Brief Summary of Minutes of Annual Meeting

George Rottinghaus called the meeting to order at 8:30 AM. This was followed by an address from David Jackson, NC 1183 administrator. Dr. Jackson described the protocols and deadline for the committee to to prepare and submit the 2012 annual report. He highlighted the need to maintain collaborations, and when possible to write collaborative proposals. Station Reports: The station reports were presented in the following order: Charles Woloshuk (IN); Suzanne Hendrich (IA); Kantha Channaiah, John Leslie (KS); Christopher Schardl, Lisa Vaillancourt (KY); Rafael Murarolli (MO); Johanna Takach (OK); Burt Bluhm (AR). Haibo Yao (MS) also gave a presentation. Business Meeting: George Rottinghaus called the business meeting to order at 2:30 PM. Officers for 2012 were George Rottinghaus (Chair), Charles Woloshuk (Vice Chair), and Lisa Vaillancourt (Secretary). For 2013, the officers will be Charles Woloshuk (Chair), Burt Bluhm (Vice Chair), and Suzanne Hendrich (Secretary). Charles Woloshuk will arrange the 2013 annual meeting in the fall at Turkey Run State Park in Indiana. The venue for the 2014 meeting will be in Arkansas, organized by Burt Bluhm. The venue for the 2015 meeting will be in Iowa, organized by Suzanne Hendrich. The committee discussed ways to increase the numbers of members, and to improve member participation in the annual meetings. The current funding situation has made it difficult for several members to attend. Having more than one representative from each state will help to ensure at least one will be able to attend the meetings. The committee was encouraged to invite other interested scientists from member states or from non-member states to participate on the project objectives or on the broader interests of the committee. Participants should send their suggestions of potential new members to the Chair, vice-Chair, or Secretary. Participants are also encouraged to bring students and postdoctoral scholars to the meetings whenever possible. The committee will need to prepare a new five-year proposal early in 2014. Members were asked to be prepared to discuss this new proposal at the 2013 meeting. Meeting was adjourned at 3:30 pm.

Objective 1. Develop data for use in risk assessment of mycotoxins in human and animal health. The Fusarium/Poultry Research Laboratory (FPRL) (MO) continues to produce mycotoxins in culture for use in-house, as well as for other researchers doing animal feeding trials with mycotoxins. The FPRL (MO) evaluated a number of mineral and organic adsorbents for binding mycotoxins in in vitro and in vivo studies in poultry and swine. Better performance of poultry exposed to aflatoxin B1 (AFB1) was observed when some of these materials were included in the diet. Work also continues to develop approaches for detoxification of mycotoxins in animals by enzymatic conversion (IA). The specific focus is on glucosides of deoxynivalenol (DON), DON-de-epoxidation, and DON glucuronidation. Resulting conversion products will be tested for toxicity in a mouse model. Future plans include more work with intestinal models and models for inflammatory bowel disease. The effects of AFB1 on hepatic gene expression in growing barrows were evaluated (MO). Crossbred weanling pigs fed 1.0 mg AFB1/kg feed had reduced (P<0.05) body weight gain and poorer feed conversion compared to controls. Microarray analysis was used to identify shifts in hepatic gene expression in pigs fed 0 and 1 mg AFB1/kg feed. A total of 720 genes were probed, 238 were highly correlated with treatment, with 80 genes downregulated, and 158 genes upregulated, in AFB1-treated pigs. Down-regulated genes were associated with retinol metabolism and xenobiotic metabolism by cytochrome P-450. Genes associated with proteasome, p53 signaling pathway, and antigen processing and presentation were upregulated in treated pigs. Objective 2. Establish integrated strategies to manage and to reduce mycotoxin contamination in cereal grains and distillers grains. Reducing levels of plant infection by toxigenic fungi is one approach to decrease mycotoxin contamination of grain and distillers grains. Several projects are focused on increasing plant resistance, or on identifying fungal pathogenicity factors that could serve as potential therapeutic targets. In MI, the timeframe in which resistance to DON and Fusarium graminearum occurs is being defined to assist in resistance breeding activities. In IA, the role of mycotoxins as pathogenicity factors in various crops is being studied by the use of non-toxigenic mutants. Mutants for use in these studies have been generated, and will be tested in the next year. These non-producers might be useful for biocontrol, if they can colonize and outcompete toxigenic strains. This approach is being explored to control endophytic fungi that produce alkaloid toxins that cause serious diseases in grazing animals (KY, OK). Strains are being identified or developed that can colonize forage grasses and provide fitness advantages without producing alkaloids. In one project (KY), the genes encoding the alkaloids have been identified and removed. These modified endophytes need to be tested in plants and on animals. In another project (OK), methods were developed to identify natural variants of endophytes that produce beneficial secondary metabolites, but not alkaloid mycotoxins. Several candidates have been identified and will be tested in future work. In IA an experiment was conducted to test the effect of fumonisin on ethanol production and distillers grains quality. Bt vs non-Bt corn lines were naturally or manually infested with insects in the field, and were allowed to become contaminated with fumonisins. Results demonstrated that BT hybrids provided better control of insects and also had lower levels of fumonisins in the grain. However, there were no differences in fumonisin enrichment in distillers grains, and there was no relationship between fumonisin levels and EtOH production. Levels of fumonisin were relatively low in this experiment, so work in the future will focus on testing effects over broader concentration ranges. Another approach for reduction of mycotoxin contamination is to directly treat stored grain. Studies were conducted on the application of chlorine dioxide gas, infrared radiation, and ozone to decrease the concentrations of mycotoxins and mycotoxigenic fungi in grain elevators (KS). Chlorine dioxide gas was effective in reducing levels of contaminating mold but had negative effects, at the concentrations used, on milling qualities of grain, and no effect on the amount of mycotoxin. Infrared radiation and ozone both reduced the incidence of grain mold, but infrared did not reduce mycotoxin levels. Ozone is known to destroy mycotoxins but the technology is expensive to implement, so work needs to continue to improve delivery methods and make them more economical. Another project (PA) is focused on the use of UV light for removal of fungi that produce allergens from cooling units. Methods were refined for identifying and quantifying molds from cooling units. Improvements in our ability to detect mycotoxins could lead to a reduction in grain contamination by allowing more efficient mixing of grain batches. A technology to monitor grain contamination by mycotoxigenic molds was developed based on CO2 emissions (KS). This method is more sensitive than other available methods. Use of fluorescence hypospectral imaging is being explored for the detection of aflatoxin contamination of grains (MS). The method shows promise since it appears to be highly sensitive, but challenges remain related to specificity of the results. Testing must continue with additional contaminated materials, which could be provided by collaborating with other members of the committee. Objective 3: Define the regulation of mycotoxin biosynthesis and the molecular relationships among mycotoxigenic fungi. Several ongoing projects seek to understand the extent and origins of genetic diversity among various important mycotoxigenic fungi. In KY, new types of fingerprinting markers were developed and used to characterize a diverse population of F. graminearum causing head blight on soft red winter wheat. Some states are working with international collaborators. The role of Aspergillus section Nigri in mycotoxin (fumonisin) contamination of maize in Italy was explored (IA). Results showed variation among strains in their ability to produce fumonisin. Non-fumonisin producing strains appeared to be equally pathogenic: in future the role of fumonisin in pathogenicity will be tested more directly. About 600 isolates of Fusarium from maize and rice in South Korea were characterized (KS). In maize, F. graminearum lineage 7, the same one that is found in the U.S., dominated. In rice, several different lineages were found and the population seemed to be old, while the maize population appeared to be more recent and may have been introduced. In South Africa, about 800 F. graminearum isolates, all of which belonged to lineage 7, were recovered from the major irrigated wheat regions (KS). AFLP analysis indicated the presence of two major regionally associated subpopulations. There was more variation within populations than between, and evidence for recombination was found. Other studies in Nigeria and Uganda sought to identify Fusarium species responsible for mycotoxin contamination of pearl millet, finger millet, maize, and sorghum grain (KS). Diversity of species, especially on finger millet, was very high. Subsistence crops like finger millet may act as refugia for fungal communities that predate commercial farming. A collaborative project (OK, KY) involved screening a large collection of endophyte-infected tall fescue seed, spanning 100 years and fifteen countries in Asia, the Middle East (the home of tall fescue), the Mediterranean, and Europe. The endophytes were evaluated for presence of genes involved in the production of beneficial secondary metabolites or alkaloid toxins. Several endophytes were identified that retain the potential to confer beneficial fitness traits on forage grasses, but do not have the capacity to produce toxic alkaloids. Future work will involve infecting agronomically desirable tall fescues with these potentially animal-friendly endophytes and testing their performance. Other projects focus on understanding the molecular and cytological mechanisms of pathogenicity in mycotoxigenic fungi. Unique interactions of F. graminearum with the barley surface have been characterized, and work is ongoing to characterize these at the physiological level (MI). In another project (KY), crosses of similar strains of F. graminearum resulted in generation of transgressive progeny that were significantly more aggressive and toxigenic than either parent. These traits were stably inherited in asexual progeny. Analysis of cosegregation of SNP markers revealed at least 10 genomic regions that may contain genes important in determining levels of aggressiveness and toxin production in planta. Future research will focus on testing hypotheses about the roles of the genes encoded by these regions. A collaboration between (AR) and (IN) resulted in an assembly of a draft genomic sequence of Stenocarpella maydis and a BLAST database consisting of 11,550 contigs. Insertional mutations were generated in S. maydis by Agrobacterium mediated transformation with the hygromycin resistant gene (Hyg) as a selectable marker. One thousand transformants were screen for mutations affecting development. Three mutants were identified and selected for further characterization. One mutant (174) is affected in pigment production associated with circadian rhythm. The insertion site was identified by TAIL-PCR and BLAST analysis with the S. maydis genome database indicated a 12 kb contig. A second strain (297) produces no pycnidia. The insertion site was identified in a 6.8 kb contig. A third mutant (653) is affected in melanin biosynthesis. It produces brown pycnidia instead of the normal black pycnidia. The insertion in this mutant was found in a 14.6 kb contig. About 300 transformants were screened to identify mutations affecting diplodiatoxin production. Although variability in the amount of toxin produced was observed, no non-producers of diplodiatoxin were identified. Future plans are to develop a collaborative grant proposal (KY, IN, AR) to USDA to explore pathogenicity and toxigenicity of S. maydis.

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