2013 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 toxins that contaminate cereal crops and are associated with human and livestock diseases. The cancer-causing toxins fumonisins are common contaminates of corn and are among the fungal toxins that are of most concern to food and feed safety. The goal of our research is to identify genetic mechanisms that block fumonisin production in the fungus and thereby reduce or prevent contamination. Fumonisin levels in corn are positively correlated with levels of corn ear rot, a disease caused by Fusarium. Given this, reducing ear rot should reduce fumonisin contamination. This year, we continued to identify and characterize Fusarium genes that are required by the fungus to cause disease, because it may be possible to inhibit the function of one or more of the genes in order to block development of ear rot and thereby reduce fumonisin contamination. A major focus of our efforts has been to randomly mutate individual genes in Fusarium and then screen the resulting mutant strains for a reduction in ability to cause disease. Analysis of over 400 mutant strains has identified five that are reduced in their ability to cause disease.
In a related line of research, we have continued to analyze factors that affect fumonisin contamination of corn in the field. This year, in collaboration with researchers at Iowa State University, we showed that another fumonisin-producing fungus, Aspergillus niger, caused only low levels of ear rot and fumonisin contamination.
We have also continued to identify and characterize Fusarium genes that regulate whether or not the fungus produces fumonisins and other toxins. One of these genes is involved in carbohydrate metabolism and appears to regulate fumonisin production indirectly by allowing the fungus to sense carbohydrates in the surrounding environment. Another gene encodes a protein that likely binds to and activates other genes. Because of the effects on fumonisin production,these genes have the potential to provide insights in how to interrupt the physiological processes that lead to the formation of fumonisins in Fusarium and thereby the development of strategies to reduce or eliminate fumonisin contamination in corn.
We have also continued to develop the innovative technology, known as ambient ionization mass spectrometry (AIMS), for analysis of fungal toxins in corn. In more traditional analytical methods, kernels are ground to a powder, the powder is extracted with organic solvents, and the levels of toxins are then measured in the solvent extracts. AIMS allows toxins to be measured directly from intact kernels by placing them into an instrument known as a mass spectrometer. Thus, AIMS can reduce the time necessary for analysis and safety concerns associated with use of solvents. We are examining two AIMS methods, both employ electrically charged molecules to dislodge toxins from kernels so they can be detected by the mass spectrometer. However, in one method, the charged molecules are in liquid form and in the other they are a gas. We can detect fumonisins with the liquid-based AIMS method but not with the gas-based method.
Identification of genetic and biochemical processes necessary for production of fungal toxins of concern to food safety. Fungi produce multiple toxins that can contaminate crops and are harmful to the health of humans and livestock. For example, fumonisin toxins can accumulate in corn and are associated with multiple diseases, including cancer and neural tube defects, in humans and livestock. A coordinated multidisciplinary approach is required to determine how and why fungi produce toxins. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois have identified the genetic and biochemical machinery that enable the fungus Fusarium to produce fumonisins and other toxins. In one approach, they identified the genes, enzymes, and biochemical reactions that are responsible for synthesis of fumonisins in Fusarium. In another approach, they identified mechanisms responsible for variation in fumonisin production among Fusarium species. In a third approach and in collaboration with German researchers, the scientists utilized genome sequencing technology to identify genes required for synthesis of all the different types of toxins produced by one species. In a fourth approach, they demonstrated that Fusarium species that cause high levels of fumonisin contamination in corn cause little or no contamination in wheat. This information will contribute to the development of strategies that prevent accumulation of fumonisins in crops and to the understanding of why some Fusarium species cause toxin contamination of crops and other species do not.
Lazzaro, I., Busman, M., Battilani, P., Butchko, R.A. 2012. FUM and BIK gene expression contribute to describe fumonisin and bikaverin synthesis in Fusarium verticillioides. International Journal of Food Microbiology. 160(2):94-98.
Uhlig, S., Busman, M., Shane, D.S., Ronning, H., Rise, F., Proctor, R. 2012. Identification of early fumonisin biosynthetic intermediates by inactivation of the FUM6 gene in Fusarium verticillioides. Journal of Agricultural and Food Chemistry. 60(41):10293-10301.
Busman, M., Butchko, R.A., Proctor, R. 2012. LC-MS/MS method for the determination of the fungal pigment bikaverin in maize kernels as an indicator of ear rot. Food Additives & Contaminants Part A. 29(11):1736-1742.
Wiemann, P., Sieber, C.M., Von Bargen, K.W., Studt, L., Niehaus, E.M., Michielse, C., Albermann, S., Wagner, D., Brown, D.W., Proctor, R., et al. 2013. Deciphering the cryptic genome: Genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Pathogens. 9(6):1-35.