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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

2018 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 predictive models for management of the pathogen, should it enter and become established in the U.S. Such data includes knowledge of survival characteristics of overwintering propagules of the pathogen. Under Objective 2: Soybean field soil was collected from soybean fields from three distinct geographical locations for use in studies of C. glycines sclerotia survival in soil at different temperatures. GPS coordinates and elevation for each location were recorded. Soils will be subjected to nutrient level, particle size, pH, and organic matter analysis at the Agricultural Analytical Services Laboratory at Penn State University (University Park, Pennsylvania). Also under Objective 2, preliminary experiments on survival of C. glycines sclerotia at high temperatures were performed. The results indicate no decrease in the survival of the pathogen after 4 hours at 100°C. After 5 hours, a reduction in viability was detected using two isolates of the pathogen. The studies will be repeated with additional isolates, longer times, and higher temperatures. 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. Boxwood blight: Boxwood blight, caused by two species of Calonectria, is a serious threat the US 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 applied a proteomic approach to identify proteins secreted by the boxwood blight pathogens Calonectria pseudonaviculata and C. henricotiae that could be used to develop protein-based diagnostic assays. From these, we selected protein candidates to serve as antigens for generation of Calonectria genus-level antibodies to use in the development of immunoassays to detect C. pseudonaviculata and C. henricotiae in boxwood leaf and environmental samples. To date, rabbit polyclonal antibodies have been generated and are in testing for specificity and sensitivity. Under Objective 2: The use of sterilants that can be used to kill the boxwood blight pathogens, Calonectria pseudonvabiculata and C. henricotiae were evaluated. All life states of the pathogen are killed within seconds to exposure of 70% ethanol, indicating that this is an effective sterilant that can be used for decontaminating greenhouse surfaces and equipment. Hot water was also tested for treating boxwood cuttings. Hot water treatments were found to kill spores and microsclerotia of the boxwood blight pathogen as well as infested leaf litter, but differences were seen between the two species of pathogen, C. pseudonaviculata (now spreading in the U.S.) and C. henricotiae (currently only found in Europe). Also under Objective 2: Temperature tolerances of the two species of boxwood blight pathogens were also compared, with differences observed in spore germination, colony growth and disease incidence when the pathogens were incubated at different temperatures. This research will assist growers to keep their plants healthy through best management practices tailored to both species. 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 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 germplasm for resistance that can be deployed is an essential part of the strategy to combat this disease. Under Objective 2: Data on hourly temperature conditions throughout the growing season in wheat production areas of Brazil, Arkansas, North Carolina and Kansas were obtained. This information was used to develop weekly average minimum and maximum temperature profiles for programming low temperature growth chambers to determine the effects of temperature on survival of the wheat blast pathogen in infected leaf tissue. Protocols were designed and tested to develop a method for foliar inoculation, selection of the most appropriate wheat cultivar to be used in the study, a method to quantitate sporulation from infected leaf tissue, and the number of replications needed to statistically reduce sampling variability. Under Objective 4: The 2018 Northern Regional Winter Wheat (NRPN) was evaluated for wheat blast resistance based on 10 replications of each wheat line. Of the 45 lines examined, 16 showed less than 10% spike infection, 6 with no infection observed. There was a delay in beginning the evaluation 50 lines 2018 Southern Regional Winter Wheat (SRPN) due to electrical renovations in the greenhouse used for this study. The completion of the evaluation of the SRPN will be completed in July 2018. Under an MTA with Bayer- Crop Science, we completed the evaluation of 18 spring wheat lines. All lines tested were highly susceptible to disease. Twenty-two winter wheat line previously shown to be resistant in our greenhouse evaluations were sent to Bolivia (ANAPO) for field evaluation. Results are expected in August 2018. In cooperation with CIMMYT under an incoming agreement from USAID, we evaluated germplasm originating from the 2017 Elite nurseries of India, Nepal and Mexico for wheat blast resistance. The material was screened in under greenhouse conditions in under field conditions in Paraguay. These results showed 12 of the 96 lines averaging less than 20% spike infection and a few less than 5%. All of the resistant lines were from Mexico. No significant resistance was observed in lines from Nepal and India. Historically, the majority of resistant wheat lines we have tested worldwide have the 2NS translocation. In this study, 3 of the resistant lines did not possess the 2NS translocation and therefore may contain a new source of resistance not previously known. Under Objective 1: We previously developed a PCR-based assay for the detection of the Triticum pathotype of M. oryzae using a marker called MoT3. In collaboration with researches at Kansas State University, a new detection system based MoT3 using loop-mediated isothermal amplification technology was developed. This new method, in combination with in-filed DNA extraction methods, is a deployable technology that can be used for specific detection of the pathogen in the field. 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. Developing cultivars with durable host plant resistance is the preferred means of managing the disease. Under Objective 3: A candidate soybean gene that confers immunity to the soybean rust pathogen, Phakopsora pachyrhizi, has been identified. This gene, referred to as Rpp1 R4, encodes a protein with a domain not normally associated with resistance proteins. This finding provides an opportunity to identify one or more proteins produced by the pathogen, known as effectors, which may be important for pathogenicity. To identify potential P. pachyrhizi effectors that interact with Rpp1 R4, we created and screened a yeast-two hybrid library constructed from infected soybean leaves. The construction of a second library, using germinating P. pachyrhizi spores is underway. In a separate study performed in collaboration with researchers at Iowa State University, we screened 82 putative P. pachyrhizi effector proteins for biological functions. Seventeen of the 82 tested could suppress or activate immune responses in non-host N. benthamiana, Arabidopsis, tomato, or pepper plants. Under Objective 4: Germplasm screening efforts were conducted in collaboration with the University of Georgia and USDA-ARS Soybean/Maize Germplasm Pathology and Genetics Research Unit in Urbana, Illinois. An additional gene that confers resistance to soybean rust was identified. This gene, termed Rpp7, was mapped to a 154 Kb interval on chromosome 19. Breeding lines and F2 populations created using rust resistant soybean lines from field plots in Paraguay by ARS scientists at the Crop Genetics and Production Research Unit in Stoneville, Mississippi were inoculated with P. pachyrhizi isolates at Ft. Detrick and evaluated for resistance. Vectors for expressing Rpp1 have been designed and are currently being developed for creating transgenic soybean lines. Gene-specific vectors for gene silencing Rpp1b candidate genes have been designed and constructed and are currently being tested in soybean. 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: In collaboration with the USDA-ARS Cereal Disease Laboratory in St. Paul, Minnesota we received 72 samples of various wheat rust species under APHIS PPQ permit from foreign countries including Bhutan, Kenya and Spain. We tested and increased fungal material from all viable samples 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.

4. Accomplishments
1. Mitochondrial genome of the soybean red leaf blotch pathogen sequenced. Soybean red leaf blotch is a serious foliar disease of soybeans in sub-Sahara Africa caused by the fungal pathogen Coniothyrium glycines. The pathogen produces a resting spore that can survive for many years in the soil and infect soybeans. If the pathogen was introduced and became established in the U.S., there is the potential for significant yield losses to occur, and thus it is listed as a Select Agent by APHIS. The mitochondrial genome of C. glycines was sequenced and found to contain 13 protein-encoding genes, 2 ribosomal RNA subunits, 4 hypothetical proteins, and 30 transfer RNAs. Gene order and phylogenetic comparisons to all available fungal mitochondrial genomes supports current fungal taxonomy. This study provides the first genomic information on this fungal pathogen that might be useful for plant pathologists to develop targets for rapid diagnostic assays or strain identification.

2. Germplasm screening for resistance to wheat blast accelerated wheat variety release. Wheat blast, caused by the Triticum pathotype of the fungus Magnaporthe oryzae, is a devastating disease of wheat. Although the pathogen spread locally in South America for nearly 30 years, the disease was not reported elsewhere until 2016 when an outbreak occurred in Bangladesh. The outbreak resulted in significant yield loss in the affected area and threatened the release of a biofortified wheat variety, Bari Gom-33, developed in Bangladesh. In collaboration with the Mexico-based International Maize and Wheat Improvement Center (CIMMYT) under a cooperative agreement with Kansas State University, the variety was tested at the Foreign Disease-Weed Science Research Unit for resistance to wheat blast. The testing revealed good levels of resistance and enabled the accelerated release of the variety Bari Gom-33 for farmers in Bangladesh to grow.

Review Publications
Qi, M., Grayczyk, J.P., Seitz, J.M., Youngsill, L., Link, T.I., Choi, D., Pedley, K.F., Voegele, R.T., Baum, T.J., Whitham, S.A. 2018. Suppression or activation of immune responses by predicted secreted proteins of the soybean rust pathogen Phakopsora pachyrhizi. Molecular Plant-Microbe Interactions. 31:163-174.
Gladieux, P., Condon, B., Ravel, S., Soanes, D., Maciel, J.N., Nhani, A., Chen, L., Terauchi, R., Lebrun, M., Tharreau, D., Mitchell, T., Pedley, K.F., Valent, B., Talbot, N., Farman, M., Fournier, E. 2018. Gene flow between divergent cereal- and grass-specific lineages of the rice blast fungus Magnaporthe oryzae. mBio. 9:e01219-17.
Childs, S.P., King, Z.R., Walker, D.R., Harris, D.K., Pedley, K.F., Buck, J.W., Boerma, H.R., Li, Z. 2017. Discovery of a seventh Rpp soybean rust resistance locus in soybean accession PI 605823. Theoretical and Applied Genetics. 131:27-41.
Miller, M.E., Shishkoff, N., Cubeta, M.A. 2018. Thermal sensitivity of Calonectria henricotiae and Calonectria pseudonaviculata conidia and microsclerotia. Mycologia.
Qi, M., Link, T.I., Muller, M., Hirschburger, D., Pedley, K.F., Braun, E., Voegele, R.T., Baum, T., Whitham, S.A. 2016. A small cysteine-rich protein from the Asian soybean rust fungus, Phakopsora pachyrhizi, suppresses plant immunity. PLoS Pathogens. 12(9):e1005827. doi: 10.1371/journal.ppat.1005827.
Villari, C., Mahaffee, W.F., Mitchell, T.K., Pedley, K.F., Pieck, M.L., Peduto Hand, F. 2017. Early detection of airborne inoculum of Magnaporthe oryzae in turfgrass fields using a quantitative LAMP assay. Plant Disease. 101(1):170-177. doi: 10.1094/PDIS-06-16-0834-RE.
Cruz, C.C., Bockus, W.W., Stack, J.P., Valent, B.S., Maciel, J.N., Peterson, G.L. 2016. A standardized inoculation protocol to test wheat cultivars for reaction to head blast caused by Magnaporthe oryzae (Triticum pathotype). Plant Health Progress. 17:186-187.
Shishkoff, N. 2016. Survival of microsclerotia of Calonectria pseudonaviculata and C. henricotiae exposed to sanitizers. Plant Health Progress. 17:13-17. doi:10.1094/PHP-RS-15-0038.
Shishkoff, N. 2016. Survival of Calonectria pseudonaviculata in leaves, twigs, and discrete microsclerotia in sand at two moisture levels and five temperatures. Plant Disease. 100:2018-2024.