Brief Summary of Minutes of Annual Meeting

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.