1a. Objectives (from AD-416)
The overall goal of this project is to develop strategies that reduce contamination of maize with mycotoxins produced by Fusarium and thereby improve the safety of maize for human and animal consumption. To achieve this goal, we propose research that addresses the four objectives listed below. Objective 1: Identify and characterize Fusarium genes and proteins that regulate production of fumonisin mycotoxins and other secondary metabolites; Objective 2: Identify critical components of fungal-plant-environmental interactions that affect fumonisin contamination in maize; Objective 3: Identify variation in distribution and arrangement of biosynthetic genes for mycotoxins and other secondary metabolites among Fusarium species; Objective 4: Develop new ambient ionization mass spectrometry (MS) techniques for the convenient analysis of mycotoxins from a variety of food related matrices.
1b. Approach (from AD-416)
Fumonisin mycotoxins are common maize contaminants that pose risks to food safety and public health, as well as livestock health. The risks result from the ability of fumonisins to cause diseases, including cancer and neural tube defects. Fumonisin contamination is a direct result of infection of maize (corn) by the fungus Fusarium verticillioides, which is a major cause worldwide of maize ear rot but is also present at a high frequency in healthy maize kernels. Fumonisin levels are, however, generally much higher in rotted kernels than in healthy infected kernels. Understanding the genetic regulation of fumonisin production and the pathogenesis of F. verticillioides in maize is critical for the development of strategies to prevent fumonisin contamination. We propose to use molecular genetics and functional genomics to identify and characterize F. verticillioides genes that regulate fumonisin biosynthesis or affect pathogenesis, because such genes are potential targets for strategies to prevent fumonisin contamination. We also propose to examine genes responsible for production of two less well understood Fusarium mycotoxins, fusarins and fusaric acid, as part of efforts to clarify the importance of the toxins in food safety. Essential for efforts to improve food safety are methods to reliably detect and quantify contaminants. Thus, we also propose to develop ambient mass spectrometry (MS) for fumonisin analysis directly from maize. This method will be an improvement over exiting methods because it will bypass sample preparations and chromatography that are required for current MS analyses. Together, the results of our research will elucidate genetic factors that control mycotoxin production in Fusarium, improve methods for mycotoxin analysis, and contribute to strategies that reduce fumonisin contamination of maize. The results have the potential to improve the safety of maize for consumption by humans, and will provide tools to seed and biotechnology companies, regulatory agencies, and other research scientist working to reduce mycotoxin contamination.
3. Progress Report
Our research group has identified a gene (SGE1) in the fungus Fusarium verticillioides that is necessary for the fungus to cause disease on maize seedlings. SGE1 is predicted to code for a protein that turns other genes “on and off.” When a gene is “on,” the protein it encodes is synthesized and when “off,” the protein is not synthesized. The results indicate that SGE1 is directly involved in the disease process; it does not affect the ability of F. verticillioides to cause disease by altering the growth, morphology, or development of the fungus. Thus, SGE1 has potential to provide significant insight into how F. verticillioides causes disease on maize, and thereby significant insight into factors that contribute to fumonisin contamination of maize. Secondary metabolites are metabolites that are not essential for life but contribute to survival in certain environments. These metabolites include toxins (e.g. fumonisins and fusarin) and pigments. Our research group has demonstrated that the gene LAE1 has differing effects on production of different F. verticillioides secondary metabolites. For example, when LAE1 is inactivated, F. verticillioides exhibits a 50% and 80% reduction in production of fumonisin and fusarin, respectively. These results provide insight into the mechanisms in F. verticillioides that control production of fumonisins and other secondary metabolites. The results also demonstrate that a single gene can affect production of multiple metabolites. Such knowledge will be important for developing strategies to block fumonisin production to reduce contamination in maize. Fundamental to research on toxins is an ability to accurately measure the levels of the toxins produced in crops and food/feed derived from crops. Therefore, we have continued to develop a new method to detect and quantify fumonisin mycotoxins directly from intact maize kernels. The new method is an improvement over existing methods because it bypasses the time-consuming steps of grinding kernels and extracting the ground material with organic solvents. The improved method will benefit researchers, industry, and other organizations that monitor toxin content of grain crops. Fusarins are toxic Fusarium metabolites but their contribution to toxicity of the fungus is poorly understood. We have demonstrated that genes responsible for fusarin production are present in a genetically wide range of Fusarium species, including species that produce the economically important mycotoxins fumonisins and trichothecenes. These findings indicate that the genetic potential for fusarin production occurs widely in Fusarium. The findings will provide insight into the contribution of fusarins to the overall toxicity of Fusarium. In a survey of four Fusarium species that normally produce fumonisin mycotoxins, we found that a lack of toxin production in some strains of each species is associated with the absence of a gene that is essential for fumonisin production. These results provide an explanation for why low levels of fumonisin contamination sometimes occur in maize ears with high levels of ear rot.