2013 Annual Report
1a.Objectives (from AD-416):
The overall goal of this research project is to enhance food safety by developing methods to reduce levels of trichothecenes and other mycotoxins that occur in grain crops as a result of infection by Fusarium graminearum (sexual stage, Gibberella zeae) and related trichothecene-producing species of Fusarium. FHB is a world-wide threat to grain producers and consumers, due to the loss in yield and to the presence of trichothecenes and other mycotoxins in the grain. As the world’s population continues to increase, the need to reduce mycotoxins in grain will increase. Development of methods to reduce mycotoxin contamination in grain will be enhanced through elucidation of the molecular genetic mechanisms that control mycotoxin production in F. graminearum and related fusaria, that control plant-fungal interactions, and that detoxify or otherwise modify mycotoxins. The objectives and proposed research are as follows: Objective 1: Identify and characterize mycotoxin detoxification genes as a mechanism to reduce/eliminate the toxins in grain-based food and feed; Objective 2: Determine the genetic bases and ecological significance of variation in types of trichothecene mycotoxins produced by Fusarium; Objective 3: Identify and characterize plant genes that affect biosynthesis of trichothecenes and other mycotoxins produced by Fusarium.
1b.Approach (from AD-416):
With a growing world population, access to safe food for all consumers, both domestic and international, will continue to be a global priority. In recent years, the world has experienced an increase in mycotoxin contamination of grains due to climatic and agronomic changes that encourage fungal growth during cultivation. One approach to reduce mycotoxin contamination of food and feed is to prevent preharvest infection of crop plants by mycotoxin-producing fungi. An alternative approach is to modify mycotoxins present in crops in order to render them nontoxic and safe for consumption by humans and animals. Fusarium head blight (FHB) is one of the most important diseases of wheat and other cereal grains worldwide. It reduces yield and quality and results in contamination of grain with trichothecene mycotoxins. The disease is caused by Fusarium graminearum as well as other trichothecene-producing species of Fusarium. The primary goal of the proposed research is to reduce levels of trichothecenes and other mycotoxins through studies that reveal how plants, the fungus Fusarium, and mycotoxins interact during infection. We expect to identify novel genes that modify, detoxify, or otherwise confer resistance to mycotoxins and to study the physiological and molecular role of mycotoxin production on the ability of Fusarium to infect wheat and other crops. We also will examine the genetic bases and ecological significance of variation in types of mycotoxins produced by Fusarium. Knowledge from these studies will contribute to development of strategies to control FHB, thereby protecting our food supply from mycotoxins. This technology will ultimately benefit other scientists, small grain breeders, stakeholders in the food and feed industry, and regulatory agencies such as the Center for Disease Control, U.S. Food and Drug Administration, Federal Grain Inspection Service, and Food Safety Inspection Service.
Trichothecenes are fungal toxins that block protein synthesis and are harmful to human and animal health. Contamination of wheat with the trichothecene deoxynivalenol (DON) is a food safety problem worldwide. Trichothecene contamination results from infection with Fusarium graminearum, a fungus that causes head scab. DON is a virulence factor that helps the fungus spread within wheat plants. In North America, the predominant F. graminearum strains causing wheat head scab produce 15-acetyl DON in culture. Recent surveys indicate that new Fusarium strains that produce 3-acetyl DON are emerging in North America and may be more aggressive or result in higher amounts of toxin in contaminated grain. We measured the amounts of toxin produced by North American strains. In order to determine if modifications in the type of trichothecene produced impact the severity of the disease or the amount of toxin produced in grain, we tested a set of F. graminearum mutant strains that differ only in the genes that control which toxins are produced on greenhouse grown wheat. We found that another Fusarium metabolite, culmorin, increases the toxicity of DON to plants. In order to determine if culmorin impacts the severity of disease, we produced and chemically characterized sets of Fusarium mutant strains that differ only in the genes that control DON and culmorin production. We found that a gene in F. graminearum, laeA, turns on the genes that control the production of DON and other mycotoxins, sexual and asexual development, and the ability of the fungus to cause plant disease. A way to limit head scab and its associated toxin contamination of grain is to introduce into plants genes that can detoxify the DON produced by Fusarium during infection and thereby limit the spread of the disease. Using DNA mining techniques, a set of trichothecene genes was discovered in a fungus used in biocontrol of insects, Beauveria bassiana, that suggest that the fungus may produce toxins similar to DON. Information on the type of toxins produced by this fungus will be used to discover new types of detoxification mechanisms. We collaborated with Rutgers University researchers to characterize DON detoxification genes in yeast and Arabidopsis. We found that harzianum A, a trichothecene produced by Trichoderma is not toxic to plants but can be used to control the growth of other plant disease-causing fungi. In addition, we collaborated with other scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit to produce T-2 toxin–glucoside that could be used to develop antibodies. This form of T-2 toxin has been found in contaminated grain but is considered a masked mycotoxin because it may escape detection with the analytical methods that have been used to keep T-2 toxin out of food and feed.
Yeast metabolite leads to tool for detecting masked mycotoxins. T-2 toxin is a trichothecene mycotoxin produced by Fusarium in infected small grains, especially oats. Ingestion of T-2 toxin contaminated grain can cause diarrhea, hemorrhaging, and feed refusal. Plants infected with mycotoxin-producing fungi form sugar derivatives of toxins. These sugar derivatives, sometimes called masked mycotoxins, are a food safety concern because they are not detectable by standard approaches and can be converted back to the parent toxin upon ingestion. Development of improved methods for detection has been hindered by an inability to isolate sufficient amounts of these masked toxins. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, found three yeast species that convert T-2 toxin to T-2 toxin glucoside. This research provided an efficient way to produce T-2 toxin glucoside that can be used to assess its toxicity and to develop new analytical methods for its detection and measurement.
Identification of a link between toxin type and Fusarium head blight severity. Fusarium head blight is caused by species of the fungus Fusarium which produce trichothecene mycotoxins. Previous studies have shown that the trichothecenes are factors in the severity of the disease. In this study we found that the type of trichothecene produced by the fungus may also be a factor in the severity of the disease. Although North American Fusarium usually produces the trichothecene 15-ADON, strains that produce a related toxin, 3-ADON, have been increasingly found in North America. In collaboration with scientists at Agriculture and Agri-Food Canada, Ottawa, Ontario, ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, found that wheat cultivars with moderate disease resistance had both more disease and higher toxin levels in the seed after infection with 3-ADON strains. Wheat growers can use this information to plant more highly resistant cultivars in regions where 3-ADON-producing Fusarium strains have been found.
Identification of a fungal metabolite that increases the toxicity of fungal toxins toward plants. Fusarium head blight is a devastating disease of cereal crops. The fungus causing the disease produces a trichothecene toxin, deoxynivalenol (DON), which is toxic to plants and helps the fungus spread in the plant tissue. DON is also toxic to humans and animals, and grain contaminated with DON is a significant food safety problem. ARS scientists in the Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois, found that some Fusarium strains produce a metabolite, culmorin, that enhances the toxicity of DON to plants, and are using modern molecular and genetic techniques to determine if Fusarium strains that make both DON and culmorin are more virulent on cereal crops. This research produced an improved understanding of the methods used by Fusarium to invade plant tissue and contaminate grain with toxins, and provides new targets for disease control and toxin reduction programs aimed at ensuring the quality and safety of the U.S. food supply.
McCormick, S.P., Price, N.P., Kurtzman, C.P. 2012. Glucosylation and other biotransformations of T-2 toxin by yeasts of the Trichomonascus clade. Applied and Environmental Microbiology. 78(24):8694-8702.
Malmierca, M.G., Cardoza, R., Alexander, N.J., McCormick, S.P., Collado, I.G., Hermosa, R., Monte, E., Gutierrez, S. 2013. Relevance of trichothecenes in fungal physiology: Disruption of tri5 in Trichoderma arundinaceum. Fungal Genetics and Biology. 53(2013):22-33.
Shin, S., Torres-Acosta, J.A., Heinen, S.J., McCormick, S.P., Lemmens, M., Kovalsky-Paris, P.M., Berthiller, F., Adam, G., Muehlbauer, G.J. 2012. Transgenic Arabidopsis thaliana expressing a barley UDP-glucosyltransferase exhibit resistance to the mycotoxin deoxynivalenol. Journal of Experimental Botany. 63(13):4731-4740.
Foroud, N.A., McCormick, S.P., MacMillan, T., Badea, A., Kendra, D.F., Ellis, B.E., Eudes, F. 2012. Greenhouse studies reveal increased aggressiveness of emergent Canadian Fusarium graminearum chemotypes in wheat. Plant Disease. 96(9):1271-1279.
McCormick, S.P., Alexander, N.J., Proctor, R. 2012. Trichothecene triangle: toxins, genes, and plant disease. In: Gang, D.R. editor. Phytochemicals, Plant Growth, and the Environment. New York, NY: Springer Science+Business Media. p. 1-17.
O'Connell, R.J., Thon, M.R., Hacquard, S., Van Themaat, E.V., Amyotte, S., Kleemann, J., Torres-Quintero, M., Damm, U., Buiate, E., Epstein, L., Alkan, N., Altmuller, J., Alvarado, B.L., Bauser, C., Becker, C., Birren, B.W., Chen, Z., Crouch, J., Duvick, J., Farman, M., Gan, P., Heiman, D., Henrissat, B., Howard, R.J., Kabbage, M., Koch, C., Kubo, Y., Law, A., Lebrun, M.H., Lee, Y.H., Miyara, L., Moore, N., Neumann, U., Panaccione, D.G., Panstruga, R., Place, M., Proctor, R., Prusky, D., Rech, G., Reinhardt, R., Rollins, J.A., Rounsley, S., Schardl, C., Schwartz, D.C., Shenoy, N., Shirasu, K., Stuber, K., Sukno, S.A., Sweigard, J.A., Takano, Y., Takahara, H., Vanderdoes, H.C., Voll, L., Will, I., Young, S., Zeng, Q., Zhang, J., Zhou, S., Dickman, M.B., Schulze-Lefert, P., Ma, L.J., Vaillancourt, L.J. 2012. Life-style transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses. Nature Genetics. 44:1060-1065.