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United States Department of Agriculture

Agricultural Research Service

Research Project: Emerging Foreign Fungal Plant Pathogens: Detection, Biology, and Interactions with Host Plants

Location: Foreign Disease-weed Science Research

2013 Annual Report

1a. Objectives (from AD-416):
1: Generate and utilize genomic sequence information of foreign fungal plant pathogens to develop diagnostic assays. 1.A - Develop accurate and rapid means for identification and detection of foreign fungal plant pathogens. 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. 2.A - Determine the effects of temperature and moisture on infection and development of disease. 2.B - Determine the effects of temperature and moisture on survival of foreign fungal plant pathogens. 3: Identify genes and proteins required for infection and pathogenicity of foreign fungal plant pathogens. 3.A - Determine genome organization and gene structure of foreign fungal plant pathogens. 3.B - Identify secreted proteins from foreign fungal plant pathogens. 4: Screen germplasm and identify resistance genes to foreign fungal plant pathogens. 4.A - Screen germplasm for resistance to foreign fungal plant pathogens. 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. In addition, the genomic sequences will be mined to identify unique target sequences to develop rapid DNA-based diagnostic assays. Unique or pathogen-specific proteins will be identified and used to generate antibodies to develop immunodiagnostic assays. Putative secreted proteins identified from bioinformatic analyses will be evaluated using a yeast secretion assay. Temperature-controlled dew and growth chambers will be used to determine effects of dew-period temperatures and durations on establishment of infection and on pathogen survival. The number and size of lesions will be measured, and the amount of sporulation will be determined. For some diseases, plant sections will be examined histologically using both light and transmission electron microscopy for systemic infection to determine if the pathogen overwinters in infected tissue. Pathogenicity studies will be performed on selected plant species to determine host range and if the pathogen can survive winters in the absence of the primary host. Germplasm will be inoculated foreign fungal plant pathogens and screened for resistance.

3. Progress Report:
Chrysanthemum white rust: Polyclonal antibodies were generated in rabbits to two unique proteins identified from P. horiana basidiospores by two-dimensional gel electrophoresis and mass spectrometry and are being tested for sensitivity and specificity. In growth chamber studies simulating fall, winter, and spring temperatures in the northeastern U.S., the pathogen was found to survive through winter in systemically infected chrysanthemum plants. Teliospores survived on and in soil a maximum of 16 weeks. Gladiolus rust: Various stains and methods to differentiate the pathogen from host cells using light microscopy were evaluated. In growth chamber studies, a diurnal temperature pattern with 33 degrees C high and 24 degrees C low severely limited disease development. Wheat blast: One hundred isolates of M. oryzae were obtained from Brazil, 20 from Bolivia, and 20 from Paraguay. Isolates of M. oryzae were also collected from other non-wheat grass species in the U.S. A Bolivian isolate of M. oryzae was transformed with a vector that confers resistance to geneticin. One transformant was used to inoculate barley and a highly susceptible wheat cultivar to generate the infected plant material to conduct survival studies. Forty of 175 wheat cultivars inoculated with a Brazilian isolate of M. oryzae, T-25, showed less than 25 percent infection. When these wheat cultivars were inoculated with B-2, an isolate from Bolivia, four cultivars were immune to both M. oryzae isolates. Soybean rust: ESTs of four putative secreted proteins identified in P. pachyrhizi were cloned into a yeast signal sequence trap vector to create an in-frame fusion protein with invertase. All four constructs secreted invertase in yeast confirming functional secretion signal peptide sequences. In collaboration with an ARS scientist at Beltsville, Maryland, 56 wild common bean lines from Central and South America were inoculated with P. pachyrhizi isolates and evaluated for resistance. F2 populations created using rust resistant soybean lines from field plots in Paraguay by ARS scientists at Stoneville, MS were inoculated with P. pachyrhizi isolates and evaluated for resistance. Gene specific vectors were constructed that target the rust resistance genes Rpp1, Rpp1b, and Rpp3, and putative loss-of-resistance phenotypes were detected for Rpp1 and Rpp3. Soybean accession PI 462312 contains the rust resistance gene Rpp3. Plants were inoculated P. pachyrhizi isolates that result in resistant and susceptible reactions. Metabolites were extracted from tissue samples collected at 0, 10, 24, 72, 144, 216, and 244 hours post inoculation, and sent to collaborators at Ohio State University for High Performance Liquid Chromatography. Red leaf blotch of soybeans: P. glycinicola sclerotia were tested for low temperature survival, and 100 percent survival was observed after 159 days at 0 degrees C and 104 days at -20 degrees C, indicating that P. glycinicola sclerotia can survive long periods of freezing.

4. Accomplishments
1. High temperatures slow development of soybean rust. Soybean rust is a devastating pathogen, often incurring substantial losses to producers. Experiments conducted by ARS researchers at Frederick, Maryland demonstrated that Phakopsora pachyrhizi, causal agent of soybean rust, is affected by diurnal temperature highs commonly occurring in southeastern U.S. When temperatures peaked at 35 degrees C for three or more consecutive days, disease development was greatly restricted. These temperature highs regularly occur in southeastern U.S., in some instances for 15 or more consecutive days. Because development of soybean rust in mid-western and northern states requires the inoculum to come in from southeastern states, temperatures in southeastern U.S. affect the entire U.S. soybean production. This information is important for developing soybean rust disease forecasting models.

2. Chrysanthemum white rust systemically infects plants. First discovered in the U.S. in 1977, Puccinia horiana is a quarantine-significant fungal pathogen and causal agent of Chrysanthemum white rust (CWR). The pathogen was believed to have been eradicated in the U.S. but recently has re-appeared on several occasions in northeastern U.S. It is important to understand how CWR infects plants and its potential to overwinter in order to implement effective control methods and eradication measures. Inoculated chrysanthemum plants were placed in a growth chamber simulating fall, winter, and spring temperatures in northeastern U.S. and newly formed stem tissues displaying CWR symptoms were examined scanning and transmission electron microscopy by ARS researchers at Frederick and Beltsville, Maryland. Results showed P. horiana in the crown, roots, and newly developed stems and, therefore, can systemically infect chrysanthemum plants and potentially over-winter. This information is important to growers and regulatory agencies as they evaluate control measures and quarantine options.

3. Genome sequences obtained for isolates of wheat blast. Wheat blast, caused by the Triticum pathotype of the fungus Magnaporthe oryzae, is a potential threat to wheat production. In several South American countries, wheat blast has lead to severe losses when environmental conditions favor disease. In the U.S., closely related pathotypes of M. oryzae that cause disease on rice and turf grasses are endemic. Utilizing next-generation sequencing technology, the genome sequences of three isolates of the Triticum pathotype were obtained by collaborators at Kansas State University. Comparisons of the Triticum pathotype genomic sequences to those of the endemic pathotypes will allow for the identification of unique molecular markers for the development of molecular based diagnostic assays by ARS researchers at Frederick, Maryland.

Review Publications
Zhang, X., Freire, M., Le, M., Hartman, G.L., Upchurch, R.G., Pedley, K.F., Stacey, G. 2012. Genetic diversity and origins of Phakopsora pachyrhizi isolates in the United States. Asian Journal of Plant Pathology. 6:52-65.

Morales, A., O'Rourke, J.A., Van De Mortel, M., Schneider, K., Bancroft, T., Borem, A., Nelson, R., Nettleton, D., Baum, T., Shoemaker, R.C., Frederick, R.D., Abdelnoor, R.V., Pedley, K.F., Whitham, S.A., Graham, M.A. 2013. Transcriptome analyses and virus induced gene silencing identify genes in the Rpp4-mediated Asian soybean rust resistance pathway. Functional Plant Biology. DOI:10.1071/FP12296.

Bonde, M.R., Nester, S.E., Berner, D.K. 2013. Effects of frequency of extreme temperature highs on development of soybean rust. Phytopathology. 103:708-716.

Debacker, M., Bonants, P., Pedley, K.F., Maes, M., Roldan-Ruiz, I., Van Bockstaele, E., Heugens, K., Van Der Lee, T. 2013. Genotypic diversity of Puccinia horiana based on newly identified molecular markers. Molecular Ecology.

Last Modified: 05/28/2017
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