Aflatoxin is a toxic secondary compound produced by a fungal source and can be responsible for massive agricultural losses world-wide. It is estimated that 25% of the world's crops are contaminated with some sort of fungal toxin, aflatoxin being chief among them. Worldwide there is a net loss of 16 million tons of corn due to aflatoxin contamination. In the US alone, aflatoxin contamination of food/feed results in an estimated $270M agricultural loss every year. Elimination of aflatoxin is a critical economic and health issue in the US. Aflatoxin contamination in crops, and subsequently livestock, threatens greater agricultural development, food security and human health. When aflatoxin-contaminated food/feed is ingested it can result in hepatotoxicity, liver cancer, kwashiorkor and Reye's syndrome. Due to its high toxicity over 100 countries restrict the level of aflatoxin in food and feed. The US Department of Agriculture regulates the allowable level of aflatoxin in corn for livestock feed and human consumption. Corn destined for human food and dairy cattle feed has the tightest limit of 20 parts per billion (ppb). To put this number into perspective, 1 ppb is equivalent to a single drop of water in a 21,700 gallon (82,135 liter) swimming pool or from a time perspective, 1 sec in 31.7 yrs. Current aflatoxin prevention mechanisms are inadequate. Breeding for fungal resistant crops, agronomic practices to lower the ability for the fungus to grow, biocontrol with atoxigenic fungal strains, improved storage methods post-harvest and using trapping agents to block uptake of aflatoxins are all currently used and still the US losses millions in crop losses each year to aflatoxin contamination. Among these efforts, the one that has the longest outlook is the actual breeding of a fungal resistant crop. Although a number of researchers are working on this area there has yet to be a successful fungal-resistant corn plant produced. Biotechnology seems to be a viable and necessary option to alleviate this fungal toxin.The objective of this research is to produce engineered corn expressing both a toxin suppression cassette and a cassette to suppress actual fungal growth. The simultaneous approach of both retarding the fungal growth and suppressing the biosynthesis of the toxin would be a significant step towards the development of a novel disease control strategy and a combination the fungus will not be able to overcome or easily evolve tolerance. We have taken a novel approach to suppress aflatoxin accumulation in crops by using a gene suppression strategy to target the fungal biosynthetic steps in the aflatoxin producing fungus. Our preliminary results of 9 independent corn transgenic lines with a kernel-specific gene expression cassette designed to target a key step in the toxin biosynthesis pathway are very intriguing and promising. Results indicate no detectable toxins in medium infected fungal transgenic corn kernels and at least an 80% toxin reduction in very heavily infected transgenic kernels compared to nontransgenic controls. This project will build on the toxin suppressed corn lines by adding an additional gene expression cassette that will retard/eliminate actual contaminating fungal growth. Resultant engineered corn plants will be infected at both pre- and post-harvest conditions under controlled greenhouse environments by aflatoxin-producing fungus and growth and toxin levels will be determined and compared to non-engineered plants. Significant reductions in both contaminating fungal growth and toxin loads are expected in the engineered corn plants.Aflatoxin contamination of crops is a global problem that accounts for massive crop losses each year.Corn is at highest risk of aflatoxin contamination.Reducing and/or alleviating the toxin load and fungal growth at either pre- and/or post-harvest would contribute to eradication of this toxin.This research of a dual genetic suppression strategy of targeting simultaneously contaminating fungal growth and toxin production will offer a novel solution to aflatoxin contamination. The experiments performed in this research will result in the proof-of-concept data needed to determine if this molecular strategy to reduce aflatoxin in storage (post-harvest) and/or crops growing in fields (pre-harvest) is effective.