Brief Summary of Minutes of Annual Meeting

Refer to NC129 Termination Report.

ACCOMPLISHMENTS Objective 1. Develop data for use in risk assessment of mycotoxins in human and animal health.

(IL) We reevaluated methods for analyzing Sa and So in body fluids, cells and tissues. Using Yoos extraction protocol and modified HPLC conditions, we successfully quantified Sa and So in cells (HepG2 hepatocytes, PK15 renal cells, A10 smooth muscle cells, H9C2[2-1] cardiomyocytes), serum and urine from pigs and calves, as well as fresh frozen swine tissues (liver, kidney, lung and heart). The modified method also worked well for formalin fixed swine liver, kidney and lung but not heart, due to unexplained peaks on the chromatogram interfering with interpretation of Sa and So. Modifications in HPLC conditions included a lower % of potassium phosphate buffer in the mobile phase (8%), a lower flow rate (1 mL/min) and a lower excitation wavelength (230 nm). Our modified method provides a relatively rapid and economical method for quantifying Sa and So in a variety of body fluids, cells and tissues.

To determine whether formalin fixed tissues could be used, we quantified Sa and So concentration by reverse-phase high-performance liquid chromatography (HPLC) in kidney, liver, and lung obtained 3 months previously from fumonisin B1 (FB1)-treated and control pigs, and compared the results for fresh frozen and formalin fixed tissues. Formalin fixed tissues had lower Sa and So than the corresponding fresh frozen tissues. The Sa:So ratio in formalin fixed tissues was higher because the loss for So was greater than for Sa. The correlation coefficients between fresh frozen and formalin fixed tissues was high (>0.95). These results indicate that formalin fixed liver, kidney and lung can be used to determine alterations in Sa and So concentrations, with the Sa:So ratio being the most useful.

We characterized the clinopathology and clearance of Sa and So from tissues, blood and urine in pigs in order to determine the usefulness of Sa and So as biomarkers of FB1 exposure. Male castrated pigs (15 kg body wt) were given 1 mg FB1/kg or saline (controls) IV at 0, 24, and 48 hr. Blood and urine were collected from FB1 pigs (n = 4 to 8) and from controls (n = 4) over 144 hr. Liver specific serum biochemistry and urinalysis (creatinine, protein and specific gravity) were determined at 0, 72 and 144 hr, and were not significantly different from controls though the values in the FB1 groups were often higher. Two FB1 treated pigs developed respiratory distress with one dying due to pulmonary edema. The Sa and So concentrations and Sa:So ratio in liver, kidney, lung and heart from Groups A and B pigs were significantly increased compared to controls (p < 0.05), but there was no difference in values in treated pigs at 72 and 144 hr 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. Sa and So concentrations decreased after this time but were still elevated at 144 hr in both serum and urine. The serum and urine So concentrations were also increased, though to a much lesser extent. . These results suggest that accumulated intracellular sphingolipids do not rapidly leave the cell and/or that sphingolipid metabolism continues to be inhibited after cessation of FB1 treatment. Therefore, tissue, serum and urine Sa and the Sa:So ratio are useful biomarkers of recent FB1 exposure.

(IA) Deoxynivalenol (DON)-glucuronide, a major mammalian metabolite of DON, was synthesized, 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-titre, 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.

We are applying the K562 cell assay to analysis of human plasma samples from 8 subjects fed a single meal of DON-contaminated bread (~325 microgram dose of DON per person, from a bread mix purchased in a local grocery store)(analyses underway), and further developing the K562 cell-based DON toxicity assay by screening human plasma samples from 20 subjects fed a DON-free diet for 4 days, adding DON to those plasma samples to determine to what extent individual plasma samples vary in the K562 cell response to added DON. These analyses are underway. We also plan to use a mouse model of low dose exposure to DON (0, 0.5, 1, 2 mg DON/kg diet) to attempt to determine a lowest observed adverse effect level with respect to DONs ability to suppress peripheral blood lymphocyte proliferation, as we have previously shown in mice fed 2 ppm DON for 28 days.

(MO)Two studies 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 non toxic doses of aflatoxin B1 (AFB1) and T-2 toxin (T-2) from hatch to day 21. It was concluded that 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. LPS also enhanced mortality rate in broiler chicks after the first LPS injection and decreased feed intake for 6 hours after the first injection containing LPS.

(MO) Fumonisin, moniliformin, zearalenone, ochratoxin A, and aflatoxin B1 and secalonic acid D are being produced 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.

(MO) The laboratory 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 quantitated by HPLC utilizing the Kobra cell for derivatization of aflatoxin B1 and G1.

Objective 2. Develop new techniques and improve current assays for identification and quantification of mycotoxins and mycotoxigenic fungi in cereal grains. (IN) We obtained funding for a graduate student to begin the development of a library of PCR primer for both conventional and real-time quantitative PCR (qPCR) to detect mycotoxigenic fungi. We are cooperating with Texas, who has separate funding, to coordinate the effect.

(ND) North Dakota: Polymerase chain reaction based assays were developed and used to detect and quantify deoxynivalenol and ochratoxin producing fungi in stored and processed barley. The objectives of the study included: a) To develop a multiplex real-time PCR method to simultaneously detect and quantify the trichothecene and ochratoxin producing fungi; b) To apply various methods to determine correlations between biomass of Fusarium, Penicillium and Aspergillus, mycotoxin production, and volatile production in barley stored under different moisture contents over time; c) To develop a real-time reverse transcription PCR method to measure mycotoxin production during malting of barley; and d) To develop a random amplified polymorphic DNA (RAPD) analysis method for monitoring irradiation effects on genetic mutations in F. graminearum isolates. We found that the multiplex real-time PCR method we developed can simultaneously detect ochratoxin producing A. ochraceus and P. verrucosum and trichothecene producing F. graminearum. In our second objective we found that the biomass and volatile content of these fungi increased in barley stored at various moisture contents (13%, 18%, 20% and 25%) over time. We observed that the mycotoxin producing gene (Tri5) expression occurred during the germination stage of malting in barley. In our fourth objective we found that electron beam irradiation may have caused mutations in F. graminearum and the mycotoxicity of the fungus was reduced by irradiation.

(USDA) During crop growth and at harvest, mycotoxigenic fungi often are co-isolated with other fungi that are capable of reducing grain quality. Aflatoxin and fumonisin in grain is often found concentrated at high levels in relatively few corn kernels. ARS research continued in Manhattan, KS, in partnership with NCAUR to obtain near infrared spectra for individual corn kernels infected with ear rot fungi. 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.

A sensitive, reproducible, and reliable analytical method using capillary electrophoresis to separate and quantify moniliformin was developed and applied to samples of field-inoculated maize. This method is more rapid than traditional methods and was used as part of a project 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.

Patulin MIPs and control polymers synthesized using UV photo-initiated polymerization at low temperatures in the Department of Chemistry, University of California, Irvine, 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.

Synthesis and characterization of molecularly imprinted polymers (MIPS) will continue. Conditions for optimal production of patulin will be published. Genetic probes will be developed to identify patulin-producing fungi. Moniliformin levels in samples of maize from a second year of field trials will be determined.

A real-time quantitative PCR assay was developed and optimized that allowed the quantitation of Fusarium graminearum and hygromycin resistance mutants of F. graminearum in planta. This protocol will be of general use to the plant pathology community studying the pathobiology of Fusarium head blight.

Complete training and validating a neural network capable of distinguishing individual yellow corn kernels infested with A. flavus, F. verticillioides, D. maydis, T. viride, or other molds and apply to bench-top instruments for machine classification of mold damaged grains, in collaboration with an ARS scientist at Manhattan, KS. Assess the expression of F. verticillioides genes during growth on live maize or in interactions with microbial colonists of maize by microarray analysis. Functionally characterize putative regulatory genes that appear to be differentially expressed under growth conditions including in a maize plant. Contrast extracellular chitinases and other antifungal proteins produced by protective corn endophytes versus mycotoxin-producing ear-rotting fungal pathogens. Examine the functional (metabolic) diversity of maize endophytes. Evaluate the competitive abilities of protective corn endophytes A. zeae and F. verticillioides in vitro and in planta. Determine if the protective corn endophyte A. zeae produces pyrrocidine antibiotics in asymptomatic corn kernels, seedlings, stalks, leaves, cobs and grain from planting through harvest. National Science Foundation, NIH and agrichemical company supported research will discover novel antifungal metabolites from cereal endophytes, mycoparasites of A. flavus sclerotia or the persistent sporulating structures of wood decay fungi with a university collaborator.

Objective 3. Establish integrated strategies to manage and to prevent mycotoxin contamination in cereal grains. (IN) We have continued to use the survey method, developed from previous NC129 projects, to monitor the Indiana corn crop. The survey is in collaboration with the Department of Botany and Plant Pathology, the Indiana Agricultural Statistics Service, and the Indiana Animal Disease Diagnostic Laboratory. This year we had a visiting scientist from Pakistan, funded by the FAO of the United Nations, whose objectives were to learn how to survey and analyze corn crops for mycotoxin.

(MO) The Fusarium/ Poultry Research Laboratory continues to evaluate mineral and organic adsorbents for binding mycotoxins in in vitro and in vivo studies. A number of chemically modified adsorbents are also being evaluated in vitro for binding of aflatoxin, vomitoxin, fumonisin, ochratoxin A, the ergot alkaloids, and zearalenone. In vivo studies, completed thus far, 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. The Fusarium/Poultry laboratory will continue to evaluate adsorbents as well as other natural feed additives for efficacy in preventing mycotoxicosis in livestock and companion animals.

(USDA) To determine whether sensitivity of corn to fumonisin mycotoxins can affect fumonisin contamination in corn, 42 genetically diverse land races of corn were screened in a laboratory assay for their sensitivity to fumonisins. Seeds of each land race were germinated on water agar containing up to 300 ¼M fumonisin B1. Only two land races were highly insensitive (ED50 200 ¼M). Genetic analysis revealed that the high level of insensitivity is an inheritable trait.

A greenhouse maize ear rot virulence assay was developed using the cultivar, Gaspe flint. This system was used to screen isolates for Fusarium for their ability to produce ear rot.

Mycoparasitic fungi are proving to be rich sources of antifungal protein/genes that can be expressed in crops, conferring resistance against fungal pathogens. Several mycoparasites isolated from A. flavus sclerotia, or as colonists of fungus-infected corn kernels at harvest, were shown 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 in a liquid minimal medium supplemented with colloidal chitin. Yields of extracellular chitinase were substantially increased by optimizing selected growth parameters.

Microbial endophytes of cereals represent under explored sources of antifungal proteins and metabolites that can suppress fungal growth or silence genes critical to mycotoxin synthesis while also being adapted to function in planta. 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. Further evaluation of the 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), in addition to the ear and kernel rotting pathogens Nigrospora oryzae, Penicillium oxalicum, and Cladosporium cladosporioides. Pyrrocidines A and B also exhibit potent antibiotic activity against Clavibacter michiganense subsp. nebraskense, an important bacterial pathogen of maize, as well as Bacillus mojaviense and Pseudomonas fluorescens, bacterial endophytes of maize used as biocontrol agents. In 2005 we developed a scalable method for the isolation and purification (crystallization) of pyrrocidines A and B.

(PA) We continued work analyzing corn silage for mycotoxins. Work this year focused on the Penicillium toxins cyclopiazonic acid, mycophenolic acid, patulin and roquefortine C. These mycotoxins were analyzed together using LC-MS. Our sample set contains collections from more than 30 dairies in Pennsylvania over two years. We found a statistically significant difference in the levels of all Penicillium toxins between the freshly chopped material and that that was ensiled. Our results indicate that Peniciliium toxins are formed in the silo. Thus, management of these toxins should focus on the silo. This data in combination with data collected earlier on the same samples shows that multi-toxin contamination is the rule. Some samples contained as many as seven different mycotoxins and many contained three or four.

Objective 4: Objective 4. Define the regulation of mycotoxin biosynthesis and the molecular relationships between mycotoxigenic fungi. (IN) A study addressed the question of whether a disk-method developed by the USDA ARS (Peoria) could be used to measure growth (ergosterol content and conidia production), polyol content and gene expression in response to various water activities. E. rubrum, Aspergillus flavus and Aspergillus nidulans were grown on disks in equilibrium with various salt solutions. The results indicated that the disk method is ideal to study gene expression and polyol production during growth under controlled moisture environments.

(IN) We 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. A mutant strain of F. verticillioides with a disrupted ±-amylase gene was impaired in its ability to produce Fumonisin B1 on starchy kernels, and both the wild-type and mutant strains produced significantly less Fumonisin B1 on a high-amylose kernel mutant of maize. When grown on a defined medium with amylose as the sole carbon source, the wild-type strain produced only trace amounts of Fumonisin B1, but it produced large amounts of Fumonisin B1 when grown on amylopectin or dextrin, a product of amylopectin hydrolysis.

(MI) We have identified the genes for biosynthesis of several mycotoxins by Fusarium graminearum. The mycotoxins include fusarin, aurofusarin, and zearalenone. From a survey of the genomic sequence, we have identified the genes encoding the major enzymes(called polyketide synthases) for each of the these mycotoxins. In addition, we have determined when these genes are expressed in each of 18 different conditions including plant infection and corn kernel colonization. Identification of the genes for zearalenone biosynthesis is particularly important, as this is an estrogenic mycotoxin of great importance to health, and an understanding of the genetics of biosynthesis has been elusive.

(WI) Define the regulation of mycotoxin biosynthesis and the molecular relationships between mycotoxigenic fungi. Keller lab uses Aspergillus nidulans as a model system to identify parameters that are global regulators of mycotoxin synthesis and spore production in mycotoxigenic fungi, specifically Aspergillus and Fusarium spp. In this past year, Keller lab has continued a collaboration with Drs. Brown and Butchko at USDA Peoria, IL to determine the conservation of the role of a mycotoxin transcriptional regulator, LaeA, and sporogenic signals derived from Ppo genes in Fusarium spp. LaeA is a transcriptional regulator of secondary metabolite gene clusters in Aspergillus spp. Disruption of this gene in A. flavus results in the loss of aflatoxin production (Keller et al. unpublished data). This gene has been transformed into F. verticilliodes to determine if it affects fumonisin production (current study, Butchko, Brown and Keller). Also, ppo genes encoding fatty acid oxygenases are conserved in filamentous fungi. Disruption of these genes results in abberant spore production, mycotoxin synthesis and loss of virulence in Aspergillus spp. Disruption of one ppo gene in F. sporotrichiodes led to loss of T2 toxin production. Current studies are aimed at determining the role of ppo and related lipoxygenase genes in F. graminearum (current study, Butchko, Brown and Keller). Efforts also continue with use of RNAi technology to silence mycotoxin production in plants (e.g. DON in wheat and aflatoxin in maize) (current study, Keller and Kaeppler). Future studies will focus on continuing with determining the role of ppo and lox genes in Fusarium pathogenicity, toxin and spore production as well as engineering wheat plants using RNAi technology to reduce DON and aflatoxin contamination.

(USDA) A significant effort of scientists in the mycotoxin research unit at NCAUR in 2005 was spent examining the metabolic pathways and regulatory mechanisms involved in mycotoxin biosynthesis, particularly for F.verticillioides, F. graminearum and Fusarium sporotrichioides.

NCAUR scientists continued to develop genomic tools in order to better 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. In FY 05, NCAUR scientists generated a F. verticillioides microarray in collaboration with the Institute for Genomic Research (TIGR). The microarray consists of >11,000 F. verticillioides gene sequences all of which are present on a glass slide. The 11,000 gene sequences present in the microarray were obtained from the F. verticillioides Expressed Sequence Tag (EST) database, which we completed during FY 04 in collaboration with TIGR. This microarray is a tool for monitoring the expression of all 11,000 gene sequences simultaneously. Previous technology allowed for analysis of expression of one gene at a time. Therefore, microarray analysis is a significant advancement in gene expression analysis that should enhance our ability to study the regulation of fumonisin biosynthesis and the interactions of F. verticillioides and corn. Such advancements should in turn aid in the development of strategies to control the presence of fumonisins in U.S. corn.

Analysis of the F. verticillioides Expressed Sequence Tags (ESTs) database 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. Conditions for production of patulin, specifically Penicillium strains and composition of culture media to use were identified. This will facilitate the production and isolation of patulin and studies of patulin biosynthesis.

Continue analysis of expression of F. verticillioides genes by microarray analysis including genes in mutant strains of F. verticillioides with defects in regulatory genes that affect fumonisin production. Examine the role of F. verticillioides polyketide synthase genes in secondary metabolism and pathogenicity with knockout mutants. Examine changes in sphingolipid metabolism in corn in response to fumonisins. Examine the role of longevity assurance factor genes in self-protection of F. verticillioides against fumonisins.

Continue the characterization of structural and regulatory genes involved mycotoxin formation in F. graminearum and involved in virulence. We will use set up and novel bioassay system in Arabidopsis thaliana to identify new toxin resistance genes. Evaluate the ability of NIV and DON producing F. graminearum strains for virulence on Gaspe flint maize.

PUBLICATIONS (Publications with authors from more than one NC-1025 location are indicated with an asterix.) Journal *Angawi, R.F., Swenson, D.C., Gloer, J.B., Wicklow, D.T. 2005. Malettins B-D: New polyketide metabolites from an unidentified fungal colonist of Hypoxylon stromata (NRRL 29110). Journal of Natural Products 68 (2): 212-216.

Bluhm, B. H. and Woloshuk. 2005. Amylopectin induces fumonisin B1 production by Fusarium verticillioides during colonization of maize kernels. Mol. Plant Microbiol. Interact. 12:1333-1339.

Bok J-W, Hoffmeister D, Maggio-Hall L, Murillo R, Glassner J, Keller NP. (2006) Genomic mining for Aspergillus natural products. Chemistry & Biology 13: 31-7. Bok J.-W., Balajee S. A., Marr K. A., Andes D., Fog Nielsen K., Frisvad J. C., Keller N. P. (2005) LaeA, a regulator of morphogenetic fungal virulence factors. Euk Cell 4: 1574-82.

Brown, D.W., Cheung, F., Proctor, R. H., Butchko, R.A.E. Zheng, L., Lee, Y., Utterback, T., Smith, S., Feldblyum, T., Glenn, A., Plattner, R.D., Butchko, R., Kendra, D.F., Town, C.D. and Whitelaw, C.A. 2005. Analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides. Fungal Genetic and Biology. 42:848-861.

Cooley, C., Bluhm, B. H., Reuhs, B. L., and Woloshuk, C. P. 2005. Glass-fiber disks provide suitable medium to study polyol production and gene expression in Eurotium rubrum. Mycologia 97:743-750.

Dakovic A, Tomasevic-Can0vic M, Dondur V, Rottinghaus GE, Medakovic V, and Zaric S. Adsorption of mycotoxins by organozeolites. Colloids and Surfaces B: Biointerfaces 46:20-25, 2005.

Deshmukh S, Asrani RK, Jindal N, Ledoux DR, Rottinghaus GE, Sharma M, and Singh SP. Effects of Fusarium moniliforme culture material, containing known levels of fumonisin B1 on progress of Salmonella gallinarum infection in Japanese quail: Clinical signs and hematological studies. Avian Diseases 49:274-280, 2005.

Deshmukh S, Asrani RK, Ledoux DR, Jindal N, Rottinghaus GE, Sharma M, and Singh SP. Individual and combined effects of Fusarium moniliforme culture material, containing known levels of fumonisin B1, and Salmonella gallinarum infection on liver of Japanese quail. Avian Diseases 49:592-600, 2005.

*Desjardins, A. E., Jarosz, A. M., Plattner, R. D., Alexander, N. J., Brown, D. W., and Jurgenson, J. E. 2004. Patterns of trichothecene production, genetic variability, and virulence to whear of Fusarium graminearum from smallholder farms in Nepal. Journal of Agricultural and Food Chemistry. 52:6341-6346. Dombrink-Kurtzman, M.A, Blackburn, J.A. 2005. Evaluation of several culture media for production of patulin by Penicillium species. International Journal of Food Microbiology 98: 241-248.

Dyer, R. B., Plattner, R. D., Kendra, D. F., and Brown, D. B. 2005. Fusarium graminearum TRI14 is required for high virulence and DON production on wheat but not for DON synthesis in vitro. Journal of Agricultural and Food Chemistry. 53:9281-9287.

*Fernandez-Surumay, G.,Osweiler, G.D., Yaeger, M.J., Rottinghaus, G.E., Hendrich, S., Buckley, L.K., Murphy. P.A. Fumonisin B-glucose reaction products are less toxic when fed to swine. J. Agric. Food Chem. 2005, 53: 4264-4271.

Foreman, J. H., Peter D. Constable, A.L. Waggoner, M. Levy, R.M. Eppley, G.W. Smith, M.E. Tumbleson, W.M. Haschek. (2004) Equine leukoencephalomalacia: Neurological abnormalities and cerebrospinal fluid changes in horses administered fumonisin B1 intravenously. Am. J. Vet Int. Med. 18: 223-230.

Gaffoor, I., and F. Trail. 2006. Characterization of two polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae. Appl. and Environ. Microbiol. 72:1793-1799.

*Gaffoor, I., Brown, D. W., Plattner, R., Proctor, R. H., Qi, W., and Trail, F. 2005. Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum). Euk Cell. 4:1926-1933.

Gupta S, Jindal N, Khokhar RS, Gupta AK, Ledoux DR, and Rottinghaus GE. Effect of ochratoxin A on Salmonella gallinarum challenged broiler chicks. British J Poultry Science 46(4): 443-450, 2005.

Hammond TM, Keller NP. (2005) RNA silencing in Aspergillus nidulans is independent of RNA dependent RNA polymerases. Genetics 169: 607-17.

*Javed, T., Bunte, R.M., Dombrink-Kurtzman, M.A., Richard, J.L., Bennett, G.A., Cote, L.M., Buck, W.B. 2005. Comparative pathologic changes in broiler chicks on feed amended with Fusarium proliferatum culture material or purified fumonisin B1 and moniliformin. Mycopathologia. 159(4):553-564.

Keller N.P, Turner G., Bennett J.W. (2005) Fungal secondary metabolism: from biochemistry to genomics. Nature Reviews Microbiology 3:937-47.

*Kleinschmidt, C.E., Clements, M.J., Maragos, C.M., Pataky, J.K., White, D.G. 2004. Evaluation of food-grade dent corn hybrids for severity of Fusarium ear rot and fumonisin accumulation in grain. Plant Disease. 89(3):291-297.

Kottapalli, B., C.E. Wolf-Hall, and P. Schwarz. 2005. Evaluation of Gaseous Ozone and Hydrogen Peroxide Treatments for Reducing Fusarium Survival in Malting Barley. J. Food Protection. 6:1236-1240.

Landgren C. 2005. Murine immunomodulation by low-dose dietary deoxynivalenol and improved detection methods for deoxynivalenol. Ph. D. Dissertation, Iowa State University Library, Ames, IA.

*Li, C., Gloer, J.B., Wicklow, D.T., Dowd, P.F. 2005. Antiinsectan decaturin and oxalicine analogs from Penicillium thiersii. Journal of Natural Products. Journal of Natural Products 68 (3): 319-322.

Maggio-Hall L., Wilson R., Keller N. P. (2005) Fundamental contribution of b-oxidation to polyketide mycotoxin production in planta. Mol Plant Microbe Interactions: 18: 783-793.

Manning BB, Terhune JS, Li MH, Robinson EH, Wise DJ, and Rottinghaus GE. Exposure to feed-borne mycotoxins T-2 toxin or ochratoxin A causes increased mortality of channel catfish challenged with Edwardsiella ictaluri. J Aquatic Animal Health 17(2):147-152, 2005.

Mansfield, M. A., De Wolf, E. D., and Kuldau, G. A. 2005. Effect of weather conditions, cultural practices, and feed quality on deoxynivalenol content in maize silage. Plant Disease, 89:1151-1157.

Maragos, C.M. 2004. Detection of moniliformin in maize using capillary electrophoresis. Food Additives and Contaminants. 21(8):803-810.

Maragos, C.M. 2004. Emerging technologies for mycotoxin detection. Journal of Toxicology Toxin Reviews. 23(2-3):317-344.

McAlpin, C.E., Horn, B.W., Wicklow, D.T. 2005. DNA fingerprinting analysis of vegetative compatibility groups in Aspergillus caelatus. Mycologia 97 (1): 70-76.

*McDonald T., Brown D., Keller N. P., Hammond TM. (2005) Inverted Repeat Transgenes Silence Mycotoxin Production in Aspergillus and Fusarium species. Mol Plant Microbe Interactions 18:539-545.

McDonald T, Noordermeer D, Zhang Y-Q, Hammond T, Keller NP (2005) The ST cluster revisited: Lessons from a genetic model. Book Chapter in Aflatoxin and Food Safety, Marcel Dekker, Inc.

Neirman et al. (many authors) (2005). Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438:1151-6.

Naudè TW, Botha CJ, Vorster JH, Roux C, van der Linde E, van der Walt SI, Rottinghaus GE, van Jaarsveld L, and Lawrence AN. Claviceps cyperi, a new cause of serious ergotism in dairy cattle consuming maize silage and tef hay contaminated with ergotised Cyperus esculentus (nut sedge) on the Highveld of South Africa. Onderstepoort J Vet Tox 72(1):23-37, 2005.

Ogunbo SO, Ledoux DR, Bermudez AJ, and Rottinghaus GE. Effects of fusaric acid in broiler chicks and turkey poults. International J Poultry Science 4(6): 356-359, 2005.

Peterson, S.W., Bayer, E.B., Wicklow, D.T. 2004. Penicillium thiersii, Penicillium angularae and Penicillium decaturense, new species isolated from wood-decay fungi in North America and their phylogenic placement from multilocus DNA sequence analysis. Mycologia 96 (6): 1280-1293.

*Ravi S. Bojja, Cerny, R.L., Proctor, R.H. and Du, L. 2004. Determining the biosynthetic sequence in the early steps of the fumonisin pathway by use of three gene-disruption mutants of Fusarium verticillioides. Journal of Agricultural and Food Chemistry. 52:2855-2860.

Rooney, A.P., Swezey, J.L., Wicklow, D.T., McAtee, M.J. 2005. Bacterial species diversity in cigarettes linked to an investigation of severe pneumonitis in U.S. military personnel deployed in operation Iraqi freedom. Current Microbiology [serial online] Available: http://www.springerlink.com.

Tessari ENC, Oliveira CAF, Cardoso ALSP, Ledoux DR, and Rottinghaus GE. Effects of aflatoxin B1 and fumonisin B1 on the levels of serum aspartate amino-transferase and total protein of broilers. Arquivos do Instituto Biol 72 (2):185-189, 2005.

Tsitsigiannis D I and Keller NP. Oxylipins act as determinants of natural product biosynthesis and seed colonization in Aspergillus nidulans (2006) Mol Microbiol 59:882-92.

Tsitsigiannis D. I., Kunze S, Willis D K, Feussner I, Keller NP. (2005) Aspergillus infection inhibits the expression of peanut 13S-HPODE-forming seed lipoxygenases. Mol Plant Microbe Interactions 18: 1081-1089.

Tsitsigiannis D. I., Koweiski, T., Zarnowski R., Keller N.P. (2005) Three putative oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans. Microbiology 151:1809-21.

Tsitsigiannis D. I., Bok J.-W., Andes D., Fog Nielsen K., Frisvad J. C., Keller N. P. (2005) Aspergillus cyclooxygenase-like enzymes are associated with prostaglandin production and virulence. Infect Immunol: 73:4548-59.

Verkley, G.J.M., da Silva, M., Wicklow, D.T., Crous, P.W. 2004. Paraconiothyrium, a new genus to accommodate the mycoparasite Coniothyrium minitans, anamorphs of Paraphaeosphaeria, and four new species. Studies in Mycology (Centenary Volume) 50: 323-335.

Wicklow, D.T., Roth, S., Deyrup, S.T., Gloer, J.B. 2005. A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides. Mycological Research 109 (5): 610-618.

Yu J-H, Keller N.P. Regulation of secondary metabolism in filamentous fungi. (2005) Ann Rev Phytopathology: 43:437-58.

Abstracts and Proceedings Boddeda, A. and C.E. Wolf-Hall. 2005. Effect of Storage Time and Moisture on Deoxynivalenol and Ochratoxin A in Raw Barley. Annual meeting of the Midwest Section American Society for Microbiology. Ames, Iowa, September 22-23.

Boddeda, A. and C.E. Wolf-Hall. 2005. Multiplex real-time PCR method to simultaneously detect and quantify deoxynivalenol and ochratoxin A producing fungi. Poster at the National Fusarium Head Blight Forum. Milwaukee, Wisconsin, December 11-13.

Haschek, W. M. (2004) Cardiovascular effects of chronic fumonisin B1 ingestion in Sinclair minipigs. Tox Sci 78(1-S):Abs 828.

Hsiao S-H, Constable P, Tumbleson M, Haschek, W. M. (2004) Both sphinganine and sphingosine are cytotoxic to H9C2[2-1] cardiomyocytes and HEPG2 hepatocytes. Tox Sci 78(1-S):Abs 823.

Kuldau, G. and C. Bacon. 2005. Clavicipitaceous endophytes, their ability to enhance plant resistance to multiple stresses. Phytopathology 95:S138.

Kuldau, G. A., and M. A. Mansfield. 2006. Mycotoxins and mycotoxigenic fungi in silages. Silage for Dairy Farms Conference, pp. 91-99, January 23-25, 2006, Camp Hill, PA, Natural Resource, Agriculture and Engineering Service, Ithaca, NY.

Mansfield, M. A. and Kuldau, G. A. 2005. Levels and frequencies of Penicillium mycotoxins in fresh and ensiled maize. Phytopathology 95:S64.

Mansfield, M. A., Archibald, D. D.,Jones,A. D., and Kuldau, G. A. 2005. Frequency and levels of two classes of sphinganine analog mycotoxins in maize silage. Phytopathology 95:S171.

Nita, M., Tilley, K., DeWolf, E., and Kuldau, G. 2005. Effects of moisture during and after anthesis on the development of Fusarium Head Blight of wheat and mycotoxin production. In: Canty, S. M., Lewis, J., Siler, L., and Ward, R. W. (Eds.), Proceedings of the National Fusarium Head Blight Forum; 2005 Dec. 11-13, Milwaukee, MN. East Lansing: Michigan State University. pp. 125-128.

Smith, G. W., Constable, P. D., Fredrickson, R. L., Tumbleson, M. E., Eppley, R. M., Haschek, W. M. (2004) Cardiovascular effects of chronic fumonisin B1 ingestion in Sinclair minipigs. Tox Sci 78(1-S):Abs 828.

Smith, G. W., W. M. Haschek, R. L.. Fredrickson, M. E. Tumbleson, R. M. Eppley, J. James, P. D. Constable (2004). Effect of fumonisin B1 ingestion on serum folate, homocyteine, and methionine concentrations in Sinclair minipigs. Experimental Biology, Abs. 8456, APS  American Physiological Soc., Topic 1009  APS Blood pressure regulation (Cardiovascular Section Posters)

Riley, R.T., Gelineau-van Waes, J. ,Voss, K.A., Sharma, R.P., Haschek, W. M. and Constable, P. D. Toxicology of the fumonisins and neural tube defects. XI International IUPAC Symposium, May 17-21, 2004, O-20, p 32-33.

Vijayakumar, C., C.E. Wolf-Hall, and F. Manthey. 2005. Fusarium infection, DON content and microbial loads in durum wheat from the Northern Plains: 2001-2004. Poster at the National Fusarium Head Blight Forum. Milwaukee, Wisconsin, December 11-13.

Zhang, H., C.E. Wolf-Hall, and C.A. Hall III. 2005. Deoxynivalenol and 15-acetyldeoxynivalenol production by Fusarium graminearum strains grown in semi-defined media with different carbohydrate sources. Poster at the National Fusarium Head Blight Forum. Milwaukee, Wisconsin, December 11-13.

Zitomer, N. C., Geiser, D. M., Archibald, D. D., Jimenez-Gasco, M. M., ODonnell, K., and Kuldau, G. A. 2005. The phylogenetics of fumonisin production in the Gibberella fujikuroi species complex. Phytopathology 95(6): S173.