Location: Foreign Disease-Weed Science Research2019 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Red leaf blotch of soybeans: Critical data on the biology of Coniothyrium glycines, the causal agent of red leaf blotch, is required for the development of best management practices, should the pathogen enter and become established in the U.S. Such data includes knowledge on the efficacy of sterilants on the overwintering propagules (sclerotia) of the pathogen. Under Objective 2: Two experiments were performed to evaluate the performance of selected sterilants in inactivating sclerotia of two isolates of Coniothyrium glycines. Oxidate (0.13% benzalkonium chloride) was used at full strength and ethanol was used at a concentration of 70%. C. glycines sclerotia were removed from 10 day-old cultures and immersed in one of the sterilants for 0, 5, 10, 15, 20, 25, or 30 minutes. At the designated time interval, each sterilant was removed and the sclerotia were washed twice with sterile water and then plated on 2% water agar. The treated sclerotia were incubated at 20°C in darkness for 5 days and then sclerotia germination was assessed. Percent germination was measured on two groups of 30 sclerotia in each replication, and there were two replications per treatment per experiment. Controls (time 0) germinated at 100% in both experiments. We found that 70% ethanol was effective in killing all sclerotia after a 5 minute treatment. Oxidate was much less effective in reducing germination, allowing germination percentages between 65% and 75% after a 30 minute treatment depending on the isolate used. Additional isolates, time intervals, and sterilants will be assessed in further experiments. Under Objective 3: Total RNA was extracted from C. glycines mycelium growth on solid media in the laboratory using commercial RNA extractions kits and evaluated for RNA yield and integrity. Gene expression studies will follow using RNA extracted from C. glycines grown under differing cultural conditions and from infected soybean plants after re-registration of the Biological Safety Level 3 Plant Pathogen containment facility at Ft. Detrick following several years of renovations to the facility. Boxwood blight: Boxwood blight, caused by two species of Calonectria, is a serious threat 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 antibody-based assays to detect C. pseudonaviculata and C. henricotiae in boxwood leaf and environmental samples. Antibodies are currently being tested for specificity and sensitivity with extracts from boxwood leaves inoculated with one of over 30 isolates of the two pathogen species. A commercial U.S. diagnostic company has expressed interest in using the antibodies to develop and market an assay kit for the boxwood blight pathogens. Under Objective 2: Twenty-five isolates of Trichoderma, a common fungus found in soil, were evaluated for efficacy as biological control agents of the boxwood blight pathogen Calonectria. Trichoderma isolates were evaluated for fast growth, ability to colonized leaf litter, ability to inhibit the development of overwintering structures in the pathogen, and ability to kill propagules of the pathogen. We found a number of promising isolates and selected the best for further studies. Current studies will determine the effect of the biocontrol agent on infected leaf litter. Wheat blast: Wheat blast, caused by the Triticum pathotype of Magnaporthe oryzae, 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. Evaluating pathogen survival for risk assessment and identifying resistant wheat germplasm for resistance that can be deployed are essential components of the strategy to combat this disease. Under Objective 2: Methods were developed to measure spore production in wheat leaves infected with the wheat pathotype of Magnaporthe oryzae to facilitate evaluation of pathogen survival in infected leaves under different environments. Environmental temperature profiles were collected from 5 select U.S. locations and used to program plant growth chambers replicate those conditions. Inoculated plants are being assessed in individual growth chambers and time tables for sampling infected plants are being established. Under Objective 4: We evaluated germplasm from the 2018 USDA Southern and Northern Performance Nurseries (SRPN and NRPN) for wheat blast resistance and identified 26 cultivars out of 95 tested as disease resistant. Twenty-five of the 2019 SRPN have been evaluated and 8 found resistant cultivars. Screening of the 2018 USDA Spring nursery obtained from the ARS Cereal Disease Laboratory identified 8 cultivars out of 17 tested resistant to wheat blast. Resistant germplasm from the 2018 USDA Spring nursery was sent to Bolivia, a location where the disease is prevalent, for field testing. To date, all resistance found in USDA nurseries can be traced to a single source, the 2NS chromosomal translocation from the wild wheat relative Aegilops ventricosa. Another wild wheat relative Aegilops tauschii, also known as goatgrass, that was previously found to be resistant to wheat blast was crossed at Kansas State University with Danby wheat. The progeny of this cross was further back crossed to the hard red winter wheat variety KanMark and 93 progeny were evaluated for a potential new source of disease resistance. However, the results of these crosses yielded no resistant lines. Additionally, in cooperation with the International Maize and Wheat Improvement Center (CIMMYT), 100 wheat cultivars from Bangladesh were evaluated for resistance and 11 were found 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 is underway. Although no interacting proteins from the pathogen were found, two soybean proteins that interact with Rpp1 R4 were identified. We are currently assessing whether these proteins play significant roles in the defense response to this pathogen. Under Objective 4: Soybean breeding lines created by ARS scientists at the Crop Genetics and Production Research Unit (Stoneville, Mississippi) were inoculated with 16 P. pachyrhizi isolates at Ft. Detrick and evaluated for resistance. Also, to further characterize the Rpp1 resistance gene transgenic soybean lines expressing Rpp1 are being developed. The requisite vectors have been designed and are currently being developed. An additional gene, Rpp1b, maps to an overlapping region of the soybean gene where Rpp1 is located, but confers a distinctly different type of resistance. To assess the function of Rpp1b, an initial set of gene-specific vectors for gene silencing Rpp1b candidate genes were tested in soybean, but failed to produce a loss of resistance phenotype. We are currently sequencing the region of the soybean genome where Rpp1b has been mapped to identify additional candidate genes for silencing. 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 tested and increased fungal isolates from viable material received in FY2018 and shipped 220 samples, originating from countries including Egypt, Kenya, Hungary, Italy, Kyrgyzstan and Kazakhstan, to the ARS Cereal Disease Laboratory, St Paul, Minnesota for genotyping and wheat resistance screens in December of 2019. To date in FY2019, we have received 72 samples of various wheat rust species under APHIS PPQ permit from foreign countries including Bhutan, Kenya and Spain.
Stone, C.L., Frederick, R.D., Tooley, P.W., Luster, D.G., Campos, B., Winegar, R.A., Melcher, U., Fletcher, J., Blagden, T. 2018. Annotation and analysis of the mitochondrial genome of Coniothyrium glycines, causal agent of red leaf blotch of soybean, reveals an abundance of homing endonucleases. PLoS One. 13(11):e0207062.
Yasuhara-Bell, J., Pedley, K.F., Farman, M., Valent, B., Stack, J.P. 2018. Specific detection of the wheat blast pathogen (Magnaporthe oryzae Triticum) by loop-mediated isothermal amplification. Plant Disease. 102:2550-2559. https://doi.org/10.1094/PDIS-03-18-0512-RE.
Pedley, K.F., Pandey, A.K., Ruck, A.L., Lincoln, L.M., Whitham, S.A., Graham, M.A. 2018. Rpp1 encodes a ULP1-NBS-LRR protein that controls immunity to Phakopsora pachyrhizi in soybean. Molecular Plant-Microbe Interactions. 32:120-133.
Harvey, R.J., Davis, D.D., Shishkoff, N., Pecchia, J.A. 2019. Impact of ammonia on Calonectria pseudonaviculata and C. henricotiae, causal agents of boxwood blight. Compost Science and Utilization. https://doi.org/10.1080/1065657X.2019.1586595.
Yasuhara-Bell, J., Pieck, M.L., Ruck, A.L., Farman, M.L., Peterson, G.L., Stack, J.P., Valent, B., Pedley, K.F. 2019. A response to Gupta et al. 2018 regarding the MoT3 assay. Phytopathology. 109:509-511. https://doi.org/10.1094/PHYTO-10-18-0397-LE.
Salgado-Salazar, C., Shishkoff, N., LeBlanc, N., Ismaiel, A.A., Collins, M., Cubeta, M.A., Crouch, J. 2018. Coccinonectria pachysandricola, causal agent of a new foliar blight disease of Sarcococca hookeriana. Plant Disease. 103(6):1337-1346. https://doi.org/10.1094/PDIS-09-18-1676-RE.
Harvey, R.J., Davis, D.D., Shishkoff, N., Pecchia, J.A. 2019. Survival of lab grown Calonectria pseudonaviculata microsclerotia during small scale composting. Bioresource Technology. 27(1):24-34. https://doi.org/10.1080/1065657X.2018.1536865.