2005 Annual Report
1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter?
Problem: Farmers need effective antimicrobial agents to control fungal and bacterial diseases of fruits, vegetables, and ornamental crops.
Since the early 1970s, both the medical and agricultural communities have struggled with the evolution of pathogen resistance to antimicrobial agents. Effective control of resistant pathogens has required increased necessity for repeated chemical applications and aggressive disease resistance management strategies. Increasing incidence of chemical resistance in fungal plant pathogens and loss of available fungicides for disease control in minor crops are two factors that drive the need for new fungicides.
Our approach to solving disease control, resistance issues, and the decreasing availability of agrochemicals in the market place is to identify naturally occurring chemicals from plants, pathogens, and marine and terrestrial organisms. Natural products often have new chemical structures that have not been realized or synthesized by organic chemists. Particularly desirable is the discovery of novel prototype antimicrobial agents representing new chemical classes that operate by different modes of action than existing antifungal agents and, consequently, lack cross-resistance to chemicals currently used. Following natural product leads offers an efficient approach to discovering and optimizing new agrochemicals for disease control.
How serious is the problem? Why does it matter?
Increasing incidence of resistance to fungicides by plant pathogens and loss of existing chemistry for disease control are two factors that drive the need to search for new agricultural fungicides. In addition, the desire for safer agrochemicals with less environmental and mammalian toxicity is a major concern. Particularly desirable is the discovery of novel prototype antimicrobial agents representing new chemical classes that lack cross-resistance to chemicals currently used. Following natural product leads offers an efficient approach to discovering and optimizing new agrochemicals for disease control. Due to the continuing development of microbial resistance in agriculture, discovery of new antimicrobial substances is an important, if not urgent, research objective.
Natural product derived disease control agents and those compounds considered Generally Regarded as Safe (GRAS) should essentially reduce registration costs, and therefore, provide for many more crops as potential benefactors and allow for the advancement of more potential candidate chemicals. Therefore, many minor crops are potential benefactors of natural product-based fungicides. Implementation of disease control measures that utilize low application rates of effective chemicals is imperative to maintaining cost-effective disease controls and farm profitability.
How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned?
The discovery and development of new natural product fungicides fulfills both major components of the NPS 306 mission for US agriculture. Quality Characterization, Preservation, and Enhancement (Component.
1)are specifically addressed under Preservation and/or Enhancement of Quality and Marketability through the discovery and development of environmentally friendly strategies for plant and animal pathogen control. New Processes, New Uses, and Value-Added Foods and Biobased Products (Component.
2) is specifically addressed under New Uses for Agricultural by-Products through research in natural product fungicides as new nutraceuticals, pharmaceuticals, biopesticides, or other high value uses. Identification and characterization of natural fungicides, particularly from plants, will identify new uses for phytochemicals for potential value-added products.
2.List the milestones (indicators of progress) from your Project Plan.
The process described below is performed for every compound that we evaluate. Compounds may enter the evaluation process early on with little or no supporting information or may come from a collaborator with a significant amount of preliminary supporting data and start with 96-well plate studies. As compounds continue through the evaluation process only a few get to the greenhouse phase, and only two compounds were evaluated in experimental field plots studies in the past 5 years.
Year 1 - Identify active compounds for evaluation (a continuing process).
- Internal NPURU submitted samples.
- Internal University of Mississippi submitted samples.
- External USDA ARS samples.
- External non-USDA samples.
Year 2 - Each active pure compound is studied progressively.
- Actives are evaluated in 96-well plate system -> IC50, IC95 or MIC.
- Invention disclosures filed.
- Re-isolation of actives initiated.
- Detached leaf and greenhouse studies initiated.
- Patent application is prepared if appropriate.
- Microscopic studies initiated.
- Re-isolation of actives initiated.
Year 4 - Active compounds is either moved on or eliminated.
- Detached leaf and greenhouse studies repeated.
- Compound scale up, recollection, or fermentation initiated.
- Microscopic studies repeated.
- Mode of action studies initiated.
- Compound scale up, recollection, or fermentation initiated.
- Mode of action studies are repeated.
- Greenhouse studies repeated.
- Experimental field plots initiated.
4a.What was the single most significant accomplishment this past year?
Patent Issued: Wedge, D. E. and D. Nagle. FUNGICIDAL PROPERTIES OF SAMPANGINE AND ITS ANALOGS TO AGRICULTURALLY IMPORTANT FUNGAL PLANT PATHOGENS. Filed with U.S. Patent Office Serial No. 10/400,712 was granted in January 2005.
4b.List other significant accomplishments, if any.
The most important single project this year was initiated with LifePharms, Inc. Currently, we are using matrix bioautography and have initially evaluated 1,000 samples from a collection of 14,000 ascomycete and basidiomycete extracts for fungicidal activity. We have screened 2,019 samples from LifePharms, we have requested that 5 extracts be followed up on and recollected. Identification and characterization of antifungal and insecticidal compounds from Pimpinella species and selected medicinal herbs from Turkey was initiated with a headquarters funded Post doctorial research associate.
4c.List any significant activities that support special target populations.
After identification of fungicidal active extracts we are using newly acquired Optimum performance Laminar Chromatography (OPLC) technology to separate active fractions. This technology is being used to support several other discovery projects and technology transfer is taking place with other Natural Products Utilization Research Unit (NPURU) visiting scientists.
Several natural product-based antifungal agents were identified as a potential active fungicides and several are in the patent preparation phase. These compounds will undergo further evaluation as experimental fungicides. New separation technologies such as Optimum Performance Laminar Chromotography are being developed to more efficiently and effectively evaluate plant and mushroom extracts for antifungal activity. Study initiated this year of antifungal and insecticidal compounds from Pimpinella species and selected medicinal herbs from Turkey has already shown great promise and has become a top priority project.
Relative to Project Number 6408-22430-003-01M, a Memorandum of Understanding between ARS and Chiang Mai University, Chiang Mai, Thailand, the project report is as follows: the project was not funded in 2004-2005 and future milestones, goals and objectives will be initiated if funding is obtained.
Relative to Project Number 6408-22430-003-02S, a Specific Cooperative Agreement between ARS and The University of Southern Mississippi, the progress report is as follows: promising results from our greenhouse studies suggest continuing to field studies for these two natural product-based fungicides since ultimately screening under field conditions constitutes the final stage in a natural product discovery program before going into commercial development and production. The principal advantage of using new fungicides primarily isolated from natural sources over chemical synthesis or modification of existing agents is that natural products represent a vast untapped reservoir of chemical compounds. In addition, the probability of discovering new fungicides with different modes of action and different chemical structures and, therefore, dissimilar toxicities and cross-resistance is extremely beneficial for widening the options we have in pest control.
5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
FY 2004 was the first year of this new 5-year research project. During the past 5 years this project has made significant discoveries in the area of natural product fungicides. One patent was filed and four compounds of commercial interest are currently under study. This research project program has also established a fungicide development pipeline within ARS that will facilitate the movement of new experimental compounds from the laboratory, to the greenhouse, and eventually into the field setting. Through various Cooperative Agreements this research project has field-tested and evaluated two new natural product fungicides against important strawberry pathogens. This project research in collaboration with scientists at the University of Mississippi has also led to the discovery of two new natural product-based fungicides.
Over the life of the research project, several in vitro and detached leaf assays and novel microscopic techniques were developed to evaluate natural product compounds that are often available only in very low concentrations. Bioautographic and 96-well antifungal assays have identified numerous natural product compounds as effective antifungal agents against several fungal plant pathogens of strawberry and other small fruits. These assays were also capable of evaluating chemical resistance and sensitivity profiling of Botrytis cinerea, Colletotrichum spp. and Fusarium species. Through collaborations with the Small Fruit Research Station (SFRS), Poplarville, MS, a new detached leaf assay was developed to study the efficacy of natural fungicides directly on the leaf surface, establish effective concentrations for disease control, and evaluate the ability of a compound to move systemically with in the leaf. New microscopic techniques that can evaluate spore viability directly on the leaf surface were developed with University of Southern Mississippi collaborators. Evaluation of new fungicides with low mammalian and environmental toxicity to control Botrytis fruit rot and flower blight, Fusarium wilt, and anthracnose use will help assure the safety of our food supply and the sustainability of small fruit industry.
An in-depth project studying the antifungal chemistry of mushrooms has yielded several potential sources of new antifungal agents. Development of a new Optimum Performance Laminar Chromatography (OPLC) high throughput platform for fungicide discovery will progress in 2004.
6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Development of various in vitro and in vivo bioassays suitable for the discovery and development of natural products using plant pathogenic fungi is in place. Fine-tuning of microbioassay techniques for evaluating new experimental natural products will continue over the life of the project. As the importance of diseases change and new disease immerge the research project is prepared to address new problems in US agriculture. Through established collaborations agrochemical discovery and development technology is being transferred between the NPURU and other government, university, and industry scientists.
Further interaction with scientists and publications will promote the use and successful adaptation of these assay techniques and new fungicide technologies in agriculture. Sampangine mode of action is being evaluated this year through a 2 year Non-funded Cooperative Agreement with Paradigm Genetics, Inc. Technology transfer should increase during the next five years as more scientists use and report on natural product discovery and evaluation techniques. Technology constraints primarily include the increasing cost of discovering and developing new fungicides. Technology transfer in the form of CRADAs with agrochemical industry partners covering USDA-discovered fungicides is progressing slowly. Once there are successful compounds in the marketplace, durability of the technology should be long term and we expect that impact will be high.
7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
ARS articles and new releases.
Wedge’s natural products research with Blair Sampson, USDA ARS Small Fruit Research Station, Poplarville, MS and Nurhayat Tabanca, Anadolu University in Eskisehir, Turkey was used as a lead story in ARS Information and News Service (December 20, 2004) ‘Plant Oils from Turkey Prove Lethal to Vegetable Pests. http://www.ars.usda.gov/is/pr/2004/041220.htm and subsequently picked up by Science Dailey http://www.sciencedaily.com/releases/2005/01/050104110330.htm (January 12, 2005) and the US State Department (http://usinfo.state.gov/gi/Archive/2004/Dec/22-716335.html) and US Embassy (http://ankara.usembassy.gov/herbs.htm) in Ankara, Turkey.
Wedge’s research with Dale Nagle resulted in a new patent -- US No. 6,844,353 -- sampangine-based compounds can control fungi that cause gray mold, anthracnose, and vascular wilt was used as a lead story in ARS Information and News Service http://www.ars.usda.gov/News/docs.htm?docid=1261 ‘Medicinal Compound Gets New Life as Fungicide’ (February 23, 2005).
Tabanca, N., Bedir, E., Ferreira, D., Slade, D., Wedge, D.E., Jacob, M.R., Khan, S.I., Kirimer, N., Can Baser, K., Khan, I.A. 2005. Bioactive constituents from turkish pimpinella species. Chemistry and Biodiversity.