Progress 11/02/99 to 09/30/04
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? Fungal infections of crops are a major factor in the post harvest spoilage of commodities rendering them unsafe for consumption. This results in very large economic losses for farmers and processors. Though man-made fungicides have been developed to protect crops from infection, plant pathogenic fungi can, and often do, develop resistance to fungicides. Synthetic fungicides can also pose a threat to the environment and the health of agricultural workers. For example, Benlate, an agricultural fungicide widely used for 33 years, was removed by the manufacturer from agricultural use after successful litigation proving damage to human health, fruit orchards, and the shrimping industry. The concern over the use of synthetic fungicides in agriculture underscores the need to discover potent fungicides
having no or low toxicity to plants, mammals, and non-target life. Natural fungicides with these characteristics would reduce or eliminate our dependency on synthetic fungicides. It is likely that plants can be the source of potent antifungal compounds. The millieu of microorganisms in their environment necessitates that plants produce potent antifungal compounds to survive. Therefore, research to discover novel, plant-produced antifungals should provide potent, non-toxic fungicides. This program to discover new, value-added biobased products from plants, particularly from low value/underutilized crops, as well as crop protection during storage and processing, are components of Problem Areas 2a (New Product Technology), 2b (New Uses for Agricultural By-products), and 2c (New and Improved Processes and Feedstocks) of National Program 306, "Quality and Utilization of Agricultural Products." This project directly addresses the discovery of bioactive products for use as preservatives
or pesticides (Problem Area 2a), discovery of antifungal compounds in crop by-products (Problem 2b), and new technologies to convert agricultural products into value-added biobased products (Problem Area 2c). There is also a secondary effort in Component V (Host Plant Resistance) of National Program 303, "Plant Diseases." This research effort addresses the need in Component V to identify antifungal plant protein genes leading to transgenic expression in host plants or upregulation in producing plants to enhance plant resistance to fungal infection. 2. List the milestones (indicators of progress) from your Project Plan. Overall Goal: Isolation and characterization of new plant derived, antifungal protectants. A. CAY-1 (active against fungi such as Aspergillus, Collectotrichum, and Phomopsis, which produce toxins on, or are pathogenic for crops): During 2003: (1) Treat field strawberries (De Lucca, Boue, Wedge, Smith [Agricultural Research Service/Mid South Area (ARS/MSA)]); (2)
Detached leaf assays (De Lucca, Boue, Wedge, Smith); (3) Materials Transfer Agreement (MTA) with Syngenta for CAY-1 testing (Wedge); (4) Testing CAY- 1 against grape fungal pathogens (De Lucca); and (5) Initial testing of CAY-1 for its ability to kill mosquito larvae. During 2004: (1) CAY-1 purification (De Lucca, Boue); (2) Continued strawberry field testing (De Lucca, Boue, Wedge Smith); (3) CRADA with Syngenta; (4) Continued testing CAY-1 against grape fungal pathogens (De Lucca); (5) Proposed determination of CAY-1 for its inhibitory properties against Eutypa, a fungus which causes serious economic losses in grape vineyards; and (6) Mosquito larvicide study grant. B. Testing of purified CAY-1 precursors (CAY-A; mol. wt. 1802; CAY-B, mol. wt. 940): During 2003: (1) Purification of CAY-A and CAY-B (De Lucca, Boue); and (2) Antifungal studies (De Lucca). C. Barley-produced binary fungicidal compounds (active against Fusarium): During 2003 (Summer/Fall): (1) Purification and initial
fungicidal assays (De Lucca, Boue). During (Winter) 2003 - (Spring) 2004: (1) Structure analyses of compounds; and (2) Purification of sufficient compound for antifungal assays and determination of toxicity for mammalian cells. D. Unknown, Semipurified Cotton Antifungal Compound (active against Aspergillus): During 2003-2004 (Winter/Spring): (1) Purification of antifungal compound (De Lucca, Boue). During 2004 (Summer/Fall): (1) Structure analysis; and (2) Antifungal, cytotoxicity studies. 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. 1) CAY-1 from cayenne pepper: a. Research scheduled during calendar year 2003 (FY 2004): We completed items 1, 2, 3, and 5 which consisted of: (a) producing sufficient quantities of our patented fungicide from cayenne pepper for (b)
testing in field plots of strawberries and detached leaf assays by our ARS colleagues, Drs. David Wedge and Barbara Smith, and (c) an MTA was signed with Syngenta allowing them to test CAY-1. Item 4, the testing of CAY-1 for activity against fungal grape pathogens, is currently being performed while item 6, testing of CAY-1 for efficacy against mosquito larvae, will be performed during July-August 2004, under a newly signed MTA with Emovations, L.L.C. (Bend, OR). b. Research scheduled during calendar year 2004 (FY 2004): Item 1, the purification of additional quantities of pure CAY-1 for testing by collaborators, was accomplished. Item 2, the continued testing of CAY-1 to determine efficacy in strawberry field plots, was changed to testing in detached leaf studies by ARS colleagues, Drs. David Wedge and Barbara Smith, because the strawberry field plots at the Louisiana State University (LSU) experimental farm in Hammond, LA, were no longer available. Item 3, a CRADA with Syngenta
to further test CAY-1, did not develop. Item 4, the testing of CAY-1 against fungal pathogens of grape, is ongoing during the summer of 2004. Item 5, the testing of CAY-1 against Eutypa, a very serious pathogen in vineyards, has not developed due to the delay in arranging collaboration with specialists who work with Eutypa. Item 6, the submission of a grant to study the CAY-1 larvicidal properties against mosquitoes, is delayed until the initial testing (see paragraph above) is completed by Emovations, L.L.C. 2) Testing of purified metabolic precursors of CAY-1 (Precursor 1: mol. mass = 1081; Precursor 2: mol. mass = 919) during calendar year 2003. The purification of precursors 1 and 2 was completed and their chemical studies determined. Precursors 1 and 2 have one and two, respectively, less glucose moieties than the final metabolic product, CAY-1. As scheduled, their antifungal properties were determined and results show that precursor 1 has significantly less fungicidal
properties than CAY-1 while precursor 2 is inactive against fungi. Results show the complete sugar substructure (five sugars) of CAY-1 is necessary for activity against fungi. 3) Unknown barley antifungal compound active against the fungus, Fusarium. As outlined in the milestones, during FY 2004 we successfully purified two compounds which are lethal to Fusarium from barley seedlings. These compounds are currently undergoing Nuclear Magnetic Resonance (NMR) structural analysis. We are also purifying sufficient quantities of both to perform antifungal and cytotoxicity studies during the Fall of 2004, as stated in the milestones. 4) Unknown, semipurified cotton antifungal compound. We have been delayed in the purification of the cotton antifungal compound due to our concentrating on the aforementioned projects. However, during the summer of 2004, we have begun again our efforts to purify this compound. B. List the milestones that you expect to address over the next 3 years (FY
2005, 2006, and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Existing Projects: 1) CAY-1: During FY 2005: (1) In vitro against dermatophytic fungal pathogens of immunocompromised patients (NIH); (2) Lethality for mosquito larvae under MTA with Emovations L.L.C.; (3) Possible additional detached leaf assays by Drs. Wedge and Smith (ARS/MSA); and (4) Production of pure CAY-1 (De Lucca, Boue, ARS/Southern Regional Research Center (SRRC).) During FY 2006: (1) Continued pure CAY-1 production (De Lucca, Boue); (2) Possible CRADA to study CAY-1 lethality to mosquito larvae and snails; and (3) Greenhouse studies (De Lucca, Boue, Wedge, Smith). During FY 2007: (1) Production of pure CAY-1, if needed (De Lucca, Boue); and (2) Possible collaborations. 2) Barley-produced Binary Fungicide: During FY 2005: (1) Production and purification for bioassays (De Lucca, Boue); (2) Patent submission; and (3) Sharing compounds with government,
university, and industrial collaborators under MTAs. During FY 2006: (1) Production of binary compounds (De Lucca, Boue); and (2) Possible CRADAs with collaborators. During FY 2007: (1) Production of binary compounds (De Lucca, Boue); and (2) Continued and new MTAs, CRADAs. 3) Fungicide Produced by Cotton Seedlings: During FY 2005: (1) Initial purification, chemical structural identification, and antifungal bioassays; and (2) Patent submission (late 2005 or early 2006). During FY 2006: (1) Production of pure compound (De Lucca, Boue); (2) Development of MTAs with interested parties; and (3) Sharing compound with ARS colleagues (Drs. Wedge and Smith). During FY 2007: (1) Production of pure compound (De Lucca, Boue); and (2) Share compound with interested parties under MTAs. New Research on Low Value/Underutilized Crops (Okra, Peanut, and Rice Hulls) to Be Initiated During FY 2005-2007: 1) FY 2005: Initial studies to determine whether aqueous extracts of okra, peanut, and rice hulls
water soluble fungicides are present. This will be exploratory in nature with more extensive purification efforts in FY 2006 and 2007 for any aqueous fungicides indicated in these tests during FY 2005. 2) FY 2006: Initial purification experiments will begin for any water soluble fungicides indicated during FY 2005 with okra, peanut, and rice hulls. Bioassays will be conducted when necessary to determine the identity of the samples containing the fungicides. Progress on purification of any fungicide from okra, peanut, and rice hulls will be partially determined by any need to continue research efforts on CAY-1, barley binary fungicide, and cotton seedling fungicides. 3) FY 2007: It is expected that purification of any fungicide from okra, peanut, and/or rice hulls will be in final phase during FY 2007. Success and speed of these experiments will depend on the complexity of the purification, as well as demands by other projects on co-workers. Purified fungicides will undergo a
series of bioassays to determine the spectrum of activity against several fungal genera. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: The single most significant accomplishment during FY 2004 was the purification of two very small molecular weight compounds, believed to be peptides, from barley. Data indicate that the pair act together to kill Fusarium, while separately both are inactive. We believe this to be the first discovery of a binary fungicidal compound. B. Other significant accomplishments: Other significant accomplishments include the testing of CAY-1 in strawberry field plots and the detached leaf assays. These data, which have not yet been statistically analyzed, will give us our first information on the potential efficacy of CAY-1 (our patented, plant-produced fungicide) on plants. Our study of the two CAY-1 precursors not only determined their chemical structure, but also showed that only
CAY-1, the final product of the metabolic pathway, is fungicidal. In collaboration with Dr. J. V. Edwards (ARS/SRRC) we are determining whether (1) CAY-1 can bind to cotton fiber and, if so, (2) does the impregnated fiber retard fungal growth. Another accomplishment is the entering into a MTA with Emovations, L.L.C. , to determine whether CAY-1, a compound of the "saponin" class having detergent-like properties, has potential utility as a mosquito larvicide and is lethal to snails. A few published reports indicate that saponins have such properties, but we have not been able to test CAY-1 against these pests until the agreement with Emovations was signed. C. Significant activities that support special target populations: No additional significant activities other than reported in item 4. D. Progress Report: During FY 2004, our patented fungicidal saponin, CAY- 1, was tested by our ARS colleagues, Drs. David Wedge (NCA Oxford, MS) and Barbara Smith (SHL, Poplarville, MS) in detached
strawberry leaf assays to determine efficacy against fungal leaf blight. The data is currently being processed. A few reports have indicated that some saponins are lethal for mosquito larvae and kill members of the "mollusk" family, such as snails and zebra mussels (which clog water intake pipes). To determine whether CAY-1 has such properties, we recently entered into a MTA with Emovations, L.L.C. (Bend, OR), which will test CAY-1 efficacy against mosquito larvae and snails. We have also purified two small molecular weight compounds, believed to be peptides from barley, which are lethal for Fusarium species. They are currently undergoing NMR strucutral analysis. By September 2004, we hope to begin spectrum of lethality studies against species of Aspergillus, Fusarium, and other fungi, as well as other studies, to characterize these compounds prior to submission of an invention disclosure. 5. Describe the major accomplishments over the life of the project, including their
predicted or actual impact. A. The first major accomplishment of the project was the discovery and patenting of the novel, fungicidal saponin, CAY-1, from cayenne pepper. This lead directly to the entering of MTAs with five companies (MycoLogics, Kemin Industries, Syngenta, Preservation Sciences, Emovations, L.L.C.) and one CRADA (MycoLogics) to determine the potential commercial utility of CAY-1 against (1) a number of problems caused by fungal infections in agriculture, food processing, and medicine; and (2) CAY-1 effectiveness against mosquito larvae and snails. B. The second major accomplishment was the discovery of two small molecular weight fungicidal compounds in barley. These compounds are not fungicidal alone but are lethal to the fungus, Fusarium oxysporum, when mixed together. The data indicates that this is the first discovery of a binary fungicidal compound set in nature. We intend to submit an invention disclosure for these compounds as soon as sufficient data is
collected. 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? A. CAY-1: This technology has been available to end-users since Winter 2001-2002. Five companies (MycoLogics, Kemin Industries, Syngenta, Preservation Sciences, Emovations, L.L.C.) have entered into MTAs with ARS to determine the potential commercial utility of CAY-1 against (1) a number of problems cause by fungal infections in agriculture, food processing, and medicine; and (2) CAY-1 effectiveness against mosquito larvae and snails. A CRADA was entered into with MycoLogics in 2002 to study in test tubes and animal studies the antifungal properties of CAY-1 for use in medicine. B. The barley binary fungicidal technology has not yet been patented so we do not foresee it being available to
end-users before the Fall of 2005.
Impacts
(N/A)
Publications
- Renault, S., De Lucca II, A.J., Boue, S.M., Bland, J.M., Vigo, C.B., Selitrennikoff, C. 2003. Cay-1, a novel antifungal compound from cayenne pepper. Medical Mycology. 41(1):75-81.
|
Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? This is an Agricultural Research Service (ARS) research project. Fungal contamination of crops may result in post harvest spoilage of the commodity. Toxins, called mycotoxins, produced by many of these fungi, can render a commodity unsafe for human or animal consumption. Either or both of these factors can result in very large economic losses for farmers and processors. Discovery, isolation, and characterization of novel, potent, natural antifungals may lead to enhanced protection against such fungi. The naturally occurring compounds being studied inhibit the growth of, or are lethal to, such fungi. Such compounds may also reduce use of synthetic compounds lethal to fungi that are harmful to the environment, affect non-target life, and are dangerous for human consumption. 2. How serious is the problem? Why does it matter? Fungal crop diseases cause very large economic losses yearly, which
can total hundreds of millions of dollars in the U.S. alone. The need for natural, safe antifungals to protect crops also grows in parallel with the increased regulation and banning of synthetic fungicides. Discovery and resultant production of natural antifungal agents and treatment technology could reduce the incidence of spoilage and mycotoxin contamination due to post-harvest fungal decay of crops. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? The research project relates to National Program 303, Plant Diseases and 306, Quality Utilization of Agricultural Products. Development of antifungal natural products as crop protectants will provide novel methods to prevent fungal infestations of post harvest crops on a commercial basis. This will improve the safety and quality of the food and feed supply and reduce economic losses associated with crop disease. Novel plant antifungals shown to effectively protect crops
will reduce the dependence on synthetic fungicides which pose an environmental threat and often kill beneficial organisms. If successful, such plant fungicides will add value to the host crop and greatly enhance the reliability of the quality of U.S. crops on international markets. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003 year: Fungal diseases of crops cause very large economic losses through post-harvest spoilage of the commodity, and with the increased regulation and banning of synthetic based pesticides, economical methods to produce natural based pesticides and crop protectants are desperately needed. Previous collaborative work between Anthony De Lucca and Stephen Boue (Food and Feed Safety Research Unit, Southern Regional Research Center [FFS, SRRC]) and David Wedge (Natural Products Utilization Research Unit [NPURU], ARS, Oxford, MS) showed CAY-1, a detergent-like compound from cayenne pepper that
kills fungi patented by SRRC scientists, was highly active against fungi pathogenic to small fruits. During this reporting period, Anthony De Lucca produced quantities of semipure CAY-1 which was tested by David Wedge and Barbara Smith (Agricultural Research Service [ARS], Poplarville, MS) on strawberries growing near Hammond, LA. Initial tests showed a low dose of semipure CAY-1 was effective against a number of diseases affecting the strawberry plants during a wet spring. Overall, the low dose of semipure CAY-1, when compared to commercial agricultural fungicides, was the fifth most active out of fourteen compounds tested. Additional tests are planned for the FY 2004 strawberry growing season. IMPACT: Successful field trials could lead to commercial use of crude or semipurified cayenne extracts as a new fungicide treatment on crops. CAY- 1 has a mode of action different from commercial fungicides, thereby making it harder for fungi to develop resistance to it. Therefore, successful
field tests would indicate to industry that CAY-1 is a viable alternative to current agricultural fungicides. B. Other Significant Accomplishment(s), if any: None at this time. C. Significant Accomplishment/Activities that Support Special Target Populations: None at this time. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. In addition to the accomplishment summarized in Question 4, a previously unreported fungicidal compound has been purified from 3 day-old barley shoots which is particularly active against a type of fungus named "Fusarium." Plans are to purify additional quantities of this compound, elucidate its structure, and perform antimicrobial tests against fungi during the remainder of FY2003 and 2004. The antimicrobial compounds, Hordatine A and B, were purified from barley shoots but found not active against Aspergillus and Fusarium species. 6. What do you expect to accomplish, year by year, over the next 3
years? FY 2004: The semipure CAY-1 will be produced at Southern Regional Research Center (SRRC) to provide the required samples for a second series of strawberry field tests to determine "real world" potential as an agricultural fungicide. Barley shoots will be extracted and the unknown antifungal compound purified in order to perform antifungal spectrum of activity studies and to elucidate its chemical structure. Additional samples will be purified and sent to collaborators at Natural Products Utilization Research Unit (NPURU), ARS, Oxford, MS and National Institutes of Health (NIH), Bethesda, MD for additional studies. If novel, we intend to prepare a Material Transfer Agreement (MTA) with MycoLogics, Denver, CO and submit a Small Business Innovation Research (SBIR) Phase 1 grant proposal. We will also attempt to purify a cottonseed antifungal which we previously semipurified. In past years, we have been unable to complete purification of the cottonseed antifungal due to shortage of
personnel and the need to complete work with CAY-1 and the barley compounds. We anticipate that the MTA's signed in FY 2003 with at least one of the three companies will lead to licensing of CAY-1 technology. Should the barley antifungal compound prove novel and efficacious in in vitro (artificial environment) studies, we intend to file an application for a patent on this compound. Should the needed personnel be available, we plan to attempt purification of another plant-derived antifungal compound we have discovered in underutilized crops during FY 2003. In FY 2004, a new five-year CRIS project will be in place and will broaden the scope of discovery of novel plant antifungal compounds. The new CRIS project will investigate low value/underutilized crops and crop waste material for novel antifungal compounds during the following years (FY 2004-2009). Discovery of such compounds may lead to value added natural products and new agricultural fungicides. FY 2005: It is anticipated that
strawberry field plot experiments begun in FY 2003 with semipure CAY-1 will continue through FY 2005 to further determine the efficacy and best conditions to use CAY-1. Additional in-house and collaborative work is expected with the barley antifungal compound and, hopefully, the cotton antifungal. We hope to begin field studies with the barley antifungal compound and complete purification of the cottonseed antifungal compound. Collaborative research with other government, university, and industrial scientists will continue. FY 2006: We plan to continue field studies with semipure CAY-1 and the barley antifungal compound (should it prove in FY 2005 to have potential as an agricultural fungicide). 7. 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? During
FY 2003, Material Transfer Agreements (MTA's) were signed with Syngenta, Kemin Industries, and Preservation Sciences, Inc., to study pure and semipure CAY-1. Samples of each have been sent to the companies and are currently undergoing evaluation. Pure CAY-1 samples sent to John Cleary, Professor, University of Mississippi Medical Center, Jackson, MS., for molecular biologic (genomic screening of Pharmaco-genomic) studies on how CAY-1 kills fungi and improved safety (human). Considering the need for further field studies and results of the ongoing tests by the aforementioned companies, the CAY-1 technology may be available to end users (i.e., industry, farmers) by approximately FY 2008. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Renault, S., De Lucca, A.J., Bland, J.M., Boue, S.B., Vigo, C.B., Sellitrennikoff, C.P.
CAY-1, a novel antifungal from cayenne pepper. Medical Mycology. 2003. v. 41. p. 1-7.
Impacts
(N/A)
Publications
- DUKE, S.O., BAERSON, S.R., DAYAN, F.E., RIMANDO, A.M., SCHEFFLER, B.E., TELLEZ, M.R., WEDGE, D.E., AKEY, D.H., ARTHUR, F.H., DE LUCCA, A.J. UNITED STATES DEPARTMENT OF AGRICULTURE - AGRICULTURAL RESEARCH SERVICE ON NATURAL PRODUCTS FOR PEST MANAGEMENT. PEST MANAGEMENT SCIENCE. 2003. V. 59. P. 708-717.
|
Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? This is an Agricultural Research Service (ARS) research project. Fungal contamination of crops may result in post harvest spoilage of the commodity. Toxins, called mycotoxins, produced by many of these fungi can render a commodity unsafe for human or animal consumption. Either or both of these factors can result in very large economic losses for farmers and processors. Discovery, isolation, and characterization of novel, potent, natural antifungals may lead to enhanced protection against such fungi. The naturally occurring compounds being studied inhibit the growth of, or are lethal to, such fungi. Such compounds may also reduce use of synthetic fungicides that are harmful to the environment, affect non- target life, and are dangerous for human consumption. 2. How serious is the problem? Why does it matter? Fungal crop diseases cause very large economic losses yearly which can total
hundreds of millions of dollars in the U.S. alone. The need for natural, safe antifungals to protect crops also grows in parallel with the increased regulation and banning of synthetic fungicides. Discovery and resultant production of natural antifungal agents and treatment technology could reduce the incidence of spoilage and mycotoxin contamination due to post-harvest fungal decay of crops. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? The research project relates to National Program 303, Plant Diseases and 306, Quality & Utilization of Agricultural Products. Development of antifungal natural products as crop protectants will provide novel methods to prevent fungal infestations of post harvest crops on a commercial basis. This will improve the safety and quality of the food and feed supply and reduce economic losses associated with crop disease. Novel plant antifungals shown to effectively protect crops will reduce
the dependence on synthetic fungicides which pose an environmental threat and often kill beneficial organisms. If successful, such plant fungicides will add value to the host crop and greatly enhance the reliability of the quality of U.S. crops on international markets. 4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002 year: Fungal diseases of crops cause very large economic losses through post-harvest spoilage of the commodity, and with the increased regulation and banning of synthetic based pesticides, economical methods to produce natural based pesticides and crop protectants are desperately needed. During this reporting period, scientists at the Agricultural Research Service (ARS), Southern Regional Research Center (SRRC) were granted a U.S. patent (6, 310,091 B1) for a novel fungicidal saponin (CAY-1) which was purified from cayenne pepper. Dr. Dave Wedge at the Natural Products Utilization Research Unit
(NPURU) determined that CAY-1 is active at low levels against Collectotrichum and Phomopsis, two economically important fungal pathogens affecting strawberries, blueberries, and grapes. As a result, cooperative research between SRRC and the NPURU will increase in FY 2003. This research will include tests to confirm the safety of impure extracts of CAY-1 on plants. If these extracts are proven safe, then formulations of impure CAY-1 will be tested in small field studies on strawberries and greenhouse trials with blueberries during late FY 2002 through FY 2003. IMPACT: Successful field and greenhouse trials could lead to commercial use of crude or semipurified cayenne extracts as a new fungicide treatment on these crops. This would provide a novel fungicide and enhance the economic value of cayenne to farmers. B. Other Significant Accomplishment(s), if any: None at this time. C. Significant Accomplishment/Activities that Support Special Target Populations: None at this time. 5. Describe
your major accomplishments over the life of the project, including their predicted or actual impact? In addition to the accomplishment summarized in Question 4, a previously unreported fungicidal compound has been semipurified from cotton cotyledons (seed halves) obtained from germinating cottonseed. It is neither present in dormant seeds nor in mature cotton leaves. During FY2003, plans are to complete the purification of this antifungal and elucidate its structure. Next antifungal studies will determine its spectrum of activity. Another major accomplishment of this project was the establishment of a Materials Transfer Agreement with MycoLogics (Denver, Colorado) to further study the antifungal properties of CAY-1. MycoLogics received a Small Business Innovation Research (SBIR) Phase I Grant from the National Institute of Health (NIH) to do so. SRRC received $12,830 to cover the cost of providing purified CAY-1 for this grant. Based on the CAY-1 work to date and that planned between
SRRC and NPURU, it is believed that impure extracts containing CAY-1 could be used on several crops to protect them against fungal infections. These crops include strawberries, blueberries, and grapes. Agricultural Research Service (ARS) scientists have also purified two antifungal compounds, hordatine A and B, from barley which are currently being tested for their antifungal properties against Aspergillus and Fusarium spp., which are two toxin-producing fungi on cotton and corn. 6. What do you expect to accomplish, year by year, over the next 3 years? FY 2003: Extracts containing CAY-1 will be tested for phytotoxicity. If shown not to be phytotoxic, Agricultural Research Service (ARS) scientists will proceed with field and greenhouse testing with strawberries and blueberries to determine the efficacy of impure CAY-1 extracts as a fungicide with commercial potential. The barley antifungals, hordatine A and B, will be studied for their antifungal properties. Another barley antifungal,
which scientists believe is novel, will be purified and studied for its chemical structure and antifungal spectrum of activity. The cotton antifungal will also be purified, its structure determined, and its antifungal spectrum of activity studied. FY 2004: It is anticipated that field plot and greenhouse studies begun in FY 2003 will be expanded during this period. It is hoped that the barley and cotton antifungals will prove effective in laboratory assays (in vitro). During FY 2004, sufficient quantities will be produced to share with other scientists in order to further elucidate their antifungal properties. Tests will be performed by NPURU to determine whether they are active in vitro against fungal plant pathogens, such as Collectrichum and Phomopsis. FY 2005: It is hoped that field and greenhouse studies will be completed with CAY-1 extracts and similar studies begun for cotton and barley antifungals studied in prior years. 7. What technologies have been transferred and to whom?
When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? A) During FY 1999, a Materials Transfer Agreement was initiated with a fungal biotechnology company to determine the fungicidal potential and safety of CAY-1. In FY2001, the company was awarded a Small Business Innovation Research (SBIR) Phase 1 grant from the Department of Health and Human Services, Public Health Service, to fund this research. The SBIR grant research was conducted in FY2002 and a report of this work will be published in Medical Mycology during 2002. Because much additional research remains (i.e., field and greenhouse studies), it is not expected that CAY-1 or its impure forms would be available for commercial use before FY2005 or 2006. CAY-1 originates in a food grade material, so it may be included under Generally Assumed As Safe (GRAS) category. However, research on the effects of
CAY-1 in animals and its impure forms must be performed to assure safety for human consumption. Also, experiments on the survivability of CAY-1 in field and stored crop environments must be performed before commercial use is begun. B) Unit scientists participated in 1 scientific society meeting, presented papers, and participated in discussions with collaborators on various projects.
Impacts
(N/A)
Publications
- 1. De Lucca, A.J., Bland, J.M., Vigo, C.B., Cushion, M., Selitrennikoff, C. P., Peter, J., Walsh, T.J. CAY-1, a fungicidal saponin from Capsicum sp. fruit. Medical Mycology. 2002. v. 40. p. 131-137.
- 2. De Lucca, A.J., Vigo, C.B., Bland, J.M., Selitrennikoff, C.P. Fungicidal saponin, CAY-1, and isolation thereof from Capsicum species fruit. 2001. U.S. Patent 6,310,091 B1.
|
Progress 10/01/00 to 09/30/01
Outputs
DUCTS FOR POST HARVEST CROP PROTECTION 1. What major problem or issue is being resolved and how are you resolving it?
This is an Agricultural Research Service (ARS) research project. Fungal contamination of crops may result in post harvest spoilage of the commodity. Toxins, called mycotoxins, produced by many of these fungi can render a commodity unsafe for human or animal consumption. Either or both of these factors can result in very large economic losses for farmers and processors. Discovery, isolation, and characterization of novel, potent, natural antifungals may lead to enhanced protection against such fungi. The naturally occurring compounds being studied inhibit the growth of, or are lethal to, such fungi. Such compounds may also reduce use of synthetic fungicides that are harmful to the environment, affect non-target life, and are dangerous for human consumption.
2. How serious is the problem? Why does it matter?
Fungal crop diseases cause very large economic losses yearly which can total hundreds of millions of dollars in the U.S. alone. The need for natural, safe antifungals to protect crops also grows in parallel with the increased regulation and banning of synthetic fungicides. Discovery and resultant production of natural antifungal agents and treatment technology could reduce the incidence of spoilage and mycotoxin contamination due to post-harvest fungal decay of crops.
3. How does it relate to the National Program(s) and National Component(s)?
The research project relates to National Program 303, Plant Diseases and 306, Quality & Utilization of Agricultural Products. Development of antifungal natural products as crop protectants will provide novel methods to prevent fungal infestations of post harvest crops on a commercial basis. This will improve the safety and quality of the food and feed supply and reduce economic losses associated with crop disease. Novel plant antifungals shown to effectively protect crops will reduce the dependence on synthetic fungicides which pose an environmental threat and often kill beneficial organisms. If successful, such plant fungicides will add value to the host crop and greatly enhance the reliability of the quality of U.S. crops on international markets.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment during FY2001 year: Fungal diseases of crops cause very large economic losses through post-harvest spoilage of the commodity, and with the increased regulation and banning of synthetic based pesticides, economical methods to produce natural based pesticides and crop protectants are desperately needed. During this reporting period, scientists at the Agricultural Research Service (ARS), Southern Regional Research Center (SRRC) submitted a patent application to the U.S. Patent Office for a novel fungicidal saponin (CAY-1) which was purified from plant material. CAY-1 killed Aspergillus flavus, A. fumigatus, A. niger, and A. parasiticus. These fungi produce several potent mycotoxins often found on contaminated crops. Non-USDA, ARS scientists determined that CAY-1 also inhibits or kills several medically important fungi. IMPACT: The possibility exists that CAY-1 could be employed in combating agricultural and medically-important fungal
pathogens. This may lead to eventual commercialization of this plant antifungal. B. Other Significant Accomplishment (s), if any: None at this time. C. Significant Accomplishment/Activities that Support Special Target Populations: None at this time. D. Progress Report. None at this time.
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
In addition to the accomplishment summarized in Question 4, a previously unreported fungicidal compound has been purified from cotton cotyledons (seed halves) obtained from germinating cottonseed. It is neither present in dormant seeds nor in mature cotton leaves. The structure of this compound from cotton is being elucidated for possible use as a fungal growth inhibitor and crop protectant. Genetic methods were utilized to further characterize the gene cluster responsible for production of nikkomycin (a small peptide or protein), which is a potent and possibly useful fungal growth inhibitor produced by certain bacteria. The goal is to genetically enhance production of this compound for use as a possible fungal growth inhibitor and crop protectant.
6. What do you expect to accomplish, year by year, over the next 3 years?
We have several compounds, believed to be novel, at different stages of purification which kill mycotoxin-producing fungi. During FY 2002, we intend to purify them, determine their chemical structure, and study their lethality for mycotoxin-producing fungi (e. g. Aspergillus flavus) which contaminate post harvest crops. We hope to enter into cooperative efforts in FY 2003 and FY 2004 to produce edible films containing these plant fungicides to protect stored crops and foods in groceries from fungal contamination. In addition, during FY 2002-2004 additional experiments to discover other novel plant fungicides will be performed.
7. 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 & durability of the technology product?
A) During FY 1999, a Materials Transfer Agreement was initiated with a fungal biotechnology company to determine the fungicidal potential and safety of CAY-1. In FY 2001, the company was awarded a Small Business Innovation Research (SBIR) Phase 1 grant from the Department of Health and Human Services, Public Health Service, to fund this research. Currently, unit scientists are purifying the CAY-1 required for this study. A U.S. Patent is pending on the composition of matter and use of CAY-1 to combat fungal pathogens of agriculture and of medical importance. A Cooperative Research and Development Agreement (CRADA) has been established with the company to transfer $12,800 into the current research project for supplies used in CAY-1 purification to be used in the SBIR Phase 1 study. Because much additional research remains, it is not expected that CAY-1 would be available for commercial use before FY 2005 or 2006. CAY-1 originates in a food grade material, so it may be included
under Generally Assumed As Safe (GRAS) category. However, research on the in vivo effects of CAY-1 must be performed to assure safety for human consumption. Also, experiments on the survivability of CAY-1 in field and stored crop environments must be performed before commercial use is begun. B) A major seed company contacted a project scientist about the possible use of CAY-1 to protect stored grains from fungal contamination and mycotoxin production. Small scale feasibility trials are underway. C) A pepper breader contacted a project scientist to obtain information on CAY-1. D) A University scientist from Alberta, Canada requested information regarding nikkomycin genes and potential chemical inducers of these genes. E) Unit scientists participated in 2 scientific society meetings, presented papers, and participated in discussions with collaborators on various projects.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
"Pepper Compound Promises Ag Benifits," by Editors, Progressive Farmer. 2001. "Study Peppered with Promise," by Andy Voung, Denver Post. 2001.
Impacts
(N/A)
Publications
- Bland, J.M., De Lucca, A.J., Jacks, T.J., Vigo, C.B. All-D-cecropin B; synthesis, secondary structure, lipopolysaccharide binding, and antibacterial activity. Molecular and Cellular Biochemistry. 2001. v. 218. p. 105-111.
- De Lucca, A.J., Walsh, T.J. Antifungal peptides: origin, activity, and therapeutic uses. Revista Iberoamericana de Micologia. 2000. v. 17. p. 116-120.
|
Progress 10/01/99 to 09/30/00
Outputs
DUCTS FOR POST HARVEST CROP PROTECTION 1. What major problem or issue is being resolved and how are you resolving it?
Fungal diseases of crops cause very large economic losses through post harvest spoilage of the commodity and can result in food safety concerns when toxin producing fungal pathogens are involved. Compounds from natural sources that inhibit these pathogens, once identified and characterized, could be used as protectants against spoilage. Many of the fungi causing crop losses through spoilage or contamination of the crop with fungal toxins known as mycotoxins have been shown to be highly sensitive to the natural product inhibitors being studied. Crops protected using microbial inhibitors could reduce the incidence of spoilage due to post-harvest decay of the plant product and contamination of the product with fungal toxins, which are known to cause hundreds of millions of dollars in losses in the U.S. each year.
2. How serious is the problem? Why does it matter?
Fungal diseases of crops cause very large economic losses through spoilage of the commodity and can result in food safety concerns when toxin producing fungal pathogens are involved. With the increased regulation and banning of synthetic based pesticides, economical methods to produce natural based pesticides and crop protectants are desperately needed. Resultant production of natural antifungal compounds and treatment technology could reduce the incidence of spoilage due to post-harvest decay of crops and contamination of the crops with fungal toxins, known to cause hundreds of millions of dollars in losses in the U.S. each year.
3. How does it relate to the National Program(s) and National Component(s)?
The research project relates to National Programs 303, Plant Diseases and 306, New Uses, Quality & Marketability of Plant & Animal Products. Development of antifungal natural products as crop protectants will provide novel ways in which to prevent fungal infestation of post harvest crops on a commercial basis. This will help ensure a safer, higher quality food and feed supply and reduce economic losses associated with crop diseases. Environmentally, such technology will reduce dependence on synthetic pesticides, provide target specificity, and will reduce application costs associated with traditional pesticides. This technology, once developed, would increase the value of the crops through finding new uses, increase the competitive ability of U.S. growers in export markets, and provide a wholesome, toxin-free crop to the marketplace.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment during FY 2000 year: Fungal diseases of crops cause very large economic losses through post-harvest spoilage of the commodity, and with the increased regulation and banning of synthetic based pesticides, economical methods to produce natural based pesticides and crop protectants are desperately needed. During this reporting period, Agricultural Research Service (ARS), Southern Regional Research Center (SRRC) scientists genetically changed a bacterial microorganism to greatly increase production of a highly potent, inhibitory peptide (small protein) active against several spoilage fungi. This research accomplishment has led to a possible method to increase production of the peptide as a crop protectant against spoilage fungi. IMPACT: The impact of this accomplishment is that technology is provided that could lead to eventual industrial production of the peptide for use as a crop protectant, thus preventing large economic losses due to
spoilage fungi. B. Other Significant Accomplishment(s), if any: None at this time. C. Significant Accomplishments/Activities that Support Special Target Populations: None at this time.
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
This new Agricultural Research Service (ARS) in-house project has only been in place since November 2, 1999, but continued progress (continuation of the previous project) has been made in increasing production of peptide (small protein) inhibitors of spoilage fungi that cause losses in post harvest commodities (see Question 4). A new inhibitory compound (designated CAY1) from cayenne pepper was chemically characterized (invention disclosure approved by ARS Patent Committee). CAY1, which inhibits certain spoilage fungi, could be useful as a protectant against post-harvest crop losses due to attack by these pathogenic fungi. When successfully implemented, the technology will prevent large economic losses in post-harvest commodities due to spoilage fungi and result in a reduction of synthetic pesticide use, which not only is expensive but adds to the environmental concerns caused by the use of intensive agriculture.
6. What do you expect to accomplish, year by year, over the next 3 years?
FY 2000-2001: Determine in small scale laboratory experiments if inhibitory peptides (small proteins) active against spoilage microbes could be potentially mass produced by the bacterial producing organism at economic levels required by industry. Test peptides for their range of inhibitory activity against a wide range of microbial/fungal plant pests as possible. Determine the mechanism of how the peptides inhibit microbes so that inhibitory activity could be improved through altering the structure of the peptide. Scale up production of protective natural products (such as CAY1 from cayenne pepper)(see Question 5). FY 2001-2002: In cooperation with scientists on Project 6435-42000-012-00D, genetically engineer higher plants (initially using a tobacco plant model system) with genes encoding new inhibitory peptides to enhance resistance to fungal spoilage organisms. Begin experiments to develop protective coatings (in cooperation with groups developing edible films) containing
natural product fungal inhibitors. FY 2002: Begin testing of antifungal technologies in combinations; e.g., test protective coatings containing various combinations of natural inhibitors on plants (initially comprising the tobacco model) which have also been genetically engineered with genes encoding inhibitory activities (e.g. peptides).
7. 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 & durability of the technology product?
Unit scientists participated in 2 scientific society meetings, presented papers, and participated in discussions with collaborators on various projects. They consulted with various scientists and groups on plant genetic engineering methods and in methods to determine the genetic coding of various genes. This is a relatively new project, but under the past project (6435-41000-062-00D), Unit scientists cooperated with sugarcane researchers and the American Sugarcane League on plant genetic engineering technologies to improve disease resistance in sugar cane. In FY 1999, a Materials Transfer Agreement with MycoLogics, Inc. was initiated with Claude Selitrennikoff of MycoLogics Inc. which relates to the current project. Subsequently, a Small Business Innovation Research Phase I grant was submitted and fully funded entitled, "A Novel Antifungal Compound from Cayenne Pepper", to the Department of Health and Human Services, Public Health Service to further study this plant-derived
fungicide as a therapeutic agent. The Agricultural Research Service (ARS) patent committee has approved the request to submit a composition of matter and use patent application on the fungicide (CAY1) to the U.S. Patent Office. Past collaborations with the Formosan Subterranean Termite Research Unit at Southern Regional Research Center (SRRC) have involved investigating possible uses of inhibitors of molting and termite gut microbes required for digestion of wood. Constraints to development of some of the above technologies: Technologies to routinely genetically engineer plants with potent microbial inhibitor genes may become available by the year 2001. However, end users will probably not be able to implement the technology until at least 2004 or 2005 because of the logistics of transformation of other crop species, field testing, and regulatory requirements of releasing genetically engineered organisms into the environment.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
Impacts
(N/A)
Publications
- Rajasekaran, K., Cary, J.W., Jacks, T.J., Cleveland, T.E. Genetic engineering for disease resistance in transgenic cotton and tobacco by introduction of antifungal proteins and peptides. XVI International Botanical Congress. 1999. p. 610. Abstr. #1903.
- De Lucca, A.J. Antifungal peptides: Potential candidates for the treatment of fungal infections. Expert Opinion on Investigational Drugs. 2000. v. 9. p. 273-299.
- De Lucca, A.J., Bland, J.M., Vigo, C.B., Cushion, M., Walsh, T.J. CAY-1, a novel fungicide from Capsicum frutescens. 40th Annual Meeting of the Interscience Conference of Antimicrobial Agents and Chemotherapy. September, 2000. Abstr. #1097.
- Engel, P., Scharfenstein, L.L. Disruption of a gene encoding a putative gamma-butyrolactone-binding protein increases nikkomycin production. Society for Industrial Microbiology. August, 1999. p. 95. Abstr. #P19.
- Rajasekaran, K., Cary, J.W., Cleveland, T.E. Expression of a gene encoding a synthetic antimicrobial peptide confers fungal resistance in vitro and in planta in transgenic plants. 6th International Conference on Plant Molecular Biology. Quebec, Canada. June, 2000. Abstr. #S03-96.
|
|