NC129: Mycotoxins in Cereal Grains

(Multistate Research Project)

Status: Inactive/Terminating

Project Menu

SAES-422 Reports

Annual/Termination Reports:

[04/22/2002] [09/17/2004] [05/30/2006]

Date of Annual Report: 04/22/2002

Report Information

Annual Meeting Dates: 04/08/2002 - 04/09/2002
Period the Report Covers: 01/01/2001 - 12/01/2001

Participants

Brief Summary of Minutes

Usefulness of Findings

Results reported for 2001 will extend existing knowledge about the toxicity and occupancy of a wide variety of Fusarium mycotoxins in grains. Progress has been made in insect/ fungi relationships beyond the issue of fumonisins in grain, for a more comprehensive model including mycotoxins in silage and stored grain. Research reported in 2001 demonstrates the potential usefulness of using ozone in managing stored maize and possibly other grains. The data indicate that, if repeated ozone treatments are needed, such treatment should not decrease the quality of grain for end-users. A number of unique molecular targets and pathways affected by fumonisin have been identified. For example, it has been shown that fumonisin induces cell cycle arrest and apoptosis in normal cells. Such studies will improve our understanding of the mechanism behind FB1 toxicity and the role of sphingolipids in signal transduction processes. Current data obtained in long term low dose feeding study using purified fumonisin B1 suggest that concerns regarding potential fumonisin B1 induced atherosclerotic effects do not appear warranted. Isolation and characterization of Fusarium genes involved in mycotoxin synthesis continues to aid efforts to control mycotoxin formation in food crops. New technologies and methods for mycotoxin analysis have provided either better, easier or quicker means to detect mycotoxins in foods. Some of our efforts have been toward developing a means to clone fusaric acid biosynthesis genes. Cloned genes would permit the construction of gene knock-out strains that would be useful for a wide range of studies involving fungal interaction with plant species and potentially in animal studies as well. The cooperative approach used by NC-129 contributors is essential to efforts to control mycotoxin formation in food crops.

Work Planned for Next Year

1. Work will continue on the evaluation of the toxic effects of fumonisins and how they might be mitigated. Studies will continue on the acute toxicity of amine-blocked forms of FB1 in swine and rats. Evaluation of potential methods for nixtamalization of corn which will not result in hydrolysis of fumonisin B1 contaminants. Evaluation of potential binders of N-fatty acyl-hydrolyzed fumonisin B1 in extracts from hot oil generated during frying of corn-derived food products. The presence of hydrolyzed FB1 and its derivatives in corn products will continue.

2. The molecular biology of fumonisin biosynthesis remains a priority. Efforts to evaluate newly identified Fusarium strains with regard to mycotoxin production, will continue.

3. Studies will continue on the potential of the adsorbent clays and bacterial enzymes to bind or destroy mycotoxins. Studies will continue on the production of Fusarium verticilloides mycotoxins and feeding studies in poultry and turkeys.

4. Work will continue on the peptide mimic of DON, including the development of transgenic plants, and a determination of the possible role the mimic may have in reducing the toxic effects of DON. Development of recombinant antibody to DON will continue, as will a study to determine the effect of sampling patterns on the estimation of DON levels in individual fields. Trichothecenes will be tested in cytotoxicity and phytotoxicity assays for structure-activity relationships. Rapid fluorescence polarization immunoassays will be developed for FB1 and DON.

5. Attempts are still in progress on methods to increase the resistance of maize to fungal disease by inserting and expressing a gene that provides antifungal proteins in the corn pedicel. The seed transmission and systemic infection of maize by Fusarium species will continue to be studied. F. verticillioides transformants will screened for defects in fusaric acid biosynthesis..

Minutes Attachment:

Attachment

Accomplishments

Publications

Publications * Indicates publications with multi-location authors. A. State Station, Agency, or On-Line Publications 1. Cardwell, K.F., Desjardins, A., Henry, S.H., Munkvold, G., and Robens, J. 2001. Mycotoxins: The Cost of Achieving Food Security and Food Quality. APSNet Feature article, Aug 2001. http://www.apsnet.org/online/feature/mycotoxin/ 2. Hart, L. P. and O. Schabenberger. 2001. Early detection of deoxynivalenol in wheat grain. 2001 National Fusarium Head Blight Proceedings. P 164-167. Cincinnati, OH. 3. Xie, W., P. Hart, P. Schwarz, and B. Tacke. 2001. Update on DON diagnostic services in 2000/2001. 2001 National Fusarium Head Blight Proceedings. P 171-174. Cincinnati, OH. 4. Ledoux DR, Rottinghaus GE, and Bermudez AJ. In vitro binding of mycotoxins by adsorbents does not always translate into in vivo efficacy. Mycotoxins and Phytotoxins in Perspective at the Turn of the Millennium, Koe WJ, Samson RA, Egmond HPV, Gilbert J, and Sabino M, eds. Proceedings of the Xth IUPAC symposium, May 21-25, 2000, Guaruja, Brazil, pp. 279-287, 2001. 5. Lui, H.-J. Role and modulation factors of natural killer cells during fumonisin B1 hepatocarcinogenesis in rats. Ph.D. thesis. Iowa State University Library, Ames, IA. 6. Mubatanhema, W. and Wilson, D.M., 2001. Detection, Control and Management of Mycotoxins in Southern Africa, A Training Manual, Published on the Peanut CRSP web page http://www.griffin.peachnet.edu/pnutcrsp.html. 71 pages. 7. Mubatanhema, W, Wilson, D.M., Widstom, N.W. and Holbrook, C.C., 2001 (abstract), A simplified HPLC method for field and research screening of aflatoxin in corn and peanut. Aflatoxin Elimination Workshop, Phoenix, AZ. 8. Munkvold, G.P., Hellmich, R.L., and Rice, L.G. 2001. Effects of Bt transformation events on Fusarium ear rot and fumonisins, 1999. Biological and Cultural Tests for Control of Plant Diseases, 2001:C2. http://www.scisoc.org/online/B&Ctests/2001/top.htm 9. Punia, R. C., Dahiya, O. S., Wilson, D. M., and Wilson, J. P. 2000. Propionic acid treatment prolongs storage and reduces rancidity of pearl millet feed grain. International Sorghum and Millets Newsletter 4180-81. 10. Wilson, D.M., Mubtanhema, W., and Jurjevic, Z. 2001. Biology and ecology of mycotoxigenic Aspergillus species as related to economic and health concerns. In Mycotoxins in Food. Eds. L.S. Jackson, M.W. Trucksess and J.W. DeVries, Plenum, New York. pp 195-204. B. Journal Articles 1. Abbas, H.K., H. Tak, C.D. Boyette, W.T. Shier, B. B. Jarvis, S. F. Hinkley, and H. L. Walker (2001) Absence of macrocyclic trichothecenes from kudzu plants treated with a high-producing isolate of Myrothecium verrucaria. Phytochemistry, 58, 269-276. 2. Bai, G.-H., Plattner, R., Desjardins, A, Kolb, F. Resistance to Fusarium Head blight and deoxynivalenol accumulation in wheat. Plant Breeding. 2001. v. 120. pp. 1-6. 3. Brown, D.W., McCormick, S.P., Alexander, N.J., Proctor, R.H., Desjardins, A.E. A genetic and biochemical approach to study trichothecene diversity in Fusarium sporotrichioides and Fusarium graminearum. Fungal Genetics and Biology. 2001. v. 32. pp. 121-133. 4. Buntin, G.D., Lee, R.D., Wilson, D.M. and McPherson, R.M. 2001. Evaluation of yeildguard transgenic resistance for control of fall armyworm and corn earworm (Lepidoptera noctuidae) on corn. Florida Entomoligist 8438-42. 5. Calvo A, Gardner H W, Keller N P (2001) Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans. J Biol Chem. 276:25766-25774. 6. Dakovic AS, Tomasevic-Canovic MR, Dondur VT, Stojsic DG, and Rottinghaus GE. In vitro adsorption of zearalenone by octadecyldimethylbenzyl ammonium exchanged clinoptilolite-heulandite tuff and bentonite. Studies Surface Science Catalysis. 135:5276-5283, 2001. 7. Dombrink-Kurtzman, M.A., Dvorak, T.J., Barron, M.E., Rooney, L.W. Effect of nixtamalization (alkaline cooking) on fumonisin-contaminated corn for production of masa and tortillas. J. Agric. Food Chem. 2000. v. 48. p. 5781-5786. 8. Dombrink-Kurtzman, M.A., Gomez-Flores, R., Weber, R.J. Activation of rat splenic macrophage and lymphocyte functions by fumonisin B1. Immunopharmacology. 2000. v. 49. p. 401-409. 9. Fakhoury, A. M. and Woloshuk, C. P. 2001. Inhibition of growth of Aspergillus flavus and fungal alpha-amylases by a lectin-like protein from Lablab purpureus. Molecular Plant-Microbe Interactions 18:955-961. 10. Gardner, H.W., Grove, M. Method to produce 9(S)-hydroperoxides of linoleic and linolenic acids by maize lipoxygenase. Lipids. 2001. v. 36. p. 529-533. 11. Gardner, H.W., Deighton, N. Effect of 4-hydroxy-2(E)-nonenal on soybean lipoxygenase-1. Lipids. 2001. v. 36. p. 623-628 12. Gumprecht, L. A., G. W. Smith, P. C. Constable, and W. M. Haschek (2001). Species and organ specificity of fumonisin-induced endothelial alterations: Potential role in porcine pulmonary edema. Toxicology 160:71-79. 13. Haschek, W. M., L. A. Gumprecht, G. Smith, M. E. Tumbleson, P. D. Constable (2001). Fumonisin toxicosis in swine: An overview of porcine pulmonary edema and current perspectives. Env Hlth Persp. 109 (suppl 2):251-257. 14. Harvey RB, Edrington TS, Kubena LF, Rottinghaus GE, Turk JR, Genovese KJ, and Nisbet DJ. Toxicity of moniliformin from Fusarium fujikuroi culture material to growing swine. J Food Protection 64:1780-1784. 15. * Haschek, W. M., K. A. Voss, V. R. Beasley. (2001) Selected Mycotoxins Affecting Animal and Human Health. In: Handbook of Toxicologic Pathology. 2nd Edn. W. M. Haschek, C. G. Rousseaux, M. A. Wallig, eds.. Academic Press. 16. Hicks J, Lockington R A, Strauss J, Dieringer D, Kubicek C, Kelly J, Keller N (2001) RcoA has pleiotropic effects on Aspergillus nidulans cellular developmental. Mol Microbiol 39:1482-1493. 17. Hill NS, Thompson FN, Stuedemann JA, Rottinghaus GE, Ju HJ, Dawe DL and Hiatt EE. Ergot alkaloid transport across ruminant digestive tissues. J Animal Sci 79:542-549, 2001. 18. Holbrook, C.C., Kvein, C.K., Rucker, K.S., Wilson, D.M., Hook, J.E. and Matheron, M.E. 2000. Preharvest aflatoxin contamination in drought-tolerant and drought-intolerant peanut genotypes. Peanut Sci. 2745-48. 19. Ito, Y., Peterson, S.W., Wicklow, D.T., Goto, T. Aspergillus pseudotamarii, a new aflatoxin producing species in Aspergillus section Flavi. Mycological Res. 2001. v. 105. p. 233-239. 20. Jindal N, Mahipal SK, Rottinghaus GE. Occurrence of fumonisin B1 in maize and poultry feeds in Haryana, India. Mycopathologia, 148(1):37-40, 2000. 21. Kells, S. A., Mason, L. J., Maier, D. E., and Woloshuk, C. P. 2001. Efficacy and fumigation characteristics of ozone in stored maize. J. Stored Prod. Res. 37:371-382. 22. Klich M, Mendoza C, Mullaney E, Keller N, Bennett J (2001) A new sterigmatoycstin-producing Emericella taxon from agricultural desert soils. Sys Appl Microbiol 24:131-138. 23. Kubena LF, Bailey RH, Byrd JA, Young CR, Corrier DE, Stanker LH,and Rottinghaus GE. Cecal volatile fatty acids and broiler chick susceptibility to Salmonella typhimurium colonization as affected by aflatoxins and T-2 toxin. Poultry Sci 80:411-417, 2001. 24. Liu, H.-J., Lu, Y., Haynes, J. S., Cunnick, J. E., Murphy, P., Hendrich, S. Reaction of fumonisin with glucose prevents promotion of hepatocarcinogenesis in female F344/N rats while maintaining normal hepatic sphinganine:sphingosine. J. Agric. Food Chem. 2001, 49, 4113 4121 25. Maragos, C.M., Jolley, M.E., Plattner, R.D., Nasir, M.S. Fluorescence polarization as a means for determination of fumonisins in maize. J. Agric. Food Chem. 2001. v. 49. p. 596-602. 26. Maragos, C.M. Novel sensors for detecting mycotoxins in foods. Mycotoxins. 2001. v. 51. p. 51-58. 27. McAlpin, C.E. An Aspergillus flavus mutant producing stipitate sclerotia and synnemata. Mycologia. 2001. v. 93. p. 552-565. 28. Momany, F.A., Dombrink-Kurtzman, M.A. Molecular dynamics simulations on the mycotoxin fumonisin B1. J. Agric. Food Chem. 2001. v. 49. p. 1056-1061. 29. * Mathur, S., P. D. Constable, R. M. Eppley, A. L. Waggoner, M. E. Tumbleson, and W. M. Haschek (2001). Fumonisin B1 is hepatotoxic and nephrotoxic in mild fed calves. Toxicol. Sci. 60:385-396. 30. * Mathur, S., P. D. Constable, R. M. Eppley, M. E. Tumbleson, G. W. Smith, W. J. Tranquilli, D. E. Morin, and W. M. Haschek (2001). Fumonisin B1 increases serum sphinganine concentration but does not alter serum sphingosine concentration or induce cardiovascular changes in milk fed calves. Toxicol. Sci. 60:379-384. 31. Ones, C., Ciacci-Zanella, J.R., Zhang, Y., Henderson, G., and Dickman, M.B. 2001. Analysis of fumonisin induced apoptosis. Environmental Health Perspectives 109:1-7. 32. Plattner, R.D. HPLC/MS analysis of Fusarium mycotoxins, fumonisins and deoxynivalenol. Natural Toxins. 2000. v. 8. p. 1-6. 33. Porter, J.K., C.W. Bacon, E.M. Wray, G.A. Kuldau, and J.F. Leslie. 2001. Fusaric acid (FA), other alkyl-pyridinecarboxylic acids (APAs), and fumonisins by Fusarium thapsinum and F. moniliforme: GC/MS analysis. In: Mycotoxins and Phycotoxins in Perspective at the Turn of the Millenium. W. J. de Koe, R. A. Samson, H. P. van Egmond, J. Gilbert and M. Sabino eds. W. J. de Koe, Wageningen, Netherlands. 34. Shier, W.T., A.C. Shier, W. Xie and C.J. Mirocha (2001) Structure-activity relationships for human estrogenic activity in zearalenone mycotoxins. Toxicon, 39, 1435-1438. 35. Shim, W-B. and Woloshuk, C. P. 2001. Regulation of fumonisin B1 biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like (C-type) gene FCC1. Appl. Environ. Microbiol. 67:1607-1612. 36. Shimizu K., Keller N. P. (2001) Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics 157: 591-600. 37. * Smith, G. W., P. D. Constable, R. M. Eppley, M. E. Tumbleson, L. A. Gumprecht, and W. M. Haschek (2000) Purified fumonisin B1 decreases cardiovascular function but does not alter pulmonary capillary permeability in swine. Toxicol. Sci. 56:240-249. 38. Soman, A.G., Gloer, J.B., Angawi, R.F., Wicklow, D.T., Dowd, P.F. Vertilecanins: new phenopicolinic acid analogs from Verticillium lecanii. J. Nat. Products. 2001. v. 64. p. 189-192. 39. Tooley, P.W., E. D. Goley, M. M. Carras, R. D. Frderick, E. L. Weber, and G. A. Kuldau. 2001. Characterization of Claviceps species pathogenic on sorghum by sequence analysis of the _-tubulin gene intron 3 region and EF-1_ gene intron 4. Mycologia 93(3): 541-551. 40. Wicklow, D.T., Kurtzman, C.P. In Memorium - Clifford W. Hesseltine (1917-1999). Mycotoxins. 2000. v. 50. p. 1-2. 41. Wilson R A, Gardner H W, Keller N P (2001) Cultivar-dependent expression of a maize lipoxygenase responsive to seed infesting fungi. Mol. Plant Microbe. Inter. 14:980-987. 42. Yu, G., L. Chen, W. Xie (2001) Correlation of Trichothecene Production and Pathogenicity in Fusarium graminearum. Journal of Nanjing Agricultural University, 24:1-5. 43. Zhang, Y., Jones, C., and Dickman, M.B. 2001. Identification of differentially expressed genes following treatment of monkey kidney cells with the mycotoxin fumonisin B1. Food Chem. Toxicol. 39:45-53.

Impact Statements

Date of Annual Report: 09/17/2004

Report Information

Annual Meeting Dates: 04/27/2003 - 04/29/2003
Period the Report Covers: 01/01/2003 - 12/01/2003

Participants

G. E. Rottinghaus (Missouri, Chair);W. M. Haschek-Hock (Illinois, Vice-Chair);D. Wilson (Georgia);C. Woloshuk (Indiana);P. A. Murphy (Iowa);J. S. Smith (Kansas);P. Hart (Michigan);Y. Dong (Minnesota);M. B. Dickman (Nebraska);Charlene Wolf-Hall (North Dakota);G. Kuldau (Pennsylvania);D. Kendra (USDA-ARS-NCAUR);N. Keller (Wisconsin)

Brief Summary of Minutes

Draft
Minutes of the NC 129 Mycotoxins in Grains Annual Meeting
April 19-20, 2004

Richard Adams Conference Center
College of Veterinary Medicine
University of Missouri,
Columbia, MO

I. Representative Participants

II. Introduction
8:25 am. Meeting called to order by Dr. Rottinghaus. Copies of the Agenda, the 2003 Annual Report and the 2003minutes were available.

Dr. Rottinghaus introduced the administrator, Dr. Durgan, Associate Dean at the University of Minnesota, for opening remarks. Dr. Durgan stated that the NC129 renewal must be completed this year. She provided the time frame and actions that needed to be taken (hard copy distributed). The major deadlines for the writing group are September 15 for submittal of a statement of issues and justification and December 1 for proposal submittal. Note that the regional committees are now being called multistate committees, so, the name for NC129 will probably change. Other states doing mycotoxin work will be given the opportunity to join the project. Dr. Durgan indicated that renewal for this project was likely based on the interdisciplinary nature of the group and documentation of collaborative research in the 2003 report.

III. Station Reports

The station reports began at 8:30 am with presentations from Pennsylvania - (Repr. Dr. Gretchen Kuldau), Illinois - (Repr. Dr. Wanda Haschek-Hock, presentation by Vincent Hsiao), Iowa - (Repr. Dr Pat Murphy, presentations by: Guillermo Fernandez and Cindy Landgren), ARS-NCAUR (Repr. Dr. Chris Maragos), Minnesota - (Repr. Dr. Yanhong Dong); Indiana - (Repr. Dr. Charles Woloshuk), Missouri - (Repr. Dr. George Rottinghaus, presentation by Angela Guaiume), North Dakota (Repr. Dr. Charlene Wolf-Hall), Kansas - (Repr. Dr. J. Scott Smith, presented by Xiarong Wu), and Nebraska (Repr. Dr. Martin Dickman). There were no representatives from Georgia, Michigan or Wisconsin.

IV. Business Meeting

The business meeting was brought to order at 4:00 pm by the Chair, Dr. Rottinghaus.

1. Minutes from the 2003 meeting were approved.

2. Future meetings: Discussion centered around an appropriate time for next year's meeting given that the renewal might not be approved till April of 2005. Most participants agreed that May was a better month for the meeting anyway because of the school year. The 2005 meeting will be held in May in Illinois (Dr Haschek-Hock.); 2006 will be hosted by Michigan (Dr. Hart), and 2007 by Pennsylvania (Dr. Kaldua).

3. New Officers: Dr. Gretchen Kaldau, Pennsylvania State University, will be Secretary for 2005, Vice-Chair in 2006 and Chair in 2007.

4. New Representative: Dr. Dave Kendra will replace Dr. Chris Maragos for ARS.

5. Annual Report: any revisions are to be provided to Dr. Haschek by close of meeting. The final report will then be sent to Dr. Durgan and posed on the website. NC129 website - www.wisc.edu/ncra contains project statement, reports, minutes and other information - posted by Dr. Woloshuk.

6. NC129 Mycotoxins in Grains project renewal. Dr. Woloshuk agreed to Co- Chair the Rewrite Committee with Dr. Wilson and to lead tomorrows meeting on project renewal planning. The Rewrite Committee consists of the following:
Co-Chairs: Drs. Wilson and Woloshuk.
Objective 1: Dr. Murphy with assistance from Dr. Haschek-Hock.
Objective 2: Dr. Maragos with assistance from Dr. Murphy.
Objective 3: Drs. Woloshuk and Kendra, with assistance from Dr. Rottinghaus.
Objective 4: Dr. Woloshuk.
The major 2004 deadlines are:
- September 15 for submittal of a statement of issues and justification and
- December 1 for proposal submittal.
Decision on renewal in March/April 2005.

7. The Internationa. IUPAC Meeting on Mycotoxins and Phycotoxins in Bethesda, May 17-21, 2004 was announced ? information is available at the AOAC website aoac.org.

Meeting Adjourned at 4:30 p.m.

V. Rewrite Meeting, April 20, 2004

Dr. Woloshuk led the discussion and submitted the following summary.

Project Title: Mycotoxins: Biosecurity and Food Safety.

Objectives: to be reworded.

1. Determine the interrelationship of mycotoxins produced in cereal grains
to human and animal health. (Old). VS Develop data for use in risk assessment of mycotoxins in human and animal health. (Proposed).
1. Leaders are Pat (IA) andWanda (IL)
2. Participants in the objective are Marty (NE), Pat (IA), Wanda (IL), Chris (ARS
support), George (MO), Scott (KS).

2. Develop new techniques and improve current assays for identification and
quantification of mycotoxins and mycotoxigenic fungi in cereal grains. (New)
1. Leader is Chris (ARS).
2. Issues and participants in the objective are
1) Analytical detection [Chris (ARS), Yanhong (MN), John (Romer Labs), Pat (IA)];
2) Molecular detection [Marty (NE), Charles (IN), Gretchen (PA)]
3) Imaging Technology [Doug (PA), ARS support].

3. Establish integrated strategies to manage and to prevent mycotoxin contamination in cereal grains. (Old)
1. Leader is George (MO).
2. Participants in the objective are Pat (IA), George (MO), Charlene (ND).

4. Define the pathways regulation of mycotoxin biosynthesis and the molecular relationships between mycotoxigenic fungi. (New, but needs help)
1. Leader is Charles (IN).
2. Participants in the objective are Gretchen (PA), Charles (IN), ARS support,
S. Du (NE), Shim (TX).

G:\vp\haschek\NC129mtg-04.doc

Minutes Attachment:

Attachment

Accomplishments

Objective 1: Determine the interrelationship of Fusarium mycotoxins from cereal grains to human and animal health. Fumonisins Illinois: Studies to determine the clearance of sphingosine (So) and sphinganine (Sa) in the blood and urine were conducted in pigs that received fumonisin B1 (FB1)at 1 mg/kg body weight IV at 0, 24 and 48 hrs, and were euthanized at 72 or 144 hrs. Sa/So analysis and biochemical evaluation will be performed on serum and urine. Tissues were collected for histopathology and Sa/So. Illinois: Serum folate, cysteine and homocysteine concentrations were determined in Sinclair minipigs fed fumonisin B1 at 0 or 10-30 ppm for 26 weeks. Treated pigs had increased homocysteine and decreased methionine concentration relative to controls. Illinois: The cytotoxic effects of exogenous So and Sa were evaluated in rat embryonic cardiomyocytes (H9C2[2-1]) and hepatocellular carcinoma cells (HepG2); Sa was more cytotoxic than So for both cell types. Iowa: Three week old swine were fed 70 ppm (97 mmole/g) FB1 (Rottinghaus culture material) or 70 ppm (97 mmole/g) FB1 processed with glucose for 14 days. At day 8, FB1 fed animals had higher AST activity and bilirubin conc.as compared to controls, while pigs fed FB1-glucose diets did not. At day 14, FB1 fed animals were not different from controls or FB1-glucose fed animals. Nebraska is developing a number of approaches to identify plant genes regulating apoptosis using fumonisin B1 as an inducer of cell death. Deoxynivalenol (DON) Iowa: To determine if acute exercise stress would exacerbate the immunosuppressive effects of DON, male BALB/c mice were fed DON at 2 ppm for 14 d and placed on a treadmill (10-20 m/min) and exercised to exhaustion (2.5 4.25 h). Only the non-exercised DON mice had suppressed lymphocyte proliferation. Fusaproliferin Kansas evaluated the mutagenic potential of fusaproliferin in 5 .S typhimurium tester strains using the standard procedure of the Ames Salmonella microsome assay. Fusaproliferin was mutagenic in some strains but not in others and required S9 in some cases. The mutagenic potency, when present, was much weaker than aflatoxin B1. Aflatoxin Missouri examined the effects of endotoxic lipopolysaccharide (LPS) in chicks and poults fed aflatoxin. Chicks were fed 0, 2 or 4 mg AFB1/kg diet and poults 0, 100, or 200 µg for 21 days. Beginning on day 7, chicks were injected ip with 0, 200, or 400 µg LPS/bird and poults with 0, 100, or 200 µg LPS/bird on alternate days. Based on mortality, results suggest a toxic synergy between AFB1 and LPS in chicks, but only an additive effect in poults. Production of mycotoxins Missouri produces fumonisin, moniliformin, zearalenone, ochratoxin A, and aflatoxin B1 and secalonic acid D for studies focused on binding or inactivation of mycotoxins in feed. Cooperation with multiple investigators, institutions and countries is ongoing. Objective 2: Develop new techniques and improve current assays for identification and quantification of mycotoxins in cereal grains. Multiplex polymerase chain reaction (PCR) assays Indiana developed a 5 fluorogenic (Taqman) real-time PCR assay for group-specific detection of trichothecene- and fumonisin-producing Fusarium spp. and to identify F. graminearum and F. verticillioides in field-collected barley and corn samples. Michigan developed DNA primers based on the tri5 gene for RT-PCR to detect Fusarium sp in small grains. There was a good correlation between DON and DNA in individual kernels (r2=74.2%), and between sample means of DON and DNA (r2=99.0%). However, DON and DNA did not necessarily correlate well with symptoms of fusarium head blight (FHB) infection. Detection of aflatoxigenicity Minnesota identified the structures of 7 yellow pigments, secreted by isolates of Aspergillus flavus which produce aflatoxins, as known biosynthetic intermediates, or side products, on the pathway to aflatoxin. These 7 pigments are the basis of culture-based rapid tests for aflatoxigenicity. A combination of the ammonium hydroxide vapor and cyclodextrin-enhanced blue fluorescence cultural methods was shown to be comparable to TLC in predicting aflatoxigenicity. Detection of fumonisins USDA developed an HPLC-fluorescence method for detection of fumonisins in corn silage combining an extraction technique using EDTA with an immunoaffinity column cleanup and derivatization with naphthalene dicarboxaldehyde. The prevalence of fumonisins in corn silage was found to be high, although levels were generally below concern. Illinois used immunoassay materials developed at USDA/NCAUR to screen maize as part of a project to improve resistance to Fusarium verticillioides and fumonisins. Zearalenone (ZEN) USDA/NCAUR developed a simple and rapid fluorescence polarization immunoassay for ZEN. Five highly sensitive monoclonal antibodies were also developed for detection of ZEN and related metabolites. Deoxynivalenol (DON) USDA/NCAUR developed a sensitive HPLC-MS method for detection of DON and nivalenol .in maize or wheat. Fusaproliferin Kansas developed a sensitive GC method to detect low levels of fusaproliferin in corn; detection limit of 0.1 ng TMS derivatized fusaproliferin and 10 ppb in corn. Preliminary survey shows that fusaproliferin is a common contaminant in corn with low levels (<9.4 ppb) generally detected (a moldy corn sample had 297 ppb). Patulin USDA/NCAUR is examining molecularly imprinted polymers (MIPs) synthesized to recognize patulin by infrared (IR) for characterization. A. flavus and F. verticillioides USDA/NCAUR: ARS (Manhattan, KS) with NCAUR seek to remove the relatively few corn kernels in which aflatoxin and fumonisin are concentrated at harvest. Near infrared spectra were obtained for kernels infected by A. flavus and F. verticillioides and applied successfully in programming a high volume commercial optical grain sorter to reject aflatoxin- and fumonisin-contaminated kernels. Macrophomina phaseolina Phytotoxin Minnesota has purified the major phytotoxin produced in culture by an isolate of the charcoal rot fungus, Macrophomina phaseolina and the determination of the chemical structure is about 90% complete. Phytotoxicity has been demonstrated in Lemna (duckweed) cultures but the phytotoxin is not phaseolinone. 8-O-metholbostrycoidin Iowa collaborated with Kansas State in supplying mycelia from Fusarium proliferatum strain 5991 for analysis of 8-O-metholbostrycoidin. Objective 3: Establish strategies for integrated management to prevent mycotoxin contamination in cereal grains. Chemical control Iowa scaled up the heat processing of glucose with fumonisin B1 (FB1) contaminated corn and FB1 corn culture material to kg level and successfully processed feed with 70 to 200 ppm FB1 with glucose and baking soda to about 10% residue free. Michigan provides DON analysis (> 4,000 samples/year) for FHB research to aid in selection of breeding lines of wheat with reduced DON, and in the evaluation of fungicides for reduction in DON. It was found that the fungicide Quadris increased levels of DON when applied at anthesis. Minnesota showed that heat stress played a major role in aflatoxin and fumonisin production in corn grown in Arkansas using natural infection with both Fusarium moniliforme and Aspergillus flavus. Missouri investigated whether the turkey could be used as a model for evaluating the efficacy of adsorbents (hydrated sodium calcium aluminosilicates, HSCAS) to ameliorate the toxic effects of aflatoxin (AF). 21-day experiments were conducted using 0. 200 or 250 ug AF/kg and 0 to 1% of HSCAS -A and B. Both adsorbents reduced some toxic effects of AF in the young turkey indicating that it is a more sensitive model for evaluating the efficacy of adsorbents. Missouri evaluated a number of adsorbents for binding mycotoxins. While several adsorbents appear to protect against zearalenone, fumonisin B1, and ochratoxin in vitro, only aflatoxin B1 is bound significantly in vivo. Biocontrol USDA/NCAUR, with the Plant Transformation Facility, Iowa State University, seeks to develop a gene expression system for maize that would allow proteins that inhibit fungal growth or reduce mycotoxin toxicity to specifically accumulate within the basal maternal tissues of the developing kernel. Using Agrobacterium-mediated transformation resulted in highly improved transformation efficiencies with maintanence of gene expression within the maternal tissues of the developing kernel as well as within the vascular tissues of stems. This new transformation method resulted in stable gene insertions which are heritable. USDA/NCAUR generated reporter strains of F. verticillioides that have the cyan fluorescent protein (CEFP) gene fused to the promoter of the fumonisin biosynthetic gene FUM8. The reporter strains can be used as tools to study regulatory processes that lead to the formation of fumonisins in maize plants which in turn could lead to control strategies that reduce or eliminate toxic fumonisin accumulation in maize. USDA/NCAUR complemented sexual spore-nonproducing mutants by adding a copy of the mating type (MAT) locus to further the goal of determining the role of sexual spores in the ability of Fusarium graminearum to cause wheat head blight. The complemented strains recovered the ability to produce sexual spores and were able to spread in inoculated wheat heads in greenhouse tests. Two field tests were conducted to establish the role of sexual spores in wheat head blight epidemics. USDA/NCAUR (in collaboration with Iowa City, IA) continued to investigate new sources of antifungal agents and fungi that can parasitize and kill other fungi useful to agriculture and medicine The common corn endophyte Acremonium zeae interferes with A. flavus growth and infection of preharvest corn kernels. Culture extracts of Acremonium had significant antifungal activity against A. flavus and F. verticillioides and two antibiotics were isolated and identified to account for the activity. In addition, other antifungal metabolites have been identified. Objective 4: Define the metabolic pathways and regulatory pathways of mycotoxin biosynthesis. Fumonisins Indiana cloned a PACC-like gene (PAC1) from F. verticillioides. The mutant produced more fumonisin than the wild type and produced fumonisin B1 when mycelia were resuspended in medium buffered at alkaline pH (8.4). Transcription of FUM1 was correlated with fumonisin production. Therefore, PAC1 is required for growth at alkaline pH and may have a role as a repressor of fumonisin biosynthesis under alkaline conditions. Indiana constructed DNA microarrays with expressed sequence tags (ESTs) from F. verticillioides. To identify genes with patterns of expression similar to the fumonisin biosynthetic (FUM) genes, the microarray was probed with labeled cDNAs synthesized from RNA isolated from a wild-type strain and a fcc1 mutant grown on maize and in a defined medium adjusted to either pH 3 or pH 8. The comparative analyses revealed differential expression of genes corresponding to 116 ESTs when the fungal strains were grown on maize. Under different pH conditions, 166 ESTs were differentially expressed, and 19 ESTs were identified that displayed expression patterns similar to 15 FUM ESTs. Pennsylvania completed a phylogenetic tree of isolates from the Gibberella fujikuroi species complex based on EF-1a sequences from over 500 isolates. This analysis has revealed numerous new phylogenetic species which are now being tested for their ability to mate sexually and produce fumonisins. USDA/NCAUR explored the metabolic pathways and regulatory mechanisms for mycotoxin biosynthesis, particularly for F. erticillioides, but also for F. raminearum and F. sporotrichioides. They completed a gene knockout analysis of all 15 genes in the fumonisin biosynthetic gene cluster and determined 1) the functions of eight of these genes, 2) that three other genes are required for normal fumonisin production in F. verticillioides and 3) that the remaining four genes are not essential for fumonisin biosynthesis. USDA/NCAUR has continued to generate a F. verticillioides Expressed Sequence Tag (EST) library with three additional complementary DNA (cDNA) libraries generated and submitted to The Institute for Genomic Research (TIGR) for DNA sequence analysis which has generated over 54,000 good quality sequences corresponding to over 6,500 unique genes in the F. verticillioides genome. Efforts have also begun to use the EST sequence data to identify genes that regulate fumonisin production. Trichothecenes Pennsylvania has developed methodologies for growing the fusaria for type A trichothecene production in small volumes. They were detected by an HPLC-MS method which separates 8 trichothecenes by liquid chromatography, enables collection of mass spectra for all of them, and allows quantitative determination. Analysis of trichothecene production in some isolates has begun as well as sequencing of the EF-1a gene for phylogenetic analysis. USDA/NCAUR, with Agriculture Canada, identified a cytochrome P450 monooxygenase gene that resides outside the trichothecene core cluster. Gene disruption was used to define the steps in trichothecene biosynthesis by F. graminearum .and to inactivate the gene in order to determine its function. Gene expression was required for oxygenation of the DON molecule at carbon positions 7 and 8. Wisconsin investigated the role of propionate metabolism in polyketide biosynthesis by manipulating the methyl citrate cycle by deletion of mcsA (DmcsA). The hypothesis is that accumulation of propionyl CoA is interfering with ST PKS function and that increasing propionyl CoA content through manipulation of the methyl citrate synthase cycle could reduce production of polyketide mycotoxins in other fungi. Patulin USDA/NCAUR found that all Penicillium sp tested were capable of producing patulin.. Ribosomal DNA (rDNA) sequences identified from two isolates from commercial apple juice have tentatively been identified as Byssochlamys species.

Publications

Peer Reviewed Journal Articles and Book Chapters. Al-Tamimi H.J., Rottinghaus G.R., Spiers D.E., Spain J., Chatman D., Eichen P.A., Carson T.L. Thermoregulatory response of dairy cows fed ergotized barley during summer heat stress. J Vet Diagn Invest 2003. 15:355-360. Butchko, R.A.E., Plattner, R.D., Proctor, R.H. FUM13 encodes a short chain dehydrogenase/reductase required for C-3 carbonyl reduction during fumonisin biosynthesis in Gibberella moniliformis. Journal of Agricultural and Food Chemistry. 2003. 51:3000-3006. Cleveland, T.E., Dowd, P.F., Desjardins, A.E., Bhatnagar, D., Cotty, P.J. United States Department of Agriculture - Agricultural Research Service research on pre-harvest prevention of mycotoxins and mycotoxigenic fungi in US crops. Pest Management Science. 2003. 59:629-642. *Clements, M.J., Kleinschmidt, C.E., Maragos, C.M., Pataky, J.K., White, D.G. Evaluation of inoculation techniques for Fusarium ear rot and fumonisin contamination of corn. Plant Disease. 2003.87:147-153. *Clements, M.J., Campbell, K.W., Maragos, C.M., Pilcher, C., Headrick, J.M., Pataky, J.K., White, D.G. Influence of CryIAb and hybrid genetics on fumonisin contamination and Fusarium ear rot of corn. Crop Science. 2003.43:1283-1293. *Constable, P.D., Smith, G.W., Rottinghaus, G.E., Tumbleson, M.E., Haschek, W.H. Fumonisin-induced blockade of ceramide synthase in sphingolipid biosynthetic pathway alters aortic input impedance spectrum of pigs. Am J Physiol Heart Circulation Physiol 2003. 284:H2034-44. Desjardins, A.E. Gibberella from A(venaceae) to Z(eae). Annual Review of Phytopathology. 2003.41:177-198. Dokovic, A., Tomasevi-Anovi, M., Rottinghaus, G., Dondur, V., Magi, Z. Adsorption of ochratoxin A on octadecyldimethyl benzyl ammonium exchanged-clinoptilolite-heulandite tuff. Colloids and Surfaces B: Biointerfaces. 2003. 30: 157-165, 2003. Evans, T.J., Rottinghaus, G.E., Casteel, S.W. Ergot. Clinical Veterinary Toxicology, Ed. Plumlee KH, Mosby/ Elsevier, 2003. pp 239-243. Evans, T.J., Rottinghaus, G.E., Casteel, S.W. Fescue. Clinical Veterinary Toxicology, Ed. Plumlee KH, Mosby/ Elsevier, 2003. pp 243-243-250. Flaherty, J.E., Pirttilä, A.M., Bluhm, B.H., Woloshuk, C.P. Role of PAC1, a pH regulatory gene from Fusarium verticillioides, in growth and fumonisin biosynthesis. Appl. Environ. Microbiol. 2003. 69:5222-5227. Fotso, J., Smith, J.S. Evaluation of beauvericin toxicity with the bacterial bioluminescence assay and the Ames mutagenicity bioassay. J Food Sci. 2003. 68:1938 1941. *Gillespie, J., Schwarz, P., Mostrom, M.S., Tacke, B., Dong, Y., Hart, P., Munn, B. Update on the USWBSI DON diagnostic laboratories. Proceedings 2003 Fusarium Head Blight Forum. 2003. p. 187-190. Goto, T., Wicklow, D.T., McAlpin, C.E., Peterson, S.W. Aspergillus bombycis genotypes (RFLP) from silkworm cultivation. Mycoscience. 2003. 44:209-215. Hart, P., Catal, M. Application of real time polymerase chain reaction to the detection and quantification of Fusarium in wheat. Proceedings 2003 Fusarium Head Blight Forum. 2003. pp191-194. Kumar, A., Jindal, N., Shukla, C.L., Pal, Y., Ledoux, D.R., Rottinghaus, G.E. Effects of ochratoxin A. on Escherichia coli challenged broiler chicks. Avian Diseases. 2003. 47:415-424. Ledoux, D.R., Broomhead, J.N., Bermudez, A.J., Rottinghaus, G.E. Individual and combined effects of the Fusarium mycotoxins fumonisin B1 and moniliformin, in broiler chicks. Avian Diseases, 2003. 47:1368-1375. Lewis, J.M., Jiang, G-L., Shi, R.R., Hart, L.P., Ward, R.W. Bioassay vs conventional characterization of FHB resistance in Ning 7840. Proceedings 2003 Fusarium Head Blight Forum. 2003. pp. 142-147. Manning, B.B., Ulloa, R.M., Li, M.H., Robinson, E.H., Rottinghaus, G.E. Ochratoxin A fed to channel (Ictalurus punctatus) causes reduced growth and lesions of hepatopancreatic tissue. Aquaculture 2003. 219:739-750. Mobio, T.A., Tavan, E., Baudrimont, I., Anane, R., Carratu, M.-R., Sanni, A., Gbeassor, M.F., Shier, T.W., Narbonne, J.-F., Creppy, E.E. Comparative study of the toxic effects of fumonisin B1 in rat C6 glioma cells and p53-null mouse embryo fibroblasts. Toxicology 2003. 183:65-75. Muhitch, M.J. Distribution of the glutamine synthetase isozyme GSp1 in maize (Zea mays). Journal of Plant Physiology. 2003. 160:610-605. Muhitch, M.J., Liang, H., Sollenberger, K.G. Transformation efficiencies and expression patterns of a series of truncated GS1-2 promoter/GUS transgenes in maize. Physiologia Plantarum. 2003. 118:346-351. Plattner, R.D., Maragos, C.M. Determination of deoxynivalenol and nivalenol in corn and wheat by liquid chromatography with electrospray mass spectrometry. Journal of the AOAC International. 2003. 86:61-65. Proctor, R.H., Brown, D.W., Plattner, R.D., Desjardins, A.E. Co-expression of fifteen contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis. Fungal Genetics and Biology. 2003. 38:237-249. Rottinghaus, G.E., Ledoux, D.R., Bermudez, A.J. Contributors to: Mycotoxins: Risks in plant, animal, and human systems. Council for Agricultural Science and Technology (CAST) Task Force report no. 139, Ames, Iowa, January, 2003. Shier, W.T., Abbas, H.K., Abou-Karam, M., Badria, F.A., Resch, P.A. Fumonisins: Abiogenic conversions of an environmental tumor promoter and common food contaminant. J. Toxicol.-Toxin Rev. 2003. 22:591-616. Shim, W-B., Flaherty, J.E., Woloshuk, C.P. Comparison of fumonisin B1 biosynthesis in maize germ and degermed kernels by Fusarium verticillioides. J. Food Protect. 2003. 66:2116-2122. Tomasevi-Anovic, M., Dakovic, A., Rottinghaus, G., Matijsevi, S., Duricic, M. Surfactant modified zeolites? New efficient adsorbents for mycotoxins. Microporous and Mesoporous Materials, 2003. 61:173-180, 2003. Tomasevi-Anovic, M., Dokovi, A., Rottinghaus, G., Arov-Stan, A. Adsorption of ochratoxin A by octadecyldimethylbenzyl-heulandite tuff. Microporous and Mesoporous Materials 2003. Tuan, N.A., Manning, B., Lovell, R.T., Rottinghaus, G.E. Responses of Nile Tilapia (Oreochromis niloticus) fed diets containing different concentrations of moniliformin or fumonisin B1. Aquaculture 2003. 217:515-528. Watts, C.M., Chen, Y.C., Ledoux, D.R., Bermudez,A.J., Rottinghaus, G.E. Effects of multiple mycotoxins and a hydrated sodium calcium aluminosilicate in poultry. International J Poultry Science 2003. 2:372-378. Wicklow, D.T., Bobell, J., Palmquist, D. Evaluation of intraspecific competition (Aspergillus flavus Link) and aflatoxin formation in suspended disc culture. Mycological Research. 2003. 107:617-623. Wu, X., Leslie, J.F., Thakur, R.A., Smith, J.S. Purification of fusaproliferin from cultures of Fusarium subglutinans by preparative high-performance liquid chromatography. J. Agric. Food Chem. 2003, 51:383-388. *Zhang, Y., Li, C., Swenson, D.C., Gloer, J.B., Wicklow, D.T., Dowd, P.F. Novel antiinsectan oxalicine alkaloids from two undescribed fungicolous Penicillium spp. Organic Letters. 2003. 5:773-776. Abstracts *Fernandez-Surumay, G., G.D. Osweiler, P.A. Murphy. Protection against fumonisin B1-induced liver toxicity by the fumonisin B1-glucose mixture in a swine model. 9th International Congress of the European Associations of Veterinary Pharmacology and Toxicology, July 2003, Lisbon, Portugal, J. Vet. Pharmacol. Therap. 22 (Suppl 1):278-9, 2003 Haschek, W. M., A. L. Waggoner, S-H Hsiao, G. W. Smith, M. E. Tumbleson, R. M. Eppley, J. H. Foreman, and P. D. Constable (2003). Clinicopathologic characterization of fumonisin B1 (FB1) induced hepato-, nephro- and neuro-toxicity in horses. Tox Sci 72(S-1):252 (Abs 1224). Hsiao, S-H, P. D. Constable, and W. M. Haschek (2003). In vitro effects of sphinganine, sphingosine, and sphingosine-1-phosphate on porcine thoracic aorta and pulmonary artery vascular rings. Tox Path 31:142 (P15). Kuldau, G. A. 2003. Mycotoxins in corn silage. Phytopathology 93:S98. Nagy, M. and G. A. Kuldau. 2003. Mycotoxigenic fungi and mycotoxins in Pennsylvanian corn silage. Phytopathology 93:S97. Tumbleson, M. E., W. M. Haschek, A. L. Waggoner, P. E. Constable, G. W. Smith, J. H. Foreman, and R. Eppley (2003). Fumonisin B1 (FB1) alters sphinganine (Sa) and sphingosine (So) concentrations in serum, tissue, urine and cerebrospinal fluid (CSF) of horses. Tox. Sci. 72(S-1):254 (Abs 1235). Wu, X. and J. S. Smith. A Sensitive GC Method for Detection of Fusaproliferin in Corn. 2003. Annual Food Safety Consortium Meeting, University of Arkansas, Fayetteville, Arkansas. October 12-14, 2003. Journal Articles In Press Abbas, H.K., R.M. Zablotowicz, M.A. Weaver, B.W. Horn, W. Xie, and W.T. Shier Comparison of cultural and analytical methods for determination of aflatoxin production by Mississippi Delta Aspergillus isolates. Can. J. Microbiol. Abou-Karam, M., H.K. Abbas and W.T. Shier (2004) N-Fatty acylation of hydrolyzed fumonisin B1, but not of intact fumonisin B1, strongly enhances in vitro mammalian toxicity. J. Toxicol. Toxin Rev., 23. Bluhm, B. H., Cousin, M.A., and Woloshuk, C. P. 2004. Multiplex real-time PCR detection of fumonisin-producing and trichothecene-producing groups of Fusarium species. J. Food Protect. Foreman, J. H., Peter D. Constable, A.L. Waggoner, M. Levy, R.M. Eppley, G.W. Smith, M.E. Tumbleson, W.M. Haschek. Neurological abnormalities and cerebrospinal fluid changes in horses with fumonisin-induced leukoencephalomalacia. Am. J. Vet. Int. Med. Geiser, D. M., Jimenez-Gasco, M. M., Kang, S., Makalowska, I., Zhang, N., Kuldau, G. A., and O?Donnell, K. L.. FUSARIUM-SEQv.1.0: A DNA sequence database for identifying toxigenic and non-toxigenic Fusaria. European Journal of Plant Pathology. 2002 Journal Publications not listed previously Alexander, N.J., McCormick, S.P., Hohn, T.M. The identification of the Saccharomyces cerevisiae gene AYTI (ORF-YLL063c) encoding an acetyltransferase. Yeast. 2002.19: 1425-1430. Desjardins, A.E., Munkvold, G.P., Plattner, R.D., Proctor R.H. FUM1--a gene required for fumonisin biosynthesis but not for maize ear rot and ear infection by Gibberella moniliformis in field tests. Molecular Plant-Microbe Interactions. 2002. 15:1157-1164. *Joshi, B. K., Gloer, J.B., Wicklow, D.T. Antifungal natural products from a sclerotium-colonizing isolate of Humicola fuscoatra. Journal of Natural Products. 2002. 65:1734-1737. Okubara, P.A., Blechl, A.E., McCormick, S.P., Alexander, N.J., Dill-Macky, R., Hohn, T.M. Engineering deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene acetyltransferase gene. Theoretical and Applied Genetics. 2002. 106: 74-83. Proctor, R.H., Desjardins, A.E., McCormick, S.P., Plattner, R.D., Alexander, N.J., Brown, D.W. Genetic analysis of the role of trichothecene and fumonisin mycotoxins in the virulence of Fusarium. European Journal of Plant Pathology. 2002. 108:691-698. Steenkamp, E.T., Wingfield, B.D., Desjardins, A.E., Marasas, W.F.O., Wingfield, M.J. Cryptic speciation in Fusarium subglutinans. Mycologia. 2002. 94:1032-1043. Wicklow, D.T., McAlpin, C.E., Peterson, S.W. Common genotypes (RFLP) within a diverse collection of yellow-green Aspergilli used to produce traditional oriental fermented foods. Mycoscience. 2002. 43289-297.

Impact Statements

Information was developed for use in assessment of risk for human and animal health associated with Fusarium mycotoxins from cereal grains e.g.<ul><li>increased serum homocysteine concentration, an independent risk factor for cardiovascular disease in humans, was identified in a long term fumonisin B1feeding study in pigs <li>exercise stress protected against deoxynivalenol toxicity in mice <li>FB1 heat processed with glucose was less toxic to pigs than the parent compound</ul>
New technologies and methods for mycotoxin analysis will provide better, easier & quicker means to detect mycotoxins in foods e.g. <ul><li>multiplex polymerase chain reaction (PCR) assays<li>culture based rapid tests for aflatoxigenicity<li>method for fumonisins detection in corn silage<li>fluorescence polarization immunoassay for zearalenone<li>fusaproliferin, which is mutagenic, identified as common contaminant in corn<li>molecularly imprinted polymers for infrared recognition of patulin</ul>
Development of strategies for integrated management to prevent mycotoxin contamination in cereal grains eg. <ul><li>a turkey model to evaluate the efficacy of adsorbents in aflatoxin toxicity <li>heat processing of glucose with fumonisin B1 (FB1) contaminated corn to decrease toxicity <li>heat stress identified as having a major role in aflatoxin and fumonisin production in corn </ul>
Defined metabolic pathways and regulatory pathways of mycotoxin biosynthesis to l assist efforts to control mycotoxin formation e.g.<ul><li>construction of DNA microarrays with expressed sequence tags (ESTs) from F. verticillioides<li>identification of genes necessary for fumominisin biosynthesis <li>a number of steps in trichothecene biosynthesis by F. graminearum were identified<li>investigation of the role of propionate metabolism in polyketide biosynthesis </ul>

Date of Annual Report: 05/30/2006

Report Information

Annual Meeting Dates: 05/22/2006 - 05/22/2006
Period the Report Covers: 10/01/2000 - 09/01/2005

Participants

Annual Report of the Cooperative Regional Project NC-129
January 1, 2004 - December 31, 2004;

I. Project: NC-129 Mycotoxins in Cereal Grains;

Personnel with full contact information for the representative;

Marty Dickman (Nebraska)
University of Nebraska
Department of Plant Pathology
Lincoln, NE 68583
mdickman@unlnotes.unl.edu;

Yanhong Dong, Ph.D. (Minnesota)
Department of Plant Pathology
University of Minnesota
St. Paul, MN 55108
612-625-2751;

Wanda Haschek-Hock, BVSc, PhD (Illinois)
Professor,
Department of Pathobiology
University of Illinois
2001 S. Lincoln
Urbana,IL 61802
Phone 217-333-3947/Fax 217-244-7421
whaschek@uiuc.edu;

Nancy Keller (Wisconsin)
npk@plantpath.wisc.edu;

David Kendra, Ph.D. (USDA-ARS)
Mycotoxin Research Unit
USDA-ARS-NCAUR
1815 N. University St.
Peoria, IL 61604 USA;

Gretchen Kuldau, PhD (Pennsylvania)
Assistant Professor
Department of Plant Pathology
321 Buckhout Lab
The Pennsylvania State University
University Park, PA 16802
phone:814-863-7232
fax:814-863-7217
lab:814-865-6986
email:kuldau@psu.edu;

Patricia A. Murphy (Iowa)
2312 Food Sciences Bldg
Iowa State University
Ames, IA 50011
Ph: 515-294-1970
Fax: 515-294-8181
Email: pmurphy@iastate.edu;

J. Scott Smith, Ph.D (Kansas)
Dept. of Animal Sci. & Ind., 208 Call Hall
Kansas State University
Manhattan, KS 66506
ph: 785.532.1219
fax: 785.532.5681
email: jsschem@ksu.edu
Web: foodsci.k-state.edu/faculty/smith.html;

George Rottinghaus (Missouri)
Veterinary Medical Diagnostic Laboratory
1600 East Rollins Street
Columbia, MO 65211
phone: 573-884-9240
FAX 573-882-1411
E-mail: rottinghausg@missouri.edu;

Frances Trail (Michigan)
Associate Professor
Departments of Plant Biology and Plant Pathology
Michigan State University
East Lansing, MI 8824
Phone: 517- 432-2939
E-mail: trail@msu.edu;

Dave Wilson (Georgia)
dwilson@tifton.cpes.peachnet.edu;

Charlene Wolf-Hall, Associate Professor (North Dakota)
Department of Veterinary and Microbiological Sciences
Great Plains Institute of Food Safety
North Dakota State University
Fargo, ND 58105
Phone 701-231-6387
Fax 701-231-7514
charlene.hall@ndsu.edu;

Charles Woloshuk (Indiana)
Botany and Plant Pathology, Purdue University
915 W. State Street
West Lafayette, Indiana 47907-2054
Phone: (765) 494-3450
FAX: (765) 494-0363
E-mail: woloshuk@purdue.edu
Web: http://www.btny.purdue.edu/faculty/woloshuk/;

Administrator

Beverly R. Durgan
Associate Dean for Research and Outreach
College of Agricultural, Food, and Environmental Sciences
277 Coffey Hall
1420 Eckles Ave
University of Minnesota
St. Paul, MN 55108
phone: 612-624-2299
FAX: 612-625-1260
email: durga001@umn.edu

Participants Attachment:

Attachment

Brief Summary of Minutes

Minutes Attachment:

Attachment

Accomplishments

Objective 1: Determine the interrelationship of Fusarium mycotoxins from cereal grains to human and animal health. In model systems, Iowa characterized at least 4 products of the FB-glucose NEB reaction, N-methyl-FB, N-carboxymethyl-FB, N-(3-hydroyacetonyl)-FB and N-(2-hydroxy, 2-carboxyethyl)-FB as well as the initial primary and very transitory, Schiff's base, FB-glucose by mass spectrometry. Illinois determined that formalin fixed tissues (kidney, liver, and lung from fumonisin B1 (FB1)-treated and control pigs) could be used to quantify Sa and So concentration. Formalin fixed tissues had lower Sa and So than the corresponding fresh frozen tissues but higher Sa:So ratio because the loss for So was greater than for Sa. The correlation coefficients between fresh frozen and formalin fixed tissues was high (>0.95). Illinois characterized the clearance of Sa and So from tissues, blood and urine in pigs given 1 mg FB1/kg or saline IV at 0, 24, and 48 h. Sa and So concentrations and Sa:So ratio in liver, kidney, lung and heart from FB1 treated pigs were significantly increased compared to controls. The serum and urine Sa concentration as well as the serum Sa:So ratio increased in FB1 treated pigs, peaking at 96 hr in serum and 120 hr in urine. Illinois continued to study the effects of sphinganine, sphingosine, and sphingosine-1-phosphate on phenylephrine contracted porcine thoracic aorta and intrapulmonary artery rings. They examined the effects of sphinganine, sphingosine, and sphingosine-1-phosphate on normal porcine aorta and pulmonary arteries using in vitro vascular ring preparations. Both sphingosine and sphinganine (>0.3 microM) significantly relaxed phenylephrine-contracted aortic rings in a dose-related fashion. A high concentration of sphingosine (30 microM), but not sphinganine, induced a vasorelaxation effect on aortic rings at baseline tension. The in vitro effects of sphingosine greater than 1.4 microM and sphinganine greater than 3.2 microM were considered as non-physiological events based on our in vivo data. This suggests that sphingosine and sphinganine mediate fumonisin B1-induced aortic hypertension in conjunction with activated alpha-adrenergic receptors, and that fumonisin B1-induced pulmonary arterial hypertension is likely mediated by sphingosine-1-phosphate. Illinois examined the difference in arterial contractility between FB1-treated (10-30 ppm) and control minipigs by using in vitro vascular ring preparations of carotid and pulmonary arteries challenged with cumulative doses of KCl and phenylephrine. The impaired arterial contractility observed in FB1-treated pigs could be due to accumulation of sphingosine and sphinganine in tissues. Iowa has demonstrated that the glucose-fumonisin adducts are far less toxic to swine dosed i.p. and by diet. Fumonisin-glucose adducts apparently protect swine from pulmonary edema at i.p. doses less than 5.5 micromole/kg BW and at dietary doses less than 528 micromole fumonisin-glucose adduct/kg BW. However, young rapidly growing swine can adapt to FB1 concentrations up to about 200 ppm in feed. Illinois studied the intravenous toxicity of fumonisin B1 in horses. Based on their work in pigs indicating fumonisin-induced cardiovascular dysfunction caused pulmonary edema, Illinois hypothesized that the pathophysiology of ELEM was related to cardiovascular dysfunction. Fumonisin B1 was administered intravenously to 8 horses (4 @ 0.20 mg fumonisin B1/kg body weight daily, 2 @ 0.10 mg fumonisin B1/kg daily, and 2 @ 0.05 mg fumonisin B1/kg daily). Four control horses were given saline. Treated horses exhibiting neurologic signs had cardiovascular dysfunction similar to those in pigs. Dose response relationships were determined and the correlation between target tissues and alterations in sphingoid bases was examined. Illinois studied fumonisin induced cardiovascular disease and atherosclerosis in swine to further examine the dose response relationship of fumonisin B1 induced hypercholesterolemia, lipid profile, So and Sa, and morphologic alterations, and to correlate these changes. They fed 35 Sinclair minipigs (barrows, 7 to 14 wk of age) fumonisin B1 at 0, 0.5, 1.0, 2.0 or 10.0 ppm (7 pigs/group) for 6 months. No significant differences were observed in serum biochemistry or lipid parameters in fumonisin treated pigs, compared to control pigs, during the 6 month study. Fumonisin-induced gross or histological alterations were not observed, nor did absolute organ weights or organ to body weight ratios differ among groups. In the highest dose (10 to 30 fumonisin B1 ppm) group, Sa and So concentrations were increased in kidney, heart, aorta, and urine; Sa was increased in liver and lung; while neither Sa nor So were increased in brain. Apparently, changes in Sa occur in some tissues at 0.5 ppm fumonisin B1, indicating that the NOEL in swine is less than 0.5 ppm. Pigs were instrumented to collect blood and urine samples in order to determine clearance of sphingosine (So), sphinganine (Sa) and So-1-phosphate (P) from blood. Mice were fed 0, 1 or 2 ppm DON for 28 days by Iowa. Blood lymphocytes were suppressed by DON. 2 ppm DON inhibited weight gain but increased feed intake; red blood cell numbers and hematocrit were reduced. DON fed at 1 ppm significantly stimulated splenocyte natural killer cytotoxicity, B-cell function, and spontaneous IFN-³ secretion. Splenocyte IL-4 was stimulated in mice fed 1 ppm DON. DON fed at 1 ppm seemed to promote T-helper cell responses in vivo, whereas the higher DON dose was immunosuppressive. Deoxynivalenol (DON)-glucuronide, a major mammalian metabolite of DON, was synthesized by Iowa, purified and assayed for its toxicity in K562 human erythroleukemia cell line. Whereas DON at 325 ng/mL caused 50% inhibition of K562 cell proliferation, as measured by Cell-titer, DON-glucuronide at >1500 ng/mL had no apparently toxic effects on K562 cells. Combining DON and DON-glucuronide in equimolar amounts within the toxic dose range of DON had no effect on DON toxicity. These results indicate that the amount of DON per se in biological fluids would most likely entirely account for DON-related toxicity signs. Kansas evaluated the mutagenic potential of fusaproliferin in 5 S. typhimurium tester strains using the standard procedure of the Ames Salmonella microsome assay. The mutagenic potency, when present, was much weaker than aflatoxin B1. Missouri determined that in rats, fusaproliferin causes a dose-related reduction in feed intake, which appears to diminish slightly with duration of exposure when fed 94, 205 or 414 mg/kg diet. There were no obvious toxicological effects on live or kidney based on organ weight/body weight ratios in rats or broiler chicks. Two studies in Missouri were conducted to determine the effects of chronic exposure to low doses of E. coli lipopolysaccharide (LPS) in chicks and poults fed diets contaminated with nontoxic doses of aflatoxin B1 (AFB1) and T-2 toxin (T-2) from hatch to day 21. Chronic exposure to low doses of LPS did not potentiate the effects of dietary non toxic doses of T-2 and AFB1 on performance of chicks and poults fed from hatch to day 21. However, LPS did potentiate the effects of T-2 on mortality rate and oral lesions in poults and, after the first LPS injection, decreased feed intake for 6 hours and enhanced mortality rate in broiler chicks. Missouri produced fumonisin, moniliformin, zearalenone, ochratoxin A, and aflatoxin B1 and secalonic acid D in culture (kg quantities) for animal feeding trials in chickens, turkeys, quail, ducks, swine, cattle, catfish and mink as well as for in vitro studies focused on identifying adsorbent materials or enzymes that will bind or inactivate mycotoxins in feed. Many of the studies involved cooperative efforts between other universities, government agencies as well as other countries. Nebraska demonstrated that fumonisin transcriptionally activates the P21 promoter. To identify genes, which are induced by FB1, a PCR-based subtraction approach was utilized. Eight genes, which showed high similarity to known mammalian genes, were identified. These genes included: tumor necrosis factor type 1 receptor associated protein 2 (TRAP2), human leukemia virus receptor (GLVR1), human Scaffold attachment factor A (SAF-A) also called heterogeneous nuclear ribonucleoprotein U (hnRNP-U), human protein kinase C-binding protein (RACK7), human oligosaccharyl transferase STT3 subunit, mouse WW-domain binding protein 2 (WBP2), human fibronectin, and unknown human clones. The ability of FB1 to alter gene expression and signal transduction pathways may be necessary for its carcinogenic and toxic effects. Kansas utilized the bacterial bioluminescence and the Salmonella typhimurium (Ames test) assays to determine the acute toxicity level and the mutagenicity potential of beauvericin. Using the Microtox® testing system, the EC50 of this mycotoxin was found to be 9 ug/mL, a moderate acute toxicity level. The Ames test was carried out on five Salmonella typhimurium standard tester strains. Beauvericin was found to be non-mutagenic with and without S9. Objective 2: Develop new techniques and improve current assays for identification and quantification of mycotoxins in cereal grains. Indiana is developing of a library of PCR primers for both conventional and real-time quantitative PCR (qPCR) to detect mycotoxigenic fungi. They developed a multiplex polymerase chain reaction (PCR) assay for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primer specificity was demonstrated by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillus spp., 9 Fusarium spp., and 10 Penicillium spp. Indiana developed a 5 fluorogenic (Taqman) real-time PCR assay for group-specific detection of trichothecene- and fumonisin-producing Fusarium spp. and to identify F. graminearum and F. verticillioides in field-collected barley and corn samples. A real-time quantitative PCR assay was developed by NCUR and optimized that allowed the quantitation of Fusarium graminearum and hygromycin resistance mutants of F. graminearum in planta. Michigan developed DNA primers based on the tri5 gene for RT-PCR to detect Fusarium sp in small grains, as a tool for diagnostics and research. Minnesota identified the structures of 7 yellow pigments, secreted by isolates of Aspergillus flavus which produce aflatoxins, as known biosynthetic intermediates, or side products, on the pathway to aflatoxin. These 7 pigments are the basis of culture-based rapid tests for aflatoxigenicity. Georgia developed an inexpensive aflatoxin screening method for use in field research. NCAUR Illinois developed an HPLC-fluorescence method for detection of fumonisins in corn silage combining an extraction technique using EDTA with an immunoaffinity column cleanup and derivatization with naphthalene dicarboxaldehyde. The prevalence of fumonisins in corn silage was found to be high, although levels were generally below concern. Illinois used immunoassay materials developed at USDA/NCAUR to screen maize as part of a project to improve resistance to Fusarium verticillioides and fumonisins. NCAUR developed a simple and rapid fluorescence polarization immunoassay for ZEN. Five highly sensitive monoclonal antibodies were also developed for detection of ZEN and related metabolites. NCAUR developed a sensitive HPLC-MS method for detection of DON and nivalenol in maize or wheat. Iowa developed a routine, rugged HPLC analysis for DON in military food stuffs. Kansas developed a sensitive GC method to detect low levels of fusaproliferin in corn with detection limit of 0.1 ng TMS derivatized fusaproliferin and 10 ppb in corn. Preliminary survey shows that fusaproliferin is a common contaminant in corn with low levels (<9.4 ppb) detected. ARS research continued in Manhattan, KS, in partnership with NCAUR to obtain near infrared spectra for individual corn kernels infected with ear rot fungi, Aspergillus flavus and Fusarium verticillioides. The spectra were applied successfully in programming a high volume commercial optical grain sorter to reject aflatoxin- and fumonisin-contaminated kernels in combine harvested corn grown in Kansas and Illinois in 2002. During 2005, a study was initiated to train and validate a neural network capable of distinguishing individual yellow corn kernels infested with Aspergillus flavus, Fusarium verticillioides, Diplodia maydis, Trichoderma viride, or other molds and apply to bench-top instruments for machine classification of mold damaged grains with the goal of producing an 'accepted grain lot' conforming to FDA guidelines for use in human food. Missouri was heavily involved in testing aflatoxin contaminated dog foods from the Eastern and Southern regions of the United States. Levels of aflatoxin contamination were in the 200-400 ppb level of aflatoxin B1 and G1. Aflatoxins were quantified by HPLC utilizing the Kobra cell for derivatization of aflatoxin B1 and G1. Kansas purified and analyzed fusaproliferin from the cultures of F. subglutinans. The purity was verified by analytical HPLC, GC, GC/MS, 1H NMR, and LC/MS. Fusaproliferin was shown to be temperature sensitive, but when dried under nitrogen and stored at -20 %C, fusaproliferin showed no sign of decomposition after a year of storage. Fusaproliferin was found to easily undergo deacetylization and form deacetyl-fusaproliferin, especially when it is dissolved in solvents at room temperature. A sensitive, reproducible, and reliable analytical method using capillary electrophoresis to separate and quantify moniliformin was developed by NCAUR and applied to samples of field-inoculated maize. This method is more rapid than traditional methods and was used to identify and characterize cryptic species of Fusarium subglutinans within the United States. Molecularly imprinted polymers (MIPs) recognizing moniliformin were developed and characterized for binding efficacy. MIPs may serve as an alternative detection platform especially for mycotoxins for which it is difficult to generate antibodies. MIPs were processed and evaluated for binding of patulin at NCAUR. When evaluated using equilibrium binding assays there were no differences between the patulin-imprinted and control polymers. NCAUR examined molecularly imprinted polymers (MIPs) synthesized to recognize patulin by infrared (IR) for characterization. Ab initio modeling and calculations have been correlated with IR spectra, indicating that patulin was present in certain of the MIPs. Pennsylvania examined the factors for and timing of fusaric acid production in the mycotoxigenic fungus Fusarium verticillioides. Liquid shake culture experiments revealed that F. verticillioides fusaric acid production is inhibited by the addition of trace elements to a minimal medium but which trace elements was responsible for the inhibition were inconclusive. A Fusarium species survey identified 10 producing fusaric acid not been previously reported and confirms that fusaric acid production can be found in all of the major groups within the genus Fusarium. Objective 3: Establish strategies for integrated management to prevent mycotoxin contamination in cereal grains. The Missouri Fusarium/ Poultry Research Laboratory continued to evaluate mineral and organic adsorbents in vitro and in vivo studies for binding of aflatoxin, vomitoxin, fumonisin, ochratoxin A, the ergot alkaloids, and zearalenone. In vivo studies have shown that only aflatoxin B1 is bound significantly in the poultry model tested. All other mycotoxins have not been bound to any appreciable degree and do not prevent mycotoxicosis. Missouri investigated whether the turkey could be used as a model for evaluating the efficacy of adsorbents to ameliorate the toxic effects of aflatoxin (AF). Adsorbents reduced some toxic effects of AF in the young turkey indicating that it is a more sensitive model for evaluating the efficacy of adsorbents. Iowa scaled up the heat processing of glucose with fumonisin B1 contaminated corn and FB1 corn culture material to kg level and successfully processed feed with 70 to 200 ppm FB1 with glucose and baking soda to about 10% residue free. The reaction is controlled by time and temperature. On farm processing would be feasible with small-scale extruders such as Instra-Pro type. Michigan provided DON analysis for FHB research throughout the United States. Over 4,000 samples are analyzed each year. The information provided is used to aid in the selection of breeding lines of wheat with reduced levels of DON, and in the evaluation of fungicides for reduction in DON. These efforts resulted in the finding that the fungicide Quadris increased levels of DON in infected grain when applied in the field at anthesis. Georgia defined the critical moisture levels for safe storage of pearl millet, has shown that post harvest treatments can help prolong storage under Georgia conditions, and demonstrated that aflatoxin can be a problem but Fusaria mycotoxins are rarely a problem in storage. Low concentrations of Fusaria mycotoxins, including some of the trichothecenes, zearalenone, beauvericin and moniliformin may occur before harvest. Iowa found that the differences in fumonisin concentrations between transgenic (Bt) and conventional corn hybrids were smaller than in 1999 report, and there were no differences in DON or aflatoxin concentrations. Michigan reported an anti-DON recombinant antibody (scFv) was used to develop transgenic Arabidopsis. The F3 generation of Arabidopsis was evaluated to identify seed homozygous for the expression of the anti-DON scFv to determine if its expression in Arabidopsis changes gene expression patterns associated with exposure of wild type Arabidopsis to DON. Gene expression patterns were determined using an Arabidopsis microarray chip and confirmed using RT-PCR by selecting 11 up regulated genes and 11 down regulated genes. Illinois developed several corn varieties showing substantial resistance to A. flavus infection or aflatoxin production. NCAUR determined that aflatoxin resistance was attributed to inbred parents that resist seed coat tearing and internal sources of kernel resistance. Kansas analyzed presence/absence alleles using amplified fragment length polymorphisms (AFLPs) at 30 loci, 24 of which are defined genetically on a linkage map of G. zeae, from > 500 isolates in 8 field populations from 7 states collected during the 1998, 1999 and 2000 cropping seasons. Observed differences are relatively small indicating that while genetic isolation by distance may occur, genetic exchange has occurred at a relatively high frequency among US populations of G. zeae. Kansas identified loci associated with pathogenicity and aggressiveness on an AFLP-based genetic map of G. zeae. Progeny that produced deoxynivalenol were 2X as aggressive as were those producing nivalenol. Simple inheritance of both traits in this interlineage cross suggests that relatively few loci for pathogenicity or aggressiveness differ between lineage 6 and 7. NCAUR determined whether 42 genetically diverse land races of corn were sensitive to fumonisins. Only two land races were highly insensitive (ED50 200 mM). Genetic analysis revealed that the high insensitivity is an inheritable trait. A greenhouse maize ear rot virulence assay was developed using the cultivar, Gaspe flint, to screen isolates for Fusarium for their ability to produce ear rot. NCAUR used the Agrobacterium-mediated transformation which resulted in highly improved transformation efficiencies with maintenance of gene expression within the maternal tissues of the developing kernel as well as within the vascular tissues of stems and resulted in stable gene insertions which are heritable. NCAUR generated reporter strains of F. verticillioides that have the cyan fluorescent protein (CEFP) gene fused to the promoter of the fumonisin biosynthetic gene FUM8 used as tools to study regulatory processes in the formation of fumonisins which could lead to control strategies that reduce or eliminate toxic fumonisin accumulation in maize. NCAUR complemented sexual spore-nonproducing mutants by adding a copy of the mating type (MAT) locus to further the goal of determining the role of sexual spores in the ability of Fusarium graminearum to cause wheat head blight. The complemented strains recovered the ability to produce sexual spores and spread in inoculated wheat heads in greenhouse tests. Several mycoparasites isolated from A. flavus sclerotia shown by NCAUR to produce extracellular proteins with potent antifungal activity against A. flavus and F. verticillioides. Gliocladium catenulatum, a soil fungus known to be a mycoparasite of Aspergillus flavus sclerotia displayed potent extracellular chitinase activity. NCAUR scientists showed that the protective corn endophyte Acremonium zeae produces pyrrocidines A and B, antibiotics that display significant antifungal activity in assays against A. flavus and F. verticillioides. Antimicrobial activity of pyrrocidine A revealed potent antibiotic activity against two major stalk and ear rot pathogens of maize F. graminearum and Stenocarpella maydis (syn. D. maydis), Nigrospora oryzae, P. oxalicum, and Cladosporium cladosporioides. Pyrrocidines A and B exhibit potent antibiotic activity against Clavibacter michiganense subsp. nebraskense, Bacillus mojaviense and Pseudomonas fluorescens, bacterial endophytes of maize used as biocontrol agents. Mycotoxigenic fungi in harvest and ensiled samples were surveyed using conventional culturing techniques and a DNA-based technique by Pennsylvannia. The DNA-based technique revealed additional species not recovered in the plating study. Few novel silage species were found. Deoxynivalenol was found in the majority of harvest and ensiled samples. Harvest samples had significantly higher deoxynivalenol concentrations than ensiled samples suggesting degradation or sequestration in the silo. Wisconsin continued to determine if RNA interference methodologies can be applied towards controlling mycotoxin contamination of cereal grains. Transformation of three Aspergilli (A. nidulans, A. flavus and A. parasiticus) and a Fusarium (F. graminearum) with inverted repeat transgenes (IRTs) containing sequences of mycotoxin-specific regulatory genes suppressed mycotoxin production in all four fungi. This atoxigenic phenotype was stable during infection on corn and wheat, and importantly, F. graminearum IRT strains were less virulent on wheat than wild type. The IRTs did not alter physiological characteristics of the fungi and results indicate that RNA silencing exists in Aspergillus and Fusarium plant pathogens and that RNA silencing technology may be a useful tool for eliminating mycotoxin contamination. Wisconsin created wheat and corn lines that contain either IRTs of GFP, F. graminearum tri6 (trichothecene regulatory gene) or A. flavus aflR (aflatoxin regulatory gene). These IRTS are promoted by plant promoters. Objective 4: Define the metabolic pathways and regulatory pathways of mycotoxin biosynthesis. Indiana found that kernels lacking starch due to physiological immaturity did not accumulate fumonisin B1. Quantitative PCR analysis indicated that kernel development also affected the expression of fungal genes involved in fumonisin B1 biosynthesis, starch metabolism, and nitrogen regulation. Indiana cloned a PACC-like gene (PAC1) from F. verticillioides that produced more fumonisin than the wild type and produced fumonisin B1 when mycelia were resuspended in medium buffered at alkaline pH (8.4). Transcription of FUM1 was correlated with fumonisin production. Therefore, PAC1 is required for growth at alkaline pH and may have a role as a repressor of fumonisin biosynthesis under alkaline conditions. Indiana constructed DNA microarrays with expressed sequence tags (ESTs) from F. verticillioides. The comparative analyses revealed differential expression of genes corresponding to 116 ESTs when the fungal strains were grown on maize. Under different pH conditions, 166 ESTs were differentially expressed, and 19 ESTs were identified that displayed expression patterns similar to 15 FUM ESTs. NCAUR continued to develop genomic tools to understand the genetic pathways that regulate fumonisin production in F. verticillioides and to identify genes that contribute to the ability of this fungus to infect corn and cause ear rot. NCAUR scientists generated a F. verticillioides microarray in collaboration with the Institute for Genomic Research (TIGR). This microarray is a tool for monitoring the expression of all 11,000 gene sequences simultaneously and should enhance our ability to study the regulation of fumonisin biosynthesis and the interactions of F. verticillioides and corn. Analysis revealed the presence of a gene, designated FUM21, which is located adjacent to the fumonisin biosynthetic gene cluster. Functional analysis of FUM21 suggests it is a transcriptional regulator of fumonisin biosynthetic genes. Identification of FUM21 is a critical break through in understanding the regulation of fumonisin biosynthesis. Pennsylvania completed a phylogenetic tree of isolates from the Gibberella fujikuroi species complex based on EF-1a sequences from over 500 isolates. This analysis has revealed numerous new phylogenetic species which are now being tested for their ability to mate sexually and produce fumonisins. NCAUR explored the metabolic pathways and regulatory mechanisms for mycotoxin biosynthesis, particularly for F. verticillioides, but also for F. raminearum and F. sporotrichioides. They completed a gene knockout analysis of all 15 genes in the fumonisin biosynthetic gene cluster and determined the functions of 8 genes, that three other genes are required for normal fumonisin production in F. verticillioides and that the remaining four genes are not essential for fumonisin biosynthesis. Pennsylvania developed methodologies for growing the Fusaria for type A trichothecene production in small volumes. Trichothecenes were detected by an HPLC-MS method which separates 8 trichothecenes allowing quantitative determination. Analysis of trichothecene production in some isolates was performed as well as sequencing of the EF-1a gene for phylogenetic analysis. NCAUR, with Agriculture Canada, identified a cytochrome P450 monooxygenase gene that resides outside the trichothecene core cluster. Gene disruption was used to define the steps in trichothecene biosynthesis by F. graminearum and to inactivate the gene in order to determine its function. Gene expression was required for oxygenation of the DON molecule at carbon positions 7 and 8. Wisconsin investigated the role of propionate metabolism in polyketide biosynthesis by manipulating the methyl citrate cycle by deletion of mcsA (DmcsA). The hypothesis is that accumulation of propionyl CoA is interfering with ST PKS function and that increasing propionyl CoA content through manipulation of the methyl citrate synthase cycle could reduce production of polyketide mycotoxins in other fungi. NCAUR found that all Penicillium sp tested were capable of producing patulin. Ribosomal DNA (rDNA) sequences identified from two isolates from commercial apple juice have tentatively been identified as Byssochlamys species. Using E. rubrum, Aspergillus flavus and Aspergillus nidulans growth, Indiana indicated that the disk method, developed by the NCAUR, is ideal to study gene expression and polyol production during growth under controlled moisture environments. Wisconsin reported a novel mechanism of gene cluster regulation by complementation of an A. nidulans ST mutant that was unable to express aflR. The complementing gene, termed laeA for loss of aflR expression, encodes a nuclear protein with closest identity to arginine and histone methyltransferases. Wisconsin determined if LaeA regulates mycotoxin biosynthesis in Fusarium spp. in fumonisin and gibberellin production. Wisconsin investigated the role of propionate metabolism in polyketide biosynthesis by manipulating the methyl citrate cycle. Wisconsin investigated the role of oxygenated fatty acids, called oxylipins, in signaling between Aspergillus spp. and host seed and found that Aspergillus spp. induce seed lipoxygenase expression resulting in the production of seed oxylipins that stimulate Aspergillus sporulation which had a profound effect on aflatoxin and ST production where 9 hydroxylated oxylipins had a stimulatory effect and 13 hydroxylated oxylipins an inhibitory effect on toxin biosynthesis. Wisconsin characterized three genes, ppoA, ppoB and ppoC, encoding dioxygenases that are required for Psi Factor formation. Wisconsin found that PpoB activity suppresses ST production whereas PpoA and PpoC activity is required for ST production. A ppoB mutant appears hypervirulent and a ppoC:ppoA double mutant hypovirulent on corn and peanuts.

Publications

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Impact Statements

Production of quantities of fumonisin, ochratoxin A, moniliformin, zearalenone, and aflatoxin B1 in culture material have made it economically feasible for research groups to do mycotoxin research in cattle, swine, poultry, equine, mink, catfish, and laboratory animals.
The Fusarium/Poultry Research Laboratory located at the University of Missouri completed a number of in vitro studies and in vivo livestock and poultry feeding studies that examined a number of proprietary adsorbents for their effectiveness in reducing the toxicity of mycotoxin contaminated feedstuffs. Information from this project is being used to advise and educate producers and grain handlers on how to safely manage the utilization of mycotoxin contaminated grains for animal feeds.
Improved animal and human health effects by reducing mycotoxin contamination of food and feed crops through genetic engineering and/or identification of fungal virulence factors.
A rapid, sensitive and reliable method for analyzing ergosterol in barley and wheat has been developed. The method can be used for both ground grains and individual kernels. The ability of detecting low levels of ergosterol in a single kernel allows the investigation of early fungal invasion and facilitates the study of pathogenesis. The method can be easily applied to handling a large number of samples, making it suitable for screening FHB resistant cultivars.
The biological impact of DON and its metabolites can be assessed in cell culture revealing that only free DON appears to be the toxic constituent. The suppression of peripheral blood lymphocytes by low dose dietary DON in mice may readily translate into a convenient method for assessment of DON toxicity in humans.
Fumonisin can apparently be detoxified by reaction with reducing sugars such as glucose, fructose or lactose at pH 7 or greater for swine and rodent feed and potentially human foods.
We developed a relatively rapid and economical method for quantifying sphingolipid biomarkers in a variety of body fluids, cells and tissues. We demonstrated that formalin fixed tissues can be used for sphingolipid determination. And that the sphingolipids, sphingosine and sphinganine are good biomarkers of fumonisin exposure in pigs.
Both conventional and real-time quantitative PCR (qPCR) to detect mycotoxigenic fungi is potentially important for monitoring the population genetics behind fungal epidemics in crops and for biosecurity.
The survey information is published in newsletter and presented during extension meeting, which allows producers, handlers and processors know the condition of the crop.
The study of kernel lacking starch provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.
All the populations of Gibberella zeae are genetically similar, have high genotypic diversity and little or no detectable genetic disequilibrium, and show evidence of extensive interpopulation genetic exchange. Geographic distance and genetic distance between populations are correlated significantly.
Isolation and characterization of Fusarium genes involved in mycotoxin synthesis and regulation continue to aid efforts to control mycotoxin formation in food crops.
New technologies and methods for fungal and mycotoxin analysis have provided either better, easier or quicker means to detect mycotoxigenic fungi and mycotoxins in foods and feeds.
For the tri-state region of North Dakota, South Dakota, and Minnesota, barley has an estimated annual impact of $1.5 billion, and represents about 40% of the U.S. malting capacity. The potential reduction of toxin-producing mold would prevent post-harvest production of mycotoxins during malting and would be beneficial for food-grade malts. Using alternative treatments such as ozone, hot water, and irradiation to reduce mycotoxin contamination, we have encouraging results with barley.
The finding that deoxynivalenol levels in corn silage are higher at harvest than after ensiling indicates that management strategies for this mycotoxin should focus on field approaches to limit development of producing fungi.
The addition of ten more species known to produce fusaric acid indicates that the possibility for contamination of feeds by this toxin is greater than previously realized. The basic information on timing and conditions for fusaric production lay the groundwork for additional studies on the impact of the toxin, its role in fungal ecology and for molecular studies.
USAID RD309-022/2265417, Keller CoPIs: D. Wilson (University of Georgia) and 5 Botswanan scientists, 1 RSA scientist, $448,000, 5/1/00-6/30/06, Basic and applied studies on aflatoxin and Aspergillus flavus management and interactions with peanut in the field and storage
USDA/ARS US Wheat and Barley Scab Initiative, Keller, $40,162 2003-4. Role of oxygenases in Fusarium graminearum pathogenicity
USDA NRI, Keller CoPI: H. Kaeppler, $475,378, RNAi-Mediated Control of Mycotoxin Contamination of Food Crops
USDA MO-ASAH 0518, Ledoux DR (PI), Bermudez AJ, Rottinghaus GE and Broomhead J: Characterization of Toxicological Effects of Multiple Mycotoxins in Poultry. USDA Animal Health Formula Funds ($17,000/year) 10/1/2001-9/30/2002 (5% effort).
Contracts with private companies: Rottinghaus GE and Ledoux DR. In vitro and in vivo testing of proprietary adsorbents and feed additives for reducing mycotoxicosis in livestock and poultry. ($50,000-$75,000/year for the five years).
US Wheat Barley Scab Initiative, $43,029, 4/16/06--4/15/07, BIOMARKERS OF LOW DOSE IMMUNOTOXICITY OF DEOXYNIVALENOL. PI: S. Hendrich, co-PI, M. Kohut.