blight disease will allow better control, but diagnosis is difficult because boxwood blight is commonly confused with Volutella blight. Therefore, ARS scientists in Beltsville and Frederick, Maryland, and Corvallis, Oregon, in partnership with university collaborators across the U.S., developed an illustrated guide to Volutella blight. The guide is intended for use by diagnosticians, regulatory officials, plant pathologists, extension personnel and growers in their efforts to minimize the spread and impact of boxwood blight and to discriminate blight from less destructive diseases.

Review Publications
Shishkoff, N., Cubeta, M.A., Miller, M.E. 2021. Rooting response of boxwood cultivars to hot water treatment and thermal sensitivity of calonectria henricotiae and C. pseudonaviculata in diseased boxwood. Journal of Environmental Horticulture. 39(1):1–10.
Cruppe, G., Silva, P., Da Silva, C.L., Peterson, G.L., Pedley, K.F., Cruz, C.D., Asif, M., Lollato, R.P., Fritz, A., Valent, B. 2020. Genome-wide association reveals limited benefits of pyramiding the 1B and 1D loci with the 2NS translocation for wheat blast control. Crop Science. 61:1089-1103.
Stone, C.L., Smith, J.R., Ray, J.D., Gillen, A.M., Frederick, R.D. 2021. Phenotypic reactions of 53 soybean genotypes to infection with each of 16 isolates of Phakopsora pachyrhizi. Journal of Crop Improvement.
Becker, L.E., Adhikari, U., May, T., Shishkoff, N., Crouch, J., Cubeta, M.A. 2021. Evaluation of boxwood cultivars for resistance to boxwood blight, 2020. Plant Disease Management Reports. 15:OT008. Available:
Castroagudin, V.L., Shishkoff, N., Stanley, O., Whitesell, R., Olson, T., Crouch, J.A. 2020. First report: Co-infection of Sarcococca hookeriana (sweetbox) by Coccinonectria pachysandricola and Calonectria pseudonaviculata causes a foliar disease of sweetbox in Pennsylvania. Plant Disease. 105(5):1568.
Tembo, B., Mulenga, R.M., Sichilima, S., M'Siska, K.K., Mwale, M., Chikoti, P., Singh, P.K., He, X., Pedley, K.F., Peterson, G.L., Singh, R.P., Braun, H.J. 2020. Detection and characterization of fungus (Magnaporthe oryzae pathotype Triticum) causing wheat blast disease on rain-fed grown wheat (Triticum aestivum L.) in Zambia. PLoS ONE. 15:e0238724.
Widmer, T.L., Mcmahon, M.B., Luster, D.G. 2020. Plant pathogenic fungi harbor as endophytes in Rhododendron spp. native to the Eastern U.S.. Fungal Ecology. Fungal Ecology 47:100949.