Progress 01/19/06 to 01/18/11
Outputs
Progress Report Objectives (from AD-416) Identify novel candidate plant and fungal genes important in virulence and mycotoxin production. Validate gene function by gene disruption and expression analyses. Develop Arabidopsis thaliana as a model system for identification of novel plant genes for mycotoxin resistance and sensitivity. Provide the scientific basis for assessing potential impact of foreign nivalenol-producing lineages of Fusarium graminearum on U.S. agriculture. Approach (from AD-416) Genes involved in mycotoxin production and virulence will be identified from Messenger RNA (mRNA) populations isolated from fungal-infected and non-infected plants. Such genes will be cloned and expressed in representative test organisms to determine the gene product and the effect on virulence. Genes that degrade mycotoxins will be tested against numerous types of mycotoxins to determine their method of degradation and effectiveness. Using the model plant system Arabidopsis, ecotypes will be screened for the ability to degrade, modify, or tolerate mycotoxins. Promising genes will be isolated and tested for toxin modification. The potential impact of foreign nivalenol-producing lines of Fusarium on United States agriculture will be assessed by measuring the level of virulence on a short-maturing corn under greenhouse conditions. We continued to screen Fusarium species trichothecene production and characterize the types of trichothecenes produced. Trichothecenes are categorized as Type A or Type B and we have characterized a biosynthetic gene that controls which type of toxin is produced. Using molecular and genetic techniques, we found that changes in a single biosynthetic gene, TRI1, determines if Type A or Type B trichothecenes are produced and determined which biosynthetic steps were controlled by this gene. We found that this gene is multifunctional in F. graminearum and controls three steps in the production of the Type B trichothecene deoxynivalenol. This is an important issue for food safety and risk assessments because type A trichothecenes tend to be more toxic than type B trichothecenes. In a collaborative study with researchers at the University of Leon, Spain, we studied the genes that control the production of mycotoxins produced by Trichoderma, a fungus that has been used for biocontrol. We characterized three genes required to produce the trichothecenes harzianum A and trichodermin and found that they differed from Fusarium trichothecene genes in both organization and function. This research will improve our understanding of the genetic basis for trichothecene toxin production in crop plants and provides targets that should be useful for preventing trichothecene contamination of food and feed. To expand our search for novel mycotoxin resistance genes, we have cooperated with scientists at Rutgers University to search for yeast and Arabidopsis genes involved in susceptibility and resistance to mycotoxins. Identification of such genes in yeast or Arabidopsis will provide us with tools to search wheat and barley genomes for genes that improve the ability of crops to resist Fusarium head blight. We continued to characterize the functions of genes from a Fusarium graminearum expressed sequence tag (EST) library including a terpene cyclase and a P450 oxygenase gene required for culmorin biosynthesis. Accomplishments 01 Genetic control of toxins in emerging fungal pathogens of cereal crops. Fusarium head blight is a devastating disease of cereal crops. The fungu causing this disease produces a mycotoxin, deoxynivalenol (DON), that contributes to the spread of disease and is a major food safety concern. In the U.S., most of the Fusarium strains that cause head blight of whea produce a form of DON called 15ADON. New Fusarium strains that produce a different form of DON, 3ADON, have been increasingly found in North America. Scientists in the Agricultural Research Service (ARS) Bacterial Foodborne Pathogens & Mycology Research Unit at the National Center for Agricultural Utilization Research in Peoria, IL, used modern molecular a genetic techniques and chemical analysis to identify changes in a single gene that determine which form of the toxin is produced. This informatio is essential to understanding why these emerging fungal pathogens are increasing in frequency in U.S. wheat fields, with the ultimate goal of developing novel disease control strategies for these emerging fungal pathogens of cereal crops. 02 Identification of toxin genes in a biocontrol fungus. Trichothecenes ar toxins produced by a number of different fungi including Fusarium, a fungus that causes disease on a wide variety of agriculturally important plants. In collaboration with researchers at the University of Leon, Spa scientists in the Agricultural Research Service (ARS) Bacterial Foodbor Pathogens & Mycology Research Unit at the National Center for Agricultur Utilization Research in Peoria, IL, found that Trichoderma, a fungus tha has been widely used for biocontrol of fungal diseases of plants, has trichothecene toxin genes that differ in both organization and function from those characterized in Fusarium. This research provides the information and molecular tools needed to evaluate the safety and utilit of fungi used as biocontrol agents.
Impacts
(N/A)
Publications
- Khatibi, P.A., Newmister, S.A., Rayment, I., McCormick, S.P., Alexander, N. J., Schmale, III, D.G. 2011. Bioprospecting for trichothecene 3-O- acetyltransferases in the fungal genus Fusarium yields functional enzymes with different abilities to modify the mycotoxin deoxynivalenol. Applied and Environmental Microbiology. 77(4):1162-1170.
- Alexander, N.J., McCormick, S.P., Waalwijk, C., Van Der Lee, T., Proctor, R. 2011. The genetic basis for 3-ADON and 15-ADON trichothecene chemotypes in Fusarium graminearum. Fungal Genetics and Biology. 48(5):485-495.
- Ismail, Y., McCormick, S.P., Hijri, M. 2011. A fungal symbiont of plant- roots modulates mycotoxin gene expression in the pathogen Fusarium sambucinum. PLoS One. 6(3):1-7.
- Cardoza, R.E., Malmierca, M.G., Hermosa, M.R., Alexander, N., Mccormick, S. P., Proctor, R., Tijerino, A.M., Rumbero, A., Monte, E., Gutierrez, S. 2011. Identification of loci and functional characterization of trichothecene biosynthesis genes in the filamentous fungus of the genus Trichoderma. Applied and Environmental Microbiology. 77(14):4867-4877.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Identify novel candidate plant and fungal genes important in virulence and mycotoxin production. Validate gene function by gene disruption and expression analyses. Develop Arabidopsis thaliana as a model system for identification of novel plant genes for mycotoxin resistance and sensitivity. Provide the scientific basis for assessing potential impact of foreign nivalenol-producing lineages of Fusarium graminearum on U.S. agriculture. Approach (from AD-416) Genes involved in mycotoxin production and virulence will be identified from mRNA populations isolated from fungal-infected and non-infected plants. Such genes will be cloned and expressed in representative test organisms to determine the gene product and the effect on virulence. Genes that degrade mycotoxins will be tested against numerous types of mycotoxins to determine their method of degradation and effectiveness. Using the model plant system Arabidopsis, ecotypes will be screened for the ability to degrade, modify, or tolerate mycotoxins. Promising genes will be isolated and tested for toxin modification. The potential impact of foreign nivalenol-producing lines of Fusarium on U.S. agriculture will be assessed by measuring the level of virulence on a short-maturing corn under greenhouse conditions. In 2010, progress was made toward the goal of identifying genes and processes that will benefit the fitness of wheat, barley, and corn in their resistance to Fusarium, a mold that causes head blight disease in small grains. As the mold infects the plant, it produces mycotoxins, which are harmful to the health of plants, animals, and humans. Progress was made on the identification of a major gene involved in the biosynthesis of a little understood mycotoxin that is produced by a number of species of Fusarium. Some species of the mold produce higher amounts of this mycotoxin and our results this year will provide a basis for searching for the genes that control the production of this mycotoxin. Progress was also made on showing that this mycotoxin is involved in causing wheat head blight disease. These results are critical for providing the information necessary for the development of disease resistant crops. We also completed a study to determine the genetic basis of the production of two types of mycotoxins that predominate in contaminated wheat in the U.S. By building on our previous work of identifying genes necessary for production of the mycotoxin called vomitoxin, we proved that changes in a single gene were responsible for the difference in the mycotoxin produced. These results will provide guidance in disease forecasting of Fusarium head blight and assist in developing tools to identify the type of mycotoxin produced by a Fusarium species. In a collaborative study with researchers at Virginia Polytech Institute and State University, we are using a bioprospecting approach to search the genomes of Fusarium species for improved genes that provide resistance to mycotoxins. We found several functional enzymes that vary in their ability to modify the mycotoxin called deoxynivalenol. We will further characterize these enzymes in an effort to find one that can be used to identify key structural features necessary for the development of commercial modification/detoxification processes. We have continued to compare mycotoxin biosynthetic genes and enzymes from various Fusarium species to better understand how mycotoxins are made. We have discovered that multiple arrangements exist for these genes and that important disease-producing species of Fusarium have a more ancestral arrangement. These results provide information to design strategies that will interfere with the production of the mycotoxin with the ultimate goal of reducing plant disease. To expand our search for novel mycotoxin resistance genes, we have cooperated with scientists at Rutgers University to search for yeast genes involved in susceptibility and resistance to mycotoxins as well as any cellular targets of mycotoxins. Identification of such genes in yeast will provide us with tools to search the genome of plants, especially in wheat or barley, to enable us to produce plants with the ability to resist Fusarium head blight. Accomplishments 01 IDENTIFICATION OF A GENE RESPONSIBLE FOR TYPE OF MYCOTOXIN PRODUCED. In the U.S., the predominant Fusarium species causing Fusarium head blight wheat produces the mycotoxin 15-acetyldeoxynivalenol (15ADON). Recent studies suggest that new lines of Fusarium that produce the mycotoxin 3- acetyldeoxynivalenol (3ADON) are emerging in North America and may be mo aggressive. Bacterial Foodborne Pathogens and Mycology Research Unit scientists at the National Center for Agricultural Utilization Research Peoria, IL, used modern genetic and rigorous chemical techniques to determine the genetic basis of the 15ADON and 3ADON production. They fou that changes in a single mycotoxin biosynthetic gene caused the producti of either 15ADON or 3ADON. This research provides the basis for continue investigation to determine the aggressiveness of each chemotype in causi disease on wheat. The aggressiveness factor will be useful in disease forecasting. 02 IDENTIFICATION OF A GENE INVOLVED IN THE BIOSYNTHESIS OF THE MYCOTOXIN CULMORIN. Fusarium head blight is a devastating disease of cereal crops and is especially aggressive during wet weather when wheat is flowering. The fungus causing the disease also produces a mycotoxin that helps the fungus spread in the wheat head. Reduction of mycotoxins in cereal grain is important for producing safe and sufficient quantities of foods for growing populations. Bacterial Foodborne Pathogens and Mycology Research Unit scientists at the National Center for Agricultural Utilization Research in Peoria, IL, identified a gene involved in the biosynthesis o the mycotoxin culmorin, a little-known mycotoxin, which may contribute t the spread of Fusarium head blight of wheat. Discovery of the gene responsible for the production of culmorin will help wheat breeders develop cultivars resistant to head blight. 03 IDENTIFICATION OF GENES INVOLVED IN MYCOTOXIN RESISTANCE. Fusarium head blight, a serious disease of cereal crops, has severe economic and healt impacts, the latter due to the presence of mycotoxins made by the invadi mold. One strategy to combat this disease is to modify genes in cereals, such as wheat and barley, to provide resistance to the mycotoxin. Bacterial Foodborne Pathogens and Mycology Research Unit scientists at t National Center for Agricultural Utilization Research in Peoria, IL, cooperated with scientists at Rutgers University to screen a library of yeast genes. They identified several previously unknown genes that are involved in mycotoxin toxicity and demonstrated, for the first time, whi parts of a cell are more sensitive to mycotoxins. These results can be used to search for plant genes that may provide novel targets for introducing resistance to mycotoxins. Also, plant researchers may be abl to target parts of the cell that are sensitive to mycotoxins and thereby produce a plant more resistant to mycotoxins. These mycotoxin resistance genes may be used by plant breeders to produce Fusarium head blight resistant lines of cereal crops. 04 RELOCATION OF GENES IN A SECONDARY METABOLITE BIOSYNTHETIC CLUSTER IN FUSARIUM. Trichothecenes are compound metabolites produced by numerous fungi, including some species of Fusarium that are pathogenic to some plants. Trichothecenes contribute to the virulence of Fusarium on small grains and are considered mycotoxins because of their toxicity to humans and animals. Bacterial Foodborne Pathogens and Mycology Research Unit scientists at the National Center for Agricultural Utilization Research Peoria, IL, discovered that the cluster of genes involved in trichothece production had increased in size during evolution of some species due t the relocation of genes into the cluster from elsewhere in the genome. This result provides tools with which scientists can examine the mycotox biosynthetic pathway and design strategies for reducing trichothecene production. 05 IDENTIFICATION OF A GENE CONTROLLING MYCOTOXIN DIFFERENTIATION. The fung Fusarium, which causes wheat head blight disease, also produces several types of mycotoxins, which are a health hazard to humans and animals. Bacterial Foodborne Pathogens and Mycology Research Unit scientists at t National Center for Agricultural Utilization Research in Peoria, IL, identified the gene in Fusarium that plays a key role in the production Type A (e.g., T-2 toxin) or Type B (e.g., nivalenol, deoxynivalenol) trichothecene mycotoxins. This information is important for agencies tha monitor the safety of food and feed supplies because type A trichothecen tend to be more toxic than type B trichothecenes. The finding that the gene exhibits a high level of variability in diverse Fusarium species provides information that can be used to design rapid methods to detect, and distinguish, between species that produce Type A and Type B trichothecenes.
Impacts
(N/A)
Publications
- McCormick, S.P. 2009. Phytotoxicity of Trichothecenes. In Appell, M., Kendra, D.F., Trucksess, M.W., editors. American Chemical Society Symposium Series 1031. Mycotoxin Prevention and Control in Agriculture. Washington DC: American Chemical Society. p. 143-155.
- Mccormick, S.P., Alexander, N.J., Harris, L.J. 2010. CLM1 of Fusarium graminearum Encodes a Longiborneol Synthase Required for Culmorin Production. Applied and Environmental Microbiology. 76(1):136-141. doi:10. 1128/AEM.02017-09.
- Mclaughlin, J.E., Bin-Umer, M.A., Tortora, A., Mendez, N., Mccormick, S.P., Turner, N.E. 2009. A Genome-Wide Screen in Saccharomyces cerevisiae Reveals a Critical Role for the Mitochondria in the Toxicity of a Trichothecene Mycotoxin. Proceedings of the National Academy of Sciences. 106(51):21883-21888. doi: 10.107/pnas.0909777106.
- Alexander, N.J., Proctor, R.H., Mccormick, S.P. 2009. Genes, Gene Clusters, and Biosynthesis of Trichothecenes and Fumonisins in Fusarium. Toxin Reviews. 28(2/3):198-215.
- Liu, Z., Palmquist, D.E., Ma, M., Liu, J., Alexander, N.J. 2009. Application of a Master Equation for Quantitative mRNA Analysis Using qRT- PCR. Journal of Biotechnology. 143:10-16.
- Proctor, R.H., McCormick, S.P., Alexander, N.J., Desjardins, A.E. 2009. Evidence that a Secondary Metabolic Biosynthetic Gene Cluster has Grown by Gene Relocation During Evolution of the Filamentous Fungus Fusarium. Molecular Microbiology. 74(5):1128-1142.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Identify novel candidate plant and fungal genes important in virulence and mycotoxin production. Validate gene function by gene disruption and expression analyses. Develop Arabidopsis thaliana as a model system for identification of novel plant genes for mycotoxin resistance and sensitivity. Provide the scientific basis for assessing potential impact of foreign nivalenol-producing lineages of Fusarium graminearum on U.S. agriculture. Approach (from AD-416) Genes involved in mycotoxin production and virulence will be identified from mRNA populations isolated from fungal-infected and non-infected plants. Such genes will be cloned and expressed in representative test organisms to determine the gene product and the effect on virulence. Genes that degrade mycotoxins will be tested against numerous types of mycotoxins to determine their method of degradation and effectiveness. Using the model plant system Arabidopsis, ecotypes will be screened for the ability to degrade, modify, or tolerate mycotoxins. Promising genes will be isolated and tested for toxin modification. The potential impact of foreign nivalenol-producing lines of Fusarium on U.S. agriculture will be assessed by measuring the level of virulence on a short-maturing corn under greenhouse conditions. Significant Activities that Support Special Target Populations In our search for factors that contribute to Fusarium Head Blight of small grains, we have identified a gene in Fusarium, using an EST library, involved in the biosynthesis of a little understood mycotoxin and have shown that this mycotoxin is likely to be involved in pathogenesis on wheat. Identification of important mycotoxin genes and pathways and their corresponding involvement in plant diseases is crucial for developing disease resistant crops. We have also examined the kinetic aspects of a specific trichothecene biosynthetic enzyme with various substrates and discovered that modification of trichothecenes is determined by conservation of active site sequences. This may offer a strategy for locking up mycotoxins in a non-reversible manner. To better understand the function of the trichothecene biosynthetic genes Tri1 and Tri101, we have compared the nucleotide sequences, flanking genes, and function of these genes from various Fusarium species. We have discovered that multiple arrangements exist for these genes and that important disease-producing species of Fusarium have a more ancestral arrangement. These results provide information to design strategies that interfere with the genes that contribute to Fusarium- induced diseases of crop plants. To understand the relationship between the type of mycotoxin produced by a Fusarium strain and the amount of disease caused, we have initiated a study to determine the chemical and genetic basis of mycotoxin production profiles, i.e. chemotypes. We have obtained the necessary Fusarium strains and completed initial genetic and chemical characterizations. Technology Transfer Number of New/Active MTAs(providing only): 10
Impacts
(N/A)
Publications
- Desjardins, A.E. 2008. Natural Product Chemistry meets Genetics: When is a Genotype a Chemotype? Journal of Agricultural and Food Chemistry. 56(17) :7587-7592.
- Desjardins, A.E. 2009. From Yellow Rain to Green Wheat Twenty-five Years of Trichothecene Biosynthesis Research. Journal of Agriculture and Food Chemistry. 57(11):4478-4484.
- Seong, K., Pasquali, M., Hilburn, K.L., Mccormick, S.P., Xu, J., Kistler, H.C. 2009. Global Gene Regulation by Fusarium Transcription Factors Tri6 and Tri10 Reveals Adaptations for Toxin Biosynthesis. Molecular Microbiology. 72:354-367.
- Alexander, N.J., Mccormick, S.P., Blackburn, J.A. 2008. Effects of Xanthotoxin Treatment on Trichothecene Production in Fusarium sporotrichioides. Canadian Journal of Microbiology. 54(12):1023-1031.
- Garvey, G.S., Mccormick, S.P., Alexander, N.J., Rayment, I. 2009. Structural and Functional Characterization of TRI3 Acetyltransferase from Fusarium sporotrichioides. Protein Chemistry. 18(4):747-761.
- Desjardins, A.E., Busman, M., Manandhar, G., Jarosz, A.M., Manandhar, H.K., Proctor, R. 2008. Gibberella Ear Rot of Maize (Zea mays) in Nepal: Distribution of the Mycotoxins Nivalenol and Deoxynivalenol in Naturally and Experimentally Infected Maize. Journal of Agricultural and Food Chemistry. 56(13):5428-5436.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Identify novel candidate plant and fungal genes important in virulence and mycotoxin production. Validate gene function by gene disruption and expression analyses. Develop Arabidopsis thaliana as a model system for identification of novel plant genes for mycotoxin resistance and sensitivity. Provide the scientific basis for assessing potential impact of foreign nivalenol-producing lineages of Fusarium graminearum on U.S. agriculture. Approach (from AD-416) Genes involved in mycotoxin production and virulence will be identified from mRNA populations isolated from fungal-infected and non-infected plants. Such genes will be cloned and expressed in representative test organisms to determine the gene product and the effect on virulence. Genes that degrade mycotoxins will be tested against numerous types of mycotoxins to determine their method of degradation and effectiveness. Using the model plant system Arabidopsis, ecotypes will be screened for the ability to degrade, modify, or tolerate mycotoxins. Promising genes will be isolated and tested for toxin modification. The potential impact of foreign nivalenol-producing lines of Fusarium on U.S. agriculture will be assessed by measuring the level of virulence on a short-maturing corn under greenhouse conditions. Significant Activities that Support Special Target Populations We have continued to define the trichothecene biosynthetic pathways in Fusarium by expressing three of the genes involved in mycotoxin production in transgenic systems. We are continuing the search for novel mycotoxin resistance genes by cloning a potential gene found in a Fusarium EST library, disrupting the gene, and transforming wild type Fusarium. Transformants have been sent to colleagues for phenotype identification. To better understand the function of the trichothecene biosynthetic gene Tri1, we have initiated a comparison of the nucleotide and deduced amino acid sequence and function of this gene from Fusarium species. We have also continued to use the Arabidopsis bioassay system to elucidate the trichothecene structural and functional relationships and to screen for plant resistance to trichothecenes. Accomplishments Arabidopsis sensitivity to trichothecene mycotoxins. The model plant Arabidopsis thaliana was used to compare the toxicity of agriculturally important trichothecenes that differ in structure from simple to complex. We showed that even simple trichothecenes can be quite toxic to plants, thus discovery of genes that confer ability to detoxify these compounds should be a high priority. This research should be of interest to maize and wheat pathologists and breeders who are trying to develop varieties that are resistant to trichothecene-producing Fusarium species that cause maize ear rot and wheat head blight. This contributes to National Program 108, Food Safety, Component 2, entitled Mycotoxins: Breeding Resistant Crops. Quantitative expression of mycotoxin genes. The quantitative measurement of the expression of genes involved in production of mycotoxins was set up. Standards were determined for measuring the housekeeping genes as well as the mycotoxin biosynthetic genes when the organism was subjected to stressful conditions. This research addresses the problem of understanding mycotoxin biochemical pathways for the improvement of crops to be resistant to fungal infection. This contributes to National Program 108, Food Safety, Component 2, entitled Mycotoxins: Breeding Resistant Crops. Mycotoxin differentiation. In the biosynthesis of trichothecene mycotoxins, the gene Tri1 plays a key role in which type of trichothecene is produced. We have examined Tri1 sequences from diverse Fusarium species and found that Tri1 exhibits a high level of variability. These results provide information that can be used to design rapid methods to detect and distinguish between species that produce trichothecenes with an 8-keto or an 8-hydroxyl group. This contributes to National Program 108, Food Safety, Component 2, entitled Mycotoxins: Breeding Resistant Crops. Technology Transfer Number of New CRADAS and MTAS: 4 Number of Active CRADAS and MTAS: 10 Number of Non-Peer Reviewed Presentations and Proceedings: 8 Number of Newspaper Articles,Presentations for NonScience Audiences: 2
Impacts
(N/A)
Publications
- Mc Cormick, S.P., Alexander, N.J. 2007. Myrothecium roridum Tri4 encodes a multifunctional oxygenase required for three oxygenation steps. Canadian Journal of Microbiology. 53:572-579.
- Desjardins, A.E. 2006. Fusarium mycotoxins: chemistry, genetics, and biology. St. Paul, MN: American Phytopathological Society Press. 260 p.
- Harris, L., Alexander, N.J., Saparno, A., Blackwell, B., Mc Cormick, S.P., Desjardins, A.E., Robert, L., Tinker, N., Hattori, J., Piche, C., Schernthaner, J., Watson, R., Ouellet, T. 2007. A novel gene cluster in Fusarium graminearum contains a gene that contributes to butenolide synthesis. Fungal Genetics and Biology. 44(4):293-306.
- Desjardins, A.E., Mc Cormick, S.P., Appell, M.D. 2007. Structure-activity relationships of trichothecene toxins in an Arabidopsis thaliana leaf assay. Journal of Agricultural and Food Chemistry. 55(16):6487-6492.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Fusarium Head Blight, a disease of wheat and barley that has severe economic impact on U.S. agriculture, is caused by invasion of the plants by a fungus (Fusarium graminearum) while the crop is growing in the field. This fungus also causes corn ear rot. These diseases cause serious losses in crop quality and yield and, to make matters worse, the fungus produces trichothecene toxins, especially deoxynivalenol, that are harmful to humans and animals that eat toxin-contaminated food or feed. The potential for the presence of fungal toxins causes extra expenses to be incurred by the grain industry for testing to assure that wheat, barley, and corn are safe for human and animal consumption and also threatens the competitiveness of U.S. agriculture in the world market. The best strategy to keep
these toxins from entering the food supply is to prevent them from being produced in the first place. To develop safe, reliable, and efficient ways to eliminate or minimize the ability of this fungus to infect wheat, barley, and corn we are determining how toxins are made and regulated, as well as the role they play in the ability of the fungus to infect crop plants and cause disease. The potential presence of toxins in the food supply means that expensive testing and remedial actions are necessary to assure that they do not reach dangerous levels in our food. The cost of testing, and the losses in crop quality and yield associated with Fusarium Head Scab have caused multi-billion dollar losses to farmers, especially the wheat and barley industries in the central U.S. during the past 10 years. This research relates directly to the mycotoxin component of the National Program 108, Food Safety. Basic knowledge from our research on plant-fungal interactions associated with the
infection process and toxin synthesis will facilitate the design and implementation of strategies to control and minimize the presence of deoxynivalenol in wheat, barley, and corn through combinations of altered agronomic practices, chemical and biological control, and improved plant resistance to mycotoxins. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2006) 1A. Characterize, genetically and biochemically, two genes co-regulated with the trichothecene cluster. 1B. Identify new candidate genes from EST libraries. 1C. Using the yeast system, screen for mycotoxin resistance genes. 2A. Construct differential libraries from Wheaton and Fundulea 201-R. 3A. Set up Arabidopsis screening program. 4A. Set up maize risk assessment system. Year 2 (FY 2007) 1B. Identify genes involved with mycotoxin biosynthesis from EST libraries. 1C. Identify potential mycotoxin resistance genes. 2B. Screen differential cDNA library. 3B. Assess the effect of
mycotoxins on the biological assay system in Arabidopsis. 3C. Obtain Arabidopsis ecotypes and begin testing on mycotoxin-amended media. 4A. Complete maize risk assessment. Year 3 (FY 2008) 1B. Continue identification and characterization of genes involved with mycotoxin biosynthesis 1D. Identify genetic regulatory events of mycotoxin biosynthesis 2C. Identify mycotoxin resistance genes from Fondulea 201-R. 3C. Continue testing of Arabidopsis ecotypes for mycotoxin resistance. 3D. Gene mapping studies of Arabidopsis mycotoxin resistance. Year 4 (FY 2009) 1D. Continue characterization of regulation of mycotoxin biosynthesis. 1E. Perform virulence testing using potential resistance genes. 2D. Test mycotoxin resistance genes from Fondulea 201-R. 3D. Continue crosses and allelism studies in Arabidopsis. Year 5 (FY 2010) 1F. Genomic/proteomic analysis of EST libraries. 2E. Distribute mycotoxin resistance genes to plant transformation community. 3D. Examine Arabidopsis databanks to obtain
gene sequences. 3E. Clone candidate genes from Arabidopsis. 4a List the single most significant research accomplishment during FY 2006. IDENTIFICATION OF THE BIOCHEMICAL FUNCTION OF THE TRI4 GENE INVOLVED IN TRICHOTHECENE PRODUCTION (NP 108, Food Safety, Component 2, entitled Mycotoxins: Breeding Resistant Crops.): Although the genetic basis of trichothecene biosynthetic pathway in F. sporotrichioides has been largely worked out by our CRIS unit, all the genes required for the oxidation steps had not been identified. Using our previously developed heterologous expression system, we were able to show that the F. graminearum Tri4 protein catalyzes four steps in the trichothecene pathway, and is therefore a multifunctional monooxygenase. This identification completes the search for most of the oxygenase genes required in this pathway. This addresses the problem of understanding mycotoxin biochemical pathways for the improvement of crops to be resistant to fungal infection. 4b List
other significant research accomplishment(s), if any. MYCOTOXIN VIRULENCE TESTING ON MAIZE IN A GREENHOUSE TEST SYSTEM. NP 108, Food Safety, Component 2, entitled Mycotoxins: Breeding Resistant Crops. We used our recently developed greenhouse virulence testing system to determine that F. graminearum strains that produce the mycotoxin deoxynivalenol, and strains that produce the mycotoxin nivalenol, are both capable of producing corn ear rot disease. The research results impact the grower and breeder because it shows that even though nivalenol producing strains are now rare in the US, they may be capable of causing ear rot disease if they are introduced into this country. This addresses the problem of understanding mycotoxins and how they relate to disease. F. PROLIFERATUM PRODUCES DISEASE IN WHEAT. NP 108, Food Safety, Component 2, entitled Mycotoxins: Breeding Resistant Crops. We determined that F. proliferatum produces blackpoint disease in wheat, and that the mycotoxin,
fumonisin, can be detected in the moldy grain. These results impact the producers by showing the need to test wheat that has blackpoint disease for the mycotoxin, fumonisin. This addresses the problem for the need to analyze, biochemically, diseased crops for the presence of mycotoxins. BIOASSAY SYSTEM IN ARABIDOPSIS FOR THE SCREENING OF MYCOTOXIN ACTIVITY. NP 108, Food Safety, Component 2, entitled Mycotoxins: Breeding Resistant Crops. We have set up a unique bioassay system for the detection of mycotoxin activity in the plant system of Arabidopsis. The assay has allowed us to assay the effect of oxygenation and esterification of the trichothecene structure on detached leaves. The result is important in the identification of the phytotoxic component of trichothecenes. This addresses the problem of understanding the mode of action by mycotoxins on plant tissue to cause disease. ANTI-MICROBIAL ACTIVITY OF A MYCOTOXIN PATHWAY GENE IN MYROTHECIUM RORIDUM. NP 108, Food Safety,
Component 2, entitled Mycotoxins: Breeding Resistant Crops. We have identified a segment of a gene in the mycotoxin biosynthetic pathway of M. roridum that has anti-microbial activity. The identification could result in the promise of a new anti- microbial compound. This addresses the problem of understanding mycotoxins and how they relate to disease. 4d Progress report. We have continued to define the trichothecene biosynthetic pathway by measuring expression of genes that are responsible for critical biosynthetic steps. We are using fungal genomics approaches to continue identification and characterization of Fusarium graminearum genes expressed during the plant-fungal interaction. 5. Describe the major accomplishments to date and their predicted or actual impact. NP 108, Component 2, entitled Mycotoxins: Breeding Resistant Crops. This is the first year of a new project continued from 3620-42000-026-00D. We showed that the F. graminearum Tri4 protein catalyzes four steps in the
trichothecene pathway and is therefore a multifunctional monooxygenase. This will be important for other researchers studying the trichothecene pathway or other mycotoxin biosynthetic pathways. We also determined that F. graminearum strains that produce the mycotoxin deoxynivalenol, and strains that produce the mycotoxin nivalenol, are both capable of producing corn ear rot disease. This will impact breeders and farmers if nivalenol producing strains are introduced into this country. We also determined that F. proliferatum produces blackpoint disease in wheat, and that the mycotoxin, fumonisin, can be detected in the moldy grain. These results impact the producers by showing the need to test wheat that has blackpoint disease for the mycotoxin, fumonisin. We also have identified a segment of a gene in the mycotoxin biosynthetic pathway of M. roridum that has anti-microbial activity. This information will be used by other researchers. 6. What science and/or technologies have been
transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Knowledge developed by our research concerning the role of toxins in Fusarium Head Blight has been transferred to researchers and plant breeders and has impacted the direction of research to find effective ways to manage the disease and develop more resistant new varieties of wheat and barley. Molecular engineered tools to study the disease and infection process and measure plant resistance have been transferred to other researchers and the seed industry. Potential genes for resistance to deoxynivalenol have been transferred to other ARS laboratories and to the seed industry to develop transgenic wheat and barley varieties that are more resistant to Fusarium Head Blight. 7. List your most important publications in the popular press and
presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). McCormick, S., Desjardins, A. 2006. Emerging Mycotoxin Issues [abstract]. Proceedings of the 47th Annual Corn Dry Milling Conference, June 1-2, 2006, Peoria, Illinois. Desjardins, A.E. 2006. Final report: integrating plant biodiversity science in Nepal. Report to the U.S. Embassy Science Fellowship in Nepal.
Impacts
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Publications
- McCormick, S.P., Alexander, N.J., Proctor, R. H. 2006. Heterologous expression of two trichothecene P450 genes in Fusarium verticillioides. Canadian Journal of Microbiology. 52:220-226.
- Desjardins, A.E., Plattner, R.D., Stessman, R.J., Mc Cormick, S.P., Millard, M.J. 2005. Identification and heritability of fumonisin insensitivity in Zea mays. Phytochemistry. 66(20):2474-2480.
- Desjardins, A.E., Busman, M. 2006. Mycotoxins in developing countries: A case study of maize in Nepal [abstract]. Mycotoxin Research. 22(2):92-95.
- McCormick, S.P., Alexander, N.J., Proctor, R.H. 2006. Fusarium Tri4 encodes a multifunctional oxygenase required for trichothecene biosynthesis. Canadian Journal of Micribiology. 52:636-642.
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