Progress 02/17/01 to 01/18/06
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? Mycotoxins, naturally occurring toxins produced by fungi, are frequent contaminants of commodities important to U.S. Agriculture. Mycotoxin contamination must be controlled for three reasons: the direct economic losses resulting from lower commodity quality (this includes international trade issues), the indirect economic losses occurring from low-level exposure of domestic animals to these toxins, and the potential impact of such toxins upon human health. To ensure adequate assessment of mycotoxin levels requires the development of rapid and reliable analytical methods for the detection and quantitation of these toxins in grains, feeds, and foods. Development of such technologies also supports the research of other scientists seeking to generate plant resistance in order to reduce
the occurrence of these toxins in the field. The USDA- ARS Mycotoxin Research Unit (MRU), National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, is focused on developing new technologies for detection of mycotoxins, using antibody-based and contemporary analytical instrumentation. Through the development of such technologies and their transfer outside of ARS, MRU contributes significantly to resolving the issues of mycotoxin analysis. There are several mycotoxins, each of which has caused severe economic losses and are a major health concern for livestock and humans. Wheat producers in several Midwestern States have experienced severe losses to Fusarium Head Blight (Deoxynivalenol). Recently, a substantial problem with aflatoxin contamination in several Southern States has re-emerged. Mycotoxin contamination is a problem of national and international scope and is a recurring one. The research conducted by MRU, when transferred to our customers, helps to
monitor the extent of the problem and provide mechanisms for protecting consumers through the diversion of contaminated commodities to alternative uses. Additionally, MRU aids in determining the effects of processing upon mycotoxin levels in foods, which may help abrogate the toxicity of contaminated material. This research relates directly to the National Program 108, Food Safety. This program is part of a larger MRU program addressing issues related to control of the fungi involved in mycotoxin production and the development of resistant commodities. The research relates directly to protection of human health (Food Safety). Because affected commodities are commonly used for feed, the research is also directly related to animal health. Lastly, because the fungi involved are common plant pathogens, the research also has a bearing upon plant disease. Because of the multifaceted nature of the mycotoxin problem, the research conducted is pertinent to several National Programs. 2.
List by year the currently approved milestones (indicators of research progress) FY02 (1) Develop rapid assays for deoxynivalenol and fumonisins using fluorescence polarization technology to improve upon the ease of and cost of screening wheat and maize for these mycotoxins. FY03 (1) Synthesis of genetic probes capable of recognizing patulin biosynthetic genes. (2) Evaluate molecular imprinted polymers (MIPS) for the mycotoxin patulin. (3) Develop fluorescence polarization assay for zearalenone in corn. (4) Develop methods for detecting mycotoxins in corn silage. FY04 (1) Characterization of anti-zearalenone antibodies in novel sensor formats. (2) The synthesis and characterization of molecularly imprinted polymers (MIPS). (3) Identification of patulin-producing fungi and the start of large- scale production. (4) Testing of antibody-based methods for detection of moniliformin. (5) Determination of moniliformin levels in samples of maize from field trials. (6) Development of a
chromatographic method for detection of fumonisins in silage. FY05 (1) Synthesis and characterization of molecularly imprinted polymers (MIPs). (2) Determination of the optimal conditions for patulin production. (3) Development of genetic probes for identification of patulin-producing fungi. (4) Determination of moniliformin levels in samples of maize from a second year of field trials. (5) Evaluation of a hand-held fluorescence polarization device for measurement of mycotoxins. FY06 (1) Development of antibodies against mycotoxins of concern. (2) Synthesis and characterization of Molecularly Imprinted Polymers (MIPs) for mycotoxins. (3) Sequencing of Penicillium and Byssochlamys species, and genewalking in order to identify additional genes involved in patulin biosynthesis. (4) Evaluation of MIPs in an equilibrium binding assay format. 4a List the single most significant research accomplishment during FY 2006. SYNTHESIS AND CHARACTERIZATION OF MOLECULARLY IMPRINTED POLYMERS FOR
MYCOTOXINS. Molecularly imprinted polymers (MIPs) capable of binding the mycotoxin moniliformin (MON) were synthesized and characterized. The MIPs were evaluated in an equilibrium binding assay format, and adsorption isotherms were determined for selected polymers, which show an excellent activity for binding of MON. This accomplishment aligns with NP 108, Food Safety, Component 2, entitled Mycotoxins: Toxin Methodology and Identification. 4b List other significant research accomplishment(s), if any. SEQUENCING OF PENICILLIUM AND BYSSOCHLAMYS SPECIES, AND GENEWALKING IN ORDER TO IDENTIFY ADDITIONAL GENES INVOLVED IN PATULIN BIOSYNTHESIS. One of the genes for patulin production was sequenced in three different patulin producing molds. Differences were detected in the amino acid sequences of these species, and these differences correlate with patulin production. These results may be of value to the apple industry for determination of these species in apple juice. A manuscript
describing this research was accepted for publication. This accomplishment aligns with NP 108, Food Safety, Component 2, entitled Mycotoxins: Toxin Methodology and Identification. 4d Progress report. Total genomic DNA of the mold Byssochlamy nivea was analyzed to determine the presence of a gene (idh gene) that is part of the pathway for producing patulin. All of the strains tested contained the idh gene. 5. Describe the major accomplishments to date and their predicted or actual impact. These accomplishments align with NP 108, Food Safety, Component 2, entitled Mycotoxins: Toxin Methodology and Identification. 1. Developed and evaluated antibodies to mycotoxins of strong economic significance and toxins of emerging importance. Three monoclonal antibodies were developed for deoxynivalenol (DON). Enzyme-linked immunosorbent assays (ELISAs) for DON were developed based upon these antibodies. The ELISAs were the most sensitive monoclonal antibody-based immunoassays yet reported for
DON. Five sensitive monoclonal antibodies were also developed for the estrogenic mycotoxin zearalenone (ZEN). Through technology transfer, several of these materials have been successfully incorporated into commercial mycotoxin test kits. Furthermore, the materials developed within the CRIS have been extensively used by other ARS researchers and university scientists in programs to help identify mycotoxin resistant varieties of maize, and the genetic basis for this resistance. 2. Developed novel sensors for mycotoxins. Rapid assays for measuring the fumonisin and zearalenone mycotoxins in maize, and DON in wheat, were developed using fluorescence polarization immunoassay. This novel format allowed for the development of tests that offer a rapid, environmentally friendly alternative to traditional instrumental methods or ELISAs. The assays can be quickly learned and are easy to perform. This, combined with the speed of the assays, will make them useful screening tools for
mycotoxins in wheat and corn. Commercial development is being undertaken by a cooperator who has received a Phase II SBIR grant. Molecularly imprinted polymers (MIPs) capable of binding the mycotoxin moniliformin (MON) were synthesized and characterized. The MIPs were evaluated in an equilibrium binding assay format, and adsorption isotherms were determined for selected polymers, which show an excellent activity for binding of MON. 3. Evaluated these methods for suitability as rapid screening methods for grain producers and processors. It was determined that nixtamalization of corn significantly reduced the amount of fumonisins present after processing. This finding is particularly important for countries where corn, especially in the form of tortillas, is a major component of the diet. Also, substantial levels of aflatoxin B1 were found in incaparina, a dietary supplement for children. This finding resulted in regulatory action by the FDA. Fumonisins were also found to be a
common, low level, contaminant of corn silage. 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? The antibodies and other immunoreagents developed in this CRIS were provided to scientists for development of new analytical technologies, to scientists for use as mycotoxin screening tools and to mycotoxin test-kit manufacturers to assist them in their efforts to improve commercial kits. Reagents developed in this research project were used by scientists with the University of Illinois as a tool in the development of fumonisin resistant corn, and by USDA-ARS scientists in Mississippi as a tool in the development of aflatoxin resistant corn.
Impacts
(N/A)
Publications
- Jackson, L.S., Dombrink Kurtzman, M. 2005. Patulin. In: Sapers, G.M., Gorny, J.R., Yousef, A.E., editors. Microbiology of Fruits and Vegetables. 1st edition. Boca Raton, FL:CRC Press. p.281-311.
- Dombrink Kurtzman, M. 2006. The isoepoxydon dehydrogenase gene of the patulin metabolic pathway differs for penicillium griseofulvum and penicillium expansum. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology. 89:1-8.
- Dombrink Kurtzman, M. 2005. The isoepoxydon dehydrogenase gene of the patulin metabolic pathway differs for penicillium griseofulvum and penicillium expansum [abstract]. International Union of Microbiological Societies Proceedings/Abstracts. p.10.
- Maragos, C.M. 2005. Mycotoxin activities at the agricultural research service-NCAUR [abstract]. Annual Meeting of the UJNR Panel on Toxic Microorganisms. p. 8.
- Maragos, C.M. 2005. Recent developments in immunochemical methods. In: Abbas, H.K., editor. Aflatoxin and Food Safety. Boca Raton, FL: Taylor & Francis Group. p. 269-290.
- Appell, M.D., Maragos, C.M., Kendra, D.F. 2005. Computational studies on the influence of solvent on the conformational preferences and selective recognition of fumonisins [abstract]. Proceedings of the 2005 Annual Multicrop Aflatoxin/Fumonisin Elimination & Fungal Genomics Workshop. p.59.
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Progress 10/01/04 to 09/30/05
Outputs
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? What does it matter? Mycotoxins, naturally occurring toxins produced by fungi, are frequent contaminants of commodities important to U.S. Agriculture. Mycotoxin contamination must be controlled for three reasons: the direct economic losses resulting from lower commodity quality (this includes international trade issues), the indirect economic losses occurring from low-level exposure of domestic animals to these toxins, and the potential impact of such toxins upon human health. To ensure adequate assessment of mycotoxin levels requires the development of rapid and reliable analytical methods for the detection and quantitation of these toxins in grains, feeds, and foods. Development of such technologies also supports the research of other scientists seeking to generate plant resistance in order to reduce the occurrence
of these toxins in the field. The USDA- ARS Mycotoxin Research Unit (MRU), National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, is focused on developing new technologies for detection of mycotoxins, using antibody-based and contemporary analytical instrumentation. Through the development of such technologies and their transfer outside of ARS, MRU contributes significantly to resolving the issues of mycotoxin analysis. There are several mycotoxins, each of which has caused severe economic losses and are a major health concern for livestock and humans. Wheat producers in several Midwestern states have experienced severe losses to Fusarium Head Blight (Deoxynivalenol). Recently, a substantial problem with aflatoxin contamination in several southern states has re-emerged. Mycotoxin contamination is a problem of national and international scope and is a recurring one. The research conducted by MRU, when transferred to our customers, helps to monitor the extent of
the problem and provide mechanisms for protecting consumers through the diversion of contaminated commodities to alternative uses. Additionally, MRU aids in determining the effects of processing upon mycotoxin levels in foods, which may help abrogate the toxicity of contaminated material. This research relates directly to the National Program 108, Food Safety. This program is part of a larger MRU program addressing issues related to control of the fungi involved in mycotoxin production and the development of resistant commodities. The research relates directly to protection of human health (Food Safety). Because affected commodities are commonly used for feed, the research is also directly related to animal health. Lastly, because the fungi involved are common plant pathogens, the research also has a bearing upon plant disease. Because of the multifaceted nature of the mycotoxin problem, the research conducted is pertinent to several National Programs. 2. List the milestones
(indicators of progress) from your Project Plan. Objective 1: Develop and evaluate mycotoxin antibodies. Objective 2: Develop novel sensors for mycotoxins; a) develop novel mycotoxin biosensors; b) develop molecularly imprinted polymers; c) novel Fourier Transform Infrared Spectrometry-Photoacoustic Spectroscopy (FTIR- PAS) sensors as presumptive tests for mycotoxins; d) Peptide Nucleic Acid (PNA) probes for patulin producing fungi. Objective 3: Evaluation of new assays. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Synthesis and characterization of Molecularly Imprinted Polymers (MIPs). Milestone Substantially Met 2. Determination of the optimal conditions for patulin production. Milestone Fully Met 3. Development of genetic probes for identification of patulin-producing fungi. Milestone Substantially Met 4. Determination of moniliformin levels in
samples of maize from a second year of field trials. Milestone Fully Met 5. Evaluation of a hand-held fluorescence polarization device for measurement of mycotoxins. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 06(Year 1): (1) Synthesis and evaluation of Molecularly Imprinted Polymers (MIPS) for mycotoxins; (2) Evaluation of MIPS in an equilibrium binding assay format; (3) Development of antibodies against mycotoxins of concern; (4) Sequencing of Penicillium and Byssochlamys species, and genewalking in order to identify additional genes involved in patulin biosynthesis. FY 07 and beyond: This project is scheduled to terminate in February, 2006, milestones for FY 07 and 08 presume the approval of submitted Project Plans. FY 07 (Year 2): (1) Further synthesis and evaluation of molecularly imprinted polymers for
mycotoxins; (2) Analyze the structures of mycotoxins by computational methods; (3) Optimize conditions for the extraction of deoxynivalenol, aflatoxin, and patulin from cereal based baby food and apple juice; (4) Develop isolation protocols for aflatoxins in meats. FY 08 (Year 3): (1) Develop antibodies recognizing nivalenol; (2) Identify relationships between mycotoxin structure and molecular imprinted polymer activities; (3) Design and synthesize additional molecular imprinted polymers; (4) Optimize conditions for extraction of fumonisin, zearalenone, and ochratoxin A from cereal based baby food and apple juice. 4a What was the single most significant accomplishment this past year? Moniliformin is a mycotoxin produced by several fungi that are pathogenic to cereal grains. Moniliformin is acutely toxic to both animals and plants, and is found in maize, wheat, rye, rice, and oats worldwide. A sensitive, reproducible, and reliable analytical method to separate and quantify
moniliformin was developed and applied to samples of field- inoculated maize. The method developed involves the separation of the moniliformin using a strong electrical field (capillary electrophoresis). The method is more rapid than traditional liquid chromatographic methods, and may find use in measurement of this mycotoxin in maize. 4b List other significant accomplishments, if any. Conditions for production of patulin, specifically Penicillium strains and composition of culture media to use were identified. This will facilitate the production and isolation of patulin and studies of patulin biosynthesis. 4d Progress report. Molecularly Imprinted Polymers (MIPs) for moniliformin were synthesized, and their binding efficacies were evaluated. Patulin MIPs and control polymers synthesized using ultraviolet (UV) photo-initiated polymerization at low temperatures in the Department of Chemistry, University of California, Irvine, were processed and evaluated for binding of patulin at
NCAUR. When evaluated using equilibrium binding assays there were no differences between the patulin-imprinted and control polymers. Partial sequences of the isoepoxydon dehydrogenase (idh) gene and the ITS1, 5.8S gene, ITS2 and 28S gene (nuclear large rDNA subunit) were identified for 8 strains of Penicillium that produce patulin. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Rapid assays for measuring the fumonisin and zearalenone mycotoxins in maize and deoxynivalenol in wheat were developed using fluorescence polarization immunoassay. The assays offer a rapid, environmentally friendly, alternative to traditional instrumental methods or enzyme- linked immunosorbent assays (ELISAs) for these mycotoxins. The assays are easy to perform and can be quickly learned. This, combined with the speed of the assays and the portability of the device will make them useful screening tools for mycotoxins in wheat and corn.
Commercial development is being undertaken by a cooperator. Genetic probes for patulin-producing fungi were identified, which may prove useful in the detection of such fungi in beverages and foods. 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? The antibodies and other immunoreagents developed in this CRIS were provided to scientists for development of new analytical technologies, to scientists for use as mycotoxin screening tools and to mycotoxin test-kit manufacturers to assist them in their efforts to improve commercial kits. Reagents developed in this research project were used by scientists with the University of Illinois as a tool in the development of fumonisin resistant corn, and by USDA-ARS scientists in Mississippi as a tool in the
development of aflatoxin resistant corn. Also during the past year, six mycotoxin test-kit manufacturers and four government and university laboratories received immunoassay reagents developed in this project. Results from this project were disseminated through presentations at four International conferences, one national workshop, and two regional conferences.
Impacts
(N/A)
Publications
- Javed, T., Bunte, R.M., Dombrink Kurtzman, M., Richard, J.L., Bennett, G.A. , Cote, L.M., Buck, W.B. 2005. Comparative pathologic changes in broiler chicks on feed amended with fusarium proliferatum culture material or purified fumonisin b1 and moniliformin. Mycopathologia. 159(4):553-564.
- Maragos, C.M. 2004. Detection of moniliformin in maize using capillary zone electrophoresis. Journal of Food Additives & Contaminants. 21(8):803- 810.
- Maragos, C.M. 2004. Emerging technologies for mycotoxin detection. Journal of Toxicology Toxins Reviews. 23:317-344.
- Kleinschmidt, C.E., Clements, M.J., Maragos, C.M., Pataky, J.K., White, D. G. 2005. Evaluation of food-grade dent corn hybrids for severity of Fusarium ear rot and fumonisin accumulations in grain. Plant Disease. 89:291-297.
- Dombrink Kurtzman, M.A., Blackburn, J.A. 2005. Evaluation of several culture media for production of patulin by Penicillium species. International Journal of Food Microbiology. 98:241-248.
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Progress 10/01/03 to 09/30/04
Outputs
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? What does it matter? Mycotoxins, naturally occurring toxins produced by fungi, are frequent contaminants of commodities important to U.S. Agriculture. Mycotoxin contamination must be controlled for three reasons: the direct economic losses resulting from lower commodity quality (this includes international trade issues), the indirect economic losses occurring from low-level exposure of domestic animals to these toxins, and the potential impact of such toxins upon human health. To ensure adequate assessment of mycotoxin levels requires the development of rapid and reliable analytical methods for the detection and quantitation of these toxins in grains, feeds, and foods. Development of such technologies also supports the research of other scientists seeking to generate plant resistance in order to reduce the occurrence
of these toxins in the field. The USDA- ARS Mycotoxin Research Unit (MRU), National Center for Agricultural Utilization Research (NCAUR), Peoria, IL, is focused on developing new technologies for detection of mycotoxins, using antibody-based and contemporary analytical instrumentation. Through the development of such technologies and their transfer outside of ARS, MRU contributes significantly to resolving the issues of mycotoxin analysis. There are several mycotoxins, each of which has caused severe economic losses and are a major health concern for livestock and humans. Wheat producers in several Midwestern states have experienced severe losses in recent years to Fusarium Head Blight (Deoxynivalenol). Recently, a substantial problem with aflatoxin contamination in several southern states has re-emerged. Mycotoxin contamination is a problem of national and international scope and is a recurring one. The research conducted by MRU, when transferred to our customers, helps to
monitor the extent of the problem and provide mechanisms for protecting consumers through the diversion of contaminated commodities to alternative uses. Additionally, MRU aids in determining the effects of processing upon mycotoxin levels in foods, which may help abrogate the toxicity of contaminated material. This research relates directly to the National Program 108, Food Safety. This program is part of a larger MRU program addressing issues related to control of the fungi involved in mycotoxin production and the development of resistant commodities. The research relates directly to protection of human health (Food Safety). Because affected commodities are commonly used for feed, the research is also directly related to animal health. Lastly, because the fungi involved are common plant pathogens, the research also has a bearing upon plant disease. Because of the multifaceted nature of the mycotoxin problem, the research conducted is pertinent to several National Programs. 2. List
the milestones (indicators of progress) from your Project Plan. Objective 1: Develop and evaluate mycotoxin antibodies. Objective 2: Develop novel sensors for mycotoxins; a) develop novel mycotoxin biosensors; b) develop molecularly imprinted polymers; c) novel FTIR-PAS sensors as presumptive tests for mycotoxins; d) PNA probes for patulin producing fungi. Objective 3: Evaluation of new assays. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004, and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. Milestones that were scheduled to be addressed in FY 2004 included (1) characterization of anti-zearalenone antibodies in novel sensor formats, (2) the synthesis and characterization of molecularly imprinted polymers (MIPS), (3) identification of patulin-producing fungi and the start of large-scale production (4) testing of
antibody-based methods for detection of moniliformin, (5) determination of moniliformin levels in samples of maize from field trials, and (6) development of a chromatographic method for detection of fumonisins in silage. Of these six milestones five were substantially or fully met and one was partially met. (1) Five monoclonal antibodies against zearalenone, developed within the CRIS unit, were characterized in a fluorescence polarization immunoassay format using a novel portable fluorescence polarization device. (2) Synthesis and characterization of MIPS against patulin and moniliformin were begun. MIPS for moniliformin have been synthesized and this project is near completion. Collaboration with the University of California was begun in order to assist in the development of MIPS for patulin. (3) Patulin producing fungi were identified and a manuscript describing this research has been accepted for publication. (4) Monoclonal antibodies with sufficient sensitivity for
moniliformin were not successfully produced in FY 2004, and this milestone was unmet. Because of technical obstacles the project has been terminated. (5) Moniliformin was determined in samples of field inoculated maize as the first year of a multi-year field study. Data from the first year were collected. (6) a chromatographic method for detection of fumonisins was successfully developed, applied to 180 silage samples, and reported in the literature. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 05 (Year 1): Synthesis and characterization of molecularly imprinted polymers (MIPS) will be continued. Conditions for optimal production of patulin will be published. Genetic probes will be developed to identify patulin-producing fungi. Moniliformin levels in samples of maize from a second year of field trials will be determined. The
hand-held fluorescence polarization device will be further evaluated using an established fluorescence polarization assay. FY 06 (Year 2): Patulin analogs will be designed and synthesized to be used in the synthesis of MIPS. Application of MIPS in solid phase extraction format will be investigated. Antibodies will be developed against mycotoxins of concern. FY 07 (Year 3): Additional genes in the patulin biosynthetic pathway will be identified for use in the development of genetic probes. MIPS will be tested as elements of biosensor devices for mycotoxins. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2004: Corn silage is a popular feed source for dairy and beef cattle. Fumonisins, toxic substances produced by certain molds, are prevalent in field corn and potentially could remain intact in corn silage. Although a number of analytical methods for the determination of fumonisins in corn and other cereals
have been developed, no chromatographic methods have been developed for silage, a notoriously difficult material to test. A sensitive, reproducible, and reliable analytical method to separate and quantify fumonisins from corn silage samples was developed and applied to samples collected from the Midwest U.S. during 2001-2002. Fumonisins were found in most of the samples, however the levels at which they were found were low enough that they are unlikely to affect cattle. B. Other Significant Accomplishment(s), if any: None. C. Significant Activities that Support Special Target Populations: None. D. Progress Report: (1) Several culture media were evaluated for production of patulin by Penicillium species. The best conditions for production of patulin were identified, as well as the strains producing the greatest amounts of patulin. (2) Capillary electrophoresis was applied to the detection of the mycotoxin moniliformin in maize. Field samples of maize inoculated with Fusarium
subglutinans were evaluated for the presence of moniliformin using the new method. (3) Molecularly imprinted polymers were developed for moniliformin and are in the process of being evaluated for binding efficacy. (4) A new, handheld, fluorescence polarization device was evaluated for use with existing fluorescence polarization assays for mycotoxins. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Rapid assays for measuring the fumonisin and zearalenone mycotoxins in maize and deoxynivalenol in wheat were developed using fluorescence polarization immunoassay. The assays offer a rapid, environmentally friendly, alternative to traditional instrumental methods or enzyme- linked immunosorbent assays (ELISAs) for these mycotoxins. The assays are easy to perform and can be quickly learned. This, combined with the speed of the assays and the portability of the device will make them useful screening tools for mycotoxins in
wheat and corn. Commercial development is being undertaken by a cooperator. 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? The antibodies and other immunoreagents developed in this CRIS were provided to scientists for development of new analytical technologies, to scientists for use as mycotoxin screening tools and to mycotoxin test-kit manufacturers to assist them in their efforts to improve commercial kits. Reagents developed in this research project were used by scientists with the University of Illinois as a tool in the development of fumonisin resistant corn, and by USDA-ARS scientists in Mississippi as a tool in the development of aflatoxin resistant corn. Also during the past year, 6 mycotoxin test-kit manufacturers received immunoassay
reagents developed in this project. Results from this project were disseminated through presentations at 4 U.S. conferences and 1 International conference.
Impacts
(N/A)
Publications
- CLEMENTS, M.J., MARAGOS, C.M., PATAKY, J.K., WHITE, D.G. SOURCES OF RESISTANCE TO FUMONISIN ACCUMULATION IN GRAIN AND FUSARIUM EAR AND KERNEL ROT OF CORN. PHYTOPATHOLOGY. 2004. V.94. P.251-260.
- Kim, E., Maragos, C.M., Kendra, D.F. 2004. Liquid chromatographic determination of fumonisin B1, B2 and B3 in corn silage. Journal of Agricultural and Food Chemistry. 52:196-200.
- Maragos, C.M. 2004. Recent advances in analytical methods for mycotoxins. In: Yoshizawa, T., editor. New Horizon of Mycotoxicology for Assuring Food Safety. Japanese Association of Mycotoxicology, November 3-5, 2003, Takamatsu, Japan. p. 215-223.
- Maragos, C.M., Kim, E. 2004. Detection of zearalenone and related metabolites by fluorescence polarization immunoassay. Journal of Food Protection. 67(5):1039-1043.
- Dombrink Kurtzman, M. 2003. Fumonisin and beauvercin induce apoptosis in turkey peripheral blood lymphocytes. Mycopathologia. 156:357-364.
- Maragos, C.M. 2004. Recent advances in screening assays for mycotoxins. Feedinfo News Service. Available http://www.feedinfo.com.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Mycotoxins, naturally occurring toxins produced by fungi, are frequent contaminants of commodities important to U.S. Agriculture. Mycotoxin contamination must be controlled for three reasons: the direct economic losses resulting from lower commodity quality (this includes international trade issues), the indirect economic losses occurring from low-level exposure of domestic animals to these toxins, and the potential impact of such toxins upon human health. To ensure adequate assessment of mycotoxin levels requires the development of rapid and reliable analytical methods for the detection and quantitation of these toxins in grains, feeds, and foods. Development of such technologies also supports the research of other scientists seeking to generate plant resistance in order to reduce the occurrence of these toxins in the field. The USDA- ARS Mycotoxin Research Unit (MRU), National Center
for Agricultural Utilization Research (NCAUR), Peoria, IL, is focused on developing new technologies for detection of mycotoxins, using antibody-based and contemporary analytical instrumentation. Through the development of such technologies and their transfer outside of ARS, MRU contributes significantly to resolving the issues of mycotoxin analysis. 2. How serious is the problem? Why does it matter? There are several mycotoxins, each of which has caused severe economic losses and are a major health concern for livestock and humans. Wheat producers in several Midwestern states have experienced severe losses in recent years to Fusarium Head Blight (Deoxynivalenol). Recently, a substantial problem with aflatoxin contamination in several southern states has re-emerged. Mycotoxin contamination is a problem of national and international scope and is a recurring one. The research conducted by MRU, when transferred to our customers, helps to monitor the extent of the problem and
provide mechanisms for protecting consumers through the diversion of contaminated commodities to alternative uses. Additionally, MRU aids in determining the effects of processing upon mycotoxin levels in foods, which may help abrogate the toxicity of contaminated material. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 108, Food Safety (100%). This program is part of a larger MRU program addressing issues related to control of the fungi involved in mycotoxin production and the development of resistant commodities. The research relates directly to protection of human health (Food Safety). Because affected commodities are commonly used for feed, the research is also directly related to animal health. Lastly, because the fungi involved are common plant pathogens, the research also has a bearing upon plant disease. Because of the multifaceted nature of the mycotoxin problem, the research conducted is
pertinent to several National Programs. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003: A rapid method was developed for measuring the mycotoxin zearalenone in corn. The method, a fluorescence polarization immunoassay, is easy to use and the reagents and labware that are required to perform the assays are minimal. Such assays are needed by the food industry as well as by scientists that are developing methods for controlling the fungi and increasing plant resistance. B. Other Significant Accomplishment(s), if any: (1) In collaboration Ron Plattner, a scientist assigned to project 3620- 42000-024-00D, a sensitive method for detection of deoxynivalenol using high performance liquid chromatography (HPLC) with mass spectrometric detection was developed and compared to a widely accepted chromatographic method. (2) Scientists at the University of Illinois used techniques and materials developed at NCAUR to
screen maize samples as part of a project to improve the resistance of maize to Fusarium verticillioides and fumonisins. C. Significant Activities that Support Special Target Populations: None. D. Progress Report: (1) Five monoclonal antibodies were developed for detection of zearalenone. The antibodies are highly sensitive and are capable of detecting estrogenic compounds analogous to zearalenone. This provides a new set of tools for the development of novel detection methods for zearalenone and related estrogenic substances in foods. (2) Molecularly imprinted polymers (MIPs) synthesized to recognize patulin are being examined by infrared (IR) for characterization. Ab initio modeling and calculations have been correlated with IR spectra, indicating that patulin was present in certain of the MIPs. Examination by IR confirmed the presence of patulin in MIPs created using an aqueous solution of polyacrylamide at room temperature. IR was also used to evaluate MIPs that had
previously been synthesized using monomers, crosslinkers, patulin and initiators in organic solvents at 65oC. (3) A number of organisms reported to be capable of producing patulin are currently being examined using different culture conditions and times of incubation. This is being done so that genetic probes can be correlated with the actual production of patulin. The strains examined to date represent different species of Penicillium. Patulin has been detected in all of the strains tested. Ribosomal DNA (rDNA) sequences have been identified from two isolates obtained from commercial apple juice. The fungi have tentatively been identified as Byssochlamys species. (4) An HPLC- fluorescence method was developed for detection of fumonisins in corn silage. The method will allow routine testing of silage samples for these mycotoxins. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Rapid assays for measuring the
fumonisin mycotoxins in maize and deoxynivalenol in wheat were developed using fluorescence polarization immunoassay. The assays offer a rapid, environmentally friendly, alternative to traditional instrumental methods or enzyme-linked immunosorbent assays (ELISAs) for these mycotoxins. The assays are easy to perform and can be quickly learned. This, combined with the speed of the assays, will make them useful screening tools for mycotoxins in wheat and corn. Commercial development is being undertaken by a cooperator. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1: Synthesis and characterization of molecularly imprinted polymers (MIPS) will be continued. Patulin-producing fungi will be identified and large-scale production begun. Antibody-based methods for detection of moniliformin will be tested. Moniliformin levels in samples of maize from field trials will be determined. Development of a chromatographic method for detection of fumonisins in
silage will be reported. Year 2: Genetic probes will be developed for patulin-producing fungi and MIPS will be tested for detection of patulin. MIPS will be tested as components in assays for detection of zearalenone. Year 3: MIPs will be used for detection of patulin in commercial products. MIPS will be combined with capillary electrophoresis for detection of mycotoxins in commodities. 7. 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? Fluorescence polarization assays developed for zearalenone were transferred to our cooperators at Diachemix Corporation, Grayslake, IL, this year. As part of assay development this research project has developed antibodies against mycotoxins. The antibodies and other immunoreagents were provided to scientists for
development of new analytical technologies, to scientists for use as mycotoxin screening tools and to mycotoxin test-kit manufacturers to assist them in their efforts to improve commercial kits. During the past year, several of the antibodies were used by cooperators to demonstrate the feasibility of using surface plasmon resonance technology for detection of the mycotoxins. Reagents developed in this research project were used by scientists with the University of Illinois as a tool in the development of fumonisin resistant corn. Also during the past year, seven mycotoxin test-kit manufacturers received immunoassay reagents developed in this project. Results from this project were disseminated through presentations at six U.S. conferences and through trips to two test kit manufacturers.
Impacts
(N/A)
Publications
- Clements, M.J., Kleinschmidt, C.E., Maragos, C.M., Pataky, J.K., White, D. G. Evaluation of inoculation techniques for Fusarium ear rot and fumonisin contamination of corn. Plant Disease. 2003. v. 87. p. 147-153.
- Clements, M.J., Campbell, K.W., Maragos, C.M., Pilcher, C., Headrick, J.M., Pataky, J.K., White, D.G. Influence of CryIAb and hybrid genetics on fumonisin contamination and Fusarium ear rot of corn. Crop Science. 2003. v. 43. p. 1283-1293.
- Plattner, R.D., Maragos, C.M. Determination of deoxynivalenol and nivalenol in corn and wheat by liquid chromatography with electrospray mass spectrometry. Journal of the AOAC International. 2003. v. 86. p. 61- 65.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Mycotoxins, naturally occurring toxins produced by fungi, are frequent contaminants of commodities important to U.S. Agriculture. Mycotoxin contamination must be controlled for three reasons: the direct economic losses resulting from lower commodity quality (this includes international trade issues), the indirect economic losses occurring from low-level exposure of domestic animals to these toxins, and the potential impact of such toxins upon human health. To ensure adequate assessment of mycotoxin levels requires the development of rapid and reliable analytical methods for the detection and quantitation of these toxins in grains, feeds, and foods. Development of such technologies also supports the research of other scientists seeking to generate plant resistance in order to reduce the occurrence of these toxins in the field. The Mycotoxin Research Unit is focused on developing new
technologies for detection of mycotoxins, using antibody-based and contemporary analytical instrumentation. Through the development of such technologies and their transfer outside of ARS the Mycotoxin Research Unit contributes significantly to resolving the issues of mycotoxin analysis. 2. How serious is the problem? Why does it matter? There are several mycotoxins, each of which has caused severe economic losses and are a major health concern for livestock and humans. Wheat producers in several Midwestern states have experienced severe losses in recent years to Fusarium Head Blight (Deoxynivalenol). Recently, a substantial problem with aflatoxin contamination in several southern states has re-emerged. Mycotoxin contamination is a problem of national and international scope and is a recurring one. The research conducted by the Mycotoxin Research Unit, when transferred to our customers, helps to monitor the extent of the problem and provide mechanisms for protecting consumers
through the diversion of contaminated commodities to alternative uses. Additionally, the Mycotoxin Research Unit aids in determining the effects of processing upon mycotoxin levels in foods, which may help abrogate the toxicity of contaminated material. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 108, Food Safety (100%). This project is part of a larger Mycotoxin Research Unit program addressing issues related to control of the fungi involved in mycotoxin production and the development of resistant commodities. The research conducted relates directly to protection of human health (Food Safety). Because affected commodities are commonly used for feed, the research is also directly related to animal health. Lastly, because the fungi involved are common plant pathogens, the research also has a bearing upon plant disease. 4. What was your most significant accomplishment this past year? A. Single
Most Significant Accomplishment during FY 2002: Rapid assays for measuring the mycotoxin deoxynivalenol in wheat were reported. The technology used, fluorescence polarization, has certain advantages over established immunoassay screening technologies and is a significant advance in that it makes screening assays for mycotoxins easier to use and potentially less expensive. Such assays are needed by the food industry as well as by scientists that are developing methods for controlling the fungi and increasing plant resistance. B. Other Significant Accomplishment(s), if any: Substantial levels of aflatoxin B1 were found in incaparina, a dietary supplement for children. This finding resulted in regulatory action by the FDA. C. Significant Accomplishments/Activities that Support Special Target Populations: none. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? Rapid assays for measuring the fumonisin mycotoxins in maize
and deoxynivalenol in wheat were developed using fluorescence polarization immunoassay. The assays offer a rapid, environmentally friendly, alternative to traditional instrumental methods or enzyme-linked immunosorbent assays (ELISAs) for these mycotoxins. The assays are easy to perform and can be quickly learned. This, combined with the speed of the assays, will make them useful screening tools for mycotoxins in wheat and corn. Commercial development is being undertaken by a cooperator. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1: Synthesis of genetic probes capable of recognizing patulin biosynthetic genes will be undertaken. Molecular imprinted polymers (MIPS) for the mycotoxin patulin will be synthesized and characterized. Fluorescence polarization assay for zearalenone in corn will be developed. Methods for detecting mycotoxins in corn silage will be developed. Year 2: New antibodies and immunoassays will be developed for moniliformin.
Genetic probes and MIPS will be tested for detection of patulin. Year 3: Improved antibodies for the fumonisins will be used to improve the sensitivity of fumonisin fluorescence polarization immunoassays. MIPs will be used for detection of patulin in foods. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Fluorescence polarization assays developed for DON were transferred to our cooperators at Diachemix Corporation, Grayslake, IL, this year. As part of assay development, this project has developed antibodies against mycotoxins. The antibodies and other immunoreagents were provided to several mycotoxin test-kit manufacturers to assist them in their efforts to improve commercial mycotoxin test kits. During the past year, seven mycotoxin test-kit manufacturers received immunoassay reagents
developed in this project. Reagents were also made available to academic scientists and plant breeders in order to assist them in attempts to develop mycotoxin resistant corn. Results from this project were disseminated at two international and three U.S. conferences. The investigators of the project also hosted the 2002 meeting of the NC-129: Fusarium Mycotoxins in Cereal Grains.
Impacts
(N/A)
Publications
- Maragos, C.M., Plattner, R.D. Rapid fluorescence polarization immunoassay for the mycotoxin deoxynivalenol in wheat. Journal of Agricultural and Food Chemistry. 2002. v. 50. p. 1827-1832.
- Maragos, C.M., Jolley, M.E., Nasir, M.S. Fluorescence polarization as a tool for the determination of deoxynivalenol in wheat. Food Additives and Contaminants. 2002. v. 19. p. 400-407.
- Trucksess, M.W., Dombrink-Kurtzman, M.A., Tournas, V.H., White, K.D. Occurrence of aflatoxins and fumonisins in incaparina in Guatemala. Food Additives and Contaminants. 2002. v. 19. p. 671-675.
- Maragos, C.M. Novel assays and sensor platforms for the detection of aflatoxins. DeVries, J.W., Trucksess, M.W., Jackson, L.S., editors. Kluwer Academic/Plenum Publishers, New York, NY. Mycotoxins and Food Safety. 2002. p. 85-93.
- Dombrink-Kurtzman, M.A., Rooney, L.W. Effect of nixtamalization on fumonisin-contaminated corn for production of tortillas. Lee, T-C., Ho, C- T., editors. American Chemical Society, Washington, DC. Bioactive Compounds in Foods; ACS Symposium Series 816. 2002. p. 206-217.
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Progress 10/01/00 to 09/30/01
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
Mycotoxins, naturally occurring toxins produced by fungi, are frequent contaminants of commodities important to U.S. Agriculture. Mycotoxin contamination must be controlled for three reasons: the direct economic losses resulting from lower commodity quality (this includes international trade issues), the indirect economic losses occurring from low-level exposure of domestic animals to these toxins, and the potential impact of such toxins upon human health. To ensure adequate assessment of mycotoxin levels requires the development of rapid and reliable analytical methods for the detection and quantitation of these toxins in grains, feeds, and foods. Development of such technologies also supports the research of other scientists seeking to generate plant resistance in order to reduce the occurrence of these toxins in the field. This unit is focused on developing new technologies for detection of mycotoxins, using antibody-based and contemporary analytical instrumentation. Through
the development of such technologies and their transfer outside of ARS this unit contributes significantly to resolving the issues of mycotoxin analysis.
2. How serious is the problem? Why does it matter?
There are several mycotoxins, each of which has caused severe economic losses and are a major health concern for livestock and humans. Wheat producers in several Midwestern states have experienced severe losses in recent years to Fusarium Head Blight (Deoxynivalenol). Recently, a substantial problem with aflatoxin contamination in several southern states has re-emerged. Mycotoxin contamination is a problem of national and international scope and is a recurring one. The research conducted by this unit, when transferred to our customers, helps to monitor the extent of the problem and provide mechanisms for protecting consumers through the diversion of contaminated commodities to alternative uses. Additionally, this unit aids in determining the effects of processing upon mycotoxin levels in foods, which may help abrogate the toxicity of contaminated material.
3. How does it relate to the National Program(s) and National Component(s)?
National Program 108, Food Safety (100%). This program is part of a larger Mycotoxin Research Unit addressing issues related to control of the fungi involved in mycotoxin production and the development of resistant commodities. The research conducted by this unit relates directly to protection of human health (Food Safety). Because affected commodities are commonly used for feed, the research is also directly related to animal health. Lastly, because the fungi involved are common plant pathogens, the research of this unit also has a bearing upon plant disease. Because of the multifaceted nature of the mycotoxin problem, the research conducted by this unit is pertinent to several national programs.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment during FY 2001: A rapid assay for measuring the fumonisin mycotoxins in maize was reported. The technology used, fluorescence polarization, has certain advantages over established immunoassay screening technologies and is a significant advance in that it makes screening assays for mycotoxins easier to use and potentially less expensive. Such assays are needed by the food industry as well as by scientists that are developing methods for controlling the fungi and increasing plant resistance. B. Other Significant Accomplishment(s), if any: None. C. Significant Accomplishments/Activities that Support Special Target Populations: None.
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
This is a new project plan certified 12/00 by the Office of Scientific Quality Review as completed peer review.
6. What do you expect to accomplish, year by year, over the next 3 years?
Year 1: Rapid assays for deoxynivalenol and fumonisins will be developed using fluorescence polarization technology to improve upon the ease of use and cost of screening wheat and maize for these mycotoxin. Year 2: The mycotoxins patulin and moniliformin will be produced to permit synthesis of novel immunogens. Genetic probes capable of recognizing patulin biosynthetic genes will be synthesized. Year 3: New immunoassays will be developed for moniliformin and genetic probes will be tested for detection of patulin.
7. 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 & durability of the technology product?
This is a new project. A deoxynivalenol (DON) antibody developed in a previous project will be provided to our CRADA partner Romer Labs, Union, MO) on an ongoing basis. Fluorescence polarization assays developed for DON will be transferred to our cooperators at Diachemix Corporation (Grayslake, IL) this year. In addition, antibodies and immunoassay reagents that result from this work will be made publically available to scientists, kit manufacturers, and plant breeders through CRADAs and Material Transfer Agreements. This CRIS unit hosted the annual meeting of the Midwest Section of the AOAC International in Peoria, IL (June, 2001). Among the topics at this 3-day meeting were technical sessions on mycotoxins, and a workshop on mycotoxin analysis. The analytical workshop gave participants the opportunity to visit NCAUR and learn state of the art analytical methods first hand. In addition, scientific presentations were made at other conferences, most notably: Presentation:
Fluorescence Polarization as a Tool for the Determination of Fumonisin and Deoxynivalenol in Cereal Grains. Gordon Research Conference on Mycotoxins & Phycotoxins. Williamstown, MA, June 25, 2001.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
Cooking process reduces toxin in corn. Agricultural Research. August, 2001. Corn toxin examined in border birth defects. The Dallas Morning News. March 4, 2001.
Impacts
(N/A)
Publications
- Maragos, C.M., Jolley, M.E., Plattner, R.D., Nasir, M.S. Fluorescence polarization as a means for determination of fumonisins in maize. Journal of Agricultural Food Chemistry. 2001. v. 49. p. 596-602.
- Maragos, C.M. Novel sensors for detecting mycotoxins in foods. Mycotoxins. 2001. v. 51. p. 51-58.
- Momany, F.A., Dombrink-Kurtzman, M.A. Molecular dynamics simulations on the mycotoxin fumonisin B1. Journal of Agricultural Food Chemistry. 2001. v. 49. p. 1056-1061.
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