NC_OLD1183: Mycotoxins: Biosecurity, Food Safety and Biofuels Byproducts (NC129, NC1025)
(Multistate Research Project)
Status: Inactive/Terminating
Date of Annual Report: 03/23/2012
Report Information
Annual Meeting Dates: 10/24/2011
- 10/24/2011
Period the Report Covers: 10/01/2010 - 12/01/2011
Period the Report Covers: 10/01/2010 - 12/01/2011
Participants
Participants (in person):Suzanne Hendrich Iowa State University,
John Leslie Kansas State University,
George Rottinghaus University of Missouri,
Lisa Vaillancourt University of Kentucky,
Charles Woloshuk Purdue University,
David Ledoux University of Missouri,
Sladana Bec University of Kentucky,
Cynthia Gaskill University of Kentucky,
Padmaja Nagabhyru University of Kentucky,
Christopher Schardl University of Kentucky,
Lori Smith University of Kentucky,
Jim Strickland USDA Forage Research Lab, Lexington KY.
Participants (by remote connection):
David Jackson University of Nebraska (AA),
Anuradha Vegi North Dakota State University.
Brief Summary of Minutes
Introduction:" Meeting called to order at 8:30 by Lisa Vaillancourt.
" Address from David Jackson, NC 1183 administrator, via the Internet. Dr. Jackson indicated that the project needed to prepare an annual report. He encouraged members to increase collaboration, especially in applying for funding. Such efforts should be highlighted in the annual reports, even if unsuccessful.
Station Reports:
" The station reports were presented in the following order: Suzanne Hendrich (IA), John Leslie (KS), George Rottinghaus (MO), David Ledoux (MO), Charles Woloshuk (IN), Sladana Bec (KY).
" Presentations were also given by Christopher Schardl, Padmaja Nagabhyru, Cynthia Gaskill, and Jim Strickland.
Business Meeting:
Lisa Vaillancourt called the business meeting to order at 4:00 PM. Dr. Vaillancourt also recorded the minutes for the meeting, and Charles Woloshuk agreed to organize the annual report. Officers were named for 2012, George Rottinghaus as Chair, Charles Woloshuk as vice Chair, and Lisa Vaillancourt as Secretary.
The committee discussed possible ways to improve member participation in the annual meetings. The current funding situation has made it difficult for several members to attend. The on-line connections are difficult to set up at both the meeting site and at members office computers. Interactive participation via internet is nearly impossible at most venues. The committee decided to invite other interested scientist to participate on the project objectives or on the broader interests of the committee such as mycotoxins and food/feed safety. Participants agreed to send their suggestions of potential new members to the Chair, vice-Chair, or Secretary.
For 2012 annual meeting, George Rottinghaus and David Ledoux will organize a symposium on mycotoxins to coincide with the American Organization of Analytical Chemists Midwest Section meeting in St. Louis, Missouri, from June 4-7, 2012. This would meet a milestone schedules for 2012.
The venue and Chair for the 2013 meeting was discussed but not determined. It was agreed that secretary Lisa Vaillancourt would send an e-mail to project participants to ask for volunteers.
Meeting was adjourned at 5:00 pm.
Accomplishments
Accomplishments: <br /> <br /> Objective 1. Develop data for use in risk assessment of mycotoxins in human and animal health.<br /> <br /> Risk assessments and in vivo dosage studies require a sufficient supply of mycotoxin-contaminated material. The Fusarium/Poultry Research Laboratory (MO) continued to produce mycotoxins in culture (aflatoxin, ochratoxin A, zearalenone, moniliformin, and fumonisin) for animal feeding trials in their own laboratories as well as other research groups.<br /> <br /> <br /> Objective 2. Establish integrated strategies to manage and to reduce mycotoxin contamination in cereal grains and distillers grains. <br /> <br /> Progress was achieved in the area of intervention strategies. The Fusarium/Poultry Research Laboratory (MO) evaluated a number of proprietary adsorbents in vitro and in vivo for their efficacy to reduce poultry and swine mycotoxicoses. <br /> <br /> With assays that included microbiological, real-time PCR, real-time RT-PCR, gas chromatography last 2 days of germination and initial stages of kilning were found to be the peak stages for Fusarium infection, Tri5 gene production, Tri5 gene expression and deoxynivalenol production during malting of barley (ND). It was also shown that also found that the gaseous ozone significantly reduced Fusarium infection in germinated barley during malting. <br /> <br /> Two studies were conducted on the application of ozone to reduce mycotoxiigenic fungi (ND, IN). Gaseous ozone did not negatively influence any aspect of malt quality and may have subtle beneficial effects on diastatic power and ²-glucans. In a modified screw conveyor to treat grain with ozone in a continuous-flow system, ozone concentrations reached 47,800 ppm with an average retention time for a corn kernel moving through the system at 1.8 min. Under these conditions, Aspergillus flavus counts were reduced by 96% in a single pass through the screw conveyor. <br /> <br /> Objective 3: Define the regulation of mycotoxin biosynthesis and the molecular relationships among mycotoxigenic fungi.<br /> <br /> Progress was made towards understanding the molecular aspects of fumonisin production during colonization of host (PA, IN). A F. verticillioides mutant lacking the ability to form hyphal-fusions was non-pathogenic on maize ears, stalks and seedlings, had development growth defects, and produced significantly less mycotoxin than wild type. Strains defective in two genes, one encoding a NADPH oxidase and the other a regulator of NADPH oxidases, had significantly reduced pathogenicity and fumonisin production. It was also demonstrated that the F. verticillioides gene Fst1 encodes a member of a class of proteins that are structurally similar to sugar transporters but may have a regulatory function. Mutants lacking a functional Fst1 are less virulent, produce less fumonisin, and are more susceptible to reactive oxygen than the wild-type strain. A hexose kinase gene Hxk1 was found to function in fructose uptake, pathogenicity, fumonisin biosynthesis, and trehalose biosynthesis. <br /> <br /> Progress was also made toward understanding the relationship between sexual reproduction in Fusarium graminearum (Gibberella zeae) and pathogenicity and deoxynivalenal (DON) production (KY). Results indicated that sexual crosses occurring among strains with similar degrees of pathogenicity could produce progeny that are potentially more damaging with respect to pathogenicity and DON. While the ability to sexual cross is controlled in part by the MAT1 locus, Results indicated that deletion the two mating specificities MAT1-1-1, and MAT1-2-1, negatively affected aggressiveness of the pathogen on wheat and DON production in planta and in vitro. This was in contrast to deletion of the complete MAT1 locus, which had no effect on disease development or on mycotoxin production. In other studies (MI) the results suggest that genes involved in DON biosynthesis are transcribed for a longer time in infecting hyphae than previously realized. Also, resistant wheat cultivars can inhibit hyphal growth of F. graminearum and expression of the DON biosynthetic genes.<br />Publications
Anuradha Vegi, Paul Schwarz and Charlene Wolf-Hall. 2011. Quanitification of Tri5 gene, expression and deoxynivalenol production during the malting of barley. International Journal of Food Microbiology 150:150-156.<br /> <br /> <br /> Anuradha Vegi. 2011. Mycotoxic bioactives in cereals and cereal-based foods, p. 253-271. In: Fruit and Cereal Bioactives: Sources, Chemistry, and Application. Tokusoglu, O., and Hall, C., (Eds). CRC Press, Taylor and Francis Group, Boca Raton, FL.<br /> <br /> <br /> Baines, D., Erb, S., Lowe, R., Turkington, K., Sabau, E., Kuldau, G., Juba, J., and Roberts, R. A. 2011. Mouldy feed, mycotoxins and Shiga-toxin-producing Escherichia coli colonization in associated with Jejunal Hemorrhage Syndrome in beef cattle. BMC Veterinary Research 2011, 7:24. <br /> <br /> <br /> Dakovic A, Kragovic M, Rottinghaus GE, Sekulic Z, Milicevic S, Milonjic S, and Zaric S. Influence of natural zeolitic tuff and organozeolites surface charge on sorption of ionizable fumonisin B1. Colloids and surfaces B, Bioiterfaces, 76:272-278, 2010.<br /> <br /> <br /> Hallen-Adams, H., Wenner, N., Kuldau, G. and Trail, F. 2011. Deoxynivalenol gene expression during wheat kernel colonization by Fusarium graminearum. Phytopathology 101: 1091-1096. <br /> <br /> <br /> James B. Dodd, Anuradha Vegi, Ashwini Vashisht, Dennis Tobias, Paul Schwarz and Charlene Wolf-Hall. 2011. Effect of ozone treatment on the safety and quality of malting barley. Accepted (in press). Journal of Food Protection.<br /> <br /> <br /> Kim, H., Smith, J. E., Ridenour, J. B., Woloshuk, C. P., and Bluhm, H. B. 2011. HXK1 regulates carbon catabolism, sporulation, fumonisin B1 production, and pathogenesis in Fusarium verticillioides. Microbiology 157:2658-2669.<br /> <br /> <br /> McDonough, M.X., Campabadal, C.A., Mason, L.J., Maier, D.E., Denvir, A., Woloshuk, C. P. 2011. Ozone application in a modified screw conveyor to treat grain for insect pests, fungal contaminants, and mycotoxins. J. Stored Prod. Res. 47:249-254.<br /> <br /> <br /> McDonough, M.X., Mason, L.J., Woloshuk, C. P. 2011. Efficacy of high concentrations of ozone on adult maize weevil, rice weevil and all life stages of red flour beetle and Indianmeal moth. J. Stored Prod. Res. 47:306-310.<br /> <br /> <br /> Reese, B. N., Payne, G. A., Nielsen, D. M., and Woloshuk, C. P. 2011. Gene expression profile and response to maize kernels by Aspergillus flavus. Phytopathology 101:797-804.<br /> <br /> <br /> Simas MMS, Albuquerque R, Oliveira CA, Rottinghaus GE, and correa B. Influence of gamma radiation on productivity parameters of chicken fed mycotoxin-contaminated corn. Applied Radiation and Isotopes 68:1903-1908, 2010( <br /> Tessari ENC, Kobashigawa E, Cardoso ALSP, Ledoux DR, Rottinghaus GE, and Oliveira CAF. Effects of aflatoxin B1 and fumonisin B1 on blood biochemical parameters in broilers. Toxins 2, 1x mqanscripts;doi:10.3390toxins20x000x, 2010<br />Impact Statements
- Project has the capacity to provide mycotoxins (fumonisin, ochratoxin A, moniliformin, zearalenone, and aflatoxin) in culture material to mycotoxin research groups that makes it economically feasible to undertake animal feeding studies that would be nearly impossible if mycotoxins had to be purchased commercially. This will help increase animal health and reduce economic losses.
- The new knowledge generated indicating a role for reactive oxygen species in F. verticillioides pathogenicity on maize opens new opportunities for deeper understanding of virulence mechanisms and potential targets for control. This will help increase plant health and reduce economic losses from plant disease.
- Quantification of mycotoxin producing genes during cereal food processing such as malting is an important contribution to identify key processing steps that need to be monitored closely to prevent mycotoxin producing fungal growth and mycotoxin production. This will improve food safety and reduce economic losses.
- Information is available to plant breeders relative to disease caused by F. graminearum and the associated mycotoxin contamination such that they can work to develop new sources of durable resistance. This will reduce significant losses to U.S. wheat and small grain growers each year.
Date of Annual Report: 01/31/2013
Report Information
Annual Meeting Dates: 12/03/2012
- 12/03/2012
Period the Report Covers: 01/01/2012 - 12/01/2012
Period the Report Covers: 01/01/2012 - 12/01/2012
Participants
Burt Bluhm, University of Arkansas; Kantha Channaiah, Kansas State University; Li Chen, University of Kentucky; Tim Evans, University of Missouri; Suzanne Hendrich, Iowa State University; David Jackson (Administrative Advisor), University of Nebraska; David Ledoux, University of Missouri; John Leslie, Kansas State University; Rafael Murarolli, University of Missouri; George Rottinghaus, University of Missouri; Christopher Schardl, University of Kentucky; Johanna Takach, Noble Foundation; Lisa Vaillancourt, University of Kentucky; Charles Woloshuk, Purdue University; Haibo Yao, Mississippi State University; Carolyn Young, Noble Foundation.Brief Summary of Minutes
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.
Accomplishments
Objective 1. Develop data for use in risk assessment of mycotoxins in human and animal health. <br /> <br /> 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.<br /> <br /> 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. <br /> <br /> 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. <br /> <br /> Objective 2. Establish integrated strategies to manage and to reduce mycotoxin contamination in cereal grains and distillers grains. <br /> <br /> 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. <br /> <br /> 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.<br /> <br /> 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. <br /> <br /> 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.<br /> <br /> Objective 3: Define the regulation of mycotoxin biosynthesis and the molecular relationships among mycotoxigenic fungi. <br /> <br /> 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.<br /> <br /> 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. <br /> 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.<br />Publications
1. Dakovic, A., M. Kragovic, G. E. Rottinghaus, D. R. Ledoux, P. Butkeraitis, D. Vojislavljevic, S. Zaric, and L. Stamenic. 2012. Preparation and characterization of zinc-exchanged montmorillonite and its effectiveness as aflatoxin B1 adsorbent. Materials Chemistry and Physics 137(2012) 213-220.<br /> <br /> 2. Dierking, R.M., Young, C.A., and Kallenbach, R.L. 2012. Mediterranean and continental tall fescue: I. Effects of endophyte status on leaf extension, proline, mono- and disaccharides, fructan, and freezing survivability. Crop Sci. 52: 451-459.<br /> <br /> 3. Johnston, S. L., L. M. Brand, D. R. Ledoux, G. R. Goss, E. D. DeBoer, and F. Chi. 2012. Effects of aflatoxin on growth performance, organ weights, and serum chemistry of broilers, and the efficacy of Calibrin-A and Calibrin-Z enterosorbents to ameliorate these effects. P198, Page 27. Abstracts 2012 IPSF, January 23-27, Atlanta, GA.<br /> <br /> 4. Khan, M.A., R. K. Asrani, A. Iqbal, R. D. Patil, G. E. Rottinghaus, and D. R. Ledoux. 2012. Fumonisin B1 and ochratoxin A nephrotoxicity in Japanese quail: an ultrastructural assessment. Comp. Clin. Pathol. DOI 10.1007/s00580-012-1486-6.<br /> <br /> 5. Landers, B. R., L. M. Brand, R. A. Murarolli, D. R. Ledoux, G. E. Rottinghaus, and A. J. Bermudez, 2011. Efficacy of curcumin, supplied by turmeric (Curcuma longa) powder, in ameliorating the toxic effects of aflatoxin in young turkeys. P167, Page 24. Abstracts 2012 IPSF, January 23-27, Atlanta, GA.<br /> <br /> 6. Ledoux, D. R. 2012. New strategies for assessing and alleviating the effects of mycotoxins. P. 15. Abstracts 31St Annual Midwest AOAC Meeting and Exposition, June 4-6, St. Louis MO.<br /> <br /> 7. Lee, J., Kim, H., Jeon, J-J., Kim, H-S., Zeller, K.A., Carter, L.L.A., Leslie, J.F., Lee, Y-H. 20012. Population structure of and mycotoxin production by Fusarium graminearum from maize in South Korea. Appl. Env. Microbiol. 78: 72161-2167<br /> <br /> 8. Martinez, E. M. and Woloshuk, C. 2012. Chapter 24. Monitoring for Spoilage and Mycotoxins. In: Stored Product Protection. Edited by D. W. Hagstrum, T. W. Phillips, G. Cuperus. Kansas State University.<br /> <br /> 9. Murarolli, R. A., D. R. Ledoux, C. Reddy, G. E. Rottinghaus, K. Wells, C. OGorman, and K. Whitworth. 2012. Effects of aflatoxin B1 on hepatic gene expression in weanling pigs. Poster 69. Page 151. In: Abstracts of Lectures and Posters, 7th Conference of the World Mycotoxin Forum and XIIIth IUPAC International Symposium on Mycotoxins and Phycotoxins.<br /> <br /> 10. Oliver WT, Miles JR, Diaz DE, Dibner JJ, Rottinghaus GE, and Harrell RJ. 2012. Zearalenone enhances reproductive tract development, but does not alter skeletal muscle signaling in prepubertal gilts. J Animal Feed Science and Technology 174(2012):79-85.<br /> <br /> 11. Parish, J.A., Parish, J.R., Best, T.F., Boland, H.T., Young, C.A., 2012. Effects of selected endophyte and tall fescue cultivar combinations on steer grazing performance, indicators of fescue toxicosis, feedlot performance, and carcass traits. J. Anim. doi.10.25277jas.2011-4725 <br /> <br /> 12. Rustemeyer, S. M., W. R. Lamberson, D. R. Ledoux, K. Wells, K. J. Austin, and K. M. Cammack. 2011. Effects of dietary aflatoxin on the hepatic expression of apoptosis genes in growing barrows. J. Anim. Sci. 89:916-925.<br /> <br /> 13. Saleh, A.A., Esele, J.P., Logrieco, A., Ritieni, A., Leslie, J.F. 2012. Fusarium verticillioides from finger millet in Uganda. Food Additives and Contaminants 29: 1762-1769.<br /> <br /> 14. Schardl, C.L., Young, C.A., Faulkner, J.R., Florea, S., Pan, J. 2012. Chemotypic diversity of epichloae, fungal symbionts of grasses. Fungal Ecology 5: 331-344<br /> <br /> 15. Sharma, D., R. K. Asrani, D. R. Ledoux, G. E. Rottinghaus, and V. K. Gupta. 2012. Toxic interaction between fumonisin B1 and moniliformin for cardiac lesions in Japanese quail. (Avian Diseases. 56(3):545-554.<br /> <br /> 16. Takach, J.E., Mittal, S., Swoboda, G., Bright, S.K., Trammell, M.A., Hopkins, A.A., and Young, C.A. 2012. Genotypic and chemotypic diversity of Neotyphodium endophytes in tall fescue from Greece. Appl. Env. Microbiol. 78: 5501-5510 <br /> <br /> 17. Trail, F., and H. Hallen-Adams. 2012. Establishing strategies for control of mycotoxins in corn. Corn Utilization Conference, Indianapolis, June.<br /> <br /> 18. Valganon de Neeff, D., D. R. Ledoux, G. E. Rottinghaus, A. Dakovic, E. Kobashigawa and C.A.F. Oliveira. 2012. In vitro efficacy of a hydrated sodium calcium aluminosilicate (HSCAS) to bind aflatoxin B1 (AFB1). XXIV Worlds Poultry Congress, Salvador, Bahia, Brazil, August 5-9, 2012. Worlds Poultry Science Journal Volume 68 (suppl. 1).<br /> <br /> 19. Valganon de Neeff, D., D. R. Ledoux, G. E. Rottinghaus, B. R. Landers, L. M. Brand, C.A.F. Oliveira. 2012. Efficacy of a hydrated sodium calcium aluminosilicate to reduce aflatoxin residues in tissues of broiler chicks fed aflatoxin B1. XXIV Worlds Poultry Congress, Salvador, Bahia, Brazil, August 5-9, 2012. Worlds Poultry Science Journal. Volume 68 (Suppl. 1).<br /> <br /> 20. Woloshuk, C. and Martinez, E. M. 2012. Chapter 6. Molds and Other Microbes in Stored Products. In: Stored Product Protection. Edited by D. W. Hagstrum, T. W. Phillips, G. Cuperus. Kansas State University.<br /> <br /> 21. Woloshuk, C.P. and Shim, W-B. (2012) Aflatoxins, Fumonisins, and Trichothecenes: A Convergence of Knowledge. FEMS Microbiology Reviews. In press DOI: 10.1111/1574-6976.12009.<br />Impact Statements
- Safe utilization of low level mycotoxin contaminated grains in animal feedstuffs can be increased with the development of new proprietary adsorbents and use of naturally occurring antioxidants to reduce or eliminate the toxicity of these mycotoxins. This information and approach will be valuable to evaluate the efficacy of dietary supplements designed to minimize oxidative damage to the liver and thus moderate the effects of mycotoxin.
- Studies of enzymatic conversions of mycotoxins could lead to probiotic therapies involving microbes that produce conversion enzymes to mitigate the effects of mycotoxin exposure in animals or humans.
- Providing mycotoxins (fumonisin, ochratoxin A, moniliformin, zearalenone, and aflatoxin B1) in culture material to mycotoxin research groups makes it economically feasible to undertake animal feeding studies that would be nearly impossible if mycotoxins were purchased commercially. Collaboration between KY and MO may allow utilization of recombinant strains that produce high levels of deoxynivalenol to make DON feeding studies with large animals (swine, cattle) practical.
- Several promising therapies for both pre- and post-harvest reduction of mycotoxin contamination of grain and distillers grains were developed and tested. Work will continue to further evaluate performance parameters, and to make them more reliable and economically viable.
- New tools and knowledge were developed to help monitor and track populations of toxic fungi, and to study the population genetics of mycotoxigenicity. This will help to improve our ability to model and predict mycotoxin outbreaks and impacts on animal and human health in the future.
- New foundational knowledge and techniques were developed for studying several important mycotoxigenic fungi including Stenocarpella maydis.
Date of Annual Report: 06/30/2014
Report Information
Annual Meeting Dates: 10/08/2013
- 10/08/2013
Period the Report Covers: 10/01/2013 - 09/01/2014
Period the Report Covers: 10/01/2013 - 09/01/2014
Participants
Brief Summary of Minutes
See attached "Copy of Minutes" file for NC1183's 2013 annual report.Accomplishments
Publications
Impact Statements
Date of Annual Report: 10/20/2015
Report Information
Annual Meeting Dates: 10/03/2014
- 10/03/2014
Period the Report Covers: 10/01/2013 - 10/01/2014
Period the Report Covers: 10/01/2013 - 10/01/2014
Participants
Hendrich, Suzanne, Munkvold, Gary, Iowa State University; Leslie, John--Kansas State University; Vaillancourt,Lisa, Schardl, Chris, Deakins,Randy--University of Kentucky; Hallen-Adams, Heather, University of Nebraska, Lincoln; Lawton, Michael, Rong, Di--Rutgers University; Kuldau, Gretchen, Pennsylvania State University; Shan, Xueyan--Mississippi State UniversityBrief Summary of Minutes
See attached "Copy of Minutes" file for NC1183's 2014 annual report.Accomplishments
Accomplishments:<br /> <br /> <br /> Investigators enhanced knowledge and practical ability regarding bioinformatic analyses of fungal genome sequencing and assembly and advanced in transition from proteomics to metabolomics via hands-on experience and interacting with respective colleagues during the American Society for Mass Spectrometry meeting. A research associate received training in operation of liquid chromatography - mass spectrometry instrumentation for analysis of small molecules and the software-based analysis of raw mass spectrometry data. We developed new bioenergy curricula with content related to mycotoxins. We have been providing trainings in molecular lab techniques and data acquisition and analysis to MSU graduate students, undergraduate students, and also high school students from the community to provide opportunities that students can be exposed to up to date molecular biology techniques in the research area. We also hosted international visiting scholars. We maintained outreach services to the community by hosting agricultural and food/feed safety experiences within our laboratory. Individuals from the community (74) participated in the following activities: “What killed my bees and contaminated my honey?” activity as a forensic experience for Bug Camp; “An Agricultural Crime Case-Focusing on Mycotoxin Analysis” activity as a forensic/feed safety experience for Winston Academy; Facility tours were provided to 4-H Participants during Summer 2014 highlighting food and feed safety. We also offered a demonstration of the practical application of analytical testing for petroleum products for environmental and mechanical engineers (50 individuals). Approximately a dozen PhD students in Toxicology and other disciplines graduated; at least that many other graduate students were mentored. Many undergraduate students were engaged in these studies as well. <br /> <br /> <br /> Professional Master's students have benefited from new curricula developed related to this project. Results have been presented at professional meetings in chemistry, plant pathology and veterinary medicine. Presentations were made at the NCC annual meetings at Purdue University, and at Iowa State University, The Genetics Society of America, Gordon Research Conference, Food Research Institute (FRI) annual meetings, the ASMS Conference on Mass Spectrometry and Allied Topics, June 2014 and at the American Society of Brewing Chemists, and informally at the annual US Wheat and Barley Scab Forum. Scientists received a report at the MSA meetings hosted by MSU; results were shared with companies. Graduate students working on these projects attended and presented material at national research conferences. Students provided extended abstracts and they were awarded ACS National Travel Grants. A number of papers were published.<br /> Obj. 1: Surveys for mycotoxins in grain storage elevator (KS) was conducted across the project. Numerous corn samples were collected and tested for the presence of aflatoxin, fumonisin, and deoxynivalenol (DON). Few samples were contaminated with fumonisin. The fumonisin levels in were very low (0.01 ppm to 7.71 ppm). Typically the samples showed a negative result for aflatoxin contamination. DON level was also below the allowable limit and ranged from 0.00 to 0.01 ppm. Generally in years lacking weather extremes, the mycotoxin analysis results were negative, also showed proper harvesting and storage of the grains. As part of this work the formation of Deoxynivalenol-3-glucoside (DON3G) during malting was investigated. DON3G is a bound, or conjugated, form of deoxynivalenol (DON). There is concern over the presence of DON3G in commercial samples of barley and malt, as it is not measured by routine analytical methods for DON. It has been proposed that DON3G may be broken down or transformed, to free DON during digestion or in food processing, and if this is the case, DON3G and other conjugated forms could make a significant contribution to the tolerable daily intake (TDI), and should be measured. DON-3-G was observed to increase by an average of 48-fold during malting. Levels ranged from the limit of detection to 65.84 mg/kg on the malt. In all cases, the levels of DON3G, greatly exceeded those of DON. The formation of DON3G during malting is attributed to the presence of UDP-glucosyl-transferase enzymes in germinating barley. A preservative/antioxidant blend mitigated DON toxicity in swine in terms of improved average daily gain. DON at 5 ppm decreased liver selenium compared with controls in swine fed for 120 d. various mycotoxin binders were tested in rodents and livestock, with generally protective results. A system was developed to study the toxicity of DON that uses Caenorhabditis elegans. The lifespan and egg-laying capacity of DON-treated nematode worms is significantly reduced compared to controls. This assay system will allow us to study mechanisms of toxicity of DON and other mycotoxins, and to test potential mitigation strategies, in a simple animal model. We performed RNA-Seq using the C. elegans system in order to identify genes that are potential targets for DON and which may also be involved in detoxification mechanisms. Genes that are up-regulated by DON include a number involved in detoxification and innate immunity, while down-regulated genes are involved in metabolism and development. The significance of these genes can now be assessed through the use of RNAi suppression or overexpression in transgenic worms.<br /> <br /> <br /> Obj. 2: Research focused on the development of new protocols for production biofuels and chemicals. Genetically modified lines of maize containing newer Bacillus thuringensis (BT) genes were assessed for content of fumonisins (FB) and susceptibility to insect damage (IA). A new BT gene was very effective in reducing FB contents compared with the older BT versions. Ethanol was made from corn containing up to 8 ppm FB, which did not adversely affect ethanol yield. Spiking ethanol fermentation with even higher levels of FB also did not affect ethanol production. Dried distillers grains had about 3 fold enrichment of FB. Batches showing lesser increase of FB are planned to be investigated further. The role of mycotoxins as virulence-enhancing factors in plant disease was studied in seedling maize, soybeans, and wheat. We have identified 4 compounds that affect the production of aflatoxin by Aspergillus and deoxynivalenol by Fusarium graminearum. We have examined their effect in culture using pure synthesized compounds. This project accomplished the first study describing a QuEChERS method for the quantitative determination of AFM1 in raw milk using HPLC-MS. The effectiveness of the binders to separate AFM1 from the milk using QuEChERS as an extraction method was possible as QuEChERS produced 3 layers separating the AFM1 in the organic layer, the middle layer contained the binder from the sample (and AFM1 if the binder was efficient), and the lower aqueous layer and the excess salts.<br /> <br /> <br /> Obj. 3: The genome sequence data for Stenocarpella maydis was refined with a more complete assembly. This work was performed in close collaboration with other members of the multi-state working group (IN, KY). This work produced a genome assembly that is comparable to published de novo genome sequences in other filamentous fungal pathogens. Work on FB production by black Aspergillus spp. (IA) showed that a good portion of these isolates produce FB2 in the laboratory, but low levels compared with F. verticillioides or F. proliferatum. Drier regions had greater black Aspergillus in maize, which co-occurred with A. flavus. The interactions between fungal species and mycotoxigenesis are planned to be further studied. We developed some novel markers and used them to analyze a population of Fusarium graminearum from Kentucky (Bec et al., 2014). We learned that most of the isolates belonged to the dominant chemotype, but that genetically they showed signs of being an isolated divergent population, suggesting there is not a lot of mixing of isolates from outside of Kentucky. We found two species that had not been previously described on symptomatic wheat heads, but these did not cause symptoms. We postulate that these colonize the tissues killed by the scab fungus. This is significant because these other species produce different types of mycotoxins. We have developed Brachypodium distachyon as a model system for studying infection by F. graminearum and the effects of DON. Detached leaves of B. distachyon can be infected with GFP-labeled wild type F. graminearum and tri5-mutant F. graminearum strains, and are also sensitive to the DON application. We have now developed B. distachyon and barley tissue culture and transformation protocols using immature and mature seeds, and have started a program to exploit CRISPR/Cas gene editing technology to engineer FHB resistance in these plants. The model plant Arabidopsis thaliana is being used to test efficacy of CRISPR/Cas gene editing to improve FHB resistance. A number of genes required for FHB susceptibility and DON detoxification are currently being targeted using this technology. A number of transgenic barley plants expressing genes involved in resistance and susceptibility have now been introduced and are currently being verified for gene expression. Functional testing against FHB will proceed once gene expression levels are known.<br /> <br /> <br /> This project’s research can potentially be used to analyze large qRT-PCR datasets in combination with the corresponding maize DNA marker data and maize phenotypic data. This is essential to the identification of aflatoxin resistance DNA markers for incorporation of maize resistance into elite commercial maize lines. A number of candidate genes differing between resistant and susceptible lines were identified. Newly found antifungal agents may have a wide array of applications – from crop protection, to wood decay prevention, to use in household cleaning products. In regards to major goals, the initial (year 1) research provided critical outcome in the form of a set of statistically significant and tentatively identified corn metabolites that are differentially abundant in fungus-resistant genotype versus fungus-susceptible genotype. <br /> <br /> <br /> We established a mechanism of bZIP regulation of fungal secondary metabolites (SMs) through RsmA, a recently discovered YAP-like bZIP protein. RsmA greatly increases SM production by binding to two sites in the Aspergillus nidulans AflR promoter region, a C6 transcription factor known for activating production of the carcinogenic and anti-predation SM, sterigmatocystin. Deletion of aflR in an overexpression rsmA (OE:rsmA) background not only eliminates sterigmatocystin production but also significantly reduces asperthecin synthesis. Furthermore, the fungivore, Folsomia candida, exhibited a distinct preference for feeding on wild type rather than an OE:rsmA strain. RsmA may thus have a critical function in mediating direct chemical resistance against predation. Taken together, these results suggest RsmA represents a bZIP pathway hardwired for defensive SM production. We also used laeA mutants as tools to elucidate virulence attributes in Aspergillus flavus. Microarray expression profiles of ?laeA and over-expression laeA (OE::laeA) were compared to wild type A. flavus. Strikingly, several nitrogen metabolism genes were oppositely mis-regulated in the ?laeA and OE::laeA mutants. One of the nitrogen regulatory genes, the bZIP encoding meaB, was up-regulated in ?laeA. Significantly, over-expression of meaB (OE::meaB) phenocopied the decreased virulence attributes of a ?laeA phenotype including decreased colonization of host seed, reduced lipase activity and loss of aflatoxin B1 production in seed. However, a double knock-down of laeA and meaB (KD::laeA,meaB) demonstrated that KD::laeA,meaB closely resembled ?laeA rather than wild type or ?meaB in growth, aflatoxin biosynthesis and sclerotia production thus suggesting that meaB does not contribute to the ?laeA phenotype. MeaB and LaeA appear to be part of regulatory networks that allow them to have both shared and distinct roles in fungal biology. <br /> <br /> <br /> The velvet genes in A. flavus are an ideal target for control strategies, as disruption of these genes can reduce the fungus ability to spread and produce toxin. We found a new member of velvet family, VelD, which only found in A. flavus and A. oryzae. We have generated vosA, velB, velC, and velD deletion mutants in A. flavus. The deletion of velB caused severely impaired (number, size and morphology) conidiation and the lack of sclerotia production. Moreover, the velB deletion mutant no longer produced AFB1. The deletion of vosA causes earlier conidiation and shows 2 fold more conidia number in 4 day culture. Besides, the vosA deletion mutant produces significantly less AFB1 comparing to WT. velB and vosA deletion mutant conidia contain only ~30% of trehalose compared to wild type spores, suggesting that both may be required for the spore viability in A. flavus. Deletion mutants of the osaA gene homologues in A. flavus show aberrations in development and aflatoxin biogenesis. For that reason, we conclude that OsaA is a key regulatory factor that participates in controlling the process of development and mycotoxin biosynthesis in Aspergillus species. We also did the first study to elucidate WetA function on mycotoxin production in A. flavus. The loss of wetA leads to reduced conidia viability and conidia autolysis in 3 days after inoculation. The wetA null mutant showed a reduced growth rate. Deletion of wetA also decreased fungal stress tolerance. The Velvet proteins, OsaA, and WetA are involved in either sporogenesis and/or mycotoxin production. Understanding the regulatory mechanisms of these proteins, we have more confidence to control both fungal dissemination and mycotoxin production in fields. <br /> We have identified several global regulators of mycotoxin production in A. nidulans, such as LaeB that regulates sterigmatocystin production. LaeA regulates all mycotoxins in all fungi. We have recently found that LaeA regulates production of spore toxins through the transcription factor known as BrlA. We found that LaeA regulates brlA expression via chromatin remodeling of the brlA promoter. A human immunotoxin in A. fumigatus, endocrocin, was found to be regulated by LaeA through BrlA. We proposed that G protein coupled receptors (GPCR) are the likely receptors involved in quorum sensing; we have published the deletion of 14 of 16 GPCRs (the 2 others were already known). We are now testing these mutants to see if they are impaired in density development, which will further advance understanding of mycotoxin regulation.<br />Publications
Wang, Y., Janssen, H. and Blaschek, H.P. Fermentative biobutanol production: An old topic with remarkable recent advances. In: Bioprocessing of Renewable Resources to Commodity Bioproducts, Bisaria and Kondo (Editors), Wiley.<br /> <br /> <br /> Amaike S, Affeldt K, Keller NP. (2013) Genetics, Biosynthesis and Regulation of Aflatoxins and Sterigmatocystin. Ed: F Kempken In The Mycota X, Springer-Verlag. In Press.<br /> <br /> <br /> Amaike S, Keller NP (2009) Distinct roles for VeA and LaeA in development and pathogenesis of Aspergillus flavus. Eukary. Cell 8(7):1051-60.<br /> <br /> <br /> Madson, DM; Ensley, SM; Patience, JE; Gauger, PC; Main, RG. (2014) Diagnostic assessment and lesion evaluation of chronic deoxynivalenol ingestion in growing swine. J Swine Health Prod 22:78-83.<br /> <br /> <br /> Susca, A; Moretti, A; Stea, G; Villani, A; Haidukowski, M; Logrieco, A; Munkvold, G. (2014) Comparison of species composition and fumonisin production in Aspergillus section Nigri populations in maize kernels from USA and Italy. Int J Food Micro 188:75-82.<br /> <br /> <br /> Patience, JF; Myers, AJ; Ensley, S; Jacobs, BM; Madson, D. (2014) Evaluation of two mycotoxin mitigation strategies in grow-finish swine diets containing corn dried distillers grains with solubles naturally contaminated with deoxynivalenol. J Animal Sci 92:620-6.<br /> <br /> <br /> Bowers, E.; Hellmich, R.; Munkvold, G. (2014) Comparison of Fumonisin Contamination Using HPLC and ELISA Methods in Bt and Near-Isogenic Maize Hybrids Infested with European Corn Borer or Western Bean Cutworm. J Agric Food Chem 62:6463-72.<br /> <br /> <br /> Miller, JD; Schaafsma, AW; Bhatnagar, D; Bondy, G; Carbone, I; Harris, LJ; Harrison, G; Munkvold, GP; Oswald, IP; Pestka, JJ (2014) Mycotoxins that affect the North American agri-food sector: state of the art and directions for the future. World Mycotoxin J 7:63-82.<br /> <br /> <br /> Ellis, ML; Munkvold G. (2014) Trichothecene Genotype of Fusarium graminearum Isolates from Soybean (Glycine max) Seedling and Root Diseases in the United States. Plant Dis. 98:1012-3.<br /> <br /> <br /> Bec, S., Ward, T., Farman, M., O'Donnell, K., Hershman, D., Van Sanford, D., and Vaillancourt, L.J. Characterization of Fusarium strains recovered from wheat with symptoms of head blight in Kentucky. Plant Disease http://dx.doi.org/10.1094/PDIS-06-14-0610-RE<br /> <br /> <br /> Womack, E.D.; *Sparks, D.L.; Reid, C.X.; Brown, A.E.; DuBien, J.L.; Ward, S. (2014) Validation of Aflatoxin M1 in Raw Milk Using QuEChERS as an Extraction Method. Journal of Chromatography A. In Review.<br /> <br /> <br /> Atkinson, C.; Pechanova, O.; Sparks, D.L.; *Brown, A.; *Rodriquez, J.M. (2014) Differentiation of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergilli by FT-IR Spectroscopy. Applied Spectroscopy. 68(8) DOI:10.1002/PMIC.201100659.<br /> <br /> <br /> Womack, E.D.; *Brown, A.E.; Sparks, D.L. (2014) A Recent Review of Non-biological Remediation of Aflatoxin-Contaminated Crops. Journal of Science, Food and Agriculture. 94:1706-1714.<br /> <br /> <br /> Baines, D., umarah, M., Kuldau, G., Juba, J., Mazza, A., Masson, L. 2013. Aflatoxin, fumonisin and shiga toxin-producing Escherichia coli infections in calves and the effectiveness of Celemanax r/Dairyman?s Choice T applications to eliminate morbidity and mortality losses. Toxins 5(10): 1872-1895.<br /> <br /> <br /> Rahman, A., Kuldau, G. A., Uddin, W. 2014. Induction of salicyclic acid-mediated defense response in perennial ryegrass against infection by Magnaporthe oryzae. Phytopathology104:614-623.<br /> <br /> <br /> Yin W, Amaike S, Wohlbach DJ, Gasch AP, Chiang Y-M, Wang CC, Bok JW, Rohlfs M, Keller NP (2012) An Aspergillus nidulans bZIP response pathway hardwired for defensive secondary metabolism operates through aflR. Mol Microbiol 83:1024-1034.<br /> <br /> <br /> Forseth RR, Amaike S, Schwenk D, Affeldt KJ, Hoffmeister D, Schroeder FC, Keller NP (2013) Homologous non-canonical NRPS gene clusters mediate redundant small-molecule biosynthesis in Aspergillus flavus. Angew Chem Int Ed Engl. 52:1590-4.<br /> <br /> <br /> Amaike S, Affeldt K, Yin WB, Franke S, Anjali Choithani A, Affeldt KJ, Keller NP (2013) The bZIP protein MeaB mediating virulence attributes in Aspergillus flavus. PLoS One. 8:e74030.<br /> <br /> <br /> Ahmed YL, Gerke J, Park H-S, Bayram ?, Neumann P, et al. (2013) The Velvet Family of Fungal Regulators Contains a DNA-Binding Domain Structurally Similar to NF-?B. PLoS Biol 11(12): e1001750. doi:10.1371/journal.pbio.1001750<br /> PLoS One. 2013 Sep 9;8(9):e74030. doi: 10.1371/journal.pone.0074030. <br /> <br /> eCollection 2013. The bZIP protein MeaB mediates virulence attributes in Aspergillus flavus. Amaike S1, Affeldt KJ, Yin WB, Franke S, Choithani A, Keller NP.<br /> <br /> <br /> Angew Chem Int Ed Engl. 2013 Jan 28;52(5):1590-4. doi: 10.1002/anie.201207456. Epub 2012 Dec 20. Homologous NRPS-like gene clusters mediate redundant small-molecule biosynthesis in Aspergillus flavus. Forseth RR1, Amaike S, Schwenk D, Affeldt KJ, Hoffmeister D, Schroeder FC, Keller NP.<br /> <br /> <br /> Springer, J., and Trail, F. 2014. Identification of natural copounds that limit aflatoxin production in Aspergillus parasiticus. Poster presented at the Mycological Society of America meeting in E. Lansing, June<br /> <br /> <br /> Asters MC, Williams WP, Perkins AD, Mylroie JE, Windham GL, Shan X (2014) Relating significance and relations of differentially expressed genes in response to Aspergillus flavus infection in maize. Sci Rep doi:10.1038/ srep04815.<br /> <br /> <br /> Shan X and Williams WP. 2014. Toward elucidation of genetic and functional genetic mechanisms in corn host resistance to Aspergillus flavus infection and aflatoxin contamination. Front. Microbiol. 5:364. doi: 10.3389/fmicb.2014.00364<br /> <br /> <br /> Erika Womack, Darrell Sparks, Cedric Reid, Ashli Brown, Jan DuBien, Stephanie Hill (2014) Validation of alfatoxin M1 in raw milk using QuEChERS as an extraction method. American Chemical Society National Meeting, San Francisco, CA, August 10-14.<br /> <br /> <br /> Cedric Reid, Erik Mylorie, Ashli Brown, Darrell Sparks, W. Paul Williams (2014) Correlating aflatoxin accumulation and fungal biomass in Aspergillus flavus inoculated maize. American Chemical Society National Meeting, San Francisco, CA, August 10-14.<br /> <br /> <br /> Erika Womack, Darrell Sparks, Ashli Brown, Stephanie Hill (2014) Evaluation of adsorbents for the removal of aflatoxin M1 from contaminated milk. The American Dairy Science Association, The American Society of Animal Science and The Canadian Society of Animal Science Joint Annual Meeting, Kansas City, MO, July 20-24.<br /> <br /> <br /> Pechanova O, Rodriguez JM, Williams WP and Pechan T. Differential metabolomics of cob tissues from maize genotypes resistant and susceptible to aflatoxin accumulation Conference Proceedings of 62nd ASMS Conference on Mass Spectrometry and Allied Topics, June 15-19, 2014, Baltimore MD, USA<br /> <br /> <br /> Schwarz, P.B. Occurrence of deoxynivalenol-3-glucoside in barley and malt from North Dakota. Proceedings of the 2013 American Society of Brewing Chemists Annual Meeting. May 19?May 22, 2013. Tucson, Arizona<br />Impact Statements
- Mycotoxin survey data have confirmed that aflatoxin, fumonisin and deoxynivalenol levels are typically minimal to zero when weather conditions are not extreme (no drought, flooding). This affects the extent to which surveillance is needed and helps to assure quality of grains for human food and animal feed.
- Malting was shown to dramatically increase the level of deoxynivalenol-3-glucoside (D3G) in barley and malt. This form of DON is not typically detected and is toxic but potentially shifts the sites of toxicity. This finding emphasizes the need to monitor for D3G and to develop methods to mitigate its formation.
- C. elegans model for assessing mycotoxin effects, especially on gene expression, has been developed and will facilitate identification of mycotoxin targets of action for mitigation strategies.
- Dried distillers grains were demonstrated to be enriched in mycotoxins, which lead to the need for continuing surveillance and developing strategies to mitigate this animal feed source for mycotoxin contamination.
- 4 compounds that affect the production of aflatoxin by Aspergillus and deoxynivalenol by Fusarium graminearum have been identified. These compounds provide opportunities to develop better mycotoxin remediation strategies that can be tested in the field in future years.
- A QuEChERS method for the quantitative determination of AFM1 in raw milk using HPLC-MS, which will aid in better detection of aflatoxin metabolites that potentially pose health risks to humans.
- We have developed Brachypodium distachyon as a model system for studying infection by F. graminearum and the effects of DON. Detached leaves of B. distachyon can be infected with GFP-labeled wild type F. graminearum and tri5-mutant F. graminearum strains, and are also sensitive to the DON application. We have now developed B. distachyon and barley tissue culture and transformation protocols using immature and mature seeds, and have started a program to exploit CRISPR/Cas gene editing technology to engineer FHB resistance in these plants, which will facilitate developing FHB resistance in grains in the field.
- This project?s research can potentially be used to analyze large qRT-PCR datasets in combination with the corresponding maize DNA marker data and maize phenotypic data. This is essential to the identification of aflatoxin resistance DNA markers for incorporation of maize resistance into elite commercial maize lines. A number of candidate genes differing between resistant and susceptible lines were identified. Newly found antifungal agents may have a wide array of applications ? from crop protection, to wood decay prevention, to use in household cleaning products.
- We established a mechanism of bZIP regulation of fungal secondary metabolites (SMs) through RsmA, a recently discovered YAP-like bZIP protein. Results suggest RsmA represents a bZIP pathway hardwired for defensive SM production, which may be important to develop plants optimized against mycotoxins that are also non-toxic to humans and animals.
- The velvet genes in A. flavus are an ideal target for control strategies, as disruption of these genes can reduce the fungus ability to spread and produce toxin. We found a new member of velvet family, VelD, which was only found in A. flavus and A. oryzae. We have generated vosA, velB, velC, and velD deletion mutants in A. flavus. The deletion of velB caused severely impaired (number, size and morphology) conidiation and the lack of sclerotia production. Moreover, the velB deletion mutant no longer produced AFB1. This work suggests an approach for mitigation of aflatoxins.
- Deletion mutants of the osaA gene homologues in A. flavus show aberrations in development and aflatoxin biogenesis. For that reason, we conclude that OsaA is a key regulatory factor that participates in controlling the process of development and mycotoxin biosynthesis in Aspergillus species, which may be useful in mitigation aflatoxins.
Date of Annual Report: 12/16/2015
Report Information
Annual Meeting Dates: 12/16/2015
- 12/16/2015
Period the Report Covers: 10/01/2014 - 09/30/2015
Period the Report Covers: 10/01/2014 - 09/30/2015
Participants
(2015) Blaschek Hans, blaschek@uiuc.edu, University of Illinois; Hendrich, Suzanne (Chair) shendric@iastate.edu, Iowa State University; Leslie, John, jfl@ksu.edu, Kansas State University; Schardl, Chris, chris.schardl@uky.edu and Vaillancourt, Lisa, (Vice-Chair) vaillan@uky.edu, University of Kentucky; Trail, Francis, trail@msu.edu, Michigan State University; Shan, Xueyan, shan@bch.msstate.edu, Mississippi State University; Rottinghaus, George, rottinghausg@missouri.edu, University of Missouri; Hallen-Adams, Heather, hhallen-adams2@unl.edu, University of Nebraska-Lincoln; Lawton, Michael, lawton@aesop.rutgers.edu, and Di, Rong, di@aesop/rutgers.edu, Rutgers University; Schwarz, Paul, Paul.Schwarz@ndsu.edu, North Dakota State University; Young, Carolyn, cayoung@noble.org, Samuel Roberts Nobel Foundation; Kuldau, Gretchen, Kuldau@psu.edu; Pennsylvania State University; Schmale, David, dscmale@vt.edu, Virginia Polytechnic Institute and State University; Yu, Jaehyuk, jyu1@wisc.edu, University of Wisconsin, Madison; Cardwell, Kitty, kcardwell@nifa.usda.gov, National Institute for Food and Agriculture, Jackson, David, david.s.jackson@unl.edu, University of Nebraska, LincolnParticipants in the previous annual meetings of the project are available at the link noted below after the summary of meeting minutes.
Brief Summary of Minutes
Institutional updates included a discussion of potential opportunities for collaboration on mycotoxin research with Feed the Future Laboratories, such as at Kansas State. Dr. Cardwell discussed NIFA funding for mycotoxin research and initiatives that may align governmental research funding to support such research. Dr. Jackson reiterated the need for the project to be more than the sum of its parts, so that subgroups within each objective should continually coordinate efforts.
Annual research progress reports were given by IA, IL, KS, KY, MI, MS, MO, NE, NJ, PA and VA.
The group went over each of the 3 objectives of the project and discussed how the different groups would meet their specific project goals. The need to update or change the project’s website was discussed and Heather Hallen-Adams volunteered to investigate this further, and potentially move the hosting of the site to UNL. There was a discussion about when and where to hold the meeting that would be associated with a national or regional meeting in either food safety or plant pathology. The consensus was to aim for a meeting that coincided with an ASPP meeting in 2017. The next meeting will be held in late September, 2016 at Rutgers, NJ. Suzanne Hendrich will step down as chair and will be succeeded by Lisa Vaillancourt, who, in turn will be succeeded as vice-chair by Michael Lawton. David Schmale will serve as secretary. The Chair reminded all participants of the need to submit an Annual Report (termination report) within 2 months of this meeting. The meeting was adjourned.
See http://www.nimss.org/lgu_v2/homepages/saes.cfm?trackID=11976 for participants and minutes from prior annual meetings (2011-2014).