Duration: 10/01/2015 to 09/30/2020

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The need, as indicated by stakeholders, and likely impacts from completion of the
work


Grain and livestock producers need to minimize mycotoxin contamination of food,
forage, and feed, and reduce the deleterious effects of mycotoxins on consumers
and livestock. In recent years, the presence of mycotoxins in the solid byproduct of
grains processed for biofuels, known as distillers grain (DG), has also become an
important issue. The sale of DGs for animal feed has become an important source of
supplemental income for biofuel producers. For grain buyers and food processors,
the primary need is a reliable method for rapid assessment of grain quality
pertaining to mycotoxins and mycotoxigenic fungi. Rapid methods to detect
mycotoxins at the first points of sale (elevators), as well as methods to detect
mycotoxigenic fungi in the commodity (e.g. DON-producing Fusarium in barley),
would prevent these stakeholders from purchasing corn contaminated with
unacceptable levels of aflatoxins and fumonisins, or wheat with excessive
concentrations of deoxynivalenol (DON), e.g. Additionally, cost-effective methods to
predict, monitor, and minimize mycotoxin production in the field, and to detoxify
mycotoxins and prevent further deterioration in contaminated feed, are needed by
producers of grain and livestock. The lowering of tolerance limits for mycotoxins in
overseas markets has increased the burden for grain buyers and food processors;
currently, levels of mycotoxins that are acceptable for some US products are
unacceptable in European and Asian markets, resulting in non-tariff trade barriers.
New methods to monitor and treat contaminated grain would benefit domestic
consumers, and would also allow American commodities to compete more
effectively in foreign markets. Finally, workers who are responsible for animal and
human health need information about the toxicity, carcinogenicity, modes of action,
and biomarkers of exposure and disease for all categories of mycotoxins. This
information would be used to train health-care providers to identify exposure and
treat related disease, as well as to develop accurate risk assessment
recommendations.


The importance of the work, and consequences if it is not done


Mycotoxins are a serious, chronic problem throughout the cereal- and forage-
producing regions of the U.S. (e.g., see http://www.youtube.com/watch?
v=JEysXbJisf0). If research is not applied broadly to address this problem, serious
negative consequences will result. First, the presence of mycotoxins is an important
health hazard. Accurate hazard assessments are essential in order to maintain
exposures by animal and human consumers within safe limits. We propose basic
research to define the toxicity of several important mycotoxins. Without this
information, it is impossible to assess the risks associated with these mycotoxins.
Additionally, the presence of mycotoxins in grain is an economic concern, especially
in the context of global markets. Without an aggressive research program to
prevent, treat, and contain outbreaks of mycotoxins in grain, U.S. grain producers
suffer the consequences of reduced marketability of their products. Furthermore,
the proposed research addresses biosecurity concerns. The natural occurrence of
mycotoxins in grain is an important security concern for producers and end-users of
the grain; mycotoxins have been used as agents of terrorism, e.g. aflatoxin in Iraq.
Without a proactive research program to find innovative ways to monitor, prevent,
and treat mycotoxin contamination of grains and forage, US agriculture will be
unprepared to deal effectively with a mycotoxin outbreak, regardless of its origin.
Finally, the production of mycotoxins by mycotoxigenic fungi in grains and forage
represents a continuing problem in agriculture. Improving our understanding of
factors relevant to allowing these fungi to colonize their hosts, and how mycotoxin
biosynthesis is regulated, will not only lead to novel treatment strategies, but will
also advance our understanding of fungal pathogenesis in general.


The advantages for doing the work as a multistate effort and the technical feasibility
of the research.


The scientists involved in this multistate, multidisciplinary research proposal work
individually on mycotoxin issues related to their respective disciplines and areas of
expertise. Just as agriculture is diverse and varies greatly from state to state (and in
many instances, within a given state), the occurrence and severity of mycotoxin
outbreaks vary widely across the US. A multistate effort ensures a thorough
approach to investigate a complex and highly variable phenomenon that has
significant impacts on both producers and consumers. Due to the wide range of
experience and expertise of the group, the proposed research should be technically
feasible.


What the likely impacts will be from successfully completing the work


The work will address the needs of the stakeholders. Outputs will include
information on the action of mycotoxins in livestock and animal models. This
information will be applicable to the risk assessment process. The work will also
address stakeholders' continuing need for new detection and monitoring methods
for grain, DGs, and forages. Information will be generated to address the need for
management practices that help prevent mycotoxin-related problems during grain
and forage production, handling, storage, processing, and consumption. Finally, we
will generate important basic knowledge about major groups of mycotoxigenic
fungi, and the biochemical and molecular factors that regulate the biosynthesis of
aflatoxins, endophyte alkaloid toxins, and Fusarium-associated mycotoxins
including deoxynivalenol, fumonisins, and zearalenone. This will reveal critical
points in the regulation where targeted controls can be developed.

Related, Current and Previous Work

Related, Current, and Previous Work

NC1183 is the only active research group that covers such a broad range of topics related to mycotoxins, including impacts on animal health, fungal biology and genetics, analytical technologies and assays, and mycotoxin mitigation in feed and resistance in plants. Methods and results have been disseminated through publication of dozens of peer-reviewed papers and abstracts, and presentations at scientific meetings and to individual stakeholders. Some highlights from the work of the group over the past five years follow:

Impacts of Mycotoxins on Human and Animal Health: Members have investigated the impacts of Fusarium and Aspergillus toxins on animal health, and tested methods for reduction of mycotoxigenicity of feed grain, in whole animal assays. New assays were also developed that use Caenorhabditis elegans and mammalian cell lines, which should facilitate future studies of the mechanisms of mycotoxigenicity.

A promising approach to the mitigation of mycotoxicosis in animals is feeding an inert binder that sequesters the mycotoxin and facilitates its harmless elimination. A binder study was completed in rats that showed efficacy of a commercially available binder against fumonisin (FB) and deoxynivalenol (DON), as indicated by significant positive effects on serum sphingolipids and body weight. A preservative/antioxidant blend also mitigated DON toxicity in swine, as indicated by significant improvements in average daily gain. DON at 5 ppm decreased liver selenium compared with controls in swine fed for 120 d. Two of three commercially available mycotoxin binders also significantly reduced (45-48%) aflatoxin (AF) levels in the milk of dairy cows fed AF.

Microarray analysis was utilized to determine the effects of dietary AF on hepatic gene expression in male broiler chicks. Results indicated that genes associated with energy production and fatty acid metabolism, growth and development, antioxidant protection, detoxification, coagulation, and immune protection were down-regulated, whereas genes associated with cell proliferation were up-regulated.

A system to study the toxicity of DON has been developed that uses Caenorhabditis elegans, the simplest multicellular animal. The lifespan and egg-laying capacity of DON-treated nematode worms is significantly reduced compared to controls. This C. elegans assay system developed by the NJ station will make it easier to study the mechanisms of toxicity of DON and other mycotoxins, and to test potential mitigation strategies, in a simple animal model. Many genes involved in the worm biological processes and molecular functions are either up- or down-regulated upon DON intoxication. Up-regulated genes include those involved in detoxification and innate immunity, while down-regulated genes are involved in metabolism and development. C. elegans gene silencing can be achieved with RNAi (RNA interference) technology, for which NJ has already obtained the C. elegans genome-wide RNAi library. C. elegans gene silencing and over-expression can also be achieved by the newest gene editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats-associated endonuclease) system (Addgene.org).

Growth of the human K562 erythroleukemic cell line is inhibited by DON, in a manner similar to inhibition of mouse splenocyte proliferation in vivo, according to preliminary data from IA. This model may be used as a biologically relevant screening assay for DON-contaminated grain and food samples; 24-h incubation of K562 cells with grain extracts containing known amounts of DON cause cell proliferation inhibition identical to the same amount of pure DON.

Specific impacts of these and other NC1183 activities include the following:

A mycotoxin binder was shown to be effective against FB and DON, two major fungal toxins that cause serious diseases in livestock and pets, and potentially cause human cancer and immune impairment.

A preservative blend was also effective against DON in swine under production conditions. These findings will serve as the basis for the development of additives for commercial pet foods and animal feeds.

Identification of genes affected by dietary AF in broiler chicks suggested possibilities for nutritional or pharmacological therapies to reduce toxic effects. For example, down-regulation of genes associated with antioxidant protection suggested that the addition of an antioxidant to diets contaminated with AF might be beneficial. A PCR analysis was used to test this prediction. The ability of curcumin, a natural antioxidant found in turmeric (Curcuma longa) powder (TMP), to ameliorate changes in hepatic gene expression of broiler chicks fed AF was evaluated. Results demonstrated partial protective effects of curcumin on changes in expression of antioxidant, biotransformation, and immune system genes in the livers of the chicks. These potential therapeutic effects of antioxidants will be further tested in the future.


Assays using model systems were developed that will greatly accelerate future research into mechanisms of mycotoxicity in animals. These assays will be made available to all members of the group in collaborative research projects.


Mycotoxin Resistance and Mitigation in Plants and Plant Products: Several groups have been working toward this objective with various approaches.


Genetically modified lines of maize containing newer Bacillus thuringensis (BT) genes were assessed for content of FB and susceptibility to insect damage. A new BT gene was found to be very effective in reducing FB contamination compared with the older BT versions. Ethanol yields from maize containing up to 8 ppm FB were not adversely affected. Spiking ethanol fermentations with even higher levels of FB also did not affect ethanol production. Dried distillers grains produced as byproducts from the contaminated grain had about 3-fold enrichment of FB in 50 out of 57 batches. The batches that did not show as significant an increase in FB will be investigated further.

DON has a significant negative impact on brewing quality, and ND is currently working on processes for the transformation of DON to the less toxic deoxynivalenol-3-glucoside during the barley malting process. Preliminary research has suggested the germinating barley seed has tremendous ability to glucosylate DON present in the sound grain, as well as that formed by Fusarium as the seed germinates.

Preliminary and ongoing maize breeding studies in MS have identified many QTLs, and genes with large and small effects that affect resistance to Aspergillus flavus infection and to AF accumulation.

The NJ station has developed a model Brachypodium distachyon (Bd21 variety) system for assessing infection by F. graminearum and the responses to application of DON. They have developed a detached leaf protocol for infection of B. distachyon with GFP-tagged wild type F. graminearum (wtFg-GFP) and tri5-mutant F. graminearum (tri5Fg-GFP), and to directly apply DON. They have previously produced transgenic wheat plants expressing yeast ribosomal protein constructs that are resistant to Fusarium head blight (FHB) with reduced DON level in the grains. They have recently developed B. distachyon and barley tissue culture and transformation protocols with immature and mature seeds, and have started using the CRISPR/Cas gene editing technology to engineer FHB resistance in both species. Additionally, the NJ station has adopted Arabidopsis thaliana as a model plant to study the effect of CRISPR/Cas gene editing to improve FHB resistance, since A. thaliana is easily transformed and has been used successfully used to study Fusarium infection.

CRISPR/Cas constructs to target the ugt (UDP- Glucuronosyl Transferase) genes in B. distachyon, A. thaliana and barley, and the B. distachyon EIN and the barley EIL1 genes, which have been implicated in disease resistant to rice, have been produced. Gene editing-out of ugt will facilitate our understanding of the DON detoxification function of this gene in these plants. Importantly, over-expressing a heterologous ugt gene such as the A. thaliana or B. distachyon ugt in barley plants will likely improve the barley resistance to FHB. Other plant expression vectors have also been produced, containing the following constructs to engineer B. distachyon and barley: FTLi to knock-down the Fusarium Transducin Beta-Like gene that is essential for Fusarium pathogenesis, GFPi to silence the green fluorescent protein gene in wtFg-GFP and tri5Fg-GFP to monitor F. graminearum infection, HPGP to over-express the hydroperoxide glutathione peroxidase gene to elevate the anti-oxidative level, and snakin-1 to over-expression this plant anti-microbial peptide.

Specific impacts of these and other NC1183 activities include the following:

Strains of the endophyte from ‘Kentucky 31’ tall fescue were generated that lack the toxic ergot alkaloid, ergovaline, by specific removal of some of the ergot alkaloid biosynthesis genes, using a novel techique that leaves no transgenes. The resulting endophyte will be useful in future development of forage cultivars with nontoxic endophyte.

The mechanism of resistance conferred by an FHB1-QTL found in a resistant variety of spring wheat was elucidated. This may make it possible to transfer this mechanism into other types of wheat, i.e. to white winter wheat, where it is not currently available.

Fusarium mycotoxins in maize carry over into animal feed components that are derived from maize-based ethanol production. Work by IA group members demonstrated that the use of genetically-modified insect resistant maize is highly effective in preventing contamination of both feed products. The work also demonstrated that mycotoxin contamination of grain had no effect on yields of ethanol, suggesting that contaminated grain could be productively diverted to this purpose as long as distiller’s grains byproducts were not used directly as animal feed.

Aspergillus flavus infection causes AF contamination in maize products and make them unsafe for food or industry purposes. The MS station has produced several inbred lines that contain significant levels of maize host plant resistance to A. flavus infection. These lines can be used in future for development of commercial resistant maize lines.

Development of model assay systems by the NJ group will greatly accelerate future studies of the effects of mycotoxins on plants, and the role of plant genetics and physiology in mycotoxin production. These assays will be made available to all group members through collaborative research.

Fungal Biology and Genetics: Several groups have made significant progress in understanding the genetics and population biology of important mycotoxigenic fungi, including generating whole genome sequences for Diplodia maydis and several Epichloë ecotypes, and developing new genetic markers for population studies. Collaborations have resulted in more rapid progression and deeper understanding of common themes.

Soil-borne fungi, like F. verticillioides, are routinely challenged by a diverse chemical environment while also encountering a variety of competing organisms. There is a striking expansion in the number of beta-lactamase encoding genes in several sequenced Fusarium species, compared with other fungi in niches that are likely to encounter limited microbial competition. This has given rise to the hypothesis that these fungi require a broad diversity of lactamase enzymes to protect against and/or produce a wide variety of xenobiotics for chemical warfare. The xenobiotic protective capacity or synthesis is hypothesized to allow for the wide-spread occurrence of Fusarium in agricultural systems.

Many black Aspergillus spp. produced FB2 in the laboratory, but at low levels compared with F. verticillioides or F. proliferatum. Drier regions had larger populations of black Aspergillus in maize, which co-occurred with A. flavus. The presence of black Aspergillus spp. might alter FB1/FB2 ratios in maize, which could somewhat reduce mycotoxicosis risk, but the correlation with the presence of A. flavus leads to the expectation of a greater co-occurrence of FB and aflatoxins.

Several aspects relevant to infection and survival of Fusarium fungi, and accumulation of FB toxins, in maize and wheat were elucidated. It was demonstrated that F. graminearum overwinters as hyphae that contain large quantities of lipids, which are used to support sporulation in the spring. The role of reactive oxygen species in Fusarium verticillioides disease development and fumonisin production on maize was established. Comparative transcriptomic analyses of Fusarium verticillioides and F. graminearum revealed differences in their approaches to survival in the field. Analyses of expression of DON genes during infection of wheat revealed how DON assists in pathogenicity, and indicated a mechanism for production of high DON, low symptom grain.

Tall fescue toxicosis causes annual losses on the order of $1 billion in the U.S. due to poor weight gain and reduced fertility in beef cattle, agalactia in dairy cattle, stillborn foals, dry gangrene of limbs, and other maladies. The complex ergot alkaloid, ergovaline, is widely believed to be the main toxin responsible, and it produced by the seed-transmitted endophyte, Epichloë coenophiala, a fungus that is key to maximum tall fescue stand survival and productivity, especially in episodically stressful environments characterizing much of U.S. pasturelands. Ergovaline belongs to a sub-class of ergot alkaloids that includes ergotamine, ergocryptine and other ergopeptines well known as potent toxins produced by the ergot fungi, Claviceps spp., which can be major contaminants of cereals. The endophytic Epichloë spp., in addition to producing ergot alkaloids, can produce alkaloids thought to be more specifically active against invertebrates (lolines and peramine), and indole-diterpenes, a diverse group of alkaloids with various activities in vertebrate and invertebrate systems. Considerable research has been conducted in KY and OK to identify and characterize genes required for alkaloid biosynthesis, and endophyte strains varying in alkaloid profiles. We now have available the draft genome sequences of five E. coenophiala strains, which are from different tall fescue ecotypes and differ in alkaloid profiles. Furthermore, targeted gene knockouts have resulted in a panel of isolates that produce key pathway intermediates spanning the spectrum of ergot alkaloid, indole-diterpene, and loline alkaloid production. For example, some produce terpendoles and/or the ergovaline, whereas others produce simple clavine alkaloids for which toxicity has not yet been assessed.

Aflatoxin B1 (AFB1), which may be formed in maize, cereals, sorghum, peanuts, and other oil-seed crops, is highly toxic and is one of the most potent naturally occurring carcinogens regulated by the US Food and Drug Administration (FDA) since 1965. Understanding the mechanisms controlling sporulation and aflatoxin production in the toxigenic fungus Aspergillus flavus is important for accomplishing the long-term goal of eliminating both fungal dispersion and AF contamination in fields. Fungal sporulation and AF production are intimately associated via bridging activities of a new class of master regulators called the velvet family proteins (VosA, VeA, VelB, VelC, and VelD), and the global regulator of secondary metabolism LaeA. A key and common property of these multi-functional regulators is that they are DNA binding proteins that function as transcription factors controlling the expression of thousands of genes associated with sporulation and toxin biosynthesis. The absence of certain velvet genes (veA, velB, and velD) results in abnormal asexual sporulation, impaired sclerotia formation, and the lack of AF production. Importantly, these regulators are only specific to fungi and conserved in most (if not all) filamentous and dimorphic fungi and their functions are confirmed (or predicted) to be conserved in many other fungi, providing the basis for using velvet as common anti-fungal targets. Characterized the fungi-specific velvet regulators that play a key role in regulating sporulation and production of mycotoxins. Work by the WI group revealed that fungal sporulation and aflatoxin production are intimately associated via bridging activities of the velvet family proteins VeA, VelB and VelD in Aspergillus flavus. Revealed that velvet proteins interact with each other, alone (“homodimers”), in various combinations (“heterodimers”), and also with other proteins including the master regulator of mycotoxins LaeA. It was further revealed that velvet proteins are a family of fungus-specific transcription factors having a NF-kB-like domain that directly binds to target DNA.

Functions of 15 G-protein coupled receptors (GPCRs) in aflatoxigenic A. flavus were characterized. It was revealed that the GprC and GprD play a crucial role in governing oxylipin signaling and quorum sensing in A. flavus.

Specific impacts of these and other NC1183 activities include the following:

Work by our group shows the need for continued vigilance by grain producers and the food industry, and for continued work to develop surveillance tools and anti-mycotoxin strategies that target fungal establishment and survival, and that consider the presence of multiple mycotoxins, especially when grains are under drought stress.

Draft genomes of nine hybrid strains representing the four species associated with Epichloë ecotypes of tall fescue were generated, enabling development of a multiplex PCR protocol for high-throughout genetic analyses. These analyses can now be performed directly with endophyte-infected plant materials to evaluate their potential for alkaloid production. A total of 170 tall fescue PI lines from the National Genetic Resources Program were evaluated using this assay for endophyte (Epichloë species) presence and diversity.

Surveys by members of the group determined that all or nearly all Nebraska, Pennsylvania, and Kentucky head scab isolates were F. graminearum of the 15-ADON chemotype. This suggested that recent episodes of increased DON contamination of grain in these states were not due chemotype shifts in the population.

Provided mechanistic insights into the novel regulatory roles of the velvet proteins in bridging fungal spore formation and mycotoxin biosynthesis, further illuminating the global functions of velvet regulators in controlling complex expression, cellular and metabolic responses in fungi. The velvet regulators are conserved in many agriculturally important fungi, thus the results of this research will aid to the development of novel ways to broadly mitigate fungal infestation and mycotoxin production in fields, thereby protecting human and plant health.

Active structures in black pepper extract were identified that inhibit aflatoxin biosynthesis. A patent was generated related to this: F. Trail and A. D. Jones. 2014. Compounds for Inhibition of Fungal Toxin Production. U.S. Patent Application Serial No.: 62/008,673 ?

Objectives

1. Objective 1: Develop data for use in risk assessment of mycotoxins in human and animal health.
   
2. Objective 2: Establish integrated strategies to manage and reduce mycotoxin contamination in cereals and in forages.
   
3. Objective 3. Better Understand the Biology and Ecology of Mycotoxigenic Fungi.
   

Methods

Objective 1: Develop data for use in risk assessment of mycotoxins in human and animal health Mycotoxin contamination of grain, grain products, and forage can have profound negative effects on human and animal health, but information is lacking that could be used to inform policy regarding minimal safe levels of exposure. This deficit will be addressed in Objective 1 via investigations of the mechanistic basis for mycotoxin-induced disease in humans and animals. Studies will utilize cell, tissue, and whole animal bioassays that have been developed by the participating stations. Data from the different bioassays will be shared and compared freely among the participants. Participants will provide mycotoxin-contaminated samples, as available, to other participants, as needed, for in vitro and in vivo toxicity testing studies using the various assays. 1.1 Dose Response Studies and Evaluation of Potential Biomarkers in a Mouse Model Dose-response assessments will be used by IA to determine and model mycotoxin toxicity, including acute toxicity, and immunomodulation in a mouse model. Exposure assessments for mycotoxins must take into account route of exposure and level and duration of exposure to the mycotoxin, as well as subpopulation vulnerability. IA will obtain dose-response data for gastrointestinal toxicity of DON, DON-glucoside and other related trichothecenes in mice, including assessment of intestinal tight junction proteins as a probable mechanism of DON toxicity. These studies will model effects of DON on very young and old individuals as vulnerable populations. Data will be generated for risk assessment of the use of mycotoxin-contaminated grains, focusing on fumonisins, with both conventional and insect-resistant transgenic maize used in the biofuels industry and its subsequent byproducts. 1.2 Mechanistic Studies Acute toxicity, carcinogenicity, teratogenicity and immunotoxicity of fumonisins and DON will be assessed in mouse and other animal models in oral feeding studies with naturally contaminated feed or purified toxin in IA. Adaptation to subchronic exposures to DON is a key observation from previous studies (IA) that will be assessed further by studying DON metabolites and DON-metabolizing gut microbial changes over time and dose of DON. The mechanisms of DON cytotoxicity in Caenorhabditis elegans will be investigated by silencing or over-expressing some of the genes previously shown to be altered by DON exposure (NJ). Conserved candidate genes identified by other NC1183 participants using other assay systems (e.g. pig, bird, mouse) will be shared with the NJ group so that they can also be subjected to gene silencing in C. elegans. 1.3 Structure Activity Relationships in Mycotoxin-treated Cell Culture and C. elegans A systematic evaluation of structure-activity relationships (SAR) has not been conducted for mycotoxins. Fumonisin analogues have been evaluated for their cytotoxicity, plant toxicity, and sphingolipid alterations, in only a few studies. Current activity with trichothecenes has focused on immune responses and cell signaling pathways. IA will use the K562 human erythroleukemia cell line as a model to evaluate SAR of DON, its major metabolites and other related trichothecenes. This cell system will also be used to model human tissue contents of trichothecenes, by comparing individual mycotoxin dose/responses with various combinations of trichothecenes and their key metabolites. The development of a rapid, inexpensive monitoring scheme for human exposures to these mycotoxins is a goal. In collaboration with IA, NJ will also test the differential toxic effects of these different forms of DON on development and life span and on gene expression patterns in the C. elegans model. The toxicity of alkaloid compounds and intermediates isolated from gene knockout strains developed in OK and KY will also be tested and compared in collaboration with NJ (C. elegans model) and IA (K562 model). 1.4 Transcriptional Profiling of Mycotoxin Response in Chicks. MO will lead an effort to explore gene expression profiles of chicks fed individual and/or mixed mycotoxins to identify candidate genes to map growth, metabolic, and regulatory pathways that control important production traits. Data on the effects of combinations of mycotoxins at the level of the whole animal, and at the cellular level are limited and are essential since mycotoxin contamination rarely involves single mycotoxins, but instead usually involves two or more mycotoxins. To meet this objective, tissues will be collected and immediately snap-frozen in liquid nitrogen, and stored at -80ºC. The extraction and purification of total RNA will be done following the RNeasy® Mini Kit protocol (Qiagen Inc., Valencia, CA). High-quality RNA will be submitted to the DNA core at MU for RNA sequencing analysis (RNA-seq). RNA-seq will be conducted on appropriate tissue (e.g. kidney, liver) samples collected from controls (chicks fed a basal diet with no mycotoxins) and chicks fed the basal diet supplemented with the mycotoxins of interest. The libraries will be sequenced following the Illumina TruSeq RNA protocol. The NextGENe- NG Release V 2.17 (beta) program will be used for mapping, alignment and quantification of the transcripts. The results of RNA-seq will be presented as the arithmetic mean of three replicates from each group. Reads per kilobase of target per million tiled reads (RPKM) will be used in the Student’s t-test analysis (P<0.05). Gene expression levels will be considered to differ significantly at 0.05 level of probability and greater than two-fold changes (down- or upregulated). The transcripts will be clustered to their functional categories according to Kyoto Encyclopedia of Genes and Genomes (KEGG Orthology) by Database for Annotation, Visualization, and Integrated Discovery- DAVID Bioinformatics Resources 6.7® (Dennis et al., 2003). MO will work with all groups interested in RNA-seq to allow them access to the DNA core at the MU where such analyses can be completed. Key genes identified as responsive to mycotoxin contamination in chicks will be shared with NJ, and C. elegans homologs (if they exist) will be tested by gene-silencing in the C. elegans assay. Objective 2: Establish integrated strategies to manage and reduce mycotoxin contamination in cereals and in forages. 2.1 Surveillance and Methods for Mycotoxin Detection Survey work will be performed periodically in KS and possibly in other states for mycotoxins, including corn fields, wheat, and other commodities. In other states, participants will work together to compile public data from surveys and evaluations conducted by various local entities and share these data with other members of the group in our annual reports. Data of this nature will provide a more precise idea about the frequency of mycotoxin contamination and would help develop contamination prevent strategies for the future. KS, in conjunction with industrial partners, will compare different sample preparation methods and rapid/easy analytical methods for different mycotoxins in a wide variety of foods/feeds. Reduction in time required for testing for contaminants in pre-harvest crops would help to predict, monitor, and minimize mycotoxin production in the field, and prevent further deterioration in contaminated grains. In addition, shorter testing times for post-harvest will address the needs of fast analysis of the contaminants prior to food distribution and to meet the ultra-fast analytical demands of possible food terrorism. 2.2 Genetic Manipulation of the Host to Reduce Mycotoxins There is a pressing need for drought tolerant and adapted maize lines for use both in the United States and in developing countries, particularly in parts of Africa where maize is a staple crop. Researchers at PA have identified maize lines with drought adaptation associated with specific root anatomical features. Contamination of maize with aflatoxins and fumonisins is associated with droughty conditions, raising the question of the mycotoxin susceptibility of these maize lines. PA will evaluate the drought-adapted maize lines for fumonisins, and if conditions are appropriate, for aflatoxins as well, under drought and moisture-adequate conditions at sites in Pennsylvania and Arizona. PA and MS will collaborate to test their ability to resist aflatoxin accumulation under inoculated and non-inoculated conditions in Mississippi. This information will inform decisions about appropriate deployment of these lines, and on a basic level provide data that may be useful in understanding drought accumulation of these important mycotoxins. MS will work to identify genes and proteins associated with resistance to Aspergillus flavus infection and aflatoxin accumulation in maize. To transfer the majority of resistance through breeding methods, the research goals are to identify the resistance-controlling genes and quantify their genetic effects individually and collectively. Genome-wide (transcriptome, proteome) research methodologies will be used for the quantification of gene/protein expression levels and DNA marker effects. Specific objectives include transcriptome and proteome profiling of genes and proteins for resistant maize inbred lines Mp715 and its offspring lines Mp718, and Mp719 under inoculated and non-inoculated conditions in multiple field environments. Maize inbred line Va35 will be used as the susceptible check. Transgenic Brachypodium distachyon, Arabidopsis thaliana, and barley plants produced by NJ will be evaluated for the transgene integrations and gene editions. Disease resistance improvement or reduction will then be assessed in these transgenic plants. Mycotoxigenic fungal strains will be received from other stations for testing in these transgenic plants. Candidate genes identified from the work in MS will be shared with NJ and plant expression vectors containing those genes will also be produced and tested for function in these model systems. 2.3 Mycotoxin Mitigation Grains with higher levels of contamination and most screenings from grain operations are unsafe for human and/or animal consumption and must be destroyed or alternate uses identified. If effective methods could be identified for treatment of grains, or for amending the feed produced from the grains, these contaminated grains and screenings could be safely and economically utilized in the livestock and poultry industry. Committee members from MO, IA, and KS will collaborate on researching technologies, including treatment with adsorbents and natural antioxidants to eliminate the mycotoxins from the feed or reduce their toxicity. The emphasis will be on in vitro and in vivo evaluation with respect to efficacy, and determining if they are effective in preventing mycotoxicosis. The key C. elegans genes affected by DON will be tested as potential targets for small molecule inhibitors to mitigate the toxicity of DON. This research will be jointly developed with the NJ group and Dr. Pang at Mayo Clinic with whom they have previously collaborated to develop Shiga toxin inhibitors. The DON toxicity mechanism and mitigation strategies delineated in C. elegans will be translated into livestock and human systems in collaboration with IA. DON metabolism in plants and animals holds some promise for mitigation of DON toxicity, through formation of DON-3-glucoside (D3G), which is known to be a minor metabolite of DON in wheat, but increasing D3G production might be feasible. From a study by Nagl et al. (2014) in piglets, DON from D3G was absorbed less than half as well as DON, and there was significant conversion of DON to DOM-1 (de-depoxy-DON), an apparently detoxified form of DON formed in the lower intestine of monogastric animals and also in some humans but only in piglets fed D3G and not DON (Nagl, Woechtl et al. 2014). This work implies that studying D3G and DOM-1 production, metabolism and toxicity further could lead to useful DON mitigation strategies (IA). This work would involve in vitro and in vivo studies. In vitro anaerobic incubations of DON with mouse or human fecal contents will be used to identify gut microbial populations capable of converting DON to DOM-1. These microbial populations (DOM-1 forming and non-DOM-1 forming) will be used to create two standardized gut microbiomes in germfree mouse colonies. Groups of mice from those 2 colonies will be used to compare toxicity of DON and D3G, with the expectation that DOM-1 forming mice fed D3G will experience less toxicity than similar mice fed DON or than non-DOM-1 forming mice fed DON or D3G. Genotypic differences in UDP glucosyl-transferease activities in germinating barley seed, and process conditions that favor their expression and activity, will continue to be investigated in ND. Explorations of physical and biological treatments that can be used control mycotoxin formation during malting will also continue. 2.4 Reducing or eliminating tall fescue toxicosis Tall fescue toxicosis is a livestock problem causing on the order of $1 billion annual losses in the U.S., for which the etiological agent or agents are alkaloids produced by the fungal endophyte, Epichloë coenophiala (Paterson et al., 1995). Based on gene sequencing and novel gene-manipulation technologies developed in KY, mutant strains of E. coenophiala will be generated that lack ergovaline, suspected to be the most important mycotoxin in the system, and possess either simpler ergot alkaloids or no ergot alkaloids. The novel technologies employed leave no transgenes in the endophyte genome, making the mutant strains suitable for field tests and, potentially, cultivar development. KY will generate plants with the mutant and wild-type endophytes, as well as isolated alkaloids, which will be tested in the mouse and C. elegans systems described above. Objective 3. Better Understand the Biology and Ecology of Mycotoxigenic Fungi. Mycotoxigenic fungi are present in essentially every agricultural field, and mycotoxin contamination represents one of the greatest continuing threats to food safety and profitability. However, the relationship between fungal contamination and mycotoxin contamination is not direct, and depends on many genetic and environmental factors. The goal of this objective is to improve our ability to predict mycotoxin contamination levels through an improved understanding of the biology and ecology of mycotoxigenic fungi. Participants in this objective have formed several collaborative teams focused on specific mycotoxins and fungal species. 3.1 Fungal Population Diversity A great amount of diversity exists among field isolates of mycotoxigenic fungi in their aggressiveness and in their ability to produce mycotoxins. There are few markers to indicate which strains are more likely to be most toxigenic. KS is leading a study of segregation of fecundity, pathogenicity and mycotoxigenicity traits in an interspecific cross between Fusarium proliferatum and F. fujikuroi (Studt et al., 2012). The long-term goal of the work is to determine how genetic exchange can occur between different species, leading to the production of strains with novel mycotoxin profiles and pathogenicity traits, and to the identification of genes outside of mycotoxin biosynthetic gene clusters that play a role in the regulation of mycotoxin production. KY is potentially a “hot spot” for mixing among mycotoxigenic F. graminearum genotypes adapted to the surrounding wheat and corn producing regions. A variety of molecular markers, including restriction fragment length polymorphism (RFLP) fingerprinting markers derived from the F. graminearum genome sequence, will be used to evaluate the genetic diversity and presence of outcrossing among Fusarium strains isolated from symptomatic wheat heads collected from across the state each year. This work will facilitate early detection of population shifts and new genotypes in KY arising from selection, migration, or recombination. NE has experience with PCR-based chemotyping Fusarium isolates, per Starkey et al. (2007) and Ward et al. (2008); additionally, they have the equipment and oligonucleotides to carry out allele-specific primer extension (including chemotyping and species identification) as in Ward et al. (2008). With input from other stations, this capacity could be expanded to detect, identify and quantify other mycotoxin-producing fungi and/or the toxigenic potential of such fungi. NE, KS, and KY will collaborate with other participants to evaluate isolates from other states by using these different tools. KY and OK have sequenced genomes of 46 Epichloë isolates representing 24 different species (12 hybrids and 12 nonhybrids). The evolution of each of these species will be tested using phylogenomics to test if isolates from each species have originated from a single origin or if some hybridizations have occurred independently more than once. 3.2 Strain Collection of Mycotoxigenic Fungi The Fungal Genetics Stock Center is a 60+ year-old collection of fungal strains with an emphasis on fungal genetics and mutants that can be used to address critical questions in fungal biology. The FGSC has recently been relocated to the KS station. A number of these strains contain classical or targeted deletions or defects in genes that specify toxin biosynthesis. Strains of interest currently held in the collection include: Fusarium spp. – Strains blocked in trichothecene biosynthesis and fumonisin biosynthesis and for self-protection against toxins; and Aspergillus spp. – Strains blocked in synthesis of aflatoxin, sterigmatocystin and norsolinic acid. The FGSC also holds many tools that are used for the genetic manipulation and analysis of these fungi. FGSC also is an acknowledged center for deposit of strains of many different genera, including those used for genome sequencing, testers for sexual cross-fertility and vegetative incompatibility, and some strains that represent genetic diversity in relevant populations. For this project the FGSC provides a location for deposit of critical strains and related genetic materials. If these materials are available for distribution to other qualified researchers, then they are added to the FGSC’s main collection for general use by the scientific community. In terms of intellectual property, strains and other materials deposited in the FGSC are considered to be in the public domain and are distributed with a requirement to cite the FGSC as the source of the strains in publications and proposals. If distribution of materials is limited, due to regulatory or other reasons, then the strains are stored as back-ups only. 3.3 Exploration into the competitive soil ecology of mycotoxigenic Fusaria Fusaria are ubiquitous soil inhabitants. They possess unusually large families of beta-lactamase encoding genes. In bacteria these enzymes are well-known to confer resistance to beta-lactam antibiotics such as penicillin that disrupt bacterial cell wall biosynthesis. The GA station will delete the F. verticillioides beta-lactamase encoding genes individually and test their altered resistance against a range of lactam-moiety containing compounds, with preference for those produced by organisms known to interact with this fungus. 3.4 Infection processes of ears and stalks by Fusarium graminearum and Diplodia maydis that lead to mycotoxin contamination Grains are a major source of food and energy Fusarium graminearum causes Gibberella ear rot and stalk rot of corn, and it is also important as the cause of a serious disease of wheat called Fusarium Head Blight (FHB). D. maydis causes Diplodia ear rot and stalk rot of corn. Both F. graminearum and D. maydis are reported to produce mycotoxins in grain and stalk residues, posing a threat to livestock fed grain, silage, or residue from infected corn. Participants from KY, IN, and AR propose to compare and contrast mechanisms of pathogenicity and mycotoxigenicity of these two pathogens in corn ears versus stalks. There have been few studies of the cytology of colonization of corn tissues by D. maydis or F. graminearum and little is known about the molecular mechanisms of their pathogenicity to corn. Researchers at KY will conduct comparative cytological analyses of colonization of corn stalks and ears by these two fungi, while researchers in IN and AR will utilize mutagenesis and genomic approaches to understand pathogenicity and mycotoxigenicity in ears. Mutant strains of both species are available in IN and will be tested for their ability to infect both ears and stalks. Interactions between the two species in inoculated stalks and ears will be studied using strains labeled with different fluorescent proteins, and tissues infected with both will be assayed for the presence of mycotoxins. 3.5 The global regulators governing aflatoxin production and sporulation in Aspergillus flavus WI will investigate the molecular mechanisms of regulating both sporulation and aflatoxin production by the master and global velvet and LaeA regulators they have previously identified in A. flavus. Expected results include better understanding the functions of these novel fungi-specific regulators in governing sporulation and AF production, identification of groups of genes that are controlled by the key master regulators and defining the common networks regulating spore formation and AF production. Understanding the mechanisms governing sporulation and AF biosynthesis will provide new insights into controlling AF contamination in food and feed. 3.6 Identification of aflatoxin and deoxynivalenol biosynthesis inhibitors While fungicides are currently in use, mycotoxigenic fungi are highly tolerant of fungicides, and, in the case of Aspergillus, little damage is incurred on the crop by the fungus other than contamination with aflatoxin. In the case of Fusarium, deoxynivalenol is required for pathogenicity in the plant, and thus targeting production of the mycotoxin by the fungus would reduce the disease. The application of compounds that halt mycotoxin biosynthesis would be a new contribution to control. MI and WI will work to understand how four novel inhibitors of mycotoxins in Aspergillus spp. and F. graminearum function to inhibit mycotoxin production and whether they can be implemented in an agricultural setting. The work will not only increase our understanding of regulation of production of mycotoxins, but provide the basis for their application to agriculture.

Measurement of Progress and Results

Outputs

• Refereed journal publications; many will be co-authored by the members from multiple states.
• Development and validation of new management tools for diagnosis and prevention of mycotoxin contamination.
• Development of new plant varieties that will have increased resistance to mycotoxigenic fungi or reduced mycotoxin contents.
• Transfer of valuable research information to clientele groups (industry, government, grain producers and food producers) through general publications, website, and extension programs.
• The Fungal Genetics Stock Center, now located at Kansas State University, will expand and publicize its collection of mycotoxigenic fungi, and work with group members to preserve and catalogue important strains for research.
• Output 6: The MS group will continue to provide mycotoxins (fumonisin, ochratoxin A, moniliformin, zearalenone, and aflatoxin B1) in culture material to all mycotoxin research groups makes it economically feasible to conduct animal feeding studies that would be nearly impossible if mycotoxins were purchased commercially.

Outcomes or Projected Impacts

• We anticipate that results from the research outlined in Objective 1 will have a major impact on government decision-making by providing a better understanding of how various environmental and food processing components affect mycotoxin biosynthesis. It will also provide critical information to the producers of animal feed for development of additives to mitigate effects of mycotoxins.
• Information generated from research Objective 2 will advance production of plant varieties with increased resistance to mycotoxins and to mycotoxigenic fungi, and development of new protocols to reduce mycotoxin contamination of plant products postharvest.
• The outcomes from Objective 3 will include new basic knowledge that can be incorporated into new management strategies to help grain growers predict and minimize mycotoxin contamination, and maximize profitability.

Milestones

(2016): We will move our website to a new host and improve the links, including adding one to the Fungal Genetics Stock Center at KSU. Research articles by group members will be linked on the website. At our 2016 annual meeting, we will explore possibilities for collaborative proposals among our membership, and develop specific targets and research groups for applications to be developed in the following year.

(2017): We will organize a mycotoxin symposium at the Midwest AOAC annual meeting. At this meeting we will present data and also develop potential collaborations outside of the committee for future research projects. We will write and submit at least one collaborative research proposal to a national funding agency (e.g. NSF, NIH, USDA). At our 2017 meeting we will continue to develop new targets for research proposals. We will submit samples of reference mycotoxigenic fungi to the Fungal Genetics Stock Center. At the end of this year we will add a section to our website highlighting our progress on deliverables (new plant lines, available strains through the fungal genetics stock center, protocols that can be made available for collaborations etc.)

(2018): At our 2018 meeting we will continue to develop new targets for joint research proposals. We will write and submit at least one collaborative research proposals or revised proposals to national funding agencies (e.g. NSF, NIH, USDA). At the end of this year we will update our website highlighting our progress on deliverables (new plant lines, available strains through the fungal genetics stock center, protocols that can be made available for collaborations, etc.)

(2019): At our 2019 meeting we will continue to develop new targets for research proposals. We will organize a mycotoxin symposium at the Midwest AOAC annual meeting. At this meeting we will present data and also develop potential collaborations outside of the committee for future research projects. We will write and submit at least one collaborative research proposal or revised proposal to a national funding agency (e.g. NSF, NIH, USDA). At the end of this year we will update our website highlighting our progress on deliverables (new plant lines, available strains through the fungal genetics stock center, protocols that can be made available for collaborations etc.)

(2020): WWe will write and submit at least one collaborative research proposal or revised proposal to a national funding agency (e.g. NSF, NIH, USDA). At our 2020 meeting we will continue to develop new targets for research proposals. At the end of this year we will update our website highlighting our progress on deliverables (new plant lines, available strains through the fungal genetics stock center, protocols that can be made available for collaborations etc.)

Projected Participation

View Appendix E: Participation

Outreach Plan

The committee will maintain a webpage, and add a twitter account, to provide information to the public. Previous website users have included news organizations, grain industry representatives, and the general public. The site incorporates contact information for members, annual reports, meeting announcements and links to all topics related to mycotoxins.
All of our outcomes derived from this research will be communicated through organized symposia. Our MWAOAC symposia have attracted researchers from states not participating in NC 1025, but have mycotoxin problems. Many of the AOAC members are involved in mycotoxin issues, and they represent industry, state and federal government. While the AOAC symposia present the work of the entire committee, each member presents his/her results at meetings specific to his/her area of expertise. Refereed journal publications will be an important outreach tool for all the listed outcomes. Many of the publications will be on applied research. Also, members who have extension activities will transfer
information to grain and food producers. With respect to the outcomes anticipated for Objective 3, several members will present their findings at the annual Fusarium Forum organized by the USDA Wheat and Barley Scab Initiative (www.scabusa.org). Attendees to the forum include growers, millers, representatives of industry, and scientists. The committee will collaborate with the international Fusarium Workshop (held in Kansas in odd years and at a prominent world site in even years), which provides training on biology, taxonomy, and toxicity of Fusarium species. We will coordinate with global efforts in Europe and Africa. Furthermore, the outcomes derived from Objective 1 will be reported at the annual meetings of the Institute of Food Technologists and Experimental Biology, which are major venues for communicating on food toxicology and nutrition toxicology.

Organization/Governance

The executive committee will consist of a chair, vice-chair, secretary and past chair. The executive committee will be elected by the technical committee. Each year a new secretary will be elected and the vice-chair will advance to chair, with the chair; becoming past chair. This committee will conduct business as necessary for the whole committee, between meetings of the technical committee.
The technical committee meeting will be called once a year by the Administrative Adviser. At these meetings, work at the participating stations will be reviewed for progress and for areas needing further effort. When advantageous, efforts will be made to provide for exchange of representatives with other technical committees. Publication of results will be in the form of scientific publications, extension reports or technical bulletins, as appropriate. Attendance at the annual meeting and participation with the group will be monitored on a yearly basis. The committee will discuss with the Administrative Advisor possible remedies for delinquent members.

Duties of Members of the Executive Committee:

Chair - establish location of meeting and coordinate the date with the Administrative Adviser. Notify technical committee members of dates, times and location of meeting and assist members in making accommodations. Call the meetings to order and preside during the
meeting. Will become past Chair following Annual Meeting adjournment.

Vice-Chair - will function as the Chair in his/her absence. Becomes chair immediately following the Annual Meeting. Is responsible for writing, getting approval and disseminating the Annual Report.

Secretary - will take minutes for all meetings of the Executive Meeting and the Annual Meeting at which he/she is elected. Is responsible for disseminating copies of the minutes to all Technical Committee members following approval by the Administrative Adviser. Becomes vice chair for the next Annual Meeting. Maintains website by soliciting and sending updates to website administrator. Maintains twitter account by soliciting and sending updates to account administrator.

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Susca, A; Moretti, A; Stea, G; Villani, A; Haidukowski, M; Logrieco, A; Munkvold, G. (2014) Comparison of species composition and fumonisin production in Aspergillus section Nigri populations in maize kernels from USA and Italy. Int J Food Micro 188: 75-82.

Trail, 2009. For Blighted Waves of Grain: Fusarium graminearum in the post-genomics era. Plant Physiology 149: 103-110

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Wu, F., Bhatnagar, D., Bui-Klimke, T., Carbone, I., Hellmich, R., Munkvold, G., Paul, P., Payne, G., and Takle. E. 2011. Climate change impacts on mycotoxins risks in US maize. World Mycotoxin J. 4:79-93.

Yarru, L. P., R. S. Settivari, E. Antoniou, D. R. Ledoux, and G. E. Rottinghaus. 2009a. Toxicological and gene expression analysis of the impact of aflatoxin B1 on hepatic function of male broiler chicks. Poultry Sci. 88:360-371.

Yarru, L. P., R. S. Settivari, N.K.S. Gowda, E. Antoniou, D. R. Ledoux, and G. E. Rottinghaus. 2009b. Effects of turmeric (Curcuma longa) on the expression of hepatic genes associated with biotransformation, antioxidant, and immune systems in broiler chicks fed aflatoxin. Poultry Sci. 88:2620-2627.

Attachments

Land Grant Participating States/Institutions

CA, IA, IL, KS, KY, MI, MO, MS, ND, NE, NJ, PA, VA, WI, WV

Non Land Grant Participating States/Institutions

Federal University of Vicosa - Brazil, Samuel Roberts Noble Foundation