Location: Foreign Disease-Weed Science Research
2023 Annual Report
Objectives
Objective 1: Generate and utilize genomic and proteomic sequence information of emerging and threatening fungal plant pathogens to develop diagnostic assays.
Sub-objective 1.A: Develop accurate and rapid means for the identification and detection of foreign fungal plant pathogens.
Subobjective 1.B: Determine origins and distribution routes of foreign and recently introduced plant pathogens.
Sub-objective 2.B: Evaluate chemical control methods for emerging fungal plant pathogens.
Objective 2: Identify factors involved in fungal pathogen infection and virulence.
Sub-objective 2.A: Determine environmental factors affecting sporulation of fungal plant pathogens.
Sub-objective 2.B: Evaluate chemical control methods for emerging fungal plant pathogens.
Sub-objective 2.C: Identify genes and proteins contributing to virulence in fungal plant pathogens.
Sub-objective 2.D: Identify alternative hosts for fungal plant pathogens.
Objective 3: Screen germplasm and identify resistance genes to emerging and threatening fungal plant pathogens.
Sub-objective 3.A: Screen germplasm for resistance to foreign fungal plant pathogens.
Sub-objective 3.B: Identify accession-specific resistance genes for emerging and threatening fungal plant pathogens.
Sub-objective 3.C: Identify new races of fungal pathogens that can be controlled by existing resistance genes.
Approach
Genomic sequence information will be generated from host plants and foreign fungal plant pathogens. Bioinformatic analyses will be conducted to identify resistance genes, pathogenicity factors, markers for evaluating pathogen populations, and unique targets for DNA-based diagnostic assay development. Pathogen proteins or isoforms will be identified and used to generate antibodies to develop immunodiagnostic assays. Fungicides will be evaluated for their efficacy in controlling emerging fungal plant pathogens. Controlled environment growth chambers will be used to determine the effect of temperature and humidity on the sporulation of fungal plant pathogens. Containment greenhouses will be utilized to screen plant germplasm with foreign fungal plant pathogens to identify sources of host resistance.
Progress Report
Under Objective 1, Sub-objective 1.A, new custom-designed antibodies for the development of immunoassays to detect the boxwood blight fungal pathogens Calonectria pseudonaviculata and C. henricotiae were synthesized from a commercial vendor. Two new antibodies were tested for sensitivity using purified peptide targets. Sensitivity was sufficiently improved over previous antibodies. The antibodies are currently being tested against the boxwood blight pathogens and closely relates species to ensure specificity. Under Sub-objective 1.B, 120 isolates of C. pseudonaviculata from Oregon nurseries were received for genotypic analysis. These isolates were shown by ARS Oregon scientists to possess unique phenotypes (temperature sensitivity) that distinguish them from the accepted cardinal growth profiles of other known isolates of C. pseudonaviculata. Our goal is to determine how these unique isolates are related on a genetic level to characterized international and North American populations. DNA extractions have been completed, and methods to perform simple sequence repeat (SSR) genetic analysis were modified to be completed on in-house sequencing equipment. The outcome of the SSR data analyses will determine whether subsequent whole genome scale SNP analysis should be pursued to provide higher levels of data signal. Additionally, 108 isolates of Colletotrichum graminicola fungi were collected from 14 countries and subjected to whole genome DNA sequencing and analyses to determine genetic diversity of the pathogen on a global scale.
Under Objective 2, Sub-objective 2.A, a system was devised to control relative humidity over a 5-day period in small chambers containing detached boxwood twigs that had been infected with boxwood blight, and a method was selected to quantify sporulation on these twigs. This assay will be used to determine if there are different reactions to humidity among boxwood cultivars and whether this can help predict severity in different regions of the U.S. Under Sub-objective 2.B, 15 isolates of Calonectria for fungicide assays were collected from New York and Oregon and are being prepared for characterization. Under Sub-objective 2.C, a collaboration with researchers in Urbana, Illinois, and Nairobi, Kenya was established, and 85 new isolates of the Select Agent plant pathogen Coniothyrium glycines were obtained from countries in eastern and southern Africa. Devitalization protocols were tested on several new C. glycines isolates. A leaf disc inoculation method for C. glycines was developed, and DNA, RNA, and protein extraction protocols were tested. Assessments of genetic diversity of all C. glycines isolates is ongoing. To identify genes expressed at different stages of infection, RNA has been extracted from C. glycines sclerotia and will soon be extracted from mycelium. A collaboration was established with researchers working on foreign peanut smut disease with scientists in Stillwater, Oklahoma, and Buenos Aires, Argentina, and a USDA-APHIS permit for the importation of the causal agent of peanut smut, Thecaphora frezzii was obtained. Using surrogate smut species, inoculation procedures were developed. Three peanut lines highly susceptible to peanut smut were obtained from the USDA germplasm collection in Griffin, Georgia. Collectively, these advances will enable gene expression studies that will contribute to our understanding of peanut smut pathogenesis. Also under Sub-objective 2.C, RNA was isolated from resting spores, germinating spores, and appressoria of the soybean rust pathogen Phakopsora pachyrhizi. The RNA was used to construct cDNA libraries for gene expression analysis that will enable the discovery of genes required by the pathogen to infect soybean.
Under Objective 3, Sub-objective 3.A, a collaboration was established with researchers working on foreign peanut smut disease in Stillwater, Oklahoma, and Buenos Aires, Argentina, and a USDA-APHIS permit for the importation of the causal agent of peanut smut, Thecaphora frezzii was obtained. Seed from six species of wild peanut were obtained from the USDA germplasm collection in Griffin, Georgia, and protocols were developed for greenhouse propagation. This work establishes a foundation to study the genetic basis of host resistance in wild peanut species. Under Sub-objective 3.B, mapping data for the soybean rust resistance gene Rpp7 and the soybean genome sequence of the susceptible soybean cultivar ‘Williams 82’ enabled the identification of at least four candidate Rpp7 genes in the resistant soybean accession PI 605823. These partial sequences of the Rpp7 candidates will assist in the identification and sequencing of the entire Rpp7 gene and will be used for loss-of-resistance assays. The identification of Rpp7 will facilitate the breeding of soybeans with durable resistance against soybean rust. Under Sub-objective 3.C, 61 samples of various cereal rust species were received from foreign countries including Spain and Paraguay under an international USDA-APHIS permit. Viable rust pathogen material was increased on wheat, barley and/or oat seedlings. Rust spores and ethanol-killed infected wheat and barberry leaves from all samples were shipped to ARS researchers in St. Paul, Minnesota, for genotyping and wheat resistance screening. Also under Sub-objective 3.C, in consultation with ARS researchers in Hilo, Hawaii, a leaf disc inoculation method was developed to study Hemileia vastatrix, the causal agent of coffee leaf rust disease. Protocols for whole plant inoculations are being developed for spore increases and experiments requiring whole plants. DNA from five historical and one contemporary coffee rust isolates were sequenced using high-throughput DNA sequencing technology. Additional contemporary isolates of coffee rust were obtained from researchers in the Dominican Republic and Kenya. The historical and contemporary isolates will be used for analysis of the pathogen’s evolution and to perform race-typing, which will provide knowledge for mitigation methods.
Accomplishments
1. Foreign variants of corn pathogen may pose risk to U.S. crops. The U.S. is the largest producer, consumer, and exporter of corn in the world, with annual production valued at more than $82 billion. In the U.S., growers minimize damage to corn caused by anthracnose disease by planting corn varieties that are resistant to the pathogen populations that reside in North America. However, outside of North America, corn anthracnose is very difficult to control, and the disease is becoming increasingly widespread. Therefore, an international team of scientists including researchers at ARS Frederick, Maryland, compared the DNA profiles of anthracnose pathogens collected worldwide. The research showed that DNA profiles varied depending on the continent where the pathogen was collected, and that U.S. isolates have different DNA profiles different than foreign isolates. Since the effectiveness of crop resistance to disease depends on knowledge of the genetic structure of pathogens, the findings from this research guides efforts to breed corn that is resistant to variants of the anthracnose pathogen residing in the U.S. and globally.
Review Publications
Avin, F.A., Liyanapathiranage, P., Shishkoff, N., Swiggart, E., Gao, Y., Baysal-Gurel, F. 2023. First report of powdery mildew of American ginseng caused by Erysiphe heraclei in Tennessee and the United States. Plant Disease. 107:1950. https://doi.org/10.1094/PDIS-10-22-2310-PDN.
Brand, T., Beltz, H., Adhikari, U., Daughtrey, M., Luster, D.G., Kong, P., Hong, C. 2022. Evaluation of fungicides for management of boxwood blight caused by Calonectria spp. under field conditions in northern Germany. Journal of Plant Diseases and Protection. 130:325–335. https://doi.org/10.1007/s41348-022-00691-8.
Proaño-Cuenca, F., Carrera-Lopez, D., Luster, D.G., Zeller, K., Cardwell, K.F. 2023. Genome sequence resources for five isolates of Coniothyrium glycines, causal pathogen of red leaf blotch of soybean. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-10-22-0113-A.
Omolehin, O., Keller, J., Gouker, F.E., Daughtrey, M., Luster, D.G., Pscheidt, J., Hong, C. 2022. Combating an invasive boxwood pathogen – Calonectria pseudonaviculata – in the United States by shifting production to less susceptible cultivars . Plant Disease. https://doi.org/10.1094/PDIS-09-22-2124-RE.
Rogério, F., Baroncell, R., Cuevas-Fernández, F., Becerra, S., Crouch, J., Bettiol, W., Azcárate-Peril, A.M., Malapi-Wight, M., Ortega, V., Betran, J., Tenuta, A., Dambolena, J.S., Esker, P.D., Revilla, P. 2022. Population genomics provide insights into the global genetic structure of Colletotrichum graminicola, the causal agent of maize anthracnose. mBio. 14(1):02878-22. https://doi.org/10.1128/mbio.02878-22.
Brand, T., Beltz, H., Ehsen, B., Adhikari, U., Daughtrey, M., Luster, D.G., Kong, P., Hong, C. 2022. Multi-year field plantings evaluating boxwood cultivars for susceptibility to the blight pathogens (Calonectria spp.) in Northern Germany. Plant Disease. 107:713-719. https://doi.org/10.1094/PDIS-05-22-1102-RE.
