2006 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? Why does it matter?
Problems: 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. To address these problems, three strategies have been used:.
1)optimizing disease resistance control strategies;.
2)discovery and development of antimicrobial agents with new modes of action; and,.
3)the development of new disease-resistant cultivars.
Our approach to solving disease control, fungicide 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 GRAS (Generally Regarded as Safe) 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.
2.List by year the currently approved milestones (indicators of research progress)
This research will focus on plants known to produce fungicidal compounds using bioassay-guided fractionation for isolation and separation of novel fungicides. Crude extracts will be tested for antifungal activity. Chromatography techniques will be used to separate, purify, and determine the number of fungicidal metabolites in the crude extract as monitored by bioautography assays. In vitro activity of pure compounds will be evaluated using a 96-well plate microbioassay to identify optimum target pathogens, standard activity, and response of fungicide-resistant pathogens. Chemical isolation, structural elucidation, structure/activity relationships, and mode of action studies will be conducted in collaboration with National Center for Natural Products Research (NCNPR) chemists. 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. Currently, we evaluate approximately 6,000 extracts and 25-50 pure compounds a year.
Year 1 (2005) 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 (2006) 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
Year 3 (2007)
- Detached leaf and greenhouse studies initiated
- Patent application is prepared if appropriate
- Microscopic studies initiated
- Re-isolation of actives initiated
Year 4 (2008) 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
Year 5 (2009)
- Compound scale up, recollection, or fermentation initiated
- Mode of action studies are repeated
- Greenhouse studies repeated
- Experimental field plots initiated
4a.List the single most significant research accomplishment during FY 2006.
Essential oils from 19 Pimpinella species were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) techniques. A total of 140 different compounds were identified and significant qualitative and quantitative differences were observed among the samples. Pimpinella essential oils were characterized as having mono-, sesqui- and trinorsesquiterpenoids, propenylphenols, and pseudoisoeugenols. Trinorsesquiterpenoids and phenylpropanoids appear to be chemical markers of Pimpinella species analyzed thus far. Essential oils obtained from Pimpinella roots share the same principal compound, epoxypseudoisoeugenyl-2-methylbutyrate at concentrations from 20 to 82.6%.
4b.List other significant research accomplishment(s), if any.
The most important single project this year was substantial progress on the LifePharms, Inc., with Edward Mena, Groton, CT, and NPURU. Approximately 6,000 of the projected 18,000 samples were evaluated in 2006. Of the extracts tested, 38 were designated primary lead candidates with activity against all the fungal isolates, 335 secondary lead candidates with activity against at least one isolate, and 216 extracts of interest with varied and unique active zones over some or all isolates. Also substantial progress in now being made with a headquarters funded Post-doctoral research associate in the identification and characterization of antifungal compounds from a wide variety of essential oils selected medicinal herbs from Turkey.
4c.List 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, especially those involving essential oils, and technology transfer is taking place with visiting scientists working in our laboratories.
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, especially those involving essential oils, and technology transfer is taking place with visiting scientists working in our laboratories.
5.Describe the major accomplishments to date and 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. Two patents were filed, and both have been granted, and several compounds of commercial interest are currently under study.
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.
The in-depth LifePharms project studying the antifungal chemistry of mushrooms has yielded several potential sources of new antifungal agents. Structure elucidation studies were conducted on three compounds. Development of a new Optimum Performance Laminar Chromatography (OPLC) high throughput platform for fungicide discovery is in progress in 2006. These OPLC techniques provided an excellent format to study and identify active compounds in essential oils.
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.
The USDA, ARS International Scientific Enhancement Program (ISEP) award to ‘Identify and characterize novel natural product based antifungal agents’ with Turkey. This award provided travel money to meet with scientists and speak at four major universities in Turkey in July 2006. The program resulted is solidified collaborations, expanded research, and has resulted in more than six peer review manuscripts with international collaborators in Turkey. Natural product discovery technology and protocols are now being transfer to international scientists. Many international visiting scientists have learned natural product separation and testing protocols, bioautography methods, and OPLC protocols for fungicide discovery.
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/2006/01/050104110330.htm (January 12, 2006) and posted on the US Embassy web site (http://ankara.usembassy.gov/herbs.htm) in Ankara, Turkey.
Five Marker Flavan-phenylpropanoid conjugates from Erythroxylum catuaba. N. Tabanca, R.S. Pawar, Z. Ali, D.E. Wedge, I.A. Khan. International Conference Quality and safety Issues Related to Botanicals, August 15-18th, 2005, University, MS.
Demirci, B., Can Baser, K., Tabanca, N., Wedge, D.E. 2006. Characterization of volatile constituents of haplopappus greenei and studies on the antifungal activity against phytopathogens. Journal of Agricultural and Food Chemistry. 54:3146-3150.
Tabanca, N., Demirci, B., Kirimer, N., Can Baser, K., Bedir, E., Khan, I.A., Wedge, D.E. 2005. Gas chromatographic-mass spectrometric analysis of essential oils from pimpinella aurea, pimpinella corymbosa, pimpinella peregrina and pimpinella puberula gathered from eastern and southern turkey. Journal of Chromatography A. 1097:192-198.
Tabanca, N., Demirci, B., Ozek, T., Kirimer, N., Can Baser, K., Bedir, E., Khan, I.A., Wedge, D.E. 2006. Gas chromatographic-mass spectrometric analysis of essential oils from pimpinella species gathered from central and northern turkey. Journal of Chromatography A. 1117: 194-205.
Wedge, D.E., Smith, B.J. Inderjit, Mukerji, K.G., editors. Springer, The Netherlands. Discovery and evaluation of natural product-based fungicides for disease control of small fruits. 2006. p. 1-15.
Wedge, D.E., Duke, S.O. Finding fungicides from natural sources. Rimando, A.M., Duke, S.O., editors. American Chemical Society, Washington, DC. Natural Products for Pest Management. 2006. pp. 152-167.