2012 Annual Report
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.
The fungus Fusarium produces multiple toxins that can contaminate cereal crops and that are associated with human and livestock diseases. The carcinogenic toxins fumonisins are common contaminates of corn and are a significant food and feed safety concern worldwide. The goal of our research is to identify genetic mechanisms that block fumonisin production in the fungus and thereby prevent contamination. Fumonisin levels in corn are positively correlated with levels of corn ear rot, a disease caused by fumonisin-producing species of Fusarium. Given this, prevention of ear rot should prevent fumonisin contamination. Thus, we are identifying Fusarium genes required by the fungus to cause ear rot, because such genes should constitute a genetic mechanism that can be obstructed to block fumonisin production. We have demonstrated that a gene required for synthesis of the carbohydrate trehalose and that mediates responses to nutritional changes is also required by Fusarium to cause disease and to produce fumonisins. In another line of research, we have demonstrated that genetic regulation of fumonisin production is linked to production of other Fusarium toxins by a gene, LAE1, that affects chromosome structure and influences whether a variety of toxin genes are expressed (i.e. turned on). To better understand the genetic control of this link between production of different toxins, we have identified regions of Fusarium chromosomes that contain groups of genes (gene clusters) that encode enzymes and other proteins required for toxin synthesis. We have used this knowledge to assess the genetic potential of Fusarium species to produce fumonisins and the mutagenic toxins fusarins. This assessment has provided valuable information on relative risks that the species pose to food and feed safety. The assessment also revealed that fumonisin biosynthetic genes can be transferred between Fusarium species. This transfer generated fumonisin-producing strains within a species that does not normally produce the toxins and thereby alerted scientists to a potential new threat to food safety. The two lines of research described above have revealed genetic mechanisms that enable Fusarium to cause disease of corn and that control toxin production in the fungus. This information will be used to develop novel control strategies to reduce fumonisin contamination of corn and improve the safety of food and feed derived from cereals.
In a third line of research, we have developed an analytical method to measure the fungal toxins fumonisins, fusaric acid, trichothecenes, and aflatoxins directly from corn kernels. The method is based on mass spectrometry but is a significant advancement in that it does not require the time-consuming and potentially hazardous protocol of extracting ground kernels with toxic organic solvents. This research has the potential to lead to innovative and highly efficient methods to measure toxin levels in corn so that contaminated grain can be kept out of the food supply.
Discovery of genetic mechanisms that control toxin production in the fungus Fusarium. The fungus Fusarium verticillioides is frequently associated with corn and is of concern to food and feed safety because it produces toxic metabolites that are common contaminants of corn. Agricultural Research Service scientists in Bacterial Foodborne Pathogens and Mycology Research Unit (BFP), National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, have identified multiple gene clusters (i.e. groups of genes located next to one another) in Fusarium verticillioides that are responsible for the production of toxins, such as the carcinogenic fumonisins and the mutagenic fusarins. The scientists have also demonstrated that a gene, LAE1, is required to activate the gene clusters and thereby induce production of the corresponding toxins in F. verticillioides. The identification of these toxin biosynthetic genes and the discovery of mechanisms used by the fungus to control toxin production provide scientists with information needed to develop novel strategies aimed at suppressing toxin production and improving the quality and safety of food and feed derived from cereals.
Brown, D.W., Butchko, R.A., Baker, S.E., Proctor, R. 2011. Phylogenomic and functional domain analysis of polyketide synthases in Fusarium. Fungal Biology. 116(2):318-331.
Choi, Y., Butchko, R.A., Shim, W. 2012. Proteomic comparison of Gibberella moniliformis in limited-nitrogen (fumonisin-inducing) and excess-nitrogen (fumonisin-repressing) conditions. Journal of Microbiology and Biotechnology. 22(6):780-787.
Busman, M., Desjardins, A.E., Proctor, R. 2012. Analysis of fumonisin contamination and the presence of Fusarium in wheat with kernel black point disease in the United States. Journal of Food Additives & Contaminants. 29(7):1092-1100.
Brown, D.W., Butchko, R.A., Busman, M., Proctor, R. 2012. Identification of gene clusters associated with fusaric acid, fusarin, and perithecial pigment production in Fusarium verticillioides. Fungal Genetics and Biology. 49(7):521-532.