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United States Department of Agriculture

Agricultural Research Service


Location: Food and Feed Safety Research

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?
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 a number of synthetic fungicides have been used for decades, plant pathogenic fungi can develop resistance to them, rendering currently employed fungicides ineffective. For example, Benlate, an agricultural fungicide widely used for 33 years, was removed by the manufacturer from agricultural use after successful litigation proved damage to human health. Concern over the use of synthetic fungicides in agriculture underscores the need to discover new, potent fungicides having no or low toxicity to plants, mammals and insects. Plants are known to produce a wide array of constitutive and induced antifungal compounds to fight infection. Plants, particularly edible ones, would be good candidates to explore for novel, fungicidal compounds to reduce or eliminate our reliance on synthetic chemicals which could be toxic to animals and beneficial insects.

The goal of this project is to discover new, value-added biobased products from plants, particularly from low value/underutilized crops. This would potentially lead to compounds that could be used to protect crops during storage and processing. Therefore, this project contains elements 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.

This project directly addresses portions of National Program 306 concerning the discovery of bioactive products for use as preservatives or pesticides (Problem Area 2a), discovery of antifungal compounds in crop by-products (Problem Area 2b) and new technologies to convert agricultural products into value-added bio-based products (Problem Area 2c). There is also a secondary effort in Component V (Host Plant Resistance) of National Program 303. This research effort addresses the need in Component V to identify antifungal plant protein genes leading to transgenic expression in host plants or up-regulation in producing plants to enhance resistance to fungal infection.

2.List by year the currently approved milestones (indicators of research progress)
Overall Goal: Isolation and characterization of new plant derived, antifungal protectants.

FY 2005: (1) Purify the antifungal compound present in barley cotyledon shoots as well as production of this compound and initial antifungal bioassays performed to determine the spectrum of activity. (2) Purify the cotton cotyledon antifungal compound with similar production of the compound and initial in vitro bioassays as with the barley compound and share compound with collaborators who would perform in vitro antifungal assays. (3) Initial extraction of peanut and rice hulls with subsequent bioassays to determine antifungal properties. (4) Production of our patented fungicide, CAY-1, for collaborative studies in greenhouse and field settings. (5) Maize antifungal purification and antifungal assays. (6) Collaborator determination of mode of action and safety of purified antifungal compounds.

FY 2006: (1) Continued production and testing of the cotton antifungal compound with collaborators. (2) Purification and structural analysis of okra fruit antifungal. Initial testing of antifungal compound by collaborators. (3) Production of CAY-1 for collaborators to test in greenhouse and fields. (4) Maize antifungal purification and antifungal assays. (5) Initial testing of barley antifungal in greenhouse by collaborators. (6) Collaborator determination of mode of action and safety of purified antifungal compounds.

FY 2007: (1) Production and in vitro testing of purified okra antifungal. (2) Purification and antifungal assays of purified peanut and rice hull antifungal. (3) Production of purified barley, cotton and okra antifungal for collaborative studies that include greenhouse and/or large lab-scale testing. (4) Begin plant transformation and engineering of crops with genome of one or more of the discovered antifungal compounds. (5) Collaborator determination of mode of action and safety of purified antifungal compounds.

FY 2008: (1) Prepare purified samples of barley, cotton and okra antifungal compounds for collaborative studies. (2) Purify rice and/or peanut hull antifungal compounds. (3) Plant transformation and engineering of crops with genome of one or more of the discovered plant antifungals. (4) Collaborator determination of mode of action and safety of purified antifungal compounds.

FY 2009: (1) Purified peanut and/or rice antifungal for collaborators. (2) Greenhouse or field testing of peanut and/or rice antifungal by collaborators.

4a.List the single most significant research accomplishment during FY 2006.
Partial purification of a potent fungicide, particularly active against the fungus, Fusarium, in water extracts of bay leaf leaves.

This work is relevant and directly addresses Problem Area 2a (the discovery of bioactive compounds for use as preservatives or pesticides), and Problem Area 2c (conversion of agricultural products into value-added biobased products of National Program 306 (Quality and Utilization of Agricultural Products). It has a secondary effort of Component V (identification of antifungal protein genes leading to transgenic expression in host plants or up-regulation in producing plants to enhance plant resistance to fungal infection) of National Program 303 (Plant Diseases).

The single most significant accomplishment during FY 2006 was the partial purification of a potent fungicide, particularly active against Fusarium, a serious plant pathogen, in water extracts of bay leaf. Our work addresses the problem of the development of resistance to fungicides by plant pathogenic fungi and the need of new, safe preservatives. These problems require the discovery of new fungicides that are non-toxic to plants, animals and humans. The possible impact is the development of a new agricultural fungicide effective against Fusarium, a serious phytopathogen of grains, as well as a new preservative for use in foods (e.g., bread) and post harvest crops such as citrus.

4b.List other significant research accomplishment(s), if any.
a) Hurricane Katrina’s damage to SRRC resulted in personnel dispersal and loss of access to the high pressure liquid chromatograph (HPLC) and none to the mass spectrometer (MS) instrumentation vital to our project. We could not continue our vital chemical separation work with the barley synergistic fungicidal compounds. Therefore, we studied two non-plant compounds for their antifungal activity. Compounds studied included EDTA and piperlongumine (chelators), and silver nitrate (source of silver ions). No studies have been reported showing the fungicidal or fungal-inhibitory properties of these compounds. We found that EDTA inhibits, but does not kill, fungi. Piperlongumine was inactive. However, our current work with silver ions shows that they are highly inhibitory to Aspergillus and Fusarium at low concentration. As soon as these studies are completed and pure CAY-1 is available in FY 2007, synergy studies will commence between CAY-1 and these compounds.

Related National Program Component: National Program 306, Problem Area 2a. This research comes under this problem area because it will determine whether EDTA and silver nitrate can act synergistically with CAY-1 (our patented fungicide), in vitro, against plant pathogenic fungi.

b) Development of Non Funded Cooperative Agreements. A cooperative agreement was developed with the Department of Natural Renewable Resources to study plant-produced antimicrobials. We are currently performing the initial separation studies of an antifungal compound from a Tibetan plant. The second agreement was set up with the Department of Entomology to determine whether our plant extracts have anti-insect properties).

Related National Program Component: National Program 303, Problem Areas 2a (discovery of bioactive compound for use as preservatives or pesticides) and 2c (new technologies to convert agricultural products into value-added bio-based products).

4c.List significant activities that support special target populations.

4d.Progress report.

5.Describe the major accomplishments to date and their predicted or actual impact.
A. Discovery of two saponins (A and B) structurally related to our patented (in 2001) fungicidal saponin (a compound with detergent properties), CAY-1, isolated from cayenne pepper. Saponin “A” has one less sugar, while saponin “B” has two less sugars, than CAY-1 in their structures. It is possible that these two saponins are metabolic precursors of CAY-1. Research on these saponins showed that (1) sugars are vital to the solubility of CAY-1 in water and (2) fungicidal properties are related to the number of sugars present. Fungicidal activity is greatest with CAY-1, less with “A” and absent in saponin “B.”

Predicted impact: This information could influence theories on the synthesis of fungicidal compounds.

B. Barley-produced fungicides. The second major accomplishment was the discovery of three small molecular weight, synergistic, 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 three fungicidal compound synergistic set in nature.

Predicted impact: (1) Successful isolation of these compounds could lead to up-regulation of these compounds in barley and subsequent enhanced protection against fungi. (2) Novel fungicides commercially used.

C. Discovery of potent fungicide in bay leaf. We discovered that bay leaf contains a potent fungicide(s) active against Fusarium species.

Predicted impact: Potential enhanced value of a low value crop (bay leaf) yielding a potent fungicide for commercial uses.

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.) entered into Material Transfer Agreements 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 Cooperative Research and Development Agreement 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 synergy fungicidal compounds, as well as bay leaf fungicidal compound, have not yet been patented so we do not foresee it being available to end-users before FY 2008-2009.

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).

Review Publications
De Lucca II, A.J., Cleveland, T.E., Wedge, D.E. 2005. Plant-derived antifungal proteins and peptides. Canadian Journal of Microbiology. 51:1001-1014.

De Lucca II, A.J., Klich, M.A., Sien, T., Cleveland, T.E., Walsh, T.J. 2005. Fungicidal properties of CAY-1, a plant saponin, for emerging fungal pathogens. 45th Interscience Conference on Antimicrobial Agents and Chemotherapy, December 2005, Washington, D.C. Abstract F-490. p. 180.

De Lucca II, A.J., Boue, S.M., Palmgren, M.S., Maskos, K., Cleveland, T.E. 2004. Reduced fungicidal properties of CAY-1 precursors. Interscience Conference on Antimicrobial Agents & Chemotherapy Proceedings. Abstract F-845. p. 208.

De Lucca II, A.J., Boue, S.M., Palmgren, M.S., Maskos, K., Cleveland, T.E. 2006. Fungicidal properties of two saponins from Capsicum frutescens and the relationship of structure and fungicidal activity. Canadian Journal of Microbiology. 52:336-342.

Last Modified: 9/22/2014
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