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United States Department of Agriculture

Agricultural Research Service


Location: Foodborne Toxin Detection and Prevention

2011 Annual Report

1a.Objectives (from AD-416)
Objectives of the research .
1)Release a promising insect resistant, premium quality almond variety, and evaluate other advanced breeding selections..
2)Characterize almond seed coat tannins at the genetic and biochemical levels and examine associaion of specific antioxidants with seed coat ink staining..
3)Identify and integrate multiple resistances into regionally adapted, high commercial quality breeding selections. Reduce mycotoxin contamination of agricultural commodities focusing on tree nuts (almonds, pistachios and walnuts) by identifying natural constituents or biocompetitive organisms that inhibit growth of fungi and aflatoxin production. Identify target genes in fungi that trigger mycotoxin biosynthesis focusing on stress response pathways.

1b.Approach (from AD-416)
Identify the natural constituents responsible for resistance of certain varieties of tree nuts to growth of aflatoxigenic strains of aspergillus. Isolate and identify novel metabolites in sclerotia of Aspergillus and develop analytical methods for such compounds in order to assess exposure levels of tree nut orchards to the fungus. Identify genes involved in triggering mycotoxin biosynthesis using high-through put bioassays. Assays involve use of deletion mutants, gene knockouts and complementation analysis. Discover natural compounds that disrupt functionality of gene targets identified. Develop biosensors for detecting toxic fungi in pre- and post harvest environments. Combining 5325-32000-006-00D (1/09).

3.Progress Report
This is the final report for the project 5325-42000-035-00D terminated January 2011. There was a period between the beginning of FY2011 and the termination date for the project in Fall and Winter. All planned experimentation for this project was completed. Some of this work served as a springboard for further research in a new project salient to the efforts of this project. The continuing efforts resulting from this project are outlined in the newly numbered project 5325-42000-037-00D. Major discoveries were made over this 5-year project. The core goal was to control mycotoxin, mainly aflatoxin, contamination of foods. The target crop was tree nuts (i.e., almonds, pistachios and walnuts). But, fundamental discoveries were made applicable to all crops. The most notable discovery was that natural antioxidants (mainly phenolic type compounds) prevent aflatoxin biosynthesis and also ochratoxin. This is a fundamental discovery because it provides insight for all crops that are to be bred for natural compounds that inhibit aflatoxin production by aflatoxin-producing fungi. This approach would enable crops to be resistant to contamination, innately, not requiring chemical treatment. A noteworthy example of this type of resistance we found in walnuts. This particular tree nut, though commonly infected with aflatoxin-producing fungi, is rarely contaminated with aflatoxin at levels that require rejection by our overseas trading partners. Ancillary to this discovery was that oxidative stress (such as drought, heat, sunlight, etc.) actually triggers aflatoxin production. We discovered natural chemicals that could be used to suppress this trigger, and the genetic basis of how this works. As a result of this genetic work, we discovered, perhaps as equal an important discovery as the one outlined above, how to kill fungi, in general, more effectively. We termed the process “chemosensitization.” The genetic research showed us that the system used by fungi to protect themselves against oxidative stress is an extremely good target for increasing vulnerability of fungi to antifungal agents. We have identified chemosensitizing agents that disrupt this protective response of fungi resulting in vastly improving the efficacy of commercial agricultural fungicides and medical antifungal drugs. Chemosensitization results in reducing levels of antifungal agents required for control and can result in overcoming resistance in strains that have become resistant to antifungal agents. This reduces costs of treatment and potential negative side effects associated with use of antifungal agents in nature and in treating patients. The results of this research have had an international reach, with major collaborations and ongoing research established with scientists in Russia, Portugal, and Finland. Moreover, the potential medical and veterinary applications resulted in ongoing studies with collaborators at the Stanford Univ. Medical School and Montana State Veterinary School. Practical application is currently being tested with collaborators on the Almond Board of California.

1. Natural volatile prevents aflatoxin contamination of almonds. This project was to develop methods of reducing mycotoxin contamination of food commodities by either inhibiting mycotoxin production and/or killing mycotoxin-producing fungi. Salicylaldehyde, a vaporizable natural chemical compound related to the active component of aspirin, was found by ARS scientists in Albany, CA, to be a potential antifungal fumigant. This compound has some antifungal activity, alone, but greatly improves fungicidal activity of the commercial fungicide, strobilurin. Salicylaldehyde has potential use as a natural fungicidal fumigant and is currently being tested on stockpiled almonds as a method to prevent aflatoxin contamination.

2. Natural compounds improve activity of antifungal drugs. A number of compounds showing antifungal activity were tested against strains of fungi that cause a severe human fungal disease called aspergillosis. ARS scientists in Albany, CA, were able to show that by simply altering the structure of these compounds slightly, antifungal activity was significantly augmented as well. A natural chemical in the spice thyme, called thymol, when co-applied with the antifungal drugs vastly increased the inhibition of fungal growth at dosages far lower than the drugs alone. These compounds worked by disrupting the fungus’ anti-oxidative system. These results showed that natural compounds have the potential to be safe and have the ability to enhance efficacy of commercial antifungal agents.

Review Publications
Kim, J.H., Campbell, B.C., Mahoney, N.E., Chan, K.L., Molyneux, R.J. 2010. Chemosensitization of aflatoxigenic fungi to antimycin A and strobilurin using salicylaldehyde, a volatile natural compound targeting cellular antioxidation system. Mycopathologia. 171(4):291-298.

Kim, J.H., Campbell, B.C., Mahoney, N.E., Chan, K.L., Molyneux, R.J., Balajee, A. 2010. Augmenting the activity of antifungal agents against aspergilli using structural analogues of benzoic acid as chemosensitizing agents. Fungal Biology. 114:817-824.

Last Modified: 4/18/2014
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