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United States Department of Agriculture

Agricultural Research Service


Location: Foreign Disease-weed Science Research

2010 Annual Report

1a. Objectives (from AD-416)
Identify genomic and phenotypic elements to characterize emerging and foreign fungal plant pathogens. Develop an understanding of the biology, genetics and epidemiology of emerging and foreign fungal plant pathogens. Screen germplasm for resistant sources to emerging and foreign fungal plant pathogens. Per PDRAM for Research on Emerging Fungal Plant Pathogens at Frederick, MD #R07 Objective 4: Develop rapid and reliable diagnostics for new and emerging foreign fungal plant pathogens that can be transferred to regulatory agencies and the private sector.

1b. Approach (from AD-416)
Utilize the Bio-Safety Level 3 Plant Pathogen Containment facilities to investigate and characterize virulence, genetic variability, epidemiology, host range, and survival of foreign and emerging fungal plant pathogens considered to be a threat to the U.S. Establish pathogen collections, compare exotic and endemic isolates for morphology, virulence, and genomics using a variety of scientific methods. Develop detection techniques using PCR and immunological methodologies. Investigate pathogen genomics and host plant resistance using classical and molecular approaches.

3. Progress Report
Soybean rust: Transmission electron microscopy experiments of soybean infection by the soybean rust pathogen, Phakopsora pachyrhizi were completed in cooperation scientists at Western Illinois University. Experiments monitoring soybean rust gene expression during the formation of a specialized structure needed for penetration of soybean leaves known as an appressorium were concluded. Approximately 1200 clones from an appressorium-enriched cDNA library were sequenced, and 31 unique appressorium clones were identified. Representative genes from 5 different functional groups were measured at various times during the infection process using quantitative real-time reverse transcription PCR. In collaboration with ARS scientists at Stoneville, MS rust resistant soybean germplasm from field trials in Paraguay and breeding lines containing the resistance genes Rpp3 and Rpp4 were evaluated for resistance to P. pachyrhizi isolates at the FDWSRU. In collaboration with an ARS scientist in Urbana, IL and at the International Institute of Tropical Agriculture, Ibadan, Nigeria, rust resistant soybean germplasm from field trials in Nigeria were inoculated with P. pachyrhizi isolates at the FDWSRU and evaluated for resistance. Approximately 140 candidate soybean genes that contribute to Rpp2-mediated resistance to soybean rust were screened using a virus-induced gene silencing approach. Eleven genes were found that compromise rust resistance when silenced. In collaboration with scientists at the Ohio State University, metabolite profiles of susceptible and resistant soybean lines were determined, and several known and novel metabolites have been discovered that are unique to a soybean rust resistant reaction. In collaboration with scientists at the University of Georgia, germplasm was screened to further characterize rust resistance in the soybean cultivar Hyuuga. Results indicate that Hyuuga carries the Rpp3 gene and a separate resistance gene located elsewhere in the genome. Red leaf blotch: A set of 23 ancestral soybean lines from the USDA soybean germplasm collection, Urbana, IL were inoculated by spraying 2 week-old plants with Phoma glycinicola. Preliminary data was obtained for 10 of the soybean lines from the ancestral set. Wheat blast: Two hundred and eighty two wheat cultivars from the USDA regional wheat germplasm collection were tested for resistance to spike infection using a Brazilian isolate of Magnaporthe grisea. Twenty nine wheat cultivars had less than 10% infection, while 4 wheat cultivars showed less than 1.0 % infection. The effect of relative humidity on conidia survival showed germination on water agar after 12 days at 93% relative humidity and 9 days at 12, 23, 43, and 75% relative humidity at 20, 25 and 30 degrees C. The effect of 10 dew periods from 0 to 32 hours after inoculation showed significant spike infection at all dew periods. Wheat spikes inoculated with 0, 100, 1,000, 10,000 and 100,000 conidia per ml resulted in 0, 13.8, 38.6, 61.7 and 80.0% infection, respectively. These results indicate the potential for establishment and spread to a broader range of climatic regions than previously thought.

4. Accomplishments
1. Protect U.S. wheat cultivars from foreign wheat blast: Wheat blast, caused by the fungal pathogen, Magnaporthe grisea, is a relatively new and emerging disease that is spreading in South America. First described in Brazil in 1986, the disease has recently begun to spread rapidly and cause significant yield losses. Fungicidal control is ineffective and no wheat cultivars have been found in Brazil that are resistant to all isolates of M. grisea. Screening U.S. wheat germplasm is necessary in order to prepare for the possible introduction of this rapidly emerging pathogen into the U.S. ARS scientists at the Foreign Disease-Weed Science Research laboratory at Ft. Detrick, Maryland successfully produced large amounts of inoculum, developed methods for inoculating wheat, and rating susceptibility and resistance, and identified a small number of U.S. wheat cultivars which may possess resistance to M. grisea. This information will be useful to government, academic and private sector researchers and breeders interested in developing blast-resistant wheat cultivars.

2. Visualization of leaf penetration by soybean rust: Phakopsora pachyrhizi is a fungus that causes a disease known as soybean rust. The pathogen is unusual because it enters the leaf directly through the surface of the leaf without entering pores called “stomates”, the more common means of entry. In order to better understand the initiation of soybean rust, a study was conducted by ARS scientists at the Foreign Disease-Weed Science Research laboratory at Ft. Detrick, Maryland to observe the process under high magnification using an instrument called an electron microscope. Our experiments showed that the fungus breached the leaf surface by directly penetrating through the waxy layer covering the surface by means of digestion, pressure, or combination of the two. Then the fungus digested its way through the outer leaf cell walls using chemicals called enzymes. Once in the leaf, the pathogen continued to penetrate internal leaf cells by digesting holes in the cell walls. An understanding of the mechanism by which the fungus enters the leaf and continues to spread is an important step in developing a better means to control the disease.

3. Water effects soybean rust spore survival: Soybean rust, caused by the fungus Phakopsora pachyrhizi, has spread from Asia to Africa, South America and recently to North America. Greenhouse and laboratory experiments were conducted by ARS scientists at the Foreign Disease-Weed Science Research laboratory at Ft. Detrick, Maryland to determine to effects of water on spore survival and germination. Studies showed that urediniospores of P. pachyrhizi from infected plants in the greenhouse and those from liquid nitrogen storage are killed if submerged in liquid water without prior hydration in a water-saturated atmosphere. However, most urediniospores survive submersion if allowed to float on the surface of water for a period of time (e.g. 1 hour). Maximum germination is obtained by over night hydration of urediniospores in a water-saturated atmosphere prior to placing in water to produce inoculum. This information is useful to government, academic and private sector researchers for production of soybean rust inoculum for research on resistance to ASR and may lead to a better understanding of how water and water vapor interact to affect establishment of infection.

4. Unique genes identified for infection by soybean rust: Soybean rust, caused by the fungus Phakopsora pachyrhizi, is a foliar disease that has the potential to cause substantial yield loss. U.S. soybean cultivars are highly susceptible to rust infection, so fungicides are the only control option available to growers. P. pachyrhizi infects by direct penetration of the leaf surface and requires the formation of a specialized infection structure know as an appressorium. ARS scientists at the Foreign Disease-Weed Science Research laboratory at Ft. Detrick, Maryland identified 31 genes that were uniquely expressed during appressorium formation. Comparative analyses revealed that these genes fell into functional categories of metabolism, cell cycle and DNA processing, protein fate, cellular transport, cellular communication and signal transduction, and cell rescue. This information will be useful to government, academic and private sector researchers for developing resistant soybean cultivars or targeting fungicides to control soybean rust.

5. Developed monoclonal antibodies as diagnostic reagents to detect soybean rust infected plants: Soybean rust is an important foliar disease in most countries where soybeans are grown, including the U.S. Effective control of soybean rust requires the early detection of rust-infected plants before symptoms appear and rust spore production allows the pathogen to continue to spread. ARS scientists at the Foreign Disease-Weed Science Research laboratory at Ft. Detrick, Maryland have developed antibodies raised against specific rust spore proteins that are highly specific for Asian soybean rust, do not cross react with known viral, bacterial or fungal soybean pathogens, and are useful for sensitive detection of the pathogen early in the infection process, before symptoms appear in the field. These antibodies will prove useful in commercialized diagnostic kit formats for growers, extension agents and consultants to develop preemptive control strategies before rust is able to propagate and spread.

Review Publications
Pedley, K.F. 2009. PCR-based assays for the detection of Puccinia horiana on chrysanthemums. Plant Disease. 93:1252-1258.

Stone, C.L., Posada-Buitrago, M.L., Boore, J.L., Frederick, R.D. 2010. Analysis of the complete mitochondrial genome sequences of the soybean rust pathogens Phakopsora pachyrhizi and P. meibomiae. Mycologia. 102(4):887-987.

Oh, C., Pedley, K.F., Martin, G.B. 2010. Tomato 14-3-3 protein 7 (TFT7) positively regulates immunity-associated programmed cell death by enhancing accumulation and signaling ability of MAPKKKalpha. The Plant Cell. 22(1):260-272.

Tooley, P.W., Carras, M.M., Sechler, A.J., Rajasab, A.H. 2010. Real-time PCR detection of sorghum ergot pathogens Claviceps africana, C. sorghi, and C. sorghicola. Journal of Phytopathology. doi: 10.1111/j.1439-0434.2010.01683.x

Last Modified: 2/23/2016
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