2008 Annual Report 1a.Objectives (from AD-416) 1: Develop and use quantitative methodologies to detect, identify, and characterize soybean pathogens and monitor the spread of disease epidemics. 2: Identify viral and host determinants of horizontal and vertical transmission of soybean viruses. 3: Identify and improve germplasm with soybean disease resistance using phenotypic and marker-assisted selection. 4: Develop and improve integrated strategies for sustainable disease management in soybean. Sub-objectives: a. Determine the efficacy of fungicides and optimize cultural practices for the management of soybean rust. b. Develop and validate biological control protocols for bacterial agents against soybean cyst nematode. 1b.Approach (from AD-416) 1) Pathogens will be collected from diseased plants, air, soil, and water and phenotypically and molecularly characterized to identify nucleotide sequences that will be used to quantitatively identify pathogenic organisms and to determine genetic variability of pathogens. . 2)To identify viral determinants of transmission, transmission phenotypes of chimeric viruses constructed from viruses differing in transmission properties will be evaluated; regions of the soybean genome involved in seed transmission will be identified using molecular markers and populations of soybean plants differing in transmission of virus through seed.. 3)Potential sources of soybean resistance genes and populations segregating for resistance will be evaluated in nurseries and other sites appropriate for resistance screening tests.. 4)Soybean germplasm accessions with resistance will be crossed with selected cultivars and lines to create populations used for inheritance and mapping studies. Fungicide trials will evaluate fungicide efficacy, sprayer/delivery technology, timing and frequency of applications, possible interactions between fungicides and soybean genotypes, row spacing, irrigation, and plant architecture on soybean rust infection. Molecular techniques will be developed to identify and quantify obligately parasitic, biological control bacteria in soybean cyst nematode populations and in field soil and culture of bacteria will be attempted using proprietary technology. 3.Progress Report Assays to detect and quantify plant pathogens in various traps and in host tissue were developed for pathogens that cause soybean rust (SBR) and sudden death syndrome (SDS). Techniques were developed to bioassay isolate and pest collections to determine if biotypes or races could be detected. Highly sensitive fluorescence assays were used to detect virus infections in soybean, which showed that BPMV remains the most prevalent virus in Illinois commercial soybean fields followed by SbDV, AMV, SMV, TRSV and TSV. The variability in internal transcribed spacers of ribosomal RNA gene clusters was examined within and among 20 soybean cyst nematode (SCN) populations. In addition, the genetic variability within and among 24 SCN populations was examined using a single copy gene, Hg-hsp90. This addresses NP303 Problem Statements 1A: New Diagnostic Methods and Tools; 1B: Detection, Identification, Characterization, and Classification of Pathogens; and 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. A series of chimeric viruses were constructed by recombining portions of genomes of two Soybean mosaic virus (SMV) isolates that differed in their rates of seed transmission. The seed transmission of the chimeric viruses is being evaluated. To identify regions of the Soybean dwarf virus genome that are required for packaging of viral RNA into virus particles and subsequent transmission by aphids, a series of mutant viruses were generated and their characterization was initiated. The first repetition of experiments for mapping genes for resistance to seed transmission of SMV was completed. The second repetition is being analyzed phenotypically and with molecular markers. This addresses NP303 Problem Statement 2B: Plant-Microbe-Vector Interactions. Germplasm accessions were evaluated for SBR resistance at seven locations in five states in a collaborative study between ARS and university researchers. The 2007 data were compiled and compared to data from previous years to identify the most resistant accessions. This information was made available to soybean breeders. Breeding and gene mapping populations with aphid, SBR, SDS, and virus resistance were developed and/or advanced, setting the stage for a series of mapping studies that will allow resistance genes to be tagged with markers. Data from several international SBR nurseries were summarized and published. Progress also was made to identify and improve germplasm with soybean disease resistance genes using phenotypic and marker-assisted selection. This addresses NP303 Problem Statement 3B. Disease resistance in new germplasm and varieties. Experiments on fungicide application timing and yield are being reevaluated. Data were taken on differential soybean phytosensitivity to the fungicide tebuconazole (Folicur). A study on fungicides on mammalian cell cytotoxicity was completed and further biochemical assays to evaluate natural products on inhibition of spore germination are in progress. This addresses NP303 Problem Statement 4C: Application of Sustainable Disease Management Tools. 4.Accomplishments 1. Soybean aphid biotype diversity identified in North America. The soybean aphid, Aphis glycines, is an invasive insect pest of soybean that was first reported in North America in 2000. Soybean germplasm was screened for aphid resistance and several resistance sources were identified. The objective of this research was to test for aphid biotype variation. All six aphid resistance sources provided resistance to the Illinois soybean aphid biotype, however, the Ohio soybean aphid biotype defeated the aphid resistance gene Rag1 from ‘Dowling’ and Rag from 'Jackson'. PI200538 and PI567597C were resistant to both the Ohio and Illinois biotypes and will be useful sources of resistance to both isolates. These results are the first to confirm that there are at least two distinct biotypes of A. glycines in North America. This research is highly relevant to scientists involved in improving soybean productivity. This research address National Program 303 Action Plan components: 1. Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; and 2. Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors. Problem Statements 1B: Detection, Identification, Characterization, and Classification of Pathogens, and 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. 2. Defense responses in soybean against Aphis glycines. Soybean resistance to the soybean aphid depends on defense-related responses. Gene expression profiles in resistant (Dowling) and susceptible (Williams 82) soybean varieties were compared over time with and without aphid infestation using cDNA microarrays consisting of 18,000 soybean-expressed sequence tags. Expression patterns of defense-related genes were examined over time. Five genes with constitutively higher expression levels were found in Dowling, the resistant soybean variety. This study enhances genomic information about defense response in soybean and may accelerate breeding for resistance. This research addresses National Program 303 Action Plan Component 3: Plant Disease Resistance. Problem Statement 3A: Mechanisms of Plant Disease Resistance. 3. Potential soybean rust resistance sources identified and confirmed. Soybean rust causes significant yield losses in soybean in areas where it occurs regularly. In international nurseries, 534 soybean plant introductions (PIs) from maturity groups III through IX that had been selected in greenhouse seedling screens were evaluated for SBR resistance in a field trial at Centro Regional de Investigación Agrícola in Capitán Miranda, Paraguay during the 2005-2006 growing season. Two lines were immune in both the field and greenhouse evaluations. In addition, six soybean lines had consistently the lowest level of disease severity across years and locations in Nigeria. In nurseries at the North Florida Research and Education Center (NFREC), 405 PIs were evaluated for resistance to North American SBR isolates in 2007. The resulting data and ratings from a similar trial conducted in Fairhope, AL were similar and confirmed that 103 PIs showed SBR resistance at both locations and at other sites in the Southeast. Adult plant resistance to SBR must be confirmed in multiple locations and years to assess the utility of the soybean lines in breeding efforts. These PIs can be used immediately by breeders (many have already been used in crosses), and the multiple sources of resistance will permit the eventual construction of resistance gene pyramids that should provide durable resistance to SBR. This research addresses National Program 303 Action Plan Component 3: Plant Disease Resistance. Problem Statement 3B: Disease resistance in new germplasm and varieties. 4. Identified a wide range of reaction phenotypes among germplasm accessions with resistance to soybean rust. Evaluation of germplasm on the basis of disease severity and development of either no lesions or reddish brown lesions may not provide an accurate assessment of SBR resistance in some soybean lines and populations. Microscopic examination of disease reactions of germplasm tested in the field at the NFREC in 2007 revealed that lesion color, density and size can vary tremendously among resistant PIs, and similar observations were made on nine putatively resistant PIs and four susceptible cultivars grown in the greenhouse. This indicates that conventional criteria used to rate SBR resistance may not be appropriate for certain germplasm accessions or breeding populations derived from them. It also indicates that resistance mechanisms may differ among resistant soybean accessions. This information may be useful for developing more robust resistance gene pyramids comprised of genes from different PI sources. This research addresses National Program 303 Action Plan Component 3: Plant Disease Resistance. Problem Statement 3B: Disease resistance in new germplasm and varieties. 5. New Legume Hosts of the Soybean Rust pathogen. Soybean rust was first found in the continental U.S. in 2004, and has been found on soybean, kudzu, Florida beggarweed, and three Phaseolus species in the field so far in the U.S. The pathogen has been reported to occur on over 90 legume species worldwide, and it is likely to infect native and introduced legume species in the U.S. The objective of this study was to determine whether any of 176 species representing 57 genera of legumes, the majority of which are either native or naturalized to soybean growing areas of the U.S., could be hosts of Phakopsora pachyrhizi, the fungus causing soybean rust. Phakopsora pachyrhizi was confirmed by the presence of sporulating uredinia and/or immunological assay on 65 new species in 25 genera, 12 of which have not been reported previously as hosts. Many of the newly identified hosts grow in the southern U.S., and could, like kudzu, serve as over wintering hosts for P. pachyrhizi. This information is important to pathologist, epidemiologist, and others interested in knowing more about potential over wintering hosts in the U.S. This research addresses National Program 303 Action Plan Component 2: Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors. Problem Statement 2C: Population Dynamics, Spread, and Epidemiology of Pathogens. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of the New MTA (providing only) 2 Number of New Germplasm Releases 1 Number of New Commercial Licenses Executed 9 Number of Web Sites Managed 2 Review Publications Atibalentja, N., Noel, G.R. 2008. Bacterial endosymbionts of plant-parasitic nematodes. Symbiosis. 46(2):87-93. Li, S., Lygin, A., Zernova, O., Lozovaya, V., Hartman, G.L., Widholm, J.M. 2008. Genotype Response of Soybean (Glycine max) Whole Plants and Hairy Roots to Fusarium solani f. sp. glycines Infection. Soybean Science. 27:275-282. 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. Thekkeveetil, T., Hobbs, H.A., Wang, Y., Kridelbaugh, D., Donnelly, J., Hartman, G.L., Domier, L.L. 2007. First report of Soybean dwarf virus in soybean in Northern Illinois. Plant Disease. 91(12):1686. Twixeyimna, M., Ojiambo, P., Ikotun, T., Paul, C., Hartman, G.L., Bandyopadhyay, R. 2007. Comparison of field, greenhouse, and detached leaf evaluations of soybean germplasm for resistance to Phakopsora pachyrhizi. Plant Disease. 91(9):1161-1169. Twizeyimana, M., Ojiambo, P.S., Ikotun, T., Ladipo, J.L., Hartman, G.L., Bandyopadhyay, R. 2008. Evaluation of Soybean Germplasm for Resistance to Soybean Rust (Phakopsora pachyrhizi) in Nigeria. Plant Disease. 92(6):947-952. Daniel, S., Hartman, G.L., Wagner, E.D., Plewa, M.J. 2007. Mammalian cell cytotoxicity analysis of soybean rust fungicides. Bulletin of Environmental Contamination and Toxicology. 78(6):474-478. Kim, K.S., Hill, C.B., Hartman, G.L., Mian, R.M., Diers, B.W. 2008. Discovery of Soybean Aphid Biotypes. Crop Science. 48(3):923-928. 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. 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. 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. Mian, R.M., Missaoui, A.M., Walker, D.R., Phillips, D.V., Boerma, H.R. 2008. Frogeye Leaf Spot of Soybean: A Review and Proposed Race Designations for Isolates of Cercospora Sojina Hara. Crop Science. 48:14-24. Bauer, S.F., Hymowitz, T., Noel, G.R. 2007. Soybean cyst nematode resistance derived from Glycine tomentella in amphiploid (G. max x G. tomentella) hybrid lines. Nematropica. 37(2):277-285.