Location: Foreign Disease-Weed Science Research
2022 Annual Report
Objectives
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.
Approach
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
Under Objective 1, antibodies developed for a boxwood blight detection assay were evaluated for specificity and sensitivity. The assay was determined to be highly sensitive and capable of detecting boxwood blight pathogens collected from diverse geographic regions. Further development of an improved, field-deployable assay is underway. Under Objective 2, boxwood cultivars were evaluated for their response to multiple isolates of boxwood blight pathogens at four different temperatures. The results indicated that temperature contributes to the variable response of boxwood cultivars and that Calonectria henricotiae causes more severe disease than C. pseudonaviculata. Under Objective 3, growth media and environmental conditions were evaluated to determine optimal cultural conditions to promote hyphal growth and sclerotia production by the soybean red leaf blotch fungus Coniothyrium glycines. RNA isolation methods were evaluated for future RNA-Seq analysis aimed at identifying genes that are required for pathogenesis. Also under this objective, whole-genome sequence analysis was performed for two isolates of Magnaporthe oryzae, the causal agent of wheat blast, collected in Zambia. The results of this analysis indicate that the M. oryzae Zambia isolates are related to South American isolates of the pathogen, and that they are distinctly different from isolates of the pathogen discovered in Bangladesh in 2016. Optimal growth conditions and RNA isolation methods were determined for Coniothyrium glycines, the causal agent of soybean red leaf blotch. Also under this objective, whole-genome sequence analysis was performed for two isolates of Magnaporthe oryzae, the causal agent of wheat blast, collected in Zambia. The results of this analysis indicate that the M. oryzae isolates collected in Zambia originated in South America. Under Objective 4, the region of the soybean genome containing the soybean rust resistance gene Rpp1b was sequenced and used to develop molecular tools to confirm its function. Gene expression analysis of the soybean rust Rpp3-candidate genes was completed and used to confirm the identity of the gene that confers resistance. Preliminary screening for wheat blast resistance in the 2020-21 Uniform Southern Soft Red Winter Wheat and the 2020-21 Emmer Wheat nurseries was completed. Samples of various cereal rust species on wheat, barberry, and native grasses were received from Spain, Kenya and Ethiopia and propagated on wheat, oat and rye seedlings in a containment greenhouse. Sixty-one samples of the foreign rust isolates were forwarded to the ARS Cereal Disease Lab for genotyping and wheat germplasm resistance screens.
Over the life of the project (2017-2022) significant findings and resources were generated for foreign fungal pathogens that cause soybean red leaf blotch, cereal rusts, wheat blast, soybean rust, and boxwood blight diseases. Critical data on the biology of the causal agent of red leaf blotch of soybean is required for the development of predictive models for management of the pathogen, should it enter and become established in the U.S. To this end, heat and chemical sterilization methods were evaluated, leading to the development of protocols for safely handling the pathogen. Growth media and environmental conditions were also evaluated to determine optimal cultural conditions to enable further characterization of the pathogen in the laboratory. Wheat blast is a severe threat to global wheat production, and the identification of wheat lines with resistance to the disease and development detection methods were research priorities. Greenhouse and field inoculation protocols were designed, tested, and standardized to screen wheat germplasm from over 1100 lines obtained from U.S. regional and international nurseries. One potential source of resistance to wheat blast disease from the wild wheat relative Aegilops ventricose was identified in multiple wheat cultivars. For detection of the wheat blast pathogen, we built upon a previously developed assay to create a field-deployable technology that can be used for specific detection. Using our diagnostic protocols, we confirmed the presence of the wheat blast pathogen in samples collected in Zambia. Further molecular characterization of these wheat blast isolates revealed that isolates collected in Zambia originated in South America. Durable resistance to soybean rust is one of the most effective long-term strategies to combat the disease. In collaboration with university and other ARS laboratories, soybean breeding lines were screened for resistance to soybean rust and were found to contain the resistance genes and/or alleles of Rpp1, Rpp1b, Rpp2, Rpp3, or Rpp4 which will enhance breeding efforts. An additional gene, Rpp7, was also identified and mapped within the soybean genome. Molecular characterization of the soybean Rpp1 gene and proteins secreted by the soybean rust pathogen have provided new information that may lead to novel control measures. In boxwood blight research, population genetics demonstrated that only one clonal lineage of the boxwood blight pathogen was widespread in the U.S. Research on endemic and foreign boxwood blight pathogens showed that some boxwood cultivars were more susceptible to blight than others, but that environment was also an important factor in disease severity. Spores and overwintering propagules of the boxwood blight pathogens are sensitive to heat, and heat treatment of rooted boxwood cuttings was found to be feasible for some cultivars. To better equip extension personnel to correctly diagnose boxwood blight, research was performed to clarify the life cycle and host range of the pathogen. Rapid diagnostic tests were also developed that will provide growers the ability to rapidly identify and remove infected plantings. These tests will also enable breeders to rate susceptibility of breeding stock to develop new boxwood varieties with resistance to this devastating disease. Molecular studies identified more than 120 secreted proteins from the boxwood blight pathogen, paving the way for downstream breeding programs aimed at developing blight-resistant boxwood cultivars. Over the life of this project over 1300 samples of various wheat rust species were received, propagated, characterized, and archived. Samples of each isolate were then shipped to the ARS Cereal Disease Laboratory, St Paul, MN for fine level SNP-based genotyping and in-depth wheat germplasm resistance screens. Together, these efforts contribute to the global surveillance of cereal rusts with the goal of identifying new races of the pathogen in order to deploy resistant cultivars.
Accomplishments
Review Publications
Aiello, D., Guarnaccia, V., Vitale, A., LeBlanc, N.R., Shishkoff, N., Polizzi, G. 2022. Impact of Calonectria diseases on ornamental horticulture: Diagnosis and control strategies. Plant Disease. 106(7):1773-1787. https://doi.org/10.1094/PDIS-11-21-2610-FE.
Yang, X., Chastrogudin, V.L., Daughtrey, M.L., Loyd, A., Weiland, G.E., Shishkoff, N., Baysal-Gurel, F., Santamaria, L., Salgado-Salazar, C., Lamondia, J., Crouch, J., Luster, D.G. 2021. A diagnostic guide of Volutella blight affecting Buxaceae. Plant Health Progress. 22:578-590. https://doi.org/10.1094/PHP-02-21-0052-DG.
Yang, X., Mcmahon, M.B., Ramachandran, S.R., Garrett, W.M., Leblanc, N.R., Crouch, J., Shishkoff, N., Luster, D.G. 2021. Comparative analysis of extracellular proteomes reveals effectors of the boxwood blight pathogens, Calonectria henricotiae and C. pseudonaviculata. Bioscience Reports. 41(3):BSR20203544. https://doi.org/10.1042/BSR20203544.
Olivera, P.D., Villegas, D., Cantero-Martinez, C., Szabo, L.J., Rouse, M.N., Luster, D.G., Bartaula, R., Lopes, M.S., Jin, Y. 2022. A unique race of the wheat stem rust pathogen with virulence on Sr31 identified in Spain and reaction of wheat and durum cultivars to this race. Plant Pathology. 71(4):873-889. https://doi.org/10.1111/ppa.13530.
Villegas, D., Bartaula, R., Cantero-Martinez, C., Luster, D.G., Szabo, L.J., Olivera, P.D., Berlin, A., Rodriguez-Algaba, J., Hovmøller, M.S., Mcintosh, R., Jin, Y. 2022. Barberry plays an active role as an alternate host of Puccinia graminis in Spain. Plant Pathology. 71:1174-1184. https://doi.org/10.1111/ppa.13540.
Valent, B., Cruppe, G., Stack, J.P., Cruz, C.D., Farman, M.L., Paul, P.A., Peterson, G.L., Pedley, K.F. 2021. Recovery plan for wheat blast caused by Magnaporthe oryzae pathotype Triticum. Plant Health Progress. 22:182-212. https://doi.org/10.1094/PHP-11-20-0101-RP.
