Progress 09/01/07 to 08/31/09
Outputs
OUTPUTS: A total of fourteen field trials were conducted and the resulting data analyzed for statistically significant differences among treatments with BtBooster, BtB5, combined with a commercially available Bacillus thuringiensis or Bt product between spring of 2008 and summer of 2009. Nineteen field trials covering five crops were scheduled over this period but five trials had to be canceled because pest populations did not materialize. Company research on the BtBooster platform technology has continued on multiple fronts and for multiple orders of insects to enhance understanding of this technology. Because there are negative connotations and possibly disadvantageous economic factors involved with producing our product in E. coli, we intend to replace it with another suitable host, Pseudomonas fluorescens or Pf, for producing BtBooster in a commercial production system. To this end, we have negotiated an agreement with a major industry player to license their patented technology and for them to transfer the lepidopteran BtBooster gene into their modified Pf strain, which is capable of producing proteins at commercially viable rates. In preparation for submission of the regulatory registration package for BtBooster, we conducted an initial meeting with US EPA personnel and delivered a presentation to a group of EPA personnel in the Office of Pesticide Programs, Biopesticides and Pollution Prevention Division. We passed several regulatory requirements by completing a pepsin digestibility assay of BtB5 and an evaluation of BtB5 for any similarity to known toxins and allergens using bioinformatics. We also accepted an invitation from the Microbial Control Division of the Society of Invertebrate Pathology to deliver an 15 minute talk on the BtBooster technology at a workshop held during the Society's annual meeting in 2008. We participated in the USDA Commercialization Assistance Program through attendance at a Commercialization Training Workshop in Washington D.C. on January 29-30, 2008 and received our Commercialization Evaluation Report or commercialization roadmap following Workshop completion. We addressed the identified barriers to commercialization by taking either his recommended action or an alternate viable action that we believe will lower or eliminate each identified barrier to commercialization. PARTICIPANTS: The Project Director or PD devoted approx. 19.2 work-months of time to the project. He was responsible for project planning, management, and execution of the research plan. He worked closely with both the Field Trial and the Regulatory Consultants to evaluate the results of both the field trials and the product characterization testing that was conducted as part of developing the regulatory data package. He played a major role in writing reports as required by InsectiGen and the USDA-SBIR program. He presented results from the field trials at the Society of Invertebrate Pathology annual meeting. The Research Scientist devoted approx. 2.4 work months to the project and oversaw production of BtBooster for field trials including developing fermentation procedures. He was also responsible for stability testing and product analysis certification including potency bioassays, SDS page gels, and Western Blot analyses for each production batch. He prepared Certificates of Analysis, prepared and characterized test materials, assigned and recorded serial numbers for test materials, and packed and shiped them to field trial cooperators. He advised the PD on BtBooster production, formulation, and optimization of the fermentation process in conjunction with the UGA Bioexpression and Fermentation Facility. He assisted the PD in evaluating results from field trials, with product characterization testing, and with writing required reports. Two part-time student employees and one part-time PhD provided approx. 4.5 months of assistance with the project. They helped the Research Scientist and PD with day to day operations, including assisting with the production of BtBooster, product potency bioassays, characterization of test materials, insect larval rearing and production, and with plant production and maintenance. The Regulatory Consultant coordinated and participated with the PD in meeting with U.S. EPA personnel and devoted approx. 2.25 work months to the project. She was responsible for the physicochemical characterization and toxicology testing of BtB and for communicating with the EPA plus identifying and communicating with external contract laboratories. She maintained field trial files, helped develop testing protocols, reviewed study reports, and communicated with the PD regarding regulatory issues. The Field Trial Consultant coordinated field trial research across the country through a network of cooperators and worked closely with the PD to ensure test protocols were followed. He assisted the PD in evaluating results assisted with writing required reports and devoted approx. 3.5 work months to the project. TARGET AUDIENCES: Presented a 15 minute talk to members of the Society of Invertebrate Pathology as a speaker in the heavily attended Microbial Control Division Workshop. Attendees included representatives from many companies involved in microbial control of crop pests, either through production of biopesticides or through incorporation of genes into crop genomes as crop protection traits. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Nineteen field trials covering five crops were scheduled between spring 2008 and summer 2009, but five trials had to be canceled because pest populations did not materialize. Pest populations were unexpectedly and uncharacteristically low to very low, often never reaching economic damage threshold levels. We first conducted a small-scale field trial to determine an acceptable range of field rates and then scaled-up and modified our fermentation process to produce the needed quantities of BtB5. We proceeded to scale-up to 800 L batch fermentations from which we ultimately achieved a yield of 1.8 grams per liter near the production limit with E. coli. Although the results of many of our trials were not striking, field trials with more typical insect pressure did demonstrate the potential of applying BtB with biopesticides for improved control of harmful lepidopteran pests. Though the use of cost of goods analysis, we estimated economically viable production yields and defined a potential production system that could meet those goals. This led us to conduct experiments with Pseudomonas fluorescens or Pf since Pf-based technology could potentially give commercially viable yields. We conducted negotiations and put in place an agreement that will allow us to produce BtB5 commercially. From U.S. EPA, we received a determination that BtB would be classified as a biochemical and subject to the registration requirements of this class. We also completed the required pepsin digestibility assay of BtB5 and found that it was readily digestible. In addition, as required prior to environmental release, we evaluated BtB for any similarity to known toxins and allergens using bioinformatics and found no homology for either of these categories. We evaluated the enhancement activity of three batches of BtB5 produced for field trial use with an equivalent amount of purified BtB5. The three batches had been commercially spray dried and had been stored for up to 18 months at 4 degrees Celsius. All three batches of BtB5 retained activity equivalent with that of purified BtB5, increasing the activity of Cry1Ab alone from approximately 15 percent mortality to greater than 80 percent mortality. This suggests that storage of BtB5 should not affect its potency.
Publications
- No publications reported this period
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