Brief Summary of Minutes of Annual Meeting

II. Introduction Meeting called to order at 8:35 by Chair, Gretchen Kuldau. Barb Christ, department head for the Penn State Department of Plant Pathology gave welcoming comments and some history of the department. Bev Durgan, the NC-1025 administrator indicated that the project was up to date with reporting. She will work with station directors on attendance issues. III. Station Reports The station reports began at 8:50 AM, and included reports from Pat Murphy for Iowa, Wanda Haschek-Hock for Illinois, Charles Woloshuk for Indiana, John Leslie for Kansas, Iffa Gaffor for Michigan, Charlene Wolf-Hall for North Dakota, Katelyn Tilley (graduate student) & Gretchen Kuldau for Pennsylvania, and Won-Bo Shim for Texas. Additional comments on the impact of mycotoxins on biofuels were provided by Mike Tumbleson of Illinois. IV. Business Meeting The chair, Gretchen Kuldau, called the business meeting to order at 3:05 PM. The minutes of the 2006 meeting were discussed. An update on the annual report was presented by Charles Woloshuck and Bev Durgan. There is a new format to follow which is significantly more concise. Charles will send the draft out for comment. Items that should be highlighted include collaborations, especially amongst the participants. Bev pointed out that this is a diverse group, spanning plants and animals, which helps to strengthen these types of connections. Frances Trail was recommended for the position of secretary in 2008. The 2008 meeting will be held in conjunction with the Midwest AOAC meeting in Bozeman MT. The dates for the AOAC meeting are May 19-22nd. Charlene is serving as the ND rep for the SD-ND-MT AOAC organizing committee, and will organize the NC-1025 session and business meeting. Wanda volunteered to help with organizing the session. We will showcase what NC-1025 does. A central theme would be developed with input suggested from Scott Smith, George Rottinghaus and Chris Maragos. Wanda suggested we include a separate poster session of recent but possibly already presented work for the NC-1025 meeting, and others agreed this would be a good idea. The 2009 meeting will be organized by Charles Woloshuk of Indiana. Meeting was adjourned at 3:30. Minutes respectfully submitted by Charlene Wolf-Hall

Goal 1. Develop data for use in risk assessment of mycotoxins in human and animal health The group at Iowa conducted a feeding study on twenty healthy adults to evaluate the effects of consuming bread contaminated with deoxynivalenol (DON). Of the group, four men and four women ate bread containing 300 µg DON at breakfast. Blood drawn from the test group was added to K-562 cells cultures to assay for DON toxicity. The results of the study indicated that this cell culture-bioassay could provide a relatively inexpensive approach for detecting DON in human subjects exposed to DON in their diets. Several members conducted surveys to determine the potential for mycotoxins in their respective states. Areas of Iowa have experienced drought or near-drought in two of the last four years. As a result, a modest level of aflatoxin contamination was found in the analysis of over 200 samples. By contrast, relatively unfavorable weather for the Fusarium mycotoxins may be in part responsible for the low prevalence of deoxynivalenol or zearalenone from the same time periods. In 2006, Indiana experienced relatively mild weather with some areas experiencing record rainfall. As a result, surveys indicated that DON contamination in corn was wide spread. High levels of fumonisin contamination, up to 174 ppm, were also recorded. A Kansas survey, which examined corn samples from four regions in the state, found fumonisin (75% of samples), beauvericin (63%), zearalenone (34%), T-2 toxin (30%), ochratoxin (26%), deoxynivalenol (8%), fusaproliferin (8%), and aflatoxin (1%). The Illinois group investigated an outbreak of fumonisin induced porcine pulmonary edema (PPE). Fumonisins were detected in stomach contents (FB1 > 5ppm) and feed (FB1 = 23ppm, FB2 = 6ppm). Serum had markedly elevated sphinganine and sphingosine concentrations as well as sphinganine to sphingosine ratio (average 3.22; experimental control reference range 0.00-0.18), confirming fumonisin mycotoxicosis. Equine leukoencephalomalacia was also reported in Illinois from the 2006 corn crop (Diagnostic Laboratory in Centralia, IL). The Illinois group and USDA/ARS also investigated the cytotoxicity of pyrrocidine A, an antibiotic produced by the maize endophyte Acremonium zeae. The group found that pyrrocidine A induces apoptosis in HepG2 and PK15 cells with initiation through both the intrinsic and the receptor mediated apoptotic pathways. Goal 2. Develop new techniques and improve current assays to identify and measure mycotoxins and mycotoxigenic fungi in cereal grains Two groups (North Dakota and Indiana) are developing gene-based methods to detect and quantify mycotoxigenic fungi. A multiplex real-time PCR method was developed for detection of the ochratoxin-producers Aspergillus ochraceus and Penicillium verrucosum and the trichothecene-producer Fusarium graminearum. A multiplex real-time PCR method based on TaqMan technology is being developed that will detect and quantify at the genus level species of Aspergillus, Penicillium, Eurotium, and Fusarium. With the increased utilization of distillers grain products (DDGs) in animal feeds the group in Missouri examined new analytical methodology to address problems associated with this complex matrix. They found that the traditional sample clean up in combination with TLC analysis is unsuitable for detecting mycotoxins in DDGs. This group experimented with immunoaffinity columns for sample cleanup with TLC and HPLC to screen and quantify, respectively, for mycotoxins. Kansas with a collaborator at the International Institute of Tropical Agriculture (Ibadan, Nigeria) tested the efficacy of sorting of corn kernels into good and bad categories by subsistence farmers in Nigeria. The results suggest that the method could significantly reduce the exposure to fumonisins by 20-25 fold. Goal 3. Establish integrated strategies to manage and to prevent mycotoxin contamination in cereal grains The group from Michigan has focused their effort towards understanding the role polyketide-derived mycotoxins have on the pathogenicity and survival of F. graminearum under field conditions. This group followed the expression of three polyketide synthase genes involved in the biosynthesis of fusarin C, zearalenone, and aurofusarin. All three genes were expressed during colonization of wheat and barley, particularly during the early stages of head colonization. In culture media, only the polyketide synthase gene involved in aurofusarin biosynthesis is expressed to a higher level. The group from North Dakota has found that irradiation of F. graminearum with electron beam caused mutations in the fungus and reduced mycotoxin production by the fungus. The group from Missouri has led the effort to evaluate the efficacy of adsorbents in feed to reduce the detrimental effects of mycotoxins on animal health. This year they evaluated several adsorbents with minerals (copper or zinc) attached to determine if the added minerals provide beneficial growth responses in poultry while at the same time binding aflatoxins in the feed. The results showed that Montmorillonite (MONT), an aluminosilicate clay and Cu-MONT at 0.5% of the diet were partially effective in ameliorating the toxic effects of aflatoxin in broilers. Tests of several commercial adsorbents indicated that the adsorbents were effective in ameliorating the toxic effects of aflatoxin in broilers without a negative effect on chick performance. Wisconsin examined conserved signaling molecules that affect mycotoxin production and spore development in Fusarium and Aspergillus species. This group found that oxygenated lipids (e.g. oxylipins), synthesized by both plants and fungi, are important for induction or suppression of toxin genes in both genera. To understand the environmental factors and infection timing favorable for production of wheat kernels asymptomatic for head scab caused by F. graminearum, Pennsylvania conducted a field study that utilized movable greenhouse enclosures and supplemental misting to control timing of infection. Results 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. Goal 4. Define the regulation of mycotoxin biosynthesis and the molecular relationships between mycotoxigenic fungi Several groups (Texas, Indiana, and USDA/ARS) have focused their effort towards understanding the regulation of fumonisin biosynthesis in F. verticillioides. It was discovered that the amylopectin component of corn kernels induces fumonisin biosynthesis. By microarray analysis, several putative genes were identified and found to be involved in fumonisin biosynthesis and sensing of the corn kernel environment. Putative fumonisin regulatory genes also were identified via subtractive suppressive hybridization and microarray analysis. One such gene was GBP1, encoding a monomeric G protein, which negatively regulates fumonisin biosynthesis. Another gene GBB1, encoding a heterotrimeric G-protein beta subunit, was found to have a role in fumonisin biosynthesis and fungal development. Disruption of GBB1 caused drastic reduction in fumonisin production but growth and fungal virulence was not affected. The Kansas group has investigated vegetative compatibility in F. proliferatum for potential strategies to control Fusarium spp. They cloned the het-c homolog of F. proliferatum. This gene has vegetative compatibility function in Neurospora crassa, but this function is not conserved in F. proliferatum. The results suggest that genes responsible for triggering an incompatibility response, which is cell death, may be limited to one or a few fungal species.

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