Brief Summary of Minutes of Annual Meeting

Accomplishments: Objective 1. Develop data for use in risk assessment of mycotoxins in human and animal health In 2006, the NCAUR group examined the antifungal activity of the pyrrocidines antibiotics produced by the corn endophyte Acremonium zeae. The goal was to determine the potential use of A. zeae as a biocontrol agent against mycotoxigenic fungi. This past year, the Illinois group performed preliminary in vivo studies to evaluate the potential of pyrrocidines to induce whole animal toxicity. Based on clinical signs, organ weights, and gross and microscopic alterations, toxicity was not identified at doses up to 10 mg pyrrocidine A/kg body weight when given as a single intraperitoneal dose to mice. The mouse model has been used to develop effective biomarkers for exposure to mycotoxins such as deoxynivalenol (DON). One biomarker that has attracted interest is the effects on peripheral blood lymphocytes. The Iowa group has tested the hypothesis that DON suppresses peripheral blood lymphocytes at 1 ppm but does not suppress at lesser doses in BALB/c mice. Peripheral blood and spleen cell suspensions were analyzed for a group of 10 female and male BALB/c mice fed DON at 0, 0.25, 0.5, 1.0, and 2.0 ppm for 14 and 28 days. In peripheral blood, the percentage of B cells and their numbers were inhibited in both sexes of BALB/c mice after 14 days of exposure to 1.0 or 2.0 ppm DON; whereas, exposure to DON over 28 days did not inhibit B cells when compared with the control diet. Monocytes (CD11b+) in peripheral blood and numbers of spleen macrophages were decreased only in female mice fed 1.0 and 2.0 ppm DON after 28 days compared with control diet. Dietary DON did not influence hematology in males but hemoglobin was suppressed in female mice at all DON doses over 14 days but not after 28 days. BALB/c mice seemingly adapted to DON exposure because peripheral blood cellular effects of DON at 14 d disappeared by 28 d with the exception of monocyte changes in females, suggesting that female sex hormones potentiated one aspect of DON immunotoxicity in BALB/c mice. The Missouri group conducted a study to determine the effects of dietary aflatoxin (AF) on hepatic gene expression in male broiler chicks. Microarray analysis was used to identify shifts in genetic expression associated with the affected physiological processes in chicks fed 0 and 2 mg AF/kg of feed to identify potential targets for pharmacological/toxicological intervention. Microarray data were analyzed using a 2-step ANOVA model and validated by quantitative real-time PCR. Compared with controls, various genes associated with energy production and fatty acid metabolism (carnitine palmitoyl transferase), growth and development (insulin like growth factor), antioxidant protection (glutathione S transferase), detoxification (epoxide hydrolase), and immune protection (interleukins) were down-regulated, whereas genes associated with cell proliferation (ornithine decarboxylase) were up-regulated in birds fed AF. Objective 2. Develop new techniques and improve current assays to identify and measure mycotoxins and mycotoxigenic fungi in cereal grains With the increased production of ethanol from corn, utilization of the major byproduct, distillers grain, has made it necessary to modify old methods or develop new methods for sample clean up and analysis of complex matrices used as feed. As a result, the Missouri group evaluated and adapted stacked immunoaffinity column cleanup for mycotoxins along with HPLC detection to measure relevant levels of aflatoxins, deoxynivalenol, zearalenone, ochratoxin A, and fumonisin in animal feedstuffs containing distillers grain. As part of a comprehensive approach for monitoring the presence of mycotoxigenic fungi in grain and grain products, the Indiana group has developed a multiplex real-time quantitative PCR assay to detect and quantify mycotoxigenic fungi. The assay uses genus-specific probes for the important mycotoxigenic Aspergillus, Fusarium, and Penicillium species. The assay can detect at least 25 mycotoxigenic species with a linear range of quantification between 10 pg and 100 ng of DNA. The assay was validated by an analysis of fungal growth in distillers grain. This assay complements the mycotoxin-specific assays already developed. The assay was used to assist in a case of spoiling distillers grain stored at a feed facility. The analysis indicated that one sample contained Fusarium species which were subsequently determined to be trichothecene-producers. The Pennsylvania group developed an assay to detect the mycotoxins deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, and the fungus-specific sterol ergosterol in single florets of wheat. The method uses standard extraction and clean-up procedures for the trichothecene mycotoxins in conjunction with the isolation of ergosterol from the plant tissue. The cleaned extract is derivitized and the analytes of interest are detected by gas chromatography with electron capture detection. Objective 3. Establish integrated strategies to manage and to prevent mycotoxin contamination in cereal grains The NC1025 committee has approached this objective through investigations that focus on the categories: intervention, surveys and plant genetics. With respect to intervention, the Missouri group evaluated the efficacy of curcumin, an antioxidant supplied by turmeric (Curcuma longa) powder, to combat the down regulation of antioxidant protection by aflatoxin. The group made their evaluation by monitoring changes in gene expression in liver of broiler chicks fed aflatoxin. Addition of curcumin to the AFB1 diet ameliorated the negative effects of aflatoxin on growth performance and liver weight. At the end of the three-week treatment period, livers were evaluated for changes in the expression of genes involved in antioxidant function and immune response by quantitative real time PCR. Inclusion of curcumin in the diet alleviated the effects of aflatoxin on the expression of several genes. The Missouri group also continued to evaluate the efficacy of proprietary adsorbents to prevent zearalenone toxicity in a non-invasive swine model. Twelve weanling gilts (6 per treatment) averaging 10.1 kg initial body weight were used in a 3-week experiment. Dietary treatments included an NRC control diet, and a diet containing 2 mg zearalenone (ZEA)/ kg diet. On days 0, 4, 7, 14, and 21 vulva measurements (length and width) were made on individual pigs. Vulva width and length were greater in pigs fed ZEA. Reproductive tract weights (tubular, vagina, and total tract) were also greater in pigs fed ZEA. Vulva width was significantly correlated with vaginal weight, tubular weight, and total reproductive tract weight. Due to concern for the potential contamination of ergot alkaloids in the malting and brewing processes of beer production, the North Dakota group investigated the impact of thermal treatment on the fate of ergot alkaloids present in sclerotia obtained from malting barley and wheat. Individual ergopeptide alkaloids were determined by HPLC, and water-soluble alkaloids were determined by ELISA. Steeping appears to solubilize a small amount of peptide and water-soluble alkaloids, with kilning resulting in a more significant reduction (H30%). Beer was prepared from malt grist containing 0.1% (w/w) sclerotia. Mass balance calculations indicated that 32, 10, and 2 percent of the original total ergopeptide alkaloids were recovered in the spent grain, wort, and beer, respectively. Average levels measured in beer were H10 ng/mL. This was investigated further by treating aqueous extracts of sclerotia (pH 5.0) at 45, 70, and 100°C for 1 hr. Losses of up to 46% of the original ergopeptide alkaloids were observed after 1 hr at 100°C. The Wisconsin group has led an effort to use RNAi technology to control mycotoxin contamination in corn (aflatoxin) and wheat (trichothecene). This group has obtained T0 corn plants transformed with an inverted repeat of aflR, the transcription factor required by Aspergillus flavus for aflatoxin biosynthesis. Their hypothesis is that the interference-RNA expressed in the transgenic plant can actually make its way into the pathogen, where it inhibits mycotoxin biosynthesis. Inoculation of the T0 plants with A. flavus yielded preliminary data suggesting that aflatoxin production is reduced in the transgenic plants. Further breeding will produce homozygous plants that will be tested in the lab in fall of 2008 and in the field of summer 2009. The group from Pennsylvania continued a study initiated in 2006 aimed at understanding the environmental factors and infection times that are favorable for production of DON in wheat kernels asymptomatic for head scab caused by F. graminearum. The field study utilized movable greenhouse enclosures and supplemental misting to control timing of infection. Results were similar to those found in 2006 and indicated that infections during the grain fill period could result in asymptomatic kernels with greater than 2 ppm deoxynivalenol in some cultivars of winter wheat. The Michigan group has also studied Fusarium graminearum; namely, they are studying the formation and dissemination of inoculum from the sexual stage. Forcible discharge of sexual spores is essential to initiation of the disease cycle of this fungus. Through expression analysis with the Fusarium GeneChip (Affymetrix), expression of 2068 genes is specific to sexual development. Two genes were identified that are responsible for the regulation of ascospore discharge. Objective 4. Define the regulation of mycotoxin biosynthesis and the molecular relationships between mycotoxigenic fungi The groups from Texas, Indiana, and NCAUR have worked together on genes that affect fumonisin biosynthesis in Fusarium verticilliodes. Studies on GBB1, a gene encoding a putative beta subunit of a heterotrimeric G protein, were started in 2006 and completed in 2007. A GBB1 deletion mutant showed no significant differences in radial growth and mycelial mass but produced significantly less Fumonisin B1 (FB1) than its wild-type progenitor. Reduced expression of the key FB1 biosynthetic genes, FUM1 and FUM8, in provides further evidence that GBB1 is involved in FB1 regulation. Stalk rot virulence, as measured by mean lesion length and by area, was not significantly different in compared to the wild type, suggesting that GBB1 does not regulate virulence in F. verticillioides. Complementation of BM83 with GBB1 restored FB1 production and hyphal growth to wild-type strain. ZFR1 is another gene that appears to affect fumonisin biosynthesis. A ZFR1-deletion strain grew approximately 2.5 fold less than wild type on endosperm tissue and a variety of other carbon sources including glucose and amylopectin. However, the strain displayed higher alhpa -amylase activity and expression of genes involved in starch saccharification than wild type, thus indicating that the reduced growth of the ZFR1-deletion strain was not due to inhibition of amylolytic enzymes. In the wild-type strain, expression of six genes encoding putative sugar transporters was significantly greater on endosperm tissue relative to germ tissue, and expression of at least 3 of the 6 genes was negatively affected by disruption of ZFR1. Intriguingly, disruption of one of the sugar transporters, FST1, had no effect on growth, kernel colonization, or kernel pH but decreased FB1 production by approximately 82% on maize kernels. AREA-orthologues in fungi are global regulators of nitrogen metabolite as well as secondary metabolism. An AREA-deletion mutant in F. verticillioides grew poorly on mature maize kernels, but grew similar to wild type (WT) with the addition of ammonium phosphate. Fumonisin B1 was not produced by AREA-deletion strain under any condition or by the WT with added ammonium phosphate. Constitutive expression of AREA rescued the growth and FB1 defects in AREA-deletion strain. The results pf the study supported the hypothesis that FB1 biosynthesis is regulated by AREA. The Wisconsin group has explored the global regulation of mycotoxin biosynthesis through mechanisms that involve chromatin changes. This group has discovered that the protein LaeA in Aspergillus species regulates the expression of genes in multiple mycotoxin clusters. Their data suggest that regulation by LaeA works through the activation of euchromatin. The NCAUR is collaborating with the Wisconsin group to determine if a similar mechanism controls mycotoxin production in Fusarium spp. Wisconsin is investigating the regulation of mycotoxin production in Aspergillus and Fusarium species by activated oxygen species known as oxylipins. With collaborators from Texas A&M and NCAUR they are investigating the role of both plant and fungal oxylipins in stimulating mycotoxin biosynthesis.

Publications: Anderson, L. L., Y.-W. Lee, R. L. Bowden & J. F. Leslie. 2007. Relationships between alleles at lineage diagnostic loci in Fusarium graminearum. Fungal Genetics Newsletter 54(Suppl.): 67. Bandyopadhyay, R., M. Kumar & J. F. Leslie. 2007. Relative severity of aflatoxin contamination of cereal crops in West Africa. Food Additives and Contaminants 24: 1109-1114. Bouhired S, Weber M, Kempf-Sontag A, Keller NP, and Hoffmeister D. 2007. Accurate prediction of Aspergillus natural product gene cluster boundaries using the transcriptional regulator LaeA. Fungal Genetics and Biology 44:1134-45. Cuomo, C., Güldener, U., Xu, J.R., Trail, F., Turgeon, B.G., Di Pietro, A., Walton, J.D., Ma, L-J, Baker, S., Rep, M., Adam, A., Antoniw, J., Baldwin, T., Calvo, S., Chang, Y-L, DeCaprio, D., Gale, L.R., Gnerre, S., Goswami, R.S., Hammond-Kosack, K., Harris, L.J., Hilburn, K., Kennell, J.C., Kroken, S., Magnuson, J.K., Mannhaupt, G., Mauceli, E., Mewes, H-W, Mitterbauer, R., Muehlbauer, G., Münsterkötter, M., Nelson, D., O'Donnell, K., Ouellet, T., Qi, W., Quesneville, H., Roncero, M.I., Seong, K-Y, Tetko, I.V., Urban, M., Waalwijk, C., Ward, T.J., Yao, J., Birren, B.W., and Kistler, H.C. 2007. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 317:1400-1402. Dakovic, A., Matijasevic, S., Rottinghaus, G. E., Dondur, V., and Pietraz, T. 2007. Adsorption of zearalenone by organomodified natural zeolitic tuff. J. Colloid and Interface Science 311:8-13. Dakovic, A., Tomasevic-Canovic, M., Rottinghaus, G. E., and Matijasevic, S. 2007. Fumonisin B1 adsorption on modified clinoptilolite rich zeolitic tuff. Microporous & Mesoporous Materials 105:285-290. Deshmukh, S., Asrani, R. K., Gupta, V. K., Ledoux, D. R., Rottinghaus, G. E., and Bermudez, A. J. 2007. Pathologic changes in extrahepatic organs and agglutinin response to Salmonella Gallinarum infection in Japanese quail fed Fusarium verticillioides culture material containing known levels of fumonisin B1. Avian Disease 51:705-712. Gao, X., Shim, W. -B., Göbel, C., Kunze, S., Feussner, I., Meeley, R., and Kolomiets, M. V. 2007. Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin. Mol. Plant-Microbe Interact. 20: 922-933. Garscha U, Jerneren F, Chung D, Keller NP, Hamberg M, and Oliw EH. 2007. Effects of gene targeting on oxylipin biosynthesis by Aspergillus spp. and the reaction mechanism of linoleate 5,8- and 8,00-diol synthases and 10R-dioxygenases. J. Biol. Chem. 282:34707-18. Hallen, H., Huebner, M., Shiu, S.-H., Guldener, U., and F. Trail. 2007. Gene expression shifts during perithecium development in Gibberella zeae (anamorph Fusarium graminearum), with particular emphasis on ion transport proteins. Fungal Genetics and Biology 44: 1146-1156. Hammond T M, Tsitsigiannis D I, and Keller NP. 2007. Infection of Arabidopsis thaliana seed by mycotoxigenic Aspergillus species. Plant Pathology 5:848-854. Hornok, L., C. Waalwijk & J. F. Leslie. 2007. Genetic factors affecting sexual reproduction in toxigenic Fusarium species. International Journal of Food Microbiology 119: 54-58. Hsiao, S.-H., Constable, P.D., Tumbleson, M.E., and Haschek, W.M. 2007. Use of formalin-fixed tissues to determine fumonisin B1 induced sphingolipid alterations in swine. J. Vet. Diagn. Invest. 19:425-430. Hsiao S-H, Wicklow DT, and Haschek WM. (2007). Cytotoxicity of pyrrocidines in HepG2 hepatocytes and PK15 renal cells. The Toxicologist CD  a supplement to Toxicological Sciences 85 (S-1): Abs. 1447. Jeney, A., E. Béki, A. Keszthelyi, J. F. Leslie & L. Hornok. 2007. Inactivation of Fpmtr, an amino acid transporter gene causes communication disturbances in Fusarium proliferatum. Journal of Basic Microbiology 47: 16-24. Ileleji, K. E., Maier, D. E. and Woloshuk, C. P. 2007. Evaluation of different temperature management strategies for suppression of Sitophilus zeamais (Motschulsky) in stored maize. J. Stored Prod. Res. 43:480-488. Kim, J. -E., Myong, K., Shim, W. -B., Yun, S. -H. and Lee, Y. -W. 2007. Functional characterization of acetylglutamate synthase and phosphoribosylamine-glycine ligase genes in Gibberella zeae. Curr. Genet. 51: 99-108. Kim, Y.-H., Woloshuk, C. P., Cho, E. H., Bae, J. M., Song, Y.-S. and Huh, G.-H. 2007. Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus ±-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab). Plant Cell Reports 26:395-405. Lee, J., R. L. Bowden & J. F. Leslie. 2007. Pheromone functions in Gibberella zeae. Fungal Genetics Newsletter 54(Suppl.): 134. Leslie, J. F., L. L. Anderson, R. L. Bowden & Y.-W. Lee. 2007. Inter- and intra-specific genetic variation in Fusarium. International Journal of Food Microbiology 119: 25-32. Mansfield, M. A. and Kuldau, G. A. 2007. Microbiological and molecular determination of mycobiota in fresh and ensiled maize. Mycologia 99:269-278. Mansfield, M. A., Archibald, D. D., Jones, A. D. and Kuldau, G. A. 2007. Relationship of sphinganine analog mycotoxin contamination in maize silage to seasonal weather conditions, and agronomic and ensiling practices. Phytopathology 97:504-511. Maggio-Hall L, Lyne P, Wolff J, and Keller NP. 2007. A single acyl-CoA dehydrogenase is required for catabolism of isoleucine, valine and short-chain fatty acids in Aspergillus nidulans. Fung. Genet. Biology 45:180-9 Oliveira, C. A. F., Butkeraitis, P., Ledoux, D. R., and Rottinghaus, G. E. 2007. Effects of fumonisin on body weight and histopathology of Japanese quail (Coturnix japonica). Ciencia Rural, Santa Maria 37:284-287. Oliveira, C. A., Ogido, R., Ledoux, D. R., Rottinghaus, G. E., Correa, B., Reis, T. A., and Goncalez, E. 2007. The quality of eggs from Japanese quail, Coturnix japonica, fed rations containing aflatoxin B1 and fumonisin B1. J. Poultry Sci. (Japanese) 44:29-33. Ogunbo, S. O., Broomhead, J. N., Ledoux, D. R., Bermudez, A. J., and Rottinghaus, G. E. 2007. The individual and combined effects of fusaric acid and T-2 toxin in broilers and turkeys. International J. Poultry Science 6:484-488. Perrin RM, Fedorova ND, Bok JW, Cramer RA, Wortman JR, Kim HS, Nierman WC, and Keller NP. 2007. Transcriptional regulation of chemical diversity in Aspergillus fumigatus by LaeA. PloS Pathogens Apr;3(4):e50. Rohlfs M., Albert M, and Keller NP, Kempken F. 2007. Secondary chemicals protect mould from fungivory. Biol. Letters 3:523-5. Schwartz, P. B., Hill, N. S., and Rottinghaus, G. E. 2007. Fate of ergot (Claviceps purpurea) alkaloids during malting and brewing. J. Am. Soc. Brewing Chemists 65:1-8. Voss, K A., G. W. Smith and W. M. Haschek. 2007. Fumonisins: Toxicokinetics, mechanism of action and toxicity. Animal Feed Science and Technology, 137:299-325. Sagaram, U. S. and Shim, W. -B. 2007. Fusarium verticillioides GBB1, a gene encoding heterotrimeric G protein b subunit, is associated with fumonisin B1 biosynthesis and hyphal development but not with fungal virulence. Mol. Plant Pathol. 8: 375-384. Sagaram, U. S., Shaw, B. D., and Shim, W. -B.. 2007. Fusarium verticillioides GAP1, a gene encoding a putative glycolipid-anchored surface protein, participates in conidiation and cell wall structure but not virulence. Microbiology 153:2850-2861. Schwarz, P. B., Hill, N. and Rottinghaus, G. 2007. Fate of ergot (Claviceps purpurea) alkaloids during malting and brewing. J. Am. Soc. Brew. Chem. 65:1-8. Shwab E., Bok JW, Tribus M, Galehr J, Graessle S, Keller NP. 2007. Histone deacetylase activity regulates chemical diversity in Aspergillus Euk. Cell 6:1656-64. Smith, C. A., Woloshuk, C. P., Robertson, D., and Payne, G. A. 2007. Silencing of the aflatoxin gene cluster in a diploid strain of Aspergillus flavus is suppressed by ectopic aflR expression. Genetics 176:2077-2086. Swett, C., L. L. Anderson, J. F. Leslie & J. Y. Uchida. 2007. Evaluating genetic diversity of Fusarium proliferatum from orchids in Hawaii. Fungal Genetics Newsletter 54(Suppl.): 69. Tsai, W.-T., Mason, L. J., and Woloshuk, C. P. 2007. Effect of three stored-grain fungi on the development of Typhaea stercorea. J. Stored Prod. Res. 43:129-133. Voss, K A., Smith, G.W., and Haschek, W.M. 2007. Fumonisins: Toxicokinetics, mechanism of action and toxicity. Animal Feed Science and Technology, 137:299-325. Wolf-Hall, C.E. 2007. Mold and mycotoxin problems encountered during malting and brewing. Int. J. Food Microbiol. 119:89-94. Wu, X., Murphy, P., Cunnick. J., and Hendrich S. 2007. Synthesis and characterization of deoxynivalenol glucuronide: its comparative immunotoxicity with deoxynivalenol. Fd. Chem. Toxicol. 45:1846-55.