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United States Department of Agriculture

Agricultural Research Service

Research Project: IDENTIFICATION, CHARACTERIZATION, AND BIOLOGY OF EMERGING FOREIGN FUNGAL PLANT PATHOGENS
2008 Annual Report


1a.Objectives (from AD-416)
Identify genomic and phenotypic elements to characterize emerg9ing 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.


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
Chrysanthemum white rust: Fourteen isolates of Puccinia horiana were obtained. Using the sequence of the internal transcribed spacer regions, primers were designed and conventional and real-time PCR assays were developed that detect the pathogen in chrysanthemums prior to the onset of symptoms. Sorghum ergot: Primers have been tested for specificity using purified DNA samples and high specificity was observed for three sets of primers, developed to detect Claviceps africana, C. sorghi, and C. sorghicola. We confirmed the utility of these primers with October 2007 field samples from Indiana. Soybean rust: Twenty-four simple sequence repeat (SSR) markers were developed and evaluated on 28 isolates of Phakopsora pachyrhizi from 13 countries. Eighteen of these SSR markers were useful for assessing genetic variation among isolates in the ARS collection at Ft. Detrick. We have optimized the conditions for producing appressoria on polystyrene plates, extracted mRNA and sent it to a company to generate a suppressive subtraction hybridization cDNA library. We have analyzed the partial proteome of P. pachyrhizi, and have identified over 65 unique proteins expressed in germinating urediniospores. Two high titer proteins were identified that are nearly identical in length (108 and 109 amino acids, respectively) and share 64% amino acid sequence identity. To better understand the role of kudzu as alternative host, 125 kudzu accessions from 56 locations in the southeastern U.S. were inoculated with three isolates of P. pachyrhizi, and differences were observed in susceptibility depending upon the kudzu accession-isolate combination. No differences were observed in the optimal temperatures for urediniospore germination, germ tube growth and infection between U.S. and foreign isolates of P. pachyrhizi. Preliminary results on the effects of post-dew period temperatures on P. pachyrhizi colonization of soybean leaves and sporulation show that sporulation is greatly reduced above 33 C. Results on effects of temperature and moisture on P. pachyrhizi urediniospore longevity indicate that liquid moisture drastically diminishes urediniospore viability regardless of whether the urediniospores are frozen at -80 C or freshly harvested. Soybean Red leaf blotch: A screening method was developed using soybean cultivar "Williams" and a representative isolate of Phoma glycinicola from Zambia. The method involves tissue-grinder maceration of thin agar cultures of the pathogen containing hyphae and sclerotia, and spraying the suspension onto two week-old soybean seedlings followed by incubation in dew chambers. Wheat blast: Isolates of Magnaporthe grisea, the causal organism of wheat blast disease, have been obtained from collaborators at Kansas State University. Methods for culturing and storing isolates have been developed. Gladiolus rust: A study to determine the survivability of Uromyces transversalis under different temperature and humidity conditions has been completed. Gladiolus corms that were harvested from infected nursery plants in California were planted and grown out to determine if the infection was systemic. No infection was observed. NP303


4.Accomplishments
1. CHRYSANTHEMUM WHITE RUST MOLECULAR DIAGNOSTIC ASSAY: Chrysanthemum white rust, caused by Puccinia horiana, is a foliar disease of chrysanthemums that can result in severe losses to commercial chrysanthemum production. Chrysanthemum white rust is not established in the U.S., although it has been introduced and successfully eradicated. To facilitate the detection of chrysanthemum white rust in commercial nurseries, a rapid diagnostic specific to P. horiana has been developed. The internal transcribed spacer region was cloned and sequenced from 14 isolates of P. horiana, and PCR primers were designed and tested for both conventional and real-time PCR assays. Both PCR assays were capable of detecting the pathogen in chrysanthemum leaf tissue prior to the onset of symptoms and will be useful to government, academic and private researchers for detecting and identifying P. horiana in commercial nurseries. The Research was conducted under ARS National Program 303 (Plant Health), Component 1 (Disease Diagnosis, Identification and Characterization of Plant Pathogens), Problem Statement 1B (Detection, Identification, Characterization, and Classification of Pathogens).

2. SOYBEAN GENE EXPRESSION IN RESPONSE TO RUST INFECTION: Soybean rust caused by the fungal pathogen, Phakopsora pachyrhizi, is a serious foliar disease of soybeans, and the disease was discovered in the continental U.S. for the first time in November 2004. Previous research identified four single genes, Rpp1-4, in soybean for resistance to soybean rust. Using microarrays, we identified 558 soybean genes that are differentially expressed in the Rpp1-mediated resistant reaction. The majority of the upregulated genes fell into the class of defense genes, with peroxidases and lipoxygenases being the most prevalent. Down-regulated genes included cell-wall-associated proteins, such as extensins, proline-rich proteins, and xyloglucan endotransglycosylases. The genes identified in this study are candidates for gene transfer experiments for government, academic and private researchers to determine if they have a functional role in resistance to soybean rust. The Research was conducted under ARS National Program 303 (Plant Health), Component 3 (Plant Disease Resistance), Problem Statement 3A (Mechanisms of Plant Disease Resistance).

3. SOYBEAN RUST PROTEOMICS: Phakopsora pachyrhizi, the causal agent of Asian soybean rust, has become established over an expanding range in the U.S., resulting in significant annual costs to soybean producers in the Southeast. Our efforts focus on identifying extracellular fungal proteins that may play roles in adhesion or early infection processes as the rust fungus penetrates the soybean leaf. We have identified over 65 unique proteins in germinating spores (urediniospores). These proteins may provide new targets for novel fungicides, and diagnostic antibodies have been generated against two PHEP proteins to rapidly identify the disease in the field. The Research was conducted under ARS National Program 303 (Plant Health), Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors), Problem Statement 2A (Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen).

4. SOYBEAN RUST EPIDEMIOLOGY: Soybean rust caused by the fungal pathogen, Phakopsora pachyrhizi, is a serious foliar disease of soybeans and has been reported in most countries where soybeans are grown. Experiments were conducted to determine effects of temperature on soybean rust spore (urediniospore) germination, and on initiation and development of soybean rust in infected plants using P. pachyrhizi isolates collected from international sources and the initial U.S. outbreak. U.S. and foreign isolates of P. pachyrhizi collected over a period of 32 years were found to behave the same with respect to how temperature affects on spore germination and initiation of infection. This information will be valuable to government, academic and private researchers in developing accurate predictive soybean rust disease models. The Research was conducted under ARS National Program 303 (Plant Health), Component 2 (Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors), Problem Statement 2A (Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen).


5.Significant Activities that Support Special Target Populations
None


6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings10

Review Publications
Allen, T.W., Workneh, F., Steddom, K.C., Peterson, G.L., Rush, C.M. 2008. The influence of tillage on dispersal of Tilletia indica teliospores from a concentrated point source. Plant Disease. 92:351-356.

Inman, A., Magnus, H.A., Riccioni, L., Hughes, K., Coates, M., Barnes, A., Barton, V., Sansford, C.E., Valvassori, M., Di Giambattista, G., Port-Puglia, A., Razzaghian, J., Peterson, G.L. 2008. Survival of Tilletia indica teliospores under European soil conditions. Plant Pathology. 57(2):290-300.

Riccioni, L., Inman, A., Magnus, H.A., Valvassori, M., Di Giambattista, G., Porta-Puglia, A., Hughes, K., Coates, M., Bowyer, R., Barnes, A., Sansford, C.E., Razzaghian, J., Prince, A., Peterson, G.L. 2008. Susceptibility of European bread and durum wheat cultivars to Tilletia indica. Plant Pathology. doi 10.1111/j 1365-3059.2008.01830.X

Bonde, M.R., Nester, S.E., Berner, D.K., Frederick, R.D., Moore, W., Little, S. 2008. Quantitative Susceptibility of Legume Species to Infection by Phakopsora pachyrhizi. Plant Disease. 92:30-36.

Slaminko, T.L., Miles, M.R., Frederick, R.D., Bonde, M.R., Hartman, G.L. 2008. New legume hosts of Phakopsora pachyrhizi based on greenhouse evaluations. Plant Disease. 92:(5)767-771.

Bandyopadhyay, R., Ojiambo, P.S., Twizeyimana, M., Asafo-Adjei, B., Frederick, R.D., Pedley, K.F., Stone, C.L., Hartman, G.L. 2007. First Report of Soybean Rust Caused by Phakopsora pachyrhizi in Ghana. Plant Disease. 91(8):1057.

Ojiambo, P.S., Bandyopadhyay, R., Twizeyimana, M., Lema, A., Frederick, R.D., Pedley, K.F., Stone, C.L., Hartman, G.L. 2007. First Report of Rust Caused by Phakopsora pachyrhizi on soybean in Democratic Republic of Congo. Plant Disease. 91(9):1204.

Miles, M.R., Morel, W., Ray, J.D., Smith, J.R., Frederick, R.D., Hartman, G.L. 2008. Adult Plant Evaluation of Soybean Accessions for Resistance to Phakopsora pachyrhizi in the Field and Greenhouse in Paraguay. Plant Disease. 92(1):96-105.

Alvord, W.G., Roayaei, J., Quinones, O.A., Schneider, K. 2007. A microarray analysis for differential gene expression in the soybean genome using bioconductor and r. Briefings in Bioinformatics. 1093:43.

Miles, M.R., Levy, C., Morel W., Mueller, T., Steinlage, T., van Rij, N., Frederick, R.D., Hartman, G.L. 2007. International fungicide efficacy trials for management of soybean rust. Plant Disease. 91(11):1450-1458.

Choi, J.J., Alkharouf, N.W., Schneider, K., Matthews, B.F., Frederick, R.D. 2008. Expression patterns in soybean resistant to Phakopsora pachyrhizi reveal the importance of peroxidases and lipoxygenases. Functional and Integrative Genomics. 8:341-359

Last Modified: 10/23/2014
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