Progress 05/15/00 to 05/14/05
Outputs
4d Progress report. This report serves to document research conducted under Specific Cooperative Agreement between the USDA/ARS and the University of Minnesota. Additional details of the research can be found in the report for the parent project 6402-42000-001-00D, Agronomic, Environmental, and Resistant Germplasm Effects on Aflatoxins and other Mycotoxins. During the project period the mycotoxin laboratory at the University of Minnesota has investigated possible applications of earlier studies carried out on the biochemical basis of two cultural tests for aflatoxigenicity in Aspergillus flavus. Those studies showed that a series of known yellow anthraquinone pigments associated with the aflatoxin biosynthetic pathway form the biochemical basis of the tests. Preliminary studies have been conducted to determine the potential of these pigments to serve as readily-detected sentinel substances for the prediction of aflatoxin contamination in crops. A major problem with
this approach is that the crops of greatest interest, notably corn, have high levels of numerous different yellow pigments, creating a high background unrelated to aflatoxins. To be useful, a method based on yellow pigments must be simpler and less expensive than current methods, or provide an earlier prediction of contamination. The possibility of early production of yellow pigments in A. flavus-contaminated corn is currently being investigated. Samples will be assayed by High Performance Liquid Chromatograph (HPLC) using established methodology. The strategy for preventing aflatoxin contamination of agricultural products by visual monitoring of production of yellow pigments, with or without base-induced conversion to red pigments may be more promising in agricultural products that are white, and hand sorting to remove aflatoxin-contaminated material is an option. During the project period studies have been carried out on the role of mycotoxin in charcoal rot disease in soybeans.
Mississippi isolates of the fungus Macrophomina phaseolina, which causes the disease, have been shown to produce the mycotoxin (-)-botryodiplodin, previously known to be produced by Penicillium roqueforte and other fungi. However, the phytotoxin phaseolinone, which has been proposed to be produced by M. phaseolina, and to play an important role in the pathology of the disease, was not found. The studies on (-)-botryodiplodin have used natural material purified from culture filtrates and have been provided to our scientists for this investigations and other investigators. Considerable effort has also been invested in the chemical synthesis of (-)- botryodiplodin for use in further testing and as a standard for LC/MS assays. An authentic phaseolinone standard was considered essential for using LC/MS analysis to show that phaseolinone is not produced by a large number of isolates of M. phaseolina in culture or in M. phaseolina- infected tissue. A standard sample of phaseolonone was
synthesized by peroxidation of a small sample of phomenone. During the project period initial efforts have been made to carry out statistical analysis on ELISA and HPLC analytical results on a large trial of aflatoxin and fumonisin levels in corn present in four sets of physical conditions (heated or not heated, each ground or whole) stored for one year under five temperature conditions in the laboratory. The results of this study may help select the best way(s) to monitor aflatoxin contamination in stored crops. Current practice for the regulation of aflatoxin contamination in the US and other developed countries is to assay crops post-harvest, and to assume that aflatoxin levels never change between sampling time and consumption. That is, it is assumed that A. flavus, which is known to contaminate the crop, will never make any more aflatoxin, and that the aflatoxin known to be present will never decompose or be metabolized by other microorganisms. This assumption is probably
valid under the nearly ideal storage conditions typically found in large operations in the US and other developed countries. However, during shipping or in small holdings with less than ideal storage conditions often found in developing countries, it may be useful to also consider levels of contamination by aflatoxigenic fungi.
Impacts
(N/A)
Publications
- Abbas, H.K., Shier, W.T., Gronwald, J.W., Lee, Y.W. Phytoxicity and Cytotoxicity of non-trichothecene phytopathogen-derived mycotoxins. Journal of Natural Toxins. 2002. v. 11. p. 173-186.
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? D. This report serves to document research conducted under a Specific Cooperative Agreement (58-6402-0-0035) between ARS and the University of Minnesota. Additional details can be found in the report for the parent 6402-42000-002-00D, "Agricultural Practices, Ecological and Varietal Effects on Aflatoxins and Other Mycotoxins in Corn." This laboratory analyzed in field samples of corn grown in Mississippi and Arkansas under various environmental conditions for aflatoxin B1 and B2 and fumonisins B1, B2, B3 and B4. The results confirmed for field trials suggestions that high nighttime temperatures (>68 degrees F) (high low temperatures) are a key factor in corn susceptibility to aflatoxin contamination. Comparison of Bt corn and aflatoxin resistant hybrids with commercial hybrids indicated it is possible to achieve highly significant reductions in aflatoxin levels under moderately adverse growing
conditions, but the levels of reduction under highly adverse growing conditions (as in 1998) were not sufficient to keep aflatoxin levels below regulatory action levels. Chemical analyses were conducted on extracts from aflatoxigenic and non- aflatoxigenic Aspergillus flavus cultures as part of studies investigating cultural methods for use in studies on aflatoxin biocontrol methods and for inexpensive monitoring of crop contamination in developing countries. The strategy for using non-aflatoxigenic A. flavus for biocontrol of aflatoxin contamination requires the identification of a non-aflatoxigenic strain capable of successfully competing with aflatoxigenic A. flavus from the soil pool by preempting them. Developing the approach requires obtaining and screening large numbers of non-aflatoxigenic A. flavus isolates to identify strains with the most desirable properties. This laboratory has supported the program by determining the molecular basis of an empirical test for
aflatoxigenicity developed by Saito and Machida. We have demonstrated that the color change from yellow to plum-red that is the hallmark of aflatoxigenic A. flavus is due to aflatoxin biosynthetic intermediates acting as pH indicator dyes, turning red or purple with elevated pH. These studies involved isolation of 13 yellow pigments and structure elucidation of seven of them. Structure elucidation used negative ion MS/MS spectrometry, 600 MHz proton nuclear magnetic resonance spectroscopy, UV- VIS absorption spectroscopy as well as HPLC. The seven are either known aflatoxin biosynthetic intermediates or known side products of the biosynthetic pathway. These observations provide a convincing explanation for the effectiveness of the Saito and Machida test and an explanation for its very low false positive rate (i.e., it would require a mutation in one of a small number of enzymes after the yellow pigments on the aflatoxin biosynthetic pathway. Understanding of the mechanism of the
test should encourage its use in the search for non-aflatoxigenic A. flavus isolates and in low-cost monitoring of the levels of aflatoxigenic A. flavus in crops. Fractionation and chemical analysis was carried out on culture extracts of the pathogenic fungus Macrophomina phaseolina, causative agent of charcoal rot disease in soybean. The expected mycotoxin (phaseolinone) was not found in the extracts. Instead, bioassay-guided fractionation led to the isolation of a different mycotoxin, whose structure was determined to be that of (-)-botryodiplodin, a known mycotoxin produced by Penicillium roqueforte and other fungal species. This is the first identification of (-)-botryodiplodin being produced by Macrophomina phaseolina. The structure of (-)-botryodiplodin was elucidated using 500 MHz proton and 13C nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry, scanning polarimetry and UV-VIS absorption spectroscopy. Synthesis of (-)-botryodiplodin for
further testing is now underway.
Impacts
(N/A)
Publications
- Abbas, H.K., Shier, W.T., Gronwald, J.W., Lee, Y.W. Phytoxicity and Cytotoxicity of non-trichothecene phytopathogen-derived mycotoxins. Journal of Natural Toxins. 2002. v. 11. p. 173-186.
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Progress 10/01/02 to 09/30/03
Outputs
4. What were the most significant accomplishments this past year? D. Progress Report: This report serves to document research conducted under Cooperative Agreement 58-6402-0-035 between the USDA/ARS and the University of Minnesota, entitled "Chemical Determination of Mycotoxins in Field Samples and Culture Materials." Additional details of the research can be found in the report for the parent project 6402-42000-002- 00D, "Agricultural Practices, Ecological and Varietal Effects on Aflatoxins and Other Mycotoxins in Corn". During the project period the mycotoxin laboratory at the University of Minnesota analyzed corn extract samples of Bt and non-Bt corn from Arkansas provided by the Crop Genetics and Production Research Unit of USDA/ARS for contamination by fumonisins and aflatoxins. The samples were subjected to clean-up followed by liquid chromatography / electrospray mass spectrometry (LC/ES/MS) and liquid chromatography / atmospheric pressure chemical
ionization / mass spectrometry (LC/APCI/MS) following sample clean-up. The detected fumonisins included FB1, FB2, FB3 and FB4 and FC4. The detected aflatoxins were AFB1 and AFB2. In related work, data analysis, including statistical analysis, has been conducted on levels of aflatoxins and fumonisins in Arkansas corn samples measured by ELISA, including correlation analysis of mycotoxin levels with growing temperature. Extensive studies have been carried out to determine the nature and amounts of yellow pigments used in the rapid identification of toxigenic and non-toxigenic strains of Aspergillus flavus and A. parasiticus. Non- toxigenic strains of A. flavus are potentially useful for strategies to reduce aflatoxin contamination by preinoculation with non-toxigenic strains. A total of 14 yellow pigments have been isolated and structures have been identified for seven pigments, representing the majority of the color produced. All seven pigments are either known biosynthetic
intermediates on the pathway to aflatoxins, or known branch-pathway intermediates. Identification of the pigments that predict aflatoxin production by A. flavus strains as being aflatoxin biosynthetic intermediates provides a convenient rationalization for the predictive power of the rapid identification method. Thus, only strains producing pigment amounts below the level of visible detection would be expected to give false negative results, and only strains with a mutation in a biosynthetic enzyme beyond that which makes norsolorinic acid would be expected to give false positive results. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Mobio, T.A.; E. Tavan, I. Baudrimont, R. Anane, M.-R. Carratu, A. Sanni, M.F. Gbeassor, T.W. Shier, J.-F. Narbonne, E.E. Creppy (2003) Comparative study of the toxic effects of fumonisin B1
in rat C6 glioma cells and p53- null mouse embryo fibroblasts. Toxicology, 183, 65-75.
Impacts
(N/A)
Publications
- Abbas, H.K., Shier, W.T., Gronwald, J.W., Lee, Y.W. Phytoxicity and Cytotoxicity of non-trichothecene phytopathogen-derived mycotoxins. Journal of Natural Toxins. 2002. v. 11. p. 173-186.
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