Progress 10/01/03 to 09/11/04
Outputs
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? In view of the increasing evolution of pest resistance, it is imperative that new bioactive compounds be found with as wide a range of new modes of action as possible. Natural products are tapped, as they present not only a huge potential in the discovery of bioactive compounds in general, but is probably the best source of novel structures with novel modes of action (of paramount importance in the development of active compounds against organisms resistant to currently used pesticides). The need to find environmentally friendly replacements for a number of currently used and already phased out synthetic bioactive agents further underscores the need for such research. Damage caused by weeds, insects, and other pests amounts to billions of dollars every year. These pests can lead to lower quality
products, increased costs of productions, restriction of U.S. products from foreign markets, or complete crop failure. Further, the unrelenting increase in the evolution of resistance by all types of pests is well documented as is the need to find environmentally friendly replacements for a number of currently used and already phased out bioactive agents. The seriousness of the problem is well emphasized by the mandatory phasing out of methyl bromide where studies estimated the loss of this pesticide for all farming would cost about $1.045 billion and 13,345 full-time jobs. EPA already banned the use of methyl parathion (also known as Penncap-M) on all fruits and many vegetables in August 1999. Allowable residue levels for the pesticide azinphos-methyl, sold as Guthion, have also been lowered. These two pesticides (organophosphates) have been used for a long time on a large variety of commercial crops. This follows from a 1996 law mandating EPA to reassess tolerance levels for
pesticides. August 1999 was the deadline for the EPA to finish its review of approximately 3,000 pesticides which has resulted in the lowering of use thresholds and the outright ban of many other pesticides. The primary goal is to discover and develop natural products for agricultural pest management. A secondary goal is to support development of medicinal plants as alternative crops. The major objectives of this project are: 1) to identify natural products, through bioassay guided isolation and procedures, for herbicidal, insecticidal, fungicidal, antimicrobial and nutraceutical use, and 2) to analyze natural compounds in the elucidation of biosynthetic pathways and mode of action, and in determining the effects of xenobiotics and other factors and conditions on the production of the desired natural pesticide(s). The research falls within components of ARS National Program 306, Quality and Utilization of Agricultural Products: New Processes, New Uses, and Value-Added Biobased
Products (70%) and 302, Plant Biological and Molecular Processes: Mechanisms of Plant Interactions with Other Organisms (30%). The project more specifically addresses three problem areas of NP 306: 1a - Definition and basis for quality, 1b Methods to evaluate and predict quality, and 2a New product technology. Analytical techniques such as gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy will be used to identify natural products contributing to pesticidal and nutraceutical properties. Biosynthesis of the natural pesticide of interest will be elucidated, also by spectroscopy, and will involve identification of precursor, intermediates, and final product. Chromatographic and spectroscopic methods will also be used to quantitate biologically active constituents as related to end-use, and to determine conditions and factors affecting biosynthesis of natural compounds. 2. List the milestones (indicators of progress)
from your Project Plan. Year 1 (FY 2000) Identify sources of natural pesticides. Phytotoxic lignans isolated and identified from Leucophyllum frutescens. Resveratrol was first identified in some Vaccinium species. It's occurrence in muscadine grapes seeds, skin and pulp was also determined quantitatively. The composition of the essential oil of Callicarpa americana was determined and its phytotoxic, fungicidal and antialgal activities investigated. Year 2 (FY 2001) Further work to identify sources of natural pesticides. Active fractions from an allelopathic rice variety were obtained. Two compounds were determined by GC-MS to be present in the active (phytotoxic to barnyardgrass) fractions. The composition of the volatile oils of Fluorensia cernua and of Hibiscus cannabinus (kenaf) were determined, as well as their phytotoxic, antifungal, and antialgal activities. Year 3 (FY 2002) Continue work on identification of natural pesticides and nutraceuticals. Start work to determine the
biosynthesis of sorgoleone. Part of the biosynthetic pathway of the allelochemical sorgoleone was elucidated. New compounds were also isolated from Sorghum bicolor and identified as resorcinolic lipids. One of these compounds was more phytotoxic than sorgoleone. In identifying natural compounds with potential use against Coptotermes formosanus (the Formosan termite), the essential oil of Lepidium meyenii and some of its constituents were evaluated. Two of the components from the essential oil of Lepidium meyenii were found to have high antitermite activity providing 100% termite mortality within hours. The cancer chemopreventive activity of pterostilbene was demonstrated for the first time, with potency equal to that of resveratrol. Its high antioxidant activity was also demonstrated. Various berries were analyzed for their resveratrol and pterostilbene content. Crude fractions of several plants along with a number of other natural products were investigated for their possible
applications against termite pests. A fraction of Parthenium argentatum was identified as containing antitermite components as did fractions from Flourensia cernua, and Monese uniflora, with some of these components having already been identified. Additionally, a large number of natural quinones and essential oil components have also shown good activity against termites. Scale up procedures for the isolation of podophyllotoxin, an anticancer drug intermediate, from natural sources were developed. Year 4 (FY 2003) Follow up work on identification of natural compounds for activity against the Formosan subterranean termite. Continue work on the biosynthesis of sorgoleone. Develop new methods and analytical procedures to identify phytochemicals, neutraceuticals, and other biologically active agents. The biosynthesis of the potent sorghum allelochemical sorgoleone was completely elucidated using 13C-NMR spectroscopy, following feeding with various 13C-labelled precursors. From the
analysis of its 13C-NMR spectra it was confirmed that sorgoleone was synthesized from two pathways: the fatty acid pathway for the aliphatic side chain, and the polyketide pathway for the quinone ring of the molecule. Establishment of the pathway will lead to further studies aimed at manipulating the biosynthesis in order to enhance levels of sorgoleone in sorghum. A new method, continuous steam distillation concurrent continuous solid phase microextraction (SD-SPME), was developed. This new technique allowed the examination of volatiles from minute amounts of material or from large dilute sample volumes and is very complementary to existing techniques such as steam distillation and classical SPME. This new method has proven usefulness in detecting and establishing identity of Ephedra sinica from a large number of other Ephedra species. Analogues of sorgoleone with varying degree of unsaturation of the aliphatic side chain were identified. This was performed as part of the
biosynthetic studies of sorgoleone. Complete NMR analysis, together with an argentation-TLC method, enabled the identification of analogues with one-, two-, and three-double bonds, as well as one with a fully saturated aliphatic side chain. Identification of these analogues has implications as to the kinds of fatty acyl starting unit that may be utilized for the biosynthesis of sorgoleone. The amino acid citrulline was identified in watermelon rinds. Citrulline improves symptoms of well being in patients with sickle cell disease, increases resistance in fatigue states, stimulates immune system and enhances sexual function. This finding presents rinds as an utilizable agricultural by-product. Various berries were analyzed for their content of piceatannol, in addition to resveratrol and pterostilbene. Piceatannol has been reported to have greater antioxidant property than resveratrol, and to possess cancer chemopreventive activity similar to resveratrol. Identification of these
stilbenes in berries will complement the health benefits reportedly derived from consumption of these fruits, which has been attributed to other phytochemicals in the berries. Pterostilbene and resveratrol were tested for their inhibitory activity against some pathogenic organisms. This work was done to investigate other biological activities of these stilbenes. Pterostilbene was found to inhibit the growth of Candida albicans, Cryptococcus neoformans, Staphylocossus aureus, and methycillin-resistant S. aureus whereas resveratrol was found to be inactive. As a phytochemical found in grapes and berries with purported health enhancing properties, pterostilbene has added benefit of being inhibitory to common human pathogens. A large number of naturally occurring benzoquinones were studied for activity against the Formosan termite, Coptotermes formosanus. The structural features/characteristics in the benoquinone ring that are important for termiticidal activity were determined.
Additional benzoquinones obtained based on these premises have all been found to be very active against the Formosan termite. Glyphosate and its decomposition product aminomethylphosphonic acid was analyzed in glyphosate-treated, glyphosate-resistant, and determined not to have effect on the levels of beneficial isoflavones in soybean seeds. Year 5 (FY 2004) Continue work on identification of natural compounds for activity against the Formosan subterranean termite. Continue work to discover constituents for pesticidal and nutraceutical use. Develop new methods and analytical procedures to identify phytochemicals, neutraceuticals, and other biologically active agents. Develop analytical procedures to determine effects of xenobiotics and other factors affecting biosynthesis of biologically active constituents. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones
were not fully or substantially met, briefly explain why not, and your plans to do so. Continue work on identification of natural compounds for activity against the Formosan subterranean termite. Continue work to discover constituents for pesticidal and nutraceutical use. Develop new methods and analytical procedures to identify phytochemicals, neutraceuticals, and other biologically active agents. Develop analytical procedures to determine effects of xenobiotics and other factors affecting biosynthesis of biologically active constituents. All milestones have been substantially met. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? None. Note: This CRIS terminated in September 2004 and was replaced by 6408- 41000-006-00D. The following are objectives that will be conducted under new approved CRIS. 1. Isolation and identification of rice
allelochemical(s) and elucidation of the biosynthesis of the allelochemical(s). 2. Identification of other fungicidal constituents in Vaccinium species. Identification of Vaccinium species with high levels of stilbenes. Increasing resveratrol content in a selected Vaccinium species. 3. Analysis and identification of biosynthetic intermediates of sorgoleone to determine expression of specific gene(s) involved in the pathway. 4. Isolation and identification of compounds with activity against the Formosan subterranean termite. 5. Bioassay-guided isolation and the identification compounds from new sources for activity against Colletotrichum spp. and Oscillatoria perornata, and for phytotoxicity. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004 (one per Research Project): An analytical method was developed able to detect ng levels of substrate and products to determine the in vitro activity of the recombinant enzyme
of an O-methyltransferase gene from Sorghum bicolor. Successful conversion of resveratrol to pterostilbene was demonstrated using this method. Development of this method was crucial to the success of another project in this unit that deals with the genetic engineering of allelochemicals into crops. B. Other Significant Accomplishment(s), if any. Glyphosate (GLY) and its degradation product aminomethylphosphonic (AMPA) were discovered in glyphosate-resistant (GR) soybeans. GLY causes injury to GR-soybean under certain conditions. Injury was found to be due to AMPA, which is also a known phytotoxin. AMPA levels found in AMPA- treated soybean of both GR and non-GR soybean and in GLY-treated GR soybean correlated similarly with phytotoxicity. A method developed earlier in this laboratory, continuous steam distillation concurrent continuous solid phase microextraction (SD-SPME), was used to identify four sex and two trail pheromones from black imported fire ants. Sex pheromones have
proven to be useful weapons against insect pests. Distributing a sex attractant throughout an area disrupts the insects communication and prevents opposite sexes from getting together. Sex and trail pheromones are valuable in monitoring pest populations, as by using baits with the appropriate pheromone pest population can build-up. Bioactivity-guided fractionation of the ethanol extract from Verbascum songaricum led to the isolation of the active component aucubin which has a good activity against the blue green algae Oscillatoria perornata, the major cause of off-flavor problem in drinking water and fresh water raised fish. Allelochemicals from Nepeta pannonica and Artemisia terrae albae were isolated and identified. Work has led to semipure fractions, which will lead to eventual identification of antitermite components from Simaruba amara wood extract. One hundred and forty-four extracts representing 49 species of plants endemic to Greece and Kazakhstan were screened for various
pesticidal activities. Thirty-two extracts were found to have antifungal activity against one or more of the pathogenic fungi Colletotrichum accutatum, C. fragariae, and C. gloeosporioides. Extract from Echinops ritro was very active against the Formosan subterranean termite. Two fractions of Verbascum songoricum demonstrated very good activity against the cyanobacterium Oscillatoria perornata. Extracts from Bellis longifolia, Leontice leontopetalum and Artemisia terrae-albae were selectively phytotoxic to bentgrass but not to lettuce. Several halogenated 1,4-benzoquinone derivatives were tested for their antitermitic properties. Some of these compounds showed promise as termiticides and/or termite repellents. C. Significant activities that support special target populations. None D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house (D) projects and the projects listed in Appendix A; mandatory for all
other subordinate projects). None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A number of sorgoleone analogues were identified (including new ones) and a resorcinolic analougues was determined to be more phytotoxic than sorgoleone. The biosynthesis of sorgoleone was completely elucidated using 13C-NMR spectroscopy, and this particular resorcinolic analogue was found to be synthesized in the same biosynthetic pathway as that of sorgoleone, branching off the pathway in one step. This presents a possibility to manipulate the pathway which will favor the synthesis of the resorcinolic lipid, a more stable constituent that is also more phytotoxic than sorgoleone. 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? None, other than results of our studies shared with other scientists via oral and poster presentations at national and international meetings. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Press release: Berry Bonanza. Washington Post. June 16, 2004. p.F01.
Impacts
(N/A)
Publications
- Rimando, A.M., Kalt, W., Magee, J.B., Ballington, J.R., Dewey, J. 2004. Resveratrol, pterostilbene and piceatannol in Vaccinium berries. Journal of Agricultural and Food Chemistry. 52:4713-4719.
- TELLEZ, M.R., KHAN, I.A., SCHANEBERG, B.T., CROCKETT, S.L., RIMANDO, A.M., KOBAISY, M.M. STEAM DISTILLATION-SOLID PHASE MICRO EXTRACTION (SD-SPME) FOR THE DETECTION OF EPHEDRA SINICA IN HERBAL PREPARATIONS. JOURNAL OF CHROMATOGRAPHY. 2004. V. 1025. P. 51-56.
- Rimando, A.M., Duke, S.O. 2003. Studies on rice allelochemicals. In: Smith, C.W., Dilday, R.H., editors. Rice: Origin, History, Technology, and Production. Hoboken, NJ: John Wiley & Sons, Inc. P. 221-244.
- Singletary, K.W., Stansbury, M.J., Giusti, M., Van Breeman, R.B., Wallig, M.A., Rimando, A.M. 2003. Inhibition of rat mammary tumorigenesis by Concord grape juice constituents. Journal of Agricultural and Food Chemistry. 51:7280-7286.
- Reddy, K.N., Rimando, A.M., Duke, S.O. 2004. Aminomethylphosphonic acid, a metabolite of glyphosate, causes injury in glyphosate-treated, glyphosate- resistant soybean. Journal of Agriculture and Food Chemistry. 52:5139-5143.
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