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United States Department of Agriculture

Agricultural Research Service

2009 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.

3.Progress Report
Aflatoxin is a highly carcinogenic compound produced by certain fungi that can infect a wide variety of edible agricultural products, such as peanuts, corn, tree nuts, and in some countries, wheat. Aflatoxin contamination is a major food safety issue and is strictly regulated, resulting in it also being a significant international trade issue involving US agricultural products. USDA scientists in the Plant Mycotoxin Research Unit, Albany, CA, continued to provide breakthroughs on research on preventing aflatoxin production and fungal growth in 2008/09. MU scientists discovered, a number of compounds, mainly classified as antioxidants, that could prevent aflatoxin biosynthesis. MU molecular biologists showed that these anti-aflatoxigenic compounds affect genes involved in the oxidative stress response systems of fungi. In collaboration with scientists in the Food and Feed Safety Research Unit, New Orleans, LA, MU scientists were able to show that these compounds fully suppress all the genes in the aflatoxin biosynthetic gene cluster. Interestingly, although the phenolic antioxidants applied suppress aflatoxin biosynthesis, they are "recognized" by the fungus as being oxidizing agents. This was discovered by virtue of the fact that the fungus increases expression of genes involved in the oxidative stress response (peroxiredoxins) when exposed to these phenolics.

In a second major discovery, MU scientists showed that aflatoxin producing and catabolizing cultures communicate via some yet to be identified signal. Fungi that are stressed to produce more aflatoxin "signal" unstressed colonies. This signaling results in the unstressed colonies to produce more aflatoxin. The same type of signaling was discovered when fungi are placed under conditions to catabolize aflatoxin.

The third major breakthrough was discovery of safe natural compounds that enhance antifungal activity of commercial fungicides and antifungal drugs. This process was defined as chemosensitization. Chemosensitization was found to be useful against fungi that are both agricultural pests and those that are of medical importance, too. The natural compounds (chemosensitizing agents) are used to disrupt the signaling system that coordinates fungal response to antifungal agents. The use of these agents augments acitivity of commercial products from 100 to 1000 fold. This work was done in collaboration with scientists at the Centers for Disease Control, Atlanta, GA and the Institute of Hygiene and Tropical Medicine, Lisbon, Portugal, on medical fungi. Additional tests of chemosensitizing agents are now being performed in the field on apples, by collaborators at Washington State University, and wheat, rice and potatoes, by collaborators at the All Russian Research Institute, Golitsino, Russia.

Collaboration with the University of Valencia, Institute of Agricultural Chemistry and Food Technology, Department of Food Science, Fungal Biotechnology Group initiated an investigation on the effects of antioxidant components of wine grapes on ochratoxin production and the expression of ochratoxin-specific genes in Aspergillus carbonarius.

1. Safe Natural Compounds Enhance Antifungal Activity of Commercial Fungicides and Antifungal Drugs. Fungal resistance to commercial antifungal agents presents a serious problem to both agriculture and medicine. USDA scientists in the Plant Mycotoxin Research Unit, Albany, CA identified a number of safe, natural products that significantly enhance effectiveness of commercial fungicides, such as strobilurin and fludioxonil. These natural compounds weaken the ability of fungi to build their cell walls or to respire normally. Once fungi are weakened by “chemosensitization” with the natural products, the commercial products are anywhere from 100 to 1000 fold more effective. ARS scientists in Albany, CA, in collaboration with scientists at the Centers for Disease Control, Atlanta, GA, found chemosensitization to be effective against a number of human pathogenic fungi that cause aspergillosis. These natural products could potentially reduce the amounts of fungicides and drugs necessary to control fungi.

2. Aflatoxin producing fungi "communicate" with each other. USDA scientists in the Plant Mycotoxin Research Unit, Albany, CA discovered that fungi that are exposed to oxidative stress produce more aflatoxin. Moreover, these stressed fungi "communicate" with unstressed fungi which, in turn, also produce more aflatoxin. To date, the molecular basis of this "communication" has not been identified. Attempts to identify an organic compound that is the "signaling" agent have not been fruitful. Thus, it appears the communicating signal is probably a very small, non-organic compound. MU scientists are in the process of examining those types of compounds.

5.Significant Activities that Support Special Target Populations
Aspergillosis is mainly a disease of people living in agrarian environments. The discovery of chemosensitizing agents that can improve chemotherapy for aspergillosis could have an impact on residents of rural communities.

6.Technology Transfer

Number of Active CRADAs1
Number of New Patent Applications Filed1

Review Publications
Yu, J., Payne, G.A., Nierman, W.C., Machida, M., Bennett, J.W., Campbell, B.C., Robens, J.F., Bhatnagar, D., Dean, R.A., Cleveland, T.E. 2008. Aspergillus flavus Genomics as a Tool for Studying the Mechanism of Aflatoxin Formation. Journal of Food Additives & Contaminants. 25(9):1152-1157.

Yu, J., Payne, G.A., Campbell, B.C., Guo, B., Cleveland, T.E., Robens, J.F., Keller, N.P., Bennett, J.W., Nierman, W.C. 2008. Mycotoxin production and prevention of aflatoxin contamination in food and feed. In: Osmani, S. and Goldman, G. (eds). The Aspergilli: Genomics, Medical Aspects, Biotechnology, and Research Methods, CRC Press, Boca Raton, FL. pp.457-472.

Molyneux, R.J., Mahoney, N.E., Kim, J.H., Campbell, B.C. 2008. Bioassay-directed Isolation and Identification of Anti-aflatoxigenic Constituents of Walnuts. In: Colegate, S.M., Molyneux, R.J. Editors, Bioactive Natural Products: Detection, Isolation and Structural Identification. Boca Raton, FL: CRC Press. p. 421-437

Molyneux, R.J., Mahoney, N.E., Kim, J.H., Campbell, B.C. 2009. Health Aspects and Anti-Aflatoxigenic Activity of Phytochemicals in Tree Nuts. In: Alasalvar, C., Shahidi, F., Editor Tree Nuts: Composition, Phytochemicals and Health Effects, Edition. Boca Raton, FL: CRC Press. p. 95-107.

Last Modified: 12/1/2015
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