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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

2020 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
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. Growth media and environmental conditions were evaluated to determine optimal cultural conditions for C. glycines hyphal growth and the production of sclerotia on solid and liquid media. 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 mouse monoclonal and rabbit polyclonal antibodies to use in the development of immunoassays to detect C. pseudonaviculata and C. henricotiae in boxwood leaf and environmental samples. Antibodies were tested for specificity and sensitivity with fungal culture extracts from boxwood leaves inoculated with multiple isolates of the two pathogen species. Although antibodies reacted strongly with fungal culture and secreted extracts, reactions against inoculated leaves were not sufficiently sensitive for utility in pathogen diagnostic assays. Two additional protein targets have been identified for future antibody generation. Also under this objective, more than 120 pathogenesis-related secreted proteins (known as effector proteins) from C. henricotiae and C. pseudonaviculata were identified. These proteins provide insight into the molecular basis of boxwood blight and may ultimately assist in the development of blight-resistant boxwood cultivars. Under Objective 2: Experiments designed to evaluate the biocontrol potential of an isolate of Trichoderma koningii against boxwood blight were completed. Infected boxwood leaves were treated with a suspension of T. koningii spores and incubated at temperatures conducive to the survival of Calonectria and sampled monthly. A significant reduction in sporulation of the boxwood blight pathogen over a six-month period was found when treated with T. koningii. Control of both Calonectria pathogens was the same and the reduction of sporulation was observed at each temperature tested. Experiments designed to evaluate potential foliar and stem endophytes that can be introduced to boxwood cuttings to reduce their susceptibility to boxwood blight are continent on ARS collaborators supplying promising endophytes; no endophytes were supplied this year. Wheat blast: Wheat blast, caused by the fungal pathogen Magnaporthe oryzae Triticum (MoT) pathotype, is an emerging disease. Although the pathogen was restricted to South America for nearly 30 years, in 2016 the pathogen was discovered in Bangladesh where it is now causing significant losses. The disease is expected to spread to other surrounding areas in Asia and there is concern that it may be imported to other parts of the world including the U.S. Identifying resistant wheat germplasm for resistance that can be deployed is an essential component of the strategy to combat this disease. Under Objective 4: Evaluation of the 2019 Northern Regional Performance Nurseries (NRPN) and Southern Regional Performance Nurseries (SRPN) was completed with 20 entries identified as resistant to wheat blast. Initial testing of the 2019 USDA Uniform Eastern Soft Red Wheat nurseries (UESR) and Southern Soft Red Wheat nurseries (USSR) was completed with four entries identified as potentially resistant to wheat blast. In cooperation with BASF, 23 winter and 49 spring wheat lines were evaluated with three and four lines, respectively, showing blast resistance. In cooperation with International Maize and Wheat Improvement Center (CIMMYT) and partially funded by the U.S. Agency for International Development, 100 spring wheat lines from the Bangladesh Agricultural Research Institute were evaluated, with seven determined to be resistant. In addition, 16 lines from the 100 spring wheat lines of the 2018 Elite Indian Nursery were found to be resistant. Evaluation of the 2020 NRPN and SRPN are underway. In collaboration with the ARS scientists at St.Paul, Minnessota, 17 Bolles- (hard red spring wheat) derived crosses were tested for resistance to blast at ARS greenhouses at Fort Detrick. In addition, under an Agreement with Kansas State University and the Association of Oilseeds and Wheat Producers (ANAPO), the Bolles crosses were also subjected to greenhouse and field testing in Santa Cruz and Okinawa, Bolivia, respectively. Seven of the 17 crosses were resistant in both greenhouse and field studies. These results highlighted the value of the greenhouse screening program at ARS Fort Detrick, MD for rapidly and reliably identifying potential sources of blast resistance in wheat. Due to the 2019 Government shutdown, the evaluation of the 2019 NRPN and SRPN was not completed in time to identify the potentially resistant wheat lines that would be sent to Bolivia for the 2020 planting season. In all of the studies above, with the exception of the UESR and ESSR nurseries, all 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 an effort to find additional genetic sources of resistance, we completed the testing of 260 synthetic hexaploid wheat lines derived from crosses between numerous geologically diverse Aegilops tauschii cultivars (a progenitor of wheat) and bread wheat. None of these crosses were resistant to wheat blast. 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 3: Previously, we identified a candidate soybean gene that confers immunity to the soybean rust pathogen, Phakopsora pachyrhizi. This gene, referred to as Rpp1 R4, encodes a protein with a domain not normally associated with resistance proteins. This finding provided an opportunity to identify interacting soybean proteins and one or more proteins produced by the pathogen, known as effectors, that may be important for pathogenicity. To identify potential proteins that interact with Rpp1 R4, we created and screened yeast-two hybrid libraries constructed from infected soybean leaves and germinating P. pachyrhizi spores. Although no interacting proteins from the pathogen were found, two soybean proteins that interact with Rpp1 R4 were identified. We are currently using virus-induced gene silencing to assess whether these interacting proteins play a role in the resistance response. Under Objective 4: The resistance gene Rpp1b maps to an overlapping region of the soybean gene where Rpp1 is located, but it confers a distinctly different type of resistance. To identify the Rpp1b gene, we constructed a bacterial artificial chromosome (BAC) library from a soybean line harboring Rpp1b. We have now completed sequencing a set of overlapping BACs spanning the region where Rpp1b has been mapped. We are now using this sequence information to design and test virus induced gene silencing constructs to determine the identity of Rpp1b. Also under this objective, soybean breeding lines created by ARS scientists at Stoneville, Mississippi were inoculated with 16 P. pachyrhizi isolates at Ft. Detrick and evaluated for rust resistance. The soybean lines were found to contain the resistance genes Rpp1, Rpp1b, Rpp2, Rpp3, or Rpp4. 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 37 samples of various wheat rust species under APHIS PPQ permit from foreign countries. We inoculated fungal material from all viable samples onto wheat seedlings, increased and shipped spores from 257 samples, originating from countries including Spain, Kenya, Tunisia, Hungary, Ethiopia, Hungary, and Italy to ARS scientists at St Paul, Minnesota for genotyping and further wheat resistance screens.


Review Publications
Delong, J.A., Stewart, J.E., Valencia-Botin, A., Pedley, K.F., Buck, J.W., Brewer, M.T. 2019. Invasions of gladiolus rust in North America are caused by a widely-distributed clone of Uromyces transversalis. PeerJ. 7:e7986.
Cruppe, G., Cruz, C.D., Peterson, G.L., Pedley, K.F., Asif, M., Fritz, A., Calderon, L., Da Silva, C.L., Todd, T., Kuhnem, P., Singh, P.K., Singh, R.P., Braun, H., Barma, N.C., Valent, B. 2019. Novel sources of wheat head blast resistance in modern breeding lines and wheat wild relatives. Plant Disease. 104(1):35-43.
Elmore, M.G., Banerjee, S., Pedley, K.F., Ruck, A.L., Whitham, S.A. 2020. De novo transcriptome of Phakopsora pachyrhizi uncovers putative effector repertoire during infection. Physiological and Molecular Plant Pathology. 110:101464.
Castroagudín, V., Weiland, J.E., Baysal-Gurel, F., Cubeta, M., Daughtrey, M., Gauthier, N., Lamondia, J., Luster, D.G., Hand, F., Shishkoff, N., Williams-Woodward, J., LeBlanc, N., Yang, X., Crouch, J.A. 2020. One clonal lineage of Calonectria pseudonaviculata is primarily responsible for the boxwood blight epidemic in the United States. Phytopathology. 110(11):1845-1853.
Olivera, P.D., Sikharulidze, Z., Dumbadze, R., Szabo, L.J., Newcomb, M., Natsarishvili, K., Rouse, M.N., Luster, D.G., Jin, Y. 2019. Presence of a sexual population of Puccinia graminisi f. sp. tritici in Georgia provides a hotspot for genotypic and phenotypic diversity. Phytopathology. 109(12):2152-2160.