Progress 03/01/04 to 02/28/09
Outputs
Progress Report Objectives (from AD-416) The objective of this agreement is technical, financial, and material cooperation for implementation of the RDA Virtual Laboratory in the United States of America. Approach (from AD-416) ARS and RDA will develop carefully selected research projects of mutual interest. Research will be conducted at ARS facilities through placement of RDA scientists for two year assignments. Research will be conducted cooperatively and results will be shared jointly. Significant Activities that Support Special Target Populations This project has terminated as of February 28, 2009. The agreement was renewed into another five year project (agreement #58-210-9-36-F), which is reported on separately. All remainaining funds for this project were rolled over to ARS account #993-0210-110. This is the final report of this agreement.
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Progress 10/01/06 to 09/30/07
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Progress Report Objectives (from AD-416) The objective of this agreement is technical, financial, and material cooperation for implementation of the RDA Virtual Laboratory in the United States of America. Approach (from AD-416) ARS and RDA will develop carefully selected research projects of mutual interest. Research will be conducted at ARS facilities through placement of RDA scientists for two year assignments. Research will be conducted cooperatively and results will be shared jointly. Significant Activities that Support Special Target Populations This report serves to document the activity of the RDA-ARS Virtual Lab (RAVL) program conducted under the cooperative agreement between ARS and the Rural Development Administration (RDA) of South Korea. Under the auspices of this agreement, an RDA scientist (Dr. Soo-Chul Park) works on cooperative research projects with ARS cooperator, Dr. Benjamin Matthews, in the Soybean Genomics & Improvement Lab (SGIL) in the Beltsville Area Research Center. The cooperative research project �Identification of genes related to plant defense and their application to enhance resistance in soybean� started in May 2006 generated great results. This project contributed to setting up the functional analysis pipeline to identify genes capable of broadening resistance of soybean to cyst nematode (SCN). First, transformation vectors, designated as pRAP, were constructed, which can be used to transform soybean with most of the candidate genes thought to induce SCN resistance in soybean plants. These modified Gateway (Invitrogen) vectors are designed for easy cloning of the target genes and for easy screening to identify real transformants because they harbor the gene encoding Green Fluorescence Protein (GFP). Second, using a promoter assay, we confirmed a suitable promoter that can induce its linked target genes under SCN infection. Third, a fast and efficient in vivo bioassay system using hairy root transformation is nearing completion. Hairy roots transformed with candidate genes can be analyzed for changes in its resistance to SCN in intact plants. Currently, a set of candidate genes, such as those encoding PR proteins, transcription factors, and those over-expressed at the SCN feeding site (syncytium), have been inserted into the pRAP vectors to be transferred into soybean plants. Over-expression or RNAi suppression of the target genes transferred into soybean using these vectors can reveal their role in the soybean defense system against SCN. The results will be reported in the 2007 Mid-Atlantic Plant Molecular Biology Society. Dr. Soo-Chul Park also works with Ms. Martha Mummey in the Office of International Research Program as the RAVL program coordinator. An annual planning meeting was held in November 2006 in Suwon, Korea to provide a framework for the RAVL program, including formalizing the official name, organization of mission, and future activities such as expansion of co- research projects, exchange of genetic resource, conduct of symposium, and developed an exchanging scientist program by Ms. Martha Mummey, ARS coordinator, and Dr Je Kyu Kim, Director of the RDA International Technical Cooperation Center (ITCC).
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Progress 10/01/05 to 09/30/06
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Progress Report 4d Progress report. This report serves to document research conducted under a trust agreement between ARS and the Rural Development Administration, Republic of Korea, established in 2003. The agreement serves to facilitate the development of research projects in areas of mutual interest, in cooperation with direct participation of RDA and ARS researchers. Since the inception of the agreement, the collaborative research between the RDA scientists and their ARS counterparts has progressed dramatically as a direct result of the opportunities provided by RDA-ARS Virtual Lab (RAVL) program. Several senior level scientists have participated in projects in ARS laboratories to examine microbial products (Bacillus thuringiensis) as insect control agents, identify genes related to plant defense and their application to enhance resistance in soybean, and develop site-specific optimum soil management strategies and in-situ soil physical and chemical property
sensors for realization of environment-friendly precision agriculture. It is anticipated that the RAVL program will continue to expand to include projects in the areas of food safety, crop and vegetable production, molecular biology, and animal health and production.
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Progress 10/01/04 to 09/30/05
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4d Progress report. This report serves to document research conducted under a trust fund cooperative agreement between ARS and the Rural Development Administration (RDA) of South Korea. Under the auspices of this agreement, an RDA scientist (Hyun Gwan Goh) works with ARS cooperators in the Insect Biocontrol Laboratory, Beltsville, MD, to Develop insect- specific microbial toxins for use as biopesticides. During the reporting period, twenty-eight strains of Bacillus thuringiensis (Bt) were screened for toxicity against he diamondback moth (DBM), Plutella xylostella, which is a serious insect pest of cruciferous plants, e.g., cabbage, cauliflower, kale, turnips, brussel sprouts and mustard greens. Bt strains were cultured on agar plates, examined for the presence of crystals and then harvested in water. Samples of Bt whole culture preparations, those treated with base [to solublize the crystal protein (pH-treated Bt)], as well as isolated spores were applied to
artificial diet prior to placing 2nd instar DBM larvae on the diet. The soluble protein concentration was 2 or 3 times higher in Bt preparations that had been treated with NaOH, centrifuged to remove spores, restored to neutrality with HCl and filtered (0.45 micrometer filter) than in supernatants of whole culture preparations. Of the 28 Bt strains tested, eight [IBL 24, 136, 156, 194, 425, 455 (Dipel, commercial strain), 465 and 745] caused high levels of larval mortality after two-three days of feeding. Whole culture Bt preparations were more toxic than samples that contained either spores alone or primarily dissolved crystal protein. With the addition of any one of the three preparations of Bt, DBM mortality increased in a dose-dependent manner. Based on LC 50 values (concentration expressed in plate equivalents required to kill 50% of the DBMs 3 or 6 days after feeding was initiated) for whole culture suspensions of IBL Bt strains, potency from greatest to least was as follows:
IBL 465, 745, 455, 425, 136, 194, 24, and 156. Spores that were damaged (shaken with beads) were no longer insecticidal, suggesting that spores must be intact to cause mortality. Previously, it had been reported that spores complemented crystal activity and that crystal protein, once digested by enzymes in the gut produced the active Cry protein that compromised the insect midgut allowing spores to enter the normally sterile body cavity. Based on electrophoretic studies, it was discovered that washed spore preparations do not contain crystal protein so that the mechanism of action by which spores alone kill DBM remains to be investigated. Preparations of pH-treated Bt were stable for ten or more weeks of refrigeration, but were not stable to boiling (therefore, not a heat-stable exotoxin) or, with the possible exception of Strain IBL 425, to extraction with methanol. From experiments in which dissolved crystal preparations of IBL strains, 425, 24, 465 and 455 (Dipel) were subjected
to gel electrophoresis, it was learned that all strains except IBL 24 produced bands that comigrated with the Cry 1 proteins. From these preliminary experiments, it was hypothesized that the toxic protein in IBL 24 either is not Cry 1 or is active in the form of a precursor or metabolite of Cry 1. When the various strains of Bt were tested on Dipel- resistant DBM, IBL 425 had the lowest LC50 (was the most toxic), followed by 24 and 194. Thus, IBL 425 was the most promising for development as a bioinsecticide against DBM. Experiments are underway to characterize the toxic Bt-produced factor(s) and to conduct field tests in the fall.
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Progress 10/01/03 to 09/30/04
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4. What were the most significant accomplishments this past year? This report serves to document research conducted under a trust fund agreement between ARS and the Rural Development Administration of South Korea. Seventy-six strains of the bacterium, Bacillus thuringiensis (Bt) have been screened for toxicity against the sweet potato whitefly (SPWF), Bemisia tabaci and 39 of these have been tested for activity against Colorado potato beetle (CPB). Bt strains were cultured on agar plates, sub-cultured to check for contamination, examined for the presence of crystals and then collected in water. In bioassays, filtered preparations were added to the whitefly and CPB diets, and percent survival after 2, 4, 6, 8 and 10 days of incubation (SPWF) or, after 24, 48, 72 and 96 hours (CPB) was recorded. Of the 76 strains of Bt tested, two were mildly toxic to CPBs and 12 produced compounds that were highly toxic to whiteflies. For these 12 Bt preparations, percent survival
of whiteflies on day 4 of treatment ranged between one and 27% while percent survival of control whiteflies was typically greater than 50%. Expressed as percent of control, whitefly survival was less than 10 % after treatment with four of the Bt strains and between 12 and 18% after treatment with five of the strains. For the remaining three toxic Bt preparations, whitefly survival was between 35 and 56 percent of control values. For active Bt strains, percent whitefly survival was also assessed in experiments in which the Bt preparations were boiled prior to addition to the whitefly diet. Boiling destroyed the toxicity of 10 of the Bt strains indicating that the active toxins in these strains were not exotoxins, ATP analogs that universally inhibit the release of energy when ATP is converted to AMP and therefore, are not suitable for use in insect control. Therefore, 10 of the strains contain compounds that may be useful as biopesticides to manage populations of whiteflies and
other sucking insects. Experiments to continue the characterization of active molecules and to test additional strains for activity against CPB are in progress.
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