Fusarium
Mycotoxins:

Vomitoxin

Nivalenol

Lycomarasmin

Fusariotoxin
T2-Toxin,

Fusaric Acid

Fumonisin B1

New! Fusarium mycotoxins: chemical names list.

Chemical Herbicides

Soil Solarization

Español

NC-129 Annual Report of Cooperative Regional Research Project

October 1, 1996 to December 31, 1997

III. Progress and Principal Accomplishments

Objective 1. Determine the interrelationship of Fusarium mycotoxins from cereal grains to human and animal health.

The full extent of the threat of the fumonisins to food safety is not known because much of the extractable and assayable fumonisins contaminating foods is converted to other unidentified forms during food processing. Structure-activity relationship studies on natural and synthetic fumonisins indicate that extensive alterations in structure are possible without loss of biological activity. FB1 is converted to other forms by several pathways under thermal food processing conditions. A major mechanism appears to be binding to protein.

Studies in Iowa using 14C-Fumonisin B1 (FB1), determined the excretion of FB1, hydrolyzed FB1 (HFB1) and FB1-fructose adduct. These data confirm previous findings in FB excretion utilizing unlabeled FB compounds. The loss of toxicity for FB1-fructose is not due to lower bioavailability.

In Missouri, a floor pen study was conducted with 270 one-week old broiler chicks to evaluate the chronic effects of low levels of moniliformin (M) in broiler chicks. Results indicate that 50 mg M/kg diet is toxic to broiler chicks and toxic effects (reduced feed intake and body weight gain) occurred as early as two weeks after chicks began to consume the diet containing 50 mg/kg diet.

A series of experiments were conducted to evaluate the immune system in turkey poultry fed dyes containing FB1. The reduction of Ab production and lymphoid organ weights, and the inhibition of lymphocyte proliferation suggest that FB1 is immunosuppressive in turkey poults. A combination of low levels of mycotoxins in the diet of broiler chicks reduced chick performance. In Nebraska, FB1 activates transcription of p21 (cdk inhibitor) in monkey cells and induces apoptosis through a p53-independent pathway. Effects of fumonisin are blocked by inhibitors of TNF, FAS and ICE proteases. Using primary chick embryo fibroblasts, FB1 did not arrest the cell cycle, nor induce apoptosis as in monkey cells.

Top of Page

Objective 2. Develop new techniques and improve current assays for identification and quantitation of Fusarium mycotoxins in cereal grains.

Deoxynivalenol (DON) produced during infection of the wheat kernel by Fusarium species is distributed throughout the wheat kernel with the highest levels generally occurring in the outer portions of the wheat kernel, the brans and shorts; and with lower levels occurring in the flour fractions, the breaks and reductions. ELISAs for Deoxynivalenol in milled wheat fractions should provide reliable results rapidly and economically in a commercial setting (Michigan).

Moniliformin, but not FB1, was acutely toxic to ducklings. Moniliformin was found to inhibit carbohydrate synthesis in cultured chicken embryo hepatocytes. This work established a new role for moniliformin as an inhibitor of carbohydrate synthesis and showed that FB1 was not involved in the inhibition of glucose production. Tissue and cyst fluid from polycystivc kidney disease (PKD) patients were examined for fungal components. Fungal DNA, Fusarium antigens and antibodies reactive with Fusarium were detected in human kidney tissue and cyst fluid from PKD patients, but not in control kidney tissue.

In the Chu lab (Wisc.) much effort during 1997 was focused on improvement of different ELISA methods for FB1 and related mycotoxins detection. Studies on the development of antibodies against FB4 were initiated.

In Minnesota, a method was developed for the analysis of DON and its derivatives in wheat and barley using GC/MS. The method is also applicable to 15-acetyldeoxynivalenol as well as nivalenol. Standard curves constructed for DON, 15-ADON and NIV are linear between 0.025 nanograms (limit of sensitivity) and 8 ng.

Iowa State (Murphy) has made the first reports of natural occurrence of fusaproliferin outside of Italy and beauvericin in the U.S. (Munkvold). This work has been done in collaboration with Antonio Logrieco and others in Italy. A method to produce >95% pure HFB1 standard has been developed and the purity confirmed by Mirocha (Minnesota). They are continuing to purify FB1 in g quantities from liquid culture fermentations using F. proliferatum 5991.

Beauvericin (BEA) is a bioactive cyclic hexadepsipeptide (C45H57N3O9; mol wt: 784 co- produced by the same Fusarium species that produce the carcinogenic fumonisin mycotoxins. On-line thermospray mass spectrometry (LC-TSP-MS) was used to analyze BEA (Smith, Kansas). This method was used successfully to analyze F. proliferatum-fermented cracked corn and meat samples.

Top of Page

Objective 3. Establish strategies for integrated management of Fusarium mycotoxins in cereal grains.

Work in Iowa demonstrated that certain genotypes of Bt transgenic corn have reduced Fusarium ear rot and reduced fumonisin concentrations compared to their non-transgenic counterparts. Exotic maize genotypes with natural resistance to F. garminearum and F. moniliforme were identified. Fusarium populations in corn seed lots can be reduced by more than 50% by removing 5% of the least dense seed, thereby reducing the potential for seed transmission.

A field test was conducted to measure the levels of fumonisins in the ears from corn plants experimentally infected with F. moniliforme (USDA). Ears infected through silks had significantly more ear rot than in water inoculated control ears. Infection at seed stage via the toothpicks introduced the specific strain into the harvested ear but ear rot symptoms were not different form untreated control ears.

To evaluated the role of stress proteins during Fusarium infection, corn cells were found to contain a large complex made up of at least six distinct stress proteins. It was further demonstrated that three of the plant stress proteins seem to cooperate to reduce temperature-induced cell damage.

In Michigan, head blight of wheat occurred at lower levels in 1997 compared to the severe epidemic in 1996. Commercial winter wheat lines were evaluated for FB1 reaction. Application of fungicide to spring wheat inoculated with F. graminearum did not result in a significant increase over untreated contols, although the incidence and severeity of infection were generally reduced.

Top of Page

Objective 4. Define the biosynthesis of Fusarium mycotoxins.

When 14C-FB1 was hydorlyzed to produce 14C-HFB, the specific activity of HFB was 50% of FB1. U-14C-acetate was used to produce labeled FB1. These data suggested that acetate carobn was incorporated into tricarbalyllic gorups of FB1 as well as all other carbons in FB1. These results complement others who suggest glutamate as final source of carbon for FB1 side chains.

Two naturally occurring structural isomers of partially HFB1 as well as N-acetyl fumonisin were detected in corn samples and screenings.

A total of 250 REM1 transformants was screened for mutations affecting FB1 biosynthesis (Woloshuk, Purdue). None of these transformants prove to be blocked in fumonisin biosynthesis. To clarify the role of fumonisins in infection and plant disease, work is underway to isolate, charcterize and disrupt a putative fumonisin biosynthetic gene (USDA). A polyketide synthase gene fragment was isolated. This gene expressed in cultures when they are producing fumonisin but was not expressed when fumonisins were not being produced.

As the first step for studying the biosynthesis of fumonisins, Chu used two immunochemical methods to screen a total of 114 strains of Fusarium cultures grown in corn for their ability to produce fumonisins. His group has also raised polyclonal antibodies against +o Tr10, a regulatory protein involved in trichothecene biosynthesis.

Top of Page

IV. Usefulness of Findings

Reproducible and realiable analytical techniques have been developed for fumonisins and beauvericin. Having such methods is important as we move further to define the mechanism of toxicity of these mycotoxins.

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. Bioassays have provided a means for isolating potentially new toxins from Fusarium culture material and for further assessment of known toxins such as the fumonisins, moniliformin, and beauvericin.

The detoxification of FB1 by a reaction with glucose or fructose continues to be studied. Resistance to FB contamination by certain corn genotypes has further been elucidated.

Studies on the pathophysiology of fumonisin toxiosis in animals and possibly humans, are essential for diagnosis, establishment of safety parameters and development of control procedures.

NC129 web site: http://www.btny.purdue.edu/NC129/NC129.html - A web site was created for transferring information generated by the NC129 project (Woloshuk, Purdue). Featured at the site is the annual project summary, links to other mycotoxins-related web sites, and a newsletter that highlights important research findings and issues concerning mycotoxins. The 1997 newsletter featured had the following titles: March 1997. 1996 Indiana Pre-Harvest Corn Ear Rot and Mycotoxin Survey Results; May 1997. Detoxificaiton of Fumonisin through Reaction with Fructose; July 1997. Two abstracts were selected from the Proceedings of the Thirty-eight Annual Corn Dry Milling Conference; October 1997. Mycotoxins as agents for biological weapons.

Top of Page

V. Work Plan for 1998

1. Continuing collecting ear rot and mycotoxin data.
2. Continue screening the region's grain for Fusarium mycotoxin contamination.
3. Provide mycotoxin analytical services for scientists who are developing crop resistance to FB1 and the livestock/feed industry.
4. Continue to investigate material with potential activity against mycotoxigenic fungi and insects.
5. Continue to study Fusarium infection and fumonisins in Bt transgenic hybrid.
6. Continue to study how fumonisin affects chicken embryos.
7. Purify gram quantities of fumonisin backbone for toxicity studies.
8. Improve analytical methodology to detect mycotoxin.
9. Improve analytical methodology for toxin purfication.
10. Continue to improve immunodetection methodologies.
11. Continue to characterize trichothecene pathway genes. Identify fumonisin pathway genes.
12. Continue animal feeding studies with culture material containing fusarium mycotoxins and examine effects under stress of temperature and pathogen attack.
    

Top of Page

VI. Publications

* Indicates publications with multi-location authors.

*Keller, N.P. and Hohn, T.M. 1997. Metabolic pathway gene clusters in filamentous fungi. Fungal Genet. Biol. 21:17-29.

*Liang, S. H., Wu, T. S., Lee, R., Chu, F. S. and Linz, J. E. 1997. Analysis of mechanisms regulating the expression of the ver-1 gene involved in aflatoxin biosynthesis. Appl. Environ. Microbiol. 63:1058-1065.

*Liu, B.H., Brewer, J. F., Flaherty, J.E., Payne, G., Bhatnagar, D. and Chu, F.S. 1997. Immunochemical identification of AFLR, a regulatory protein involved in aflatoxin biosynthesis. Food & Agric. Immun. 9:289-298.

*Meredith, F., Bacon, C., Norred, W. and Plattner, R. 1997. Purification of fumonisin B2 isolated from rice culture. J. Agric. Food Chem. 45:3143-3147.

*Morgan, M.K., Schroeder, J.J., Rottinghaus, G.E., Powell D.C., Bursian, S.J., and Aulerich, R.J. 1997. Dietary fumonisins disrupt sphingolipid metabolism in mink and increase the free sphinganine to sphingosine ration in urine but not hair. Vet. Human Tox. 39:334-336.

*Munkvold, G.P. and Desjardins, A.E. 1997. Fumonisins in maize. Can we reduce their occurrence? Plant Dis. 81:556-565.

*Norred, W.P., Plattner, R.D., Dombrink-Kurtzman, M.A., Meredith, F.I. and Riley, R.T. 1997. Mycotoxin-induced elevation of free sphingoid bases in precision-cut rat liver slices: Specificity of the response and structure-activity relationships. Toxicol. Appl. Pharmacol. 147:63-70.

Abbas, H.K., Smeda, R.J., Duke, S.O., and Shier, W.T. 1997. Fumonisin plant interactions. Bull. lnst. Compr. Agr. Sci., Kinki Univ. 5:1-11.

Abbas, H.K., Duke, S.O., Shier, W.T., Badria, F.A., Ocamb, C.M., Woodward, R.P., Xie, W., and Mirocha, C. J. 1997. Comparison of ceramide synthase inhibitors with other phytotoxins produced by Fusarium species. J. Nat. Toxins 6:163-181.

Alexander, N., Hohn, T.M. and McCormick, S.P. 1997. The TRI11 gene of Fusarium sporotrichioides encodes a cytochrome P450 monooxygenase required for C-15 hydroxylation in trichothecene biosynthesis. Appl. Environ. Microbiol. 64: Accepted Nov. 26, 1997.

Bermudez, A.Y., Ledoux, D.R., Rottinghaus, G.E., Stodsdill, P.L., and Bennett,, G.A. 1997. Effects of feeding Fusarium fujikuroi culture material, containing known levels of moniliformin, in turkey Poults. Avian Path 26:565-577.

Bermudez, A.J., Ledoux, D.R., Rottinghaus, G.E., and Bennett, G.A. 1997. The individual and combined effects of the Fusarium mycotoxins, moniliformin and fumonisin B1 in turkeys. Avian Dis. 41:304-311.

Chu, F. S. 1997. Recent development and application of immunochemistry in agriculture and food analysis. In "Proceeding of International Symposium of Biomolecular reactions and Industrial Applications of Immunology in Foods and Agriculture." (Ratih Dewanti-Hariyadi & Fransiska Zakaria, eds., ISBN 979-95046-3-5) pp. 21-40, Nov. 8, 1997, IPB, Bogor & The French Embassy, Jakarta, Indonesia.

Chu, F.S. 1997. Trichothecene mycotoxicoses. In "Encyclopedia of Human Biology". Second edition, vol. 8, 511-522. Academic Press, NY.

Desjardins, A.E., McCormick, S.P., Plaisted, R.L. and Brodie, B.B. 1997. Association between solavetivone production and resistance to Globodera rostochiensis in potato. Agric. Food Chem. 45:2322-2326.

Desjardins, A.E., Plattner, R.D. and Nelson, P.E. 1997. Production of fumonisin Bl and moniliformin by Gibberella fujikuroi from rice from various geographic areas. Appl. Environ. Microbiol. 63:1838-1842.

Desjardins, A.E., McCormick, S.P., Plaisted, R.L. and Brodie, B.B. 1997. Association between solavetivone production and resistance to Globodera rostochiensis in potato. J. Agric. Food Chem. 45:2322-2326.

Desjardins, A.E. and Hohn, T.M. 1997. Mycotoxins in plant pathogenesis. Mol. Plant-Microbe Interact. 10:147-152.

Dombrink-Kurtzman, M.A. and Knutson, C.A. 1997. A study of maize endosperm hardness in relation to amylose content and susceptibility to damage. Cereal Chem. 74:776-780.

Edrington,T.S., Kubena, L.F., Harvey, R.B., and Rottinghaus, G.E. 1997. Influence of a super- activated charcoal on the toxic effects of aflatoxin or T-2 toxin in growing broilers. Poultry Sci. 76:1205-1211.

Ferguson, S.A., St Omer, V.E., Kwon, O.S., Holson, R.R., Houston, R.J., Rottinghaus, G.E., and Slikker, W. 1997. Prenatal fumonisin (FB1) treatment in rats results in minimal maternal or offspring toxicity. Neurotoxicol. 18:561-569.

Filho, Edson R., Xie, W., Mirocha, C.J., and Hogge, L.R. Fragmentation of some zearalenones by Fast-atom Bombardment Mass Spectrometry. Rapid. Comm. Mass Spectrometry. 11:1515-20.

Guzman, R.E., Casteel, S.W., Rottinghaus, G.E., and Turk, J.R. 1997. Chronic consumption of fumonisins derived from Fusarium moniliforme culture material: Clinical and pathological effects in swine. J Vet Diagn Invest 9:216-218.

Hart, L. P. and Schabenberger, 0. 1998. Variability of vomitoxin in truckloads of wheat in an wheat scab epidemic year. Plant Disease (accepted).

Ho, A.K., Peng, R., Ho., A.A., Duffield, R. and Dombrink-Kurtzman, M.A. 1996. Interactions of fumonisins and sphingoid bases with GTP-binding proteins. Biochem. Arch. 12:249-260.

Hohn, T.M. 1997. Fungal phytotoxins: Biosynthesis and activity. pp. 129-144. IN: G. Carroll and P. Tudzynski (eds.) The Mycota Vol. V. Part A: Plant Relationships. Springer-Verlag, Berlin.

Hopmans, E.C., Hendrich, S., Murphy, P.A. 1997. Excretion of fumonisin Bl, hydrolyzed fumonisin Bl and fumonisin Bl-fructose adduct in rats. J. Agric. Food Chem. 45:2618-2625.

Kubena, L.F., Edrington, T.S., Harvey R.B., Buckley, S.A., Phillips, T.D., Rottinghaus, G.E., and Casper, H.H. 1997. Individual and combined effects of fumonisin B1 present in Fusarium moniliforme culture material and T-2 toxin or deoxynivalenol in broiler chicks. Poultry Sci. 76:1239- 1247.

Kubena, L.F., Harvey, R.B., Buckley, S.A., Edrington, T.S., and Rottinghaus, G.E. 1997. Individual and combined effects of moniliformin present in Fusarium fujikuroi culture material and aflatoxin in broiler chicks. Poultry Sci. 76:265-270.

Kubena, L.F., Edrington, T.S., Harvey, R.B., Phillips, T.D., Saar, A.B., and Rottinghaus, G.E. 1997. Individual and combined effects of fumonisin B1 present in Fusarium moniliforme culture material and diacetoxyscirpenol or ochratoxin A in turkey poults. Poultry Sci .76:256-264.

Liu, B.H. and Chu, F.S. 1997. Production and characterization of monoclonal antibodies against sterigmatocystin 0-methyltransferase. Food & Agric. Immun. 9:167-176.

Lu, Z., Dantzer, W.R., Hopmans, E.C., Prisk, V., Cunnick, J.E., Murphy, P.A., Hendrich, S. 1997. Reaction with fructose detoxifies fumonisin Bl while stimulating liver-associated natural killer cell activity in rats. J. Agric. Food Chem. 45:803-809.

Maragos, C.M., Bennett, G.A. and Richard, J.L. 1997. Affinity column clean-up for the analysis of fumonisins and their hydrolysis products in corn. Food Agric. Immunol. 9:3-12.

Maragos, C.M. and GREER, J.I. 1997. Analysis of Aflatoxin Bl in corn using capillary electrophoresis with laser-induced fluorescence detection. J. Agric. Food Chem. 45:4337-4331.

Maragos, C.M. 1997. Measurement of mycotoxins in food with a fiber-optic immunosensor. J. Clin. Ligand Assay 20:136-139.

McCormick, S.P. and Hohn, T.M. 1997. Accumulation of trichothecenes in liquid cultures of a Fusarium sporotrichioides mutant lacking a functional trichothecene C-15 hydroxylase. Appl. Environ. Microbiol. 63:1685-1688.

Miernyk, J.A. 1997. The 70 kDa stress-related proteins as molecular chaperones. Trends Plant Sci. Rev. 2:180-187.

Miller-Hjelle, M.A., Hjelle, J.T., Jones, M., Mayberry, W.R., Dombrink-Kurtzman, M.A., Peterson, S.W., Nowak, D.M. and Darras, F.S. 1997. Polycystic kidney disease: An unrecognized emerging infectious disease? Emerging Infect. Dis. 3:113-127.

Muhitch, M.J. 1997. Effects of expression E. coli threonine synthase in tobacco (Nicotiana tabacum L.) suspension culture cells on free amino acid levels, aspartate pathway enzyme activities and uptake of aspartate into the cells. J. Plant Physiol. 150:16-22.

Munkvold, G.P., Carlton, W.M., Hellmich, R.L., and Rice, M.E. 1997. Effects of Bt transformation on diseases of corn. Pp. 223-234 in: Proc. 9th Annu. Integrated Crop Mngmnt Conf. Ames, IA, Nov 17-18, 1997.

Munkvold, G.P., Stahr, H.M., Logrieco, A., Moretti, A., and Ritieni, A. 1997. Occurrence of fusaproliferin in Iowa maize and maize-based livestock feed. Inoculum 48:27.

Munkvold, G.P., Hellmich, R.L., and Showers, W.B. 1997. Reduced Fusarium ear rot and symptomless infection in kernels of maize genetically engineered for European corn borer resistance. Phytopathology 87:1071-1077.

Munkvold, G.P., and Carlton, W.M. 1997. Influence of inoculation method on systemic Fusarium moniliforme infection of maize plants grown from infected seeds. Plant Dis. 81:211-216.

Munkvold, G.P., McGee, D.C., and Carlton, W.M. 1997. Importance of different pathways for maize kernel infection by Fusarium moniliforme. Phytopathology 87:209-217.

Prieto, R. and Woloshuk, C. P. 1997. ordl, an oxidoreductase gene responsible for the conversion of 0-methylsterigmatocystin to aflatoxin in Aspergillus flavus. Appl. Environ. Microbiol. 63:1661-1666.

Proctor, R.H., Desjardins, A.E. and Plattner, R.D. 1997. Analysis of a Gibberella fujikuroi mutant deficient in a hydroxylation step of fumonisin biosynthesis. Nineteenth Fungal Genet. Conf. Abstr. P.119.

Proctor, R.H., Hohn, T.M. and McCormick, S.P. 1997. Restoration of wild-type virulence to Tri5 disruption mutants of Gibberella zeae via gene reversion and mutant complementation. Microbiology 143:2583-2591.

Reams, R.Y., Thacker, B.L., Harrington, D.D., Novilla, N.K., Rottinghaus, G.E., Bennett, G.A., and Horn, J. 1997. A sudden death syndrome induced in poults and chicks fed diets containing Fusarium fujikuroi with known concentrations of moniliformin. Avian Dis. 41:20-35.

Richard, J.L. 1997. Gliotoxin, a mycotoxin associated with cases of avian aspergillosis. J. Nat. Toxins 6:11-18.

Shier, W.T., Abbas, H.K., and Badria, F.A. 1997. Structure-activity relationships of the corn fungal toxin fumonisin Bl: implications for food safety. J. Nat. Toxins 6:225-242.

Smith, J. Scott, Thakur, Rohan A., Leslie, John F. and Walter, Marasas, F.O. 1997. Chemical Analysis and Toxicity of a New Fusarium Myctoxin. Pittsburgh Conference of Analytical Chemistry and Applied Spectroscopy Abstracts. Abstr. No. 546.

Stahr, H.M., Imerman, P.M., Sun, T. A Method to Analyze Commodity Samples for Fusaproliferin. Midwest Regional AOAC Meeting.

Taylor, S.L., King, J.W., Greer, J.I. and Richard, J.L. 1997. Supercritical fluid extraction of aflatoxin Ml from beef liver. J. Food Prot. 60:698-700.

Thakur, R.A. and J.S. Smith. 1997. Liquid Chromatography/Thermospray/Mass Spectrometry Analysis of Beauvericin. J. Agric. Food Chem. 45:1234-1239.

Thompson, V.S. and Maragos, C.M. 1997. Measurement of fumonisins in corn with a fiber-optic fluoroimmunosensor. Proc. Adv. Fluorescence Sensing Technol. III. Vol. 2980:532-538.

Trapp, S.C., Hohn, T.M., McCormick, S.P. and Jarvis, B.B. 1997. Characterization of the gene cluster for biosynthesis of macrocyclic trichothecenes in Myrothecium roridum. Mol. Gen. Genet.: Accepted Nov. 26, 1997.

Woloshuk, C. P., Cavaletto, J. R., and Cleveland, T. E. 1997. Inducers of aflatoxin biosynthesis from colonized maize kernels are generated by an amylase activity from Aspergillus flavus. Phytopathology 87:164-160.

Wu, W. and Vesonder, R.F. 1997. Inhibition of gluconeogenesis in cultured chickens embryo hepatocytes by Fusarium metabolites. Nat. Toxins 5:80-85.

Xie, Weiping, Mirocha, C.J., and Chen, Junping. Detection of two naturally occurring structural isomers of partially hydrolyzed fumonisin Bl in corn by on-line capillary liquid chromatography-fast atom bombardment mass spectrometry. J. Agric. Food Chem. 45:1251-55.

Yu, Hui, Evans, C.K., Kolaczkowski, E.K., Dill-Macky, R., and Mirocha, C.J.. Chemistry, Physiology and Role of Deoxynivalenol in Pathogenicity. Bull. lnst. Compr. Agr. Sci., Kinki Univ. 5:1-11.

Yu, W. and Chu, F. S. 1998. An improved direct competitive enzyme-linked immunosorbent assay for cyclopiazonic acid in corn, peanuts and mixed feed. J. Agric. Food Chem. (Accepted).

Yuan, Q.Y., Clarke, J., Linz, J. E., Pestka, J. J., and Hart, L. P. 1997. Molecular cloning, expression, and characterization of a functional anti-zearalenone single-chain Fv fragment. Appl. Environ. Microbiol. 63:263-269.

Top of Page