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NC1025: Mycotoxins:Biosecurity and Food Safety (NC129)

Annual/Termination Reports (SAES-422): [08/07/2006] [06/25/2007] [07/07/2008] [07/22/2009]

Date of Annual Report: 08/07/2006

Report Information:
  • Annual Meeting Dates: 05/20/06 to 05/22/06
  • Period the Report Covers: 05/2005 to 05/2006

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Refer to NC129 Termination Report.


    URL: Copy of minutes

    Accomplishments:
    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.

    Impact Statements:
    1. (IL) We have improved the assay used to determine changes in tissue and body fluid sphingolipids that serve as a biomarker for fumonisin B1 exposure. We have also shown that this assay can be done on formalin fixed tissues that will allow retrospective determination of exposure. Our study in pigs demonstrated that sphingolipid changes persist after fumonisin exposure ceases, thus indicating that these sphingolipids are good biomarkers of exposure.
    2. (IA) The biological impact of deoxynivalenol and its metabolites can be assessed in cell culture revealing that only free deoxynivalenol appears to be the toxic constituent. This information will be used in developing strategies to mitigate the impacts of deoxynivalenol-contaminated grain.
    3. (MO) Poultry feeding studies examined a number of adsorbent clays and bacterial enzymes in vitro and in vivo for their effectiveness in reducing the toxicity of mycotoxin-contaminated feedstuffs. This information is used by industry for effective feed formulation and recommendations to producers.
    4. (MO) Fumonisin, ochratoxin A, moniliformin, zearalenone, and aflatoxin B1 in culture material are made available to research groups making it economically feasible to undertake animal feeding studies in cattle, swine, poultry, equine, mink, catfish, and laboratory animals. This effort facilitates numerous animal toxicology studies that might not otherwise be possible thus contributing to our knowledge of mycotoxin impacts.
    5. (MO) Utilization of our analytical facilities allowed hundreds of contaminated dog food samples to be quickly analyzed for aflatoxins and helped prevent additional animal loss.
    6. (IN) The information on PCR methodologies for detection mycotoxigenic fungi is potentially important for monitoring the population genetics behind fungal epidemics in crops and for biosecurity.
    7. (USDA) Isolation and characterization of Fusarium genes involved in mycotoxin synthesis and regulation continue to aid efforts to control mycotoxin formation in food crops.
    8. (USDA) 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.
    9. (IN) The survey information is published in newsletter and presented during extension meeting, which allows producers, handlers and processors know the condition of the crop.
    10. (ND) Barley with mild FHB may lead to the production of mycotoxins during malting. Maltsters have strict limits for malt quality that ultimately have severely affected barley production in the USA. Treatment of FHB infected barley may prevent mold growth and further mycotoxin production during malting allowing utilization of otherwise good quality barley.
    11. (PA) We found that the Penicillium toxins cyclopiazonic acid, mycophenolic acid, patulin and roquefortine C increase during ensiling. Producers wishing to manage these toxins should focus their efforts on the silo and not the field.
    12. (PA) We found that of the silage samples that contained toxin, the majority of those had three or more and up to seven different toxins present. Detailed analysis such as this has not been previously reported. Scientists working to understand how mycotoxins in dairy diets exert adverse effects will be able to use this information to perform more realistic tests.
    13. (IN) The disk-method is useful to study the potential competitiveness between fungal species in low water environments associated with stored grain.
    14. (IN) This maize kernel colonization study provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.
    15. (MI) Characterization of the expression patterns for these genes will allow us to understand how mycotoxin production is regulated. This will enable us to work on management strategies.
    16. (WI) Results from these RNAi studies should help in identifying virulence attributes of mycotoxigenic fungi, possibly providing for antifungal targets, as well as reducing mycotoxin contamination of crops.
    Last Modified: 13-Nov-2007

    Date of Annual Report: 06/25/2007

    Report Information:
  • Annual Meeting Dates: 05/22/07 to 05/23/07
  • Period the Report Covers: 01/2006 to 12/2006

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    II. Introduction

    Meeting called to order at 8:35 by Chair, Gretchen Kuldau.

    Barb Christ, department head for the Penn State Department of Plant Pathology gave welcoming comments and some history of the department.

    Bev Durgan, the NC-1025 administrator indicated that the project was up to date with reporting. She will work with station directors on attendance issues.

    III. Station Reports

    The station reports began at 8:50 AM, and included reports from Pat Murphy for Iowa, Wanda Haschek-Hock for Illinois, Charles Woloshuk for Indiana, John Leslie for Kansas, Iffa Gaffor for Michigan, Charlene Wolf-Hall for North Dakota, Katelyn Tilley (graduate student) & Gretchen Kuldau for Pennsylvania, and Won-Bo Shim for Texas.

    Additional comments on the impact of mycotoxins on biofuels were provided by Mike Tumbleson of Illinois.

    IV. Business Meeting

    The chair, Gretchen Kuldau, called the business meeting to order at 3:05 PM.

    The minutes of the 2006 meeting were discussed.

    An update on the annual report was presented by Charles Woloshuck and Bev Durgan. There is a new format to follow which is significantly more concise. Charles will send the draft out for comment. Items that should be highlighted include collaborations, especially amongst the participants. Bev pointed out that this is a diverse group, spanning plants and animals, which helps to strengthen these types of connections.

    Frances Trail was recommended for the position of secretary in 2008.

    The 2008 meeting will be held in conjunction with the Midwest AOAC meeting in Bozeman MT. The dates for the AOAC meeting are May 19-22nd. Charlene is serving as the ND rep for the SD-ND-MT AOAC organizing committee, and will organize the NC-1025 session and business meeting. Wanda volunteered to help with organizing the session. We will showcase what NC-1025 does. A central theme would be developed with input suggested from Scott Smith, George Rottinghaus and Chris Maragos. Wanda suggested we include a separate poster session of recent but possibly already presented work for the NC-1025 meeting, and others agreed this would be a good idea.

    The 2009 meeting will be organized by Charles Woloshuk of Indiana.

    Meeting was adjourned at 3:30.

    Minutes respectfully submitted by Charlene Wolf-Hall

    Accomplishments:
    Goal 1. Develop data for use in risk assessment of mycotoxins in human and animal health The group at Iowa conducted a feeding study on twenty healthy adults to evaluate the effects of consuming bread contaminated with deoxynivalenol (DON). Of the group, four men and four women ate bread containing 300 µg DON at breakfast. Blood drawn from the test group was added to K-562 cells cultures to assay for DON toxicity. The results of the study indicated that this cell culture-bioassay could provide a relatively inexpensive approach for detecting DON in human subjects exposed to DON in their diets.

    Several members conducted surveys to determine the potential for mycotoxins in their respective states. Areas of Iowa have experienced drought or near-drought in two of the last four years. As a result, a modest level of aflatoxin contamination was found in the analysis of over 200 samples. By contrast, relatively unfavorable weather for the Fusarium mycotoxins may be in part responsible for the low prevalence of deoxynivalenol or zearalenone from the same time periods. In 2006, Indiana experienced relatively mild weather with some areas experiencing record rainfall. As a result, surveys indicated that DON contamination in corn was wide spread. High levels of fumonisin contamination, up to 174 ppm, were also recorded. A Kansas survey, which examined corn samples from four regions in the state, found fumonisin (75% of samples), beauvericin (63%), zearalenone (34%), T-2 toxin (30%), ochratoxin (26%), deoxynivalenol (8%), fusaproliferin (8%), and aflatoxin (1%).

    The Illinois group investigated an outbreak of fumonisin induced porcine pulmonary edema (PPE). Fumonisins were detected in stomach contents (FB1 > 5ppm) and feed (FB1 = 23ppm, FB2 = 6ppm). Serum had markedly elevated sphinganine and sphingosine concentrations as well as sphinganine to sphingosine ratio (average 3.22; experimental control reference range 0.00-0.18), confirming fumonisin mycotoxicosis. Equine leukoencephalomalacia was also reported in Illinois from the 2006 corn crop (Diagnostic Laboratory in Centralia, IL).

    The Illinois group and USDA/ARS also investigated the cytotoxicity of pyrrocidine A, an antibiotic produced by the maize endophyte Acremonium zeae. The group found that pyrrocidine A induces apoptosis in HepG2 and PK15 cells with initiation through both the intrinsic and the receptor mediated apoptotic pathways.

    Goal 2. Develop new techniques and improve current assays to identify and measure mycotoxins and mycotoxigenic fungi in cereal grains

    Two groups (North Dakota and Indiana) are developing gene-based methods to detect and quantify mycotoxigenic fungi. A multiplex real-time PCR method was developed for detection of the ochratoxin-producers Aspergillus ochraceus and Penicillium verrucosum and the trichothecene-producer Fusarium graminearum. A multiplex real-time PCR method based on TaqMan technology is being developed that will detect and quantify at the genus level species of Aspergillus, Penicillium, Eurotium, and Fusarium.

    With the increased utilization of distillers grain products (DDGs) in animal feeds the group in Missouri examined new analytical methodology to address problems associated with this complex matrix. They found that the traditional sample clean up in combination with TLC analysis is unsuitable for detecting mycotoxins in DDGs. This group experimented with immunoaffinity columns for sample cleanup with TLC and HPLC to screen and quantify, respectively, for mycotoxins.

    Kansas with a collaborator at the International Institute of Tropical Agriculture (Ibadan, Nigeria) tested the efficacy of sorting of corn kernels into good and bad categories by subsistence farmers in Nigeria. The results suggest that the method could significantly reduce the exposure to fumonisins by 20-25 fold.

    Goal 3. Establish integrated strategies to manage and to prevent mycotoxin contamination in cereal grains The group from Michigan has focused their effort towards understanding the role polyketide-derived mycotoxins have on the pathogenicity and survival of F. graminearum under field conditions. This group followed the expression of three polyketide synthase genes involved in the biosynthesis of fusarin C, zearalenone, and aurofusarin. All three genes were expressed during colonization of wheat and barley, particularly during the early stages of head colonization. In culture media, only the polyketide synthase gene involved in aurofusarin biosynthesis is expressed to a higher level.

    The group from North Dakota has found that irradiation of F. graminearum with electron beam caused mutations in the fungus and reduced mycotoxin production by the fungus.

    The group from Missouri has led the effort to evaluate the efficacy of adsorbents in feed to reduce the detrimental effects of mycotoxins on animal health. This year they evaluated several adsorbents with minerals (copper or zinc) attached to determine if the added minerals provide beneficial growth responses in poultry while at the same time binding aflatoxins in the feed. The results showed that Montmorillonite (MONT), an aluminosilicate clay and Cu-MONT at 0.5% of the diet were partially effective in ameliorating the toxic effects of aflatoxin in broilers. Tests of several commercial adsorbents indicated that the adsorbents were effective in ameliorating the toxic effects of aflatoxin in broilers without a negative effect on chick performance.

    Wisconsin examined conserved signaling molecules that affect mycotoxin production and spore development in Fusarium and Aspergillus species. This group found that oxygenated lipids (e.g. oxylipins), synthesized by both plants and fungi, are important for induction or suppression of toxin genes in both genera.

    To understand the environmental factors and infection timing favorable for production of wheat kernels asymptomatic for head scab caused by F. graminearum, Pennsylvania conducted a field study that utilized movable greenhouse enclosures and supplemental misting to control timing of infection. Results indicated that infections during the grain fill period could result in asymptomatic kernels with greater than 2 ppm deoxynivalenol in some cultivars of winter wheat.

    Goal 4. Define the regulation of mycotoxin biosynthesis and the molecular relationships between mycotoxigenic fungi

    Several groups (Texas, Indiana, and USDA/ARS) have focused their effort towards understanding the regulation of fumonisin biosynthesis in F. verticillioides. It was discovered that the amylopectin component of corn kernels induces fumonisin biosynthesis. By microarray analysis, several putative genes were identified and found to be involved in fumonisin biosynthesis and sensing of the corn kernel environment. Putative fumonisin regulatory genes also were identified via subtractive suppressive hybridization and microarray analysis. One such gene was GBP1, encoding a monomeric G protein, which negatively regulates fumonisin biosynthesis. Another gene GBB1, encoding a heterotrimeric G-protein beta subunit, was found to have a role in fumonisin biosynthesis and fungal development. Disruption of GBB1 caused drastic reduction in fumonisin production but growth and fungal virulence was not affected.

    The Kansas group has investigated vegetative compatibility in F. proliferatum for potential strategies to control Fusarium spp. They cloned the het-c homolog of F. proliferatum. This gene has vegetative compatibility function in Neurospora crassa, but this function is not conserved in F. proliferatum. The results suggest that genes responsible for triggering an incompatibility response, which is cell death, may be limited to one or a few fungal species.

    Impact Statements:
    1. As in previous years, the immediate impact of NC1025 efforts is most visible from the surveys and testing of commercial products. Our surveys, which showed an occurrence of higher than normal mycotoxin contamination, provided needed information to the livestock and ethanol industries. As a result, the need was high for information about the efficacy of mycotoxin adsorbents and methods for analyzing DDGs. Communication among NC1025 member is good, which allowed information to easily flow to the clientele groups. The high levels of fumonisin contamination of the 2006 midwest corn crop (up to 174 ppm fumonisins reported from Indiana corn) is reflected in spontaneous outbreaks of disease in pigs and horses. We believe that this is the first documented case of PPE in the US since 1989.
    2. 1. A DON bioassay that is inexpensive, which should lead to improved health monitoring for exposure in human populations
    3. 2. Molecular assays to monitor mycotoxigenic fungi in food and feed
    4. 3. A better understanding of molecular regulation of mycotoxin biosynthesis during growth and pathogenesis
    5. 4. Pyrrocidines are in need of risk assessment.
    Last Modified: 28-Jun-2007

    Date of Annual Report: 07/07/2008

    Report Information:
  • Annual Meeting Dates: 06/10/08 to 06/11/08
  • Period the Report Covers: 01/2007 to 12/2007

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Accomplishments:
    Accomplishments: Objective 1. Develop data for use in risk assessment of mycotoxins in human and animal health In 2006, the NCAUR group examined the antifungal activity of the pyrrocidines antibiotics produced by the corn endophyte Acremonium zeae. The goal was to determine the potential use of A. zeae as a biocontrol agent against mycotoxigenic fungi. This past year, the Illinois group performed preliminary in vivo studies to evaluate the potential of pyrrocidines to induce whole animal toxicity. Based on clinical signs, organ weights, and gross and microscopic alterations, toxicity was not identified at doses up to 10 mg pyrrocidine A/kg body weight when given as a single intraperitoneal dose to mice. The mouse model has been used to develop effective biomarkers for exposure to mycotoxins such as deoxynivalenol (DON). One biomarker that has attracted interest is the effects on peripheral blood lymphocytes. The Iowa group has tested the hypothesis that DON suppresses peripheral blood lymphocytes at 1 ppm but does not suppress at lesser doses in BALB/c mice. Peripheral blood and spleen cell suspensions were analyzed for a group of 10 female and male BALB/c mice fed DON at 0, 0.25, 0.5, 1.0, and 2.0 ppm for 14 and 28 days. In peripheral blood, the percentage of B cells and their numbers were inhibited in both sexes of BALB/c mice after 14 days of exposure to 1.0 or 2.0 ppm DON; whereas, exposure to DON over 28 days did not inhibit B cells when compared with the control diet. Monocytes (CD11b+) in peripheral blood and numbers of spleen macrophages were decreased only in female mice fed 1.0 and 2.0 ppm DON after 28 days compared with control diet. Dietary DON did not influence hematology in males but hemoglobin was suppressed in female mice at all DON doses over 14 days but not after 28 days. BALB/c mice seemingly adapted to DON exposure because peripheral blood cellular effects of DON at 14 d disappeared by 28 d with the exception of monocyte changes in females, suggesting that female sex hormones potentiated one aspect of DON immunotoxicity in BALB/c mice. The Missouri group conducted a study to determine the effects of dietary aflatoxin (AF) on hepatic gene expression in male broiler chicks. Microarray analysis was used to identify shifts in genetic expression associated with the affected physiological processes in chicks fed 0 and 2 mg AF/kg of feed to identify potential targets for pharmacological/toxicological intervention. Microarray data were analyzed using a 2-step ANOVA model and validated by quantitative real-time PCR. Compared with controls, various genes associated with energy production and fatty acid metabolism (carnitine palmitoyl transferase), growth and development (insulin like growth factor), antioxidant protection (glutathione S transferase), detoxification (epoxide hydrolase), and immune protection (interleukins) were down-regulated, whereas genes associated with cell proliferation (ornithine decarboxylase) were up-regulated in birds fed AF.

    Objective 2. Develop new techniques and improve current assays to identify and measure mycotoxins and mycotoxigenic fungi in cereal grains With the increased production of ethanol from corn, utilization of the major byproduct, distillers grain, has made it necessary to modify old methods or develop new methods for sample clean up and analysis of complex matrices used as feed. As a result, the Missouri group evaluated and adapted stacked immunoaffinity column cleanup for mycotoxins along with HPLC detection to measure relevant levels of aflatoxins, deoxynivalenol, zearalenone, ochratoxin A, and fumonisin in animal feedstuffs containing distillers grain. As part of a comprehensive approach for monitoring the presence of mycotoxigenic fungi in grain and grain products, the Indiana group has developed a multiplex real-time quantitative PCR assay to detect and quantify mycotoxigenic fungi. The assay uses genus-specific probes for the important mycotoxigenic Aspergillus, Fusarium, and Penicillium species. The assay can detect at least 25 mycotoxigenic species with a linear range of quantification between 10 pg and 100 ng of DNA. The assay was validated by an analysis of fungal growth in distillers grain. This assay complements the mycotoxin-specific assays already developed. The assay was used to assist in a case of spoiling distillers grain stored at a feed facility. The analysis indicated that one sample contained Fusarium species which were subsequently determined to be trichothecene-producers. The Pennsylvania group developed an assay to detect the mycotoxins deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, and the fungus-specific sterol ergosterol in single florets of wheat. The method uses standard extraction and clean-up procedures for the trichothecene mycotoxins in conjunction with the isolation of ergosterol from the plant tissue. The cleaned extract is derivitized and the analytes of interest are detected by gas chromatography with electron capture detection. Objective 3. Establish integrated strategies to manage and to prevent mycotoxin contamination in cereal grains The NC1025 committee has approached this objective through investigations that focus on the categories: intervention, surveys and plant genetics. With respect to intervention, the Missouri group evaluated the efficacy of curcumin, an antioxidant supplied by turmeric (Curcuma longa) powder, to combat the down regulation of antioxidant protection by aflatoxin. The group made their evaluation by monitoring changes in gene expression in liver of broiler chicks fed aflatoxin. Addition of curcumin to the AFB1 diet ameliorated the negative effects of aflatoxin on growth performance and liver weight. At the end of the three-week treatment period, livers were evaluated for changes in the expression of genes involved in antioxidant function and immune response by quantitative real time PCR. Inclusion of curcumin in the diet alleviated the effects of aflatoxin on the expression of several genes. The Missouri group also continued to evaluate the efficacy of proprietary adsorbents to prevent zearalenone toxicity in a non-invasive swine model. Twelve weanling gilts (6 per treatment) averaging 10.1 kg initial body weight were used in a 3-week experiment. Dietary treatments included an NRC control diet, and a diet containing 2 mg zearalenone (ZEA)/ kg diet. On days 0, 4, 7, 14, and 21 vulva measurements (length and width) were made on individual pigs. Vulva width and length were greater in pigs fed ZEA. Reproductive tract weights (tubular, vagina, and total tract) were also greater in pigs fed ZEA. Vulva width was significantly correlated with vaginal weight, tubular weight, and total reproductive tract weight. Due to concern for the potential contamination of ergot alkaloids in the malting and brewing processes of beer production, the North Dakota group investigated the impact of thermal treatment on the fate of ergot alkaloids present in sclerotia obtained from malting barley and wheat. Individual ergopeptide alkaloids were determined by HPLC, and water-soluble alkaloids were determined by ELISA. Steeping appears to solubilize a small amount of peptide and water-soluble alkaloids, with kilning resulting in a more significant reduction (H30%). Beer was prepared from malt grist containing 0.1% (w/w) sclerotia. Mass balance calculations indicated that 32, 10, and 2 percent of the original total ergopeptide alkaloids were recovered in the spent grain, wort, and beer, respectively. Average levels measured in beer were H10 ng/mL. This was investigated further by treating aqueous extracts of sclerotia (pH 5.0) at 45, 70, and 100°C for 1 hr. Losses of up to 46% of the original ergopeptide alkaloids were observed after 1 hr at 100°C. The Wisconsin group has led an effort to use RNAi technology to control mycotoxin contamination in corn (aflatoxin) and wheat (trichothecene). This group has obtained T0 corn plants transformed with an inverted repeat of aflR, the transcription factor required by Aspergillus flavus for aflatoxin biosynthesis. Their hypothesis is that the interference-RNA expressed in the transgenic plant can actually make its way into the pathogen, where it inhibits mycotoxin biosynthesis. Inoculation of the T0 plants with A. flavus yielded preliminary data suggesting that aflatoxin production is reduced in the transgenic plants. Further breeding will produce homozygous plants that will be tested in the lab in fall of 2008 and in the field of summer 2009. The group from Pennsylvania continued a study initiated in 2006 aimed at understanding the environmental factors and infection times that are favorable for production of DON in wheat kernels asymptomatic for head scab caused by F. graminearum. The field study utilized movable greenhouse enclosures and supplemental misting to control timing of infection. Results were similar to those found in 2006 and indicated that infections during the grain fill period could result in asymptomatic kernels with greater than 2 ppm deoxynivalenol in some cultivars of winter wheat. The Michigan group has also studied Fusarium graminearum; namely, they are studying the formation and dissemination of inoculum from the sexual stage. Forcible discharge of sexual spores is essential to initiation of the disease cycle of this fungus. Through expression analysis with the Fusarium GeneChip (Affymetrix), expression of 2068 genes is specific to sexual development. Two genes were identified that are responsible for the regulation of ascospore discharge.

    Objective 4. Define the regulation of mycotoxin biosynthesis and the molecular relationships between mycotoxigenic fungi The groups from Texas, Indiana, and NCAUR have worked together on genes that affect fumonisin biosynthesis in Fusarium verticilliodes. Studies on GBB1, a gene encoding a putative beta subunit of a heterotrimeric G protein, were started in 2006 and completed in 2007. A GBB1 deletion mutant showed no significant differences in radial growth and mycelial mass but produced significantly less Fumonisin B1 (FB1) than its wild-type progenitor. Reduced expression of the key FB1 biosynthetic genes, FUM1 and FUM8, in provides further evidence that GBB1 is involved in FB1 regulation. Stalk rot virulence, as measured by mean lesion length and by area, was not significantly different in compared to the wild type, suggesting that GBB1 does not regulate virulence in F. verticillioides. Complementation of BM83 with GBB1 restored FB1 production and hyphal growth to wild-type strain. ZFR1 is another gene that appears to affect fumonisin biosynthesis. A ZFR1-deletion strain grew approximately 2.5 fold less than wild type on endosperm tissue and a variety of other carbon sources including glucose and amylopectin. However, the strain displayed higher alhpa -amylase activity and expression of genes involved in starch saccharification than wild type, thus indicating that the reduced growth of the ZFR1-deletion strain was not due to inhibition of amylolytic enzymes. In the wild-type strain, expression of six genes encoding putative sugar transporters was significantly greater on endosperm tissue relative to germ tissue, and expression of at least 3 of the 6 genes was negatively affected by disruption of ZFR1. Intriguingly, disruption of one of the sugar transporters, FST1, had no effect on growth, kernel colonization, or kernel pH but decreased FB1 production by approximately 82% on maize kernels. AREA-orthologues in fungi are global regulators of nitrogen metabolite as well as secondary metabolism. An AREA-deletion mutant in F. verticillioides grew poorly on mature maize kernels, but grew similar to wild type (WT) with the addition of ammonium phosphate. Fumonisin B1 was not produced by AREA-deletion strain under any condition or by the WT with added ammonium phosphate. Constitutive expression of AREA rescued the growth and FB1 defects in AREA-deletion strain. The results pf the study supported the hypothesis that FB1 biosynthesis is regulated by AREA. The Wisconsin group has explored the global regulation of mycotoxin biosynthesis through mechanisms that involve chromatin changes. This group has discovered that the protein LaeA in Aspergillus species regulates the expression of genes in multiple mycotoxin clusters. Their data suggest that regulation by LaeA works through the activation of euchromatin. The NCAUR is collaborating with the Wisconsin group to determine if a similar mechanism controls mycotoxin production in Fusarium spp. Wisconsin is investigating the regulation of mycotoxin production in Aspergillus and Fusarium species by activated oxygen species known as oxylipins. With collaborators from Texas A&M and NCAUR they are investigating the role of both plant and fungal oxylipins in stimulating mycotoxin biosynthesis.

    Impact Statements:
    1. The research results on the non-toxicity of pyrrocidines appear to be good news because significant levels of the metabolites have been identified in preharvest corn. The result on total dietary exposure to DON indicate that 0.5 ppm or less would not exert immunotoxicity in terms of suppression of peripheral blood lymphocyte populations. The main outcome of this study is that the current voluntary action level for US food manufacturers for DON (1.0 ppm) may need to be reevaluated based on these data. Results from the microarray study demonstrate that the physiological responses associated with aflatoxin exposure correlates with altered gene expression in chick livers, which may lead to the identification of effective biomarkers for AF exposure.
    2. Veterinary toxicology laboratories will benefit from the evaluation made of the commercial affinity column methodology for detecting mycotoxins in grains and the improved detection of mycotoxins in livestock feeds. As the first step in a monitoring system, the multiplex PCR assay will guide subsequent decisions for toxin-specific PCR and mycotoxin analysis. The method for detection of trichothecene mycotoxins and ergosterol in single wheat florets will be used by researchers to study the movement of Fusarium graminearum and the mycotoxins it produces in the wheat head during development of wheat head scab disease.
    3. Results from the evaluation of curcumin demonstrate that the chemical has protective activity in livers of chicks fed aflatoxin. Based on the results from the non-invasive swine model, the vulva width can be used as a response variable to evaluate the effects of zearalenone, and differences in vulva width between treatments were observed as early as 4 days after treatment began. The results of malting studies also suggest that cross contamination of barley with ergot alkaloids can occur. A large amount of the original ergopeptide alkaloids was lost during brewing and is believed to have resulted from thermal degradation.
    4. Impact 3 continued... The preliminary results from transgenic corn suggest that RNAi technology may have potential use in the management of mycotoxin contamination. Results from the experiments to determine the conditions for mycotoxin contamination of asymptomatic kernels indicate that a larger number of wheat varieties should be examined to determine if the results are consistent across varieties. The information obtained from the analysis of F. graminearum genechips provided a better understanding how sexual spores develop and fire.
    5. Impact 4 The results imply that cross-talk between the host and the pathogen has a significant impact on mycotoxin contamination in the field. Furthermore,the results demonstrate the important role several genes have in the regulation of mycotoxin biosynthesis during growth of the host.
    Last Modified: 21-Jul-2009

    Date of Annual Report: 07/22/2009

    Report Information:
  • Annual Meeting Dates: 05/19/09 to 05/20/09
  • Period the Report Covers: 01/2008 to 12/2008

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    NC-1025 Annual meeting minutes  20 May 2009  Turkey Run State Park, Marshall, IN

    The annual meeting of NC-1025 began at 8:00 a.m. on Wednesday morning May 20, 2009 in the Dogwood room of the Turkey Run Inn in Turkey Run State Park, Marshall, Indiana. Registra-tion of $30 per person was collected for the meeting.

    Minutes for the previous years meeting with the Midwest AOAC in Bozeman, Montana were not submitted by Dr. David Kendra and were unavailable for review.

    Dr. Beverly Durgan, the projects administrative advisor, began the meeting with a reminder that the project was in its fourth year and that a renewal proposal was due by September 1. She out-lined some of the things that needed to be decided  Does the committee want to apply for a re-newal? Does the committee want to keep the current NC number and name? She also requested that we review the current national priority areas and make sure that the new/rewritten project fits within those priority areas. Since the project underwent a major rewrite the last time, she thought that at least some of the current objectives and work plans would carry over to a renewal.

    Project reports were made by:

    George Rottinghaus  University of Missouri David LeDoux  University of Missouri Frances Trail  Michigan State University Pat Murphy  Iowa State University Suzanne Hendricks  Iowa State University Gretchen Kuldau  The Pennsylvania State University Wanda Haschek  University of Illinois Champaign-Urbana John Leslie  Kansas State University Charles Woloshuk  Purdue University Steve Hooser  Purdue University Lisa Vaillancourt  University of Kentucky

    A break for lunch was taken after Dr. Woloshuks project report.

    Following the project reports, the business meeting was held followed by a discussion of the re-newal of the project. Prior to the discussion, Dr. Haschek (IUCU) announced that she was semi-retired and that a replacement for her should be found.

    The current years (2008) annual report is being prepared by Dr. Charlene Wolf-Hall (NDSU). The current vice-chair Dr. David Kendra (NCAUR) was not present, which resulted in a discus-sion regarding the site for the meeting in 2010. Dr. Woloshuk indicated that he received no re-sponse from Dr. Kendra when queried about hosting the meeting in Peoria next year. Dr. Fran-ces Trail (MSU) volunteered to serve as chair in 2010 and to assume the responsibility for orga-nizing next years meeting. The meeting will be in Michigan at a date and location to be decided later. Either Dr. John Leslie or Dr. J. Scott Smith of KSU will serve as vice-chair of the 2010 meeting and the chair and organizer of the 2011 meeting, assuming that the project is renewed for an additional five-year term. Dr. Woloshuk will lead the effort to prepare the termination re-port for the current five-year project when it is due next year. He will be requesting lists of rele-vant publications and interactions from all project participants in the near future.

    A lengthy discussion ensued that centered on the revision of the current proposal for resubmis-sion and the renewal. To be successful the group needs to be composed of an interdisciplinary mix of chemists, food scientists, plant pathologists and toxicologists. No one university has staff in all of these areas. Integrating these efforts to keep everyone current in the related fields that are important to this committee and the field of mycotoxicology was viewed as one of the most important roles filled by the committee. USDA-ARS involvement, especially from NCAUR in Peoria, Illinois, was viewed as essential for the long-term success of the project.

    The current proposal has four objectives. All of them will be rewritten for the new proposal, with some material deleted, some refocused and some new material added. Dr. Rottinghaus agreed to be the lead for the current Objective 1, Dr. Woloshuk for the current objective 2, and Dr. Trail for the current objective 4. Current objective 3 may not survive in its current form, with the lead to rewrite it to be determined at a later date. The section on surveys contained in current Objective 3 will be deleted. This section may be replaced with work on dry distiller grains (DDGs) from commercial ethanol fermentations.

    Those writing individual pieces are to have them to Dr. Woloshuk by the end of July. Potential outcomes, outputs, milestones and the lead to rewrite objective 3, if necessary, also are to be identified at this time. Two decisions were made on the general thrust of the renewal. First, the current web site will be kept and updated. Second, occasional symposia at the Midwest AOAC meetings will continue to be scheduled concurrently in a joint meeting of the committee with this group.

    The meeting adjourned at 3:30 p.m.

    Meeting attendees: Beverly Durgan (Minnesota) Wanda Haschek (Illinois) Steve Hooser (Purdue) Suzanne Hendricks (ISU) Gretchen Kuldau (Penn. State) David LeDoux (Missouri) John Leslie (KSU) Pat Murphy (ISU) George Rottinghaus (Missouri) Frances Trail (MSU) Lisa Vaillancourt (Kentucky) Charles Woloshuk (Purdue)

    Accomplishments:
    Objective 1. Develop data for use in risk assessment of mycotoxins in human and animal health The Illinois group set up a subcontract with Purdue University to perform additional in vivo studies investigating the effects of pyrrocidine which was previously shown to be cytotoxic in mammalian cell lines. Illinois will be providing research material to Purdue University for testing of new analytical procedures for diagnosis of fumonisin toxicity.

    The Iowa group hypothesized that deoxynivalenol (DON) changed leukocyte subset numbers and their migration potential in peripheral blood, with interactions of age and sex. These leukocyte markers could be functional biomarkers of DON exposure in humans. In BALB/c mice fed DON at 0, 1.0 and 2.0 ppm, age, sex and feeding interval altered immune response of peripheral blood (PB) and splenic leukocytes, as measured by flow cytometry by staining cell surface markers. In 2.5-month old female mice, a decreased percentage of PB CD4+ cells and a decreased percentage of CD11b+ (macrophage) splenic leukocytes at 2.0 ppm DON were seen after 14 d only, whereas 2.0 ppm DON caused an increased percentage of CD19+ cells after 14 and 28 d. In 16.5-month old females, decreased PB CXCR5+ B cells were noted after feeding 2.0 ppm DON for 14 d, and 1.0 ppm but not 2.0 ppm DON decreased splenic CD11b+ cell %. In old male mice, 1.0 and 2.0 ppm DON increased granulocytes and CD29+CD11a+ neutrophils after 14 d and 2.0 ppm DON increased CCR9+ T cytotoxic cells after 28 d. DONcaused no changes to immune cell markers in young male mice.

    Objective 2. Develop new techniques and improve current assays to identify and measure mycotoxins and mycotoxigenic fungi in cereal grains

    A simple method for identifying heterozygous chromosome rearrangements in Fusarium spp. was adapted by the Kansas group from techniques proven to work with Gibberella zeae. Preliminary studies with this technique sufficed to identify the chromosomal rearrangements known in the mapping cross of G. zeae and to suggest that the lineages of G. zeae are not genetically isolated by chromosome rearrangements. The peptide sex pheromones of G. zeae have been identified and the genes encoding both the peptide pheromones and their receptors cloned. Only one of the pheromone/receptor pairs appears functional in G. zeae based on the fertility and interaction patterns of various deletion mutants. The one active pheromone is fungistatic and capable of blocking germination of both conidia and ascospores. Strains in which the receptor or the pheromone are deleted are still fertile as homothallics, but are much less fertile, in terms of both perithecia and ascospores produced, than are strains in which both the pheromone and its receptor are functional.

    The Kansas group reported that Fusarium fujikuroi is a close relative of Fusarium proliferatum capable of producing high quantities of gibberellic acid and causing the bakane disease of rice. F. fujikuroi has recently been introduced to California, where it is of great concern. The fungus can pathogenize native grass species that grow in and near the rice fields, which should make eradication of the disease from infested fields all but impossible. The California populations were clonal, with a single genotype widely dispersed across the state. Both mating types were present in the field populations but with a huge excess of MAT-1 alleles. Both AFLP and VCG strain typing gave similar results in terms of evaluating the genetic diversity present.

    The Kansas group reported that two new Fusarium species one from sorghum and maize and another from finger millet have been identified. Sexual stages have been identified for both species, and tester isolates developed to text cross-fertility are available in one species but not the other. The mycotoxin production capabilities of these species are not yet known

    The North Dakota group reported that methods were developed for the detection and quantification of Fusarium metabolites in matrices such as barley, wheat and potatoes. Covalently bound deoxynivalenol can be detected and quantified in barley with a new GC method, and in wheat with a new HPLC method, which also detects and measures pigmented metabolites of Fusarium graminearum.

    The Purdue group reported that the purpose of our research was to develop a multiplex real-time PCR assay to detect and quantify mycotoxigenic fungi. Our strategy was to utilize broad-spectrum primers to amplify the targeted genomic DNA and specific fluorescent probes to detect and quantify the different fungal genera. An analysis of PCR primer indicated PCR products from more than 40 Aspergillus species, 23 Fusarium species, and 32 Penicillium species as well as 64 other fungal genera.

    Genus-specific Taqman probes were designed from ITS sequences of rDNA to detect mycotoxigenic Fusarium, Penicillium, and Aspergillus. The specificity of the probes was established against a wide range of fungal species. To increase the utility of assay, multiplex conditions were developed. The assay was validated by analyzing fungal growth in distillers grain, an animal feedstock that accumulates as a by-product when ethanol is produced from corn.

    Objective 3. Establish integrated strategies to manage and to prevent mycotoxin contamination in cereal grains

    The Fusarium/ Poultry Research Laboratory in Missouri evaluated a number of mineral and organic adsorbents for binding mycotoxins in in vitro and in vivo studies in poultry, swine and cattle. Naturally occurring antioxidants (curcumin) were evaluated for reducing mycotoxin toxicity in poultry. The laboratory continues to produce mycotoxins in culture (kg quantities) for in house as well as for other researchers doing animal feeding trials with mycotoxins. Diagnostic testing for mycotoxins in contaminated feedstuffs was improved utilizing affinity column methodology in combination with HPLC analysis.

    A three week (hatch to 21 days) study was conducted to evaluate the efficacy of turmeric (Curcuma longa) powder (TMP), a source of the antioxidant curcumin (1.48%), and a hydrated sodium calcium aluminosilicate (HSCAS) adsorbent to ameliorate aflatoxicosis in broilers. Compared with chicks fed AFB1, the addition of TMP or HSCAS to the AFB1 diet improved (P<0.05) growth performance of chicks. Turmeric and HSCAS also ameliorated (P<0.05) aflatoxicosis in terms of serum protein, albumin, cholesterol, and calcium. The decreased antioxidant function in terms of level of peroxides, superoxide dismutase activity and total antioxidants in liver due to AFB1 were also alleviated (P<0.05) by the inclusion of TMP and/or HSCAS. The reduction in the severity of hepatic microscopic lesions due to TMP and HSCAS inclusion in the AFB1 diet demonstrated the protective action of curcumin/HSCAS used in the present study.

    A three week study (hatch to 21 days) was conducted to evaluate the efficacy of total curcuminoids (TCMN) to ameliorate the effects of aflatoxin (AF) in broilers. Food grade turmeric powder (Curcuma longa) containing 2.55% TCMN was the source of TCMN. The addition of 74 and 222 ppm TCMN to the AF diet improved (P<0.05) weight gain and feed efficiency. The increased (P<0.05) relative liver weight in chicks fed AF was reduced significantly (P<0.05) by 74, 222, and 444 ppm TCMN. The inclusion of 222 ppm TCMN ameliorated the adverse effects of AF on serum total protein, and albumin and ³-glutamyl transferase activity. Decreased antioxidant functions in liver due to AF feeding were alleviated by the inclusion of 222 ppm TCMN. Addition of 222 ppm TCMN to the AF diet provided the greatest protection against AF and demonstrated maximum antioxidant activity.

    An experiment was conducted to determine the efficacy of the adsorbent Calibrin A (CA), in ameliorating the toxic effects of aflatoxin (AF) in broiler chicks. A second objective was to determine if Calibrin A at 0.5% of the diet would negatively affect chick performance. The addition of CA to chick diets at a level of 0.50% did not negatively affect (P > 0 .05) chick performance or relative liver weight or cause any liver lesions. Compared with controls, feed intake (FI) and body weight gain (BWG) were depressed (P < 0.05) in chicks fed AF, with greater reductions in FI and BWG observed in birds fed 3 mg/kg AF compared with those fed 2 mg/kg AF. The addition of 0.25% or 0.50% CA to the AF-contaminated diets significantly (P < 0.05) improved FI and BWG. Compared with controls, relative liver weights were higher in chicks fed AF (P < 0.05), and the addition of CA (0.25% or 0.5%) to the AF diet containing 2 mg/kg AF reduced (P < .05) the increase in liver weight. Compared to controls with a lesion score of 1 (no lesions), liver lesion scores in birds fed AF averaged 2.69 (moderate aflatoxicosis). The addition of 0.25 or 0.50% CA to the 2 mg/kg AF diet reduced the lesion scores to 2.25 and 1.63, respectively. Results indicate that CA at 0.50% of the diet did not negatively affect chick performance, relative liver weight, or cause any liver lesions indicating that this level of CA did not negatively affect nutrient content of the diet. CalibrinA at 0.25 or 0.5% of the diet significantly ameliorated the toxic effects of 2 and 3 mg/kg AF in young growing chicks.

    The North Dakota group reported on natural antifungal components of flax seed were evaluated as potential additives to prevent mold growth in products such as pasta.

    The Penn State group conducted growth chamber studies to examine the role of temperature in fungal colonization and deoxynivanol development when Fusarium graminearum, the causal agent of Head Scab of Wheat, is inoculated on wheat heads. When inoculated plants are kept at 15 C fungal growth is limited but toxin is produced at significant levels and is translocated in the wheat head to areas that are not yet colonized by the fungus.

    Objective 4. Define the regulation of mycotoxin biosynthesis and the molecular relationships between mycotoxigenic fungi The Michigan group reported that the trichothecene mycotoxin deoxynivalenol (DON; vomitoxin) is produced by several Fusarium species during infection of grain crops. DON acts as a virulence factor in wheat. Previous experiments, including Affymetrix GeneChip assays, indicated that genes involved in DON biosynthesis are highly expressed during infection of wheat and barley. Notably, DON biosynthesis gene expression appears to be correlated with the infection front, and to diminish once the fungus is fully established. In order to better understand the role of DON during plant infection, we chose to monitor expression of Tri5, the trichodiene synthase gene, necessary for DON production. We dissected wheat heads for several days following inoculation with Fusarium graminearum and monitored Tri5 expression using quantitative reverse-transcript PCR (qRT-PCR). The highest proportional TRI5 expression was consistently detected at the growing front, in accordance with the microarray data. However, during the course of this study, TRI5 gene expression was never lost, i.e. hyphae in kernals nearest the inoculation point did not cease expressing TRI5. We are continuing to study expression of TRI5 through kernel development in order to understand how and when DON accumulates. In addition, we are beginning to test the correlation between green tissue and TRI5 expression by treating the wheat with the strobilurin fungicides. Strobilurins delay plant senescence, and have been implicated in increased DON accumulations. By determining the precise interaction between strobilurins and DON, we may be able to better time fungicide application to reduce or eliminate this synergism.

    The Penn State group reported that molecular genetics studies of mycotoxigenic fungi often require the use of classical genetic analysis. We confirmed that F. verticillioides isolate M-3125 (mat -) carries the spore killer sensitive allele. The genome sequence for this isolate is publically available and as such is frequently used for molecular genetic analysis. The presence of the spore killer sensitive allele in M-3125 means that in crosses with isolates carrying the killer allele (the majority of isolates in nature and in culture collections) genes with linkage to spore killer will not be recovered in the cross confounding genetic analysis. We searched a collection of fifty F. verticillioides isolates from the Penn State Fusarium Research Center for mat + isolates that were either spore killer sensitive. Two isolates, M-7815 and M-8024, meet these criteria and can be used in crosses with M-3125 without the segregation distortion that accompanies crosses with spore killer isolates

    The Purdue group reported that Expression of AREA also appears to affect the ability of F. verticillioides to colonize maize kernels. Indiana group used strains containing the reporter gene of ²-glucuronidase (GUS) fused to the promoter of NIAD (nitrate reductase) to study AREA expression and fungal growth. Because NiaD is transcriptionally regulated by AreA, it was possible to measure AREA expression by the activation of the NIAD promoter in the reporter construct. Growth, GUS expression, and fumonisin were monitored in a wild type, a disruption mutant (areA), and a strain with constitutive expression of AREA. While F. verticillioides grows equally well on each of the developmental stages of maize kernels (blister, milk, dough, and dent), AREA expression was found to be the lowest in colonized blister stage kernels and to increase with kernel maturity. Strain areA grew poorly on mature maize kernels, but grew similar to the wild type when provided with the addition of ammonium phosphate. FB1 was not produced by strain areA under any condition or by the wild type with added ammonium phosphate. However, constitutive expression of AREA rescued the growth and FB1 defects in areA. The strain with constitutive expression of AREA also produced FB1 on maize kernel with added ammonium phosphate. Growth of areA on the blister stage kernels was similar to the wild type, indicating that utilization of the available nitrogen source in these kernels does not require AREA. GUS activity was not detected in the wild type, suggesting that nitrogen metabolite repression of NIAD occurs during growth on blister kernels. GUS expression was detectable in constitutive-expression strain, which supports the conclusion that blister stage kernels are nitrogen metabolite repressive. These results provided some insight as to the effects that carbon metabolism and carbon:nitrogen ratio have on secondary metabolism during colonization

    Impact Statements:
    1. Impact 1 Given the toxicity of pyrrocidines in mammalian cell lines, the results of in vivo studies are essential for risk assessment. Development of analytical procedures that can be offered as standard diagnostic tests for fumonisin toxicity is needed for diagnosis of toxicity in field cases and monitoring of fumonisin induced diseases.
    2. Low-dose DON changed leukocyte populations in peripheral blood and spleen, and seemingly altered B cell and T cell migration in a manner suggesting that female sex hormones may potentiate some immune functional effects of DON, and that sex-related factors deserve further examination in interaction with DON in aged mice. Most of the immune cell changes were transient, supporting adaptation to DON, confirming previous observations and indicating a need to identify the mechanisms of this adaptation.
    3. These results also suggest that DON stimulated digestive system inflammation in old male mice, a more persistent effect than the other effects of DON. These studies support a need for human epidemiology of DON examining similar markers.
    4. Impact 2 Prevention and control of mycotoxins in grains and other food commodities helps keep the risk of chronic and acute foodborne illnesses to an acceptable level in the United States. The methods for detecting and measuring mycotoxins help with prevention through screening. Methods that look for previously undetectable covalently bound (masked) mycotoxins are an important contribution to risk assessment for mycotoxin dietary exposure as well as control development for resistant plants and other intervention strategies.
    5. A comprehensive scheme to analyze grain and grain products for the presence of mycotoxigenic fungi should begin with the detection and quantification of the key mycotoxigenic genera. The information derived from the preliminary analysis would then guide subsequent analyses with mycotoxin-specific or species-specific measures. The assay we have developed can be used as an initial step to evaluate the mycotoxigenic potential of distillers grain and various other agricultural commodities. The method has an advantage over the traditional plating methods that are time consuming, laborious, and require an expertise for fungal identification.
    6. A further advantage is the PCR assay can detect these mycotoxigenic fungi within distillers grain, which often contain enormous yeast populations that interfere with plate-method quantification of less numerous fungal species. The assay is applicable for the analysis of stored grains and foods and could be used as an initial step in quality assessment in conjunction with other mycotoxin-specific tests such as chemical analysis or PCR.
    7. Impact 3 Safe utilization of low level mycotoxin contaminated grains in animal feedstuffs can be increased with the development of new proprietary adsorbents and use of naturally occurring antioxidants to reduce or eliminate the toxicity of these mycotoxins. Providing mycotoxins (fumonisin, ochratoxin A, moniliformin, zearalenone, and aflatoxin B1) in culture material to mycotoxin research groups makes it economically feasible to undertake animal feeding studies that would be nearly impossible if mycotoxins were purchased commercially. The projects looking for interventions, such as antifungal treatments, help with prevention as well through control of post-harvest fungal contamination.
    8. Impact 4 Control of Head Blight has been problematic. The most important goal is to remove DON from the grain. This work investigates the interaction between DON biosynthesis and grain development.Identification of strains for crossing with M-3125 will provide a new tool for researchers to employ classical genetic analysis in the sequenced isolate of Fusarium verticillioides.
    Last Modified: 27-Jul-2009
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