Brief Summary of Minutes of Annual Meeting

Annual Report of Research Activities

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

IA Station: The group has developed and are validating assays for the detection and quantitation of aflatoxin AFB1. The group has conducted both intralab and interlab validation of the liver method.  The group continues its work on developing mycotoxin-resistant crops, and on assessing new fungal threats to grain quality, in particular conducting surveys for the presence and mycotoxigenicity of several Aspergillus species.

MO Station: The Fusarium/Poultry Research Laboratory evaluated a large number of mineral and organic adsorbents for binding mycotoxins in in vitro and in vivo studies in poultry, swine and cattle.  Naturally occurring antioxidants were evaluated for reducing mycotoxin toxicity in poultry. The laboratory continues to produce mycotoxins in culture (kg quantities aflatoxin, zearalenone, ochratoxin A, fumonisin B₁) for in-house use, as well as for other researchers doing animal feeding trials with mycotoxins. These data were published in refereed journals and/or provided to commercial companies which used the data to produce efficacious products for agriculture to prevent mycotoxicosis.

NC Station:  The NC group have monitored major mycotoxins present in a variety of crops grown in developing countries.  They are particularly focused on assessing and validating commercial kits for testing the presence of mycotoxins.  Their results indicate that ability to detect mycotoxins depends on the test kit being used as well as the sampling procedures employed.  They have shown that both pre- and post-harvest processes contribute to contamination of nuts, raisins and wheat contaminated with aflatoxins, tricothecenes and other Fusaiurm-derived toxins such as as zearalenone.  They also helped establish and trained staff to perform mycotoxin assays in a lab in Afghanistan.

NJ Station: The NJ Station has shown that lifespan of Caenorhabditis elegans is significantly reduced by treatment with DON.  RNAseq analysis identified a large number of deoxinivalenol (DON)-regulated genes, including those that encode enzymes likely to be involved in detoxification, as well as a number of novel genes whose role in the response to DON has yet to be determined. These include genes associated with innate immunity and lipid metabolism and transport and others.  The functional roles of several of these highly up-regulated genes is being investigated through RNAi suppression.  RNAi-mediated suppression of these genes reduced the viability of DON-treated C. elegans, compared to wild-type DON-treated worms.

In collaboration with Dr. Christopher Schardl at University of Kentucky, the NJ group used the C. elegans system to analyze the cytotoxicity of N-formylloline (NFL) isolated from the endophyte Epichloë. These studies revealed that NFL was far more toxic to worms than was DON, and also severely reduced fecundity.  These results shows that C. elegans can be used to study the cytotoxicity of fungal toxins other than DON and that this system may be of general use for understanding the mode of action of emerging mycotoxins.

Objective 2: Establish integrated strategies to manage and reduce mycotoxin contamination in cereals and in forages.

MI Station: The Trail lab has screened extracts of plants and fungi to identify compounds that inhibit aflatoxin and DON biosynthesis in mycotoxigenic fungi.  Compounds present in black pepper have been structurally characterized and synthesized. Their efficacy has been tested in preventing aflatoxin contamination on maize grains.  These assays have shown that the compounds are effective in inhibiting aflatoxin production. The group is now working on commercial applications. They have identified bioactive compounds with anti-mycotoxin activity from additional sources, and in several cases have characterized the structure.  These will now enter the pipeline to test for efficacy in field control of mycotoxins. 

MS Station:  The Shan lab is working to incorporate natural host plant resistance to reduce aflatoxin in corn in a sustainable manner. Since most of the QTLs identified were of low to moderate effects, pyramiding of these QTLs will lead to enhanced resistance to aflatoxin accumulation in corn. From previous studies, ten different near isogenic inbred lines (NILs) carrying one to three major resistance QTLs from Aspergillus flavus resistant corn line Mp313E have been generated and continuously evaluated for the resistance. Using next-generation sequencing techniques, the Shan group has sequenced whole genomes of Mp313E and assembled genome sequences for four major resistant QTLs. Specific objectives include genome sequence analysis of the resistance QTLs to illustrate mechanisms of resistance genes; screening of SNP markers; and development of DNA markers for breeding using the NIL-QTL lines from Mp313E x Va35.

The Brown group has developed a single maize kernel aflatoxin extraction method and correlated and characterized aflatoxin accumulation and fungal biomass for during several weeks after inoculation with Aspergillus flavus. They have also: assessed if certain storage conditions can reduce the growth of Aspergillus flavus and additional aflatoxin accumulation of harvested maize; Developed a viable method for differentiating between formulations of synthetic auxins using Fourier Transform Infrared spectroscopy (FT-IR); Investigated the use of biochar for the remediation of aflatoxin M1 from contaminated milk.

MO Station:  In parts of the world where grain is not screened for mycotoxins, the most promising and practical approach for detoxification and remediation of highly contaminated feedstuffs has been the addition of adsorbents to the contaminated feed to selectively bind the mycotoxin during the digestive process. These natural sorbents are generally recognized as safe, have been approved for use as feed additives by the European Union, and represent a multimillion dollar business. Many of these binding agents have proven to be very successful in binding the aflatoxins in in vitro and in vivo studies, but are relatively ineffective in binding other mycotoxins found in feedstuffs. The MO Station has developed assays to test the efficacy of various sorbants and additives for their ability to reduce the exposure of animal tissues to mycotoxins.  Information from this project in combination with in vivo mycotoxin detoxification studies of the most successful candidates by collaborating researchers can be used to advise producers, extension personnel, and regulatory agencies on how to safely manage the utilization of mycotoxin contaminated grains in animal feeds.

NE Station:  Trials were conducted in 2016 to quantify the impact of wheat variety, fungicide treatment, and storage conditions on DON, as measured by DON biosynthesis gene expression as well as DON quantity (measured by gas chromatography). Data analysis is underway on these experiments.

NJ Station: The NJ group have developed dedicated CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 nuclease) gene-editing platforms to disrupt genes that condition susceptibility to Fusarium graminearum (Fg) infection to engineer FHB resistance in model plants Brachypodium (Bd21 variety), Arabidopsis, and barley.  This platform has been used to construct CRISPR constructs that target the EIN2, HSK and 2OGO genes in all three species.  Homologs of these genes in other species condition susceptibility to Fusarium and other fungal pathogens.  Gene-edited AtHSK- and At2OGO-knock out (KO) mutant Arabidopsis plants have now been constructed and verified.   Disease susceptibility assays as well as Fusarium growth assays show that the gene-edited AtHSK and At2OGO Arabidopsis plants are more resistant to infection by F. graminearum-GFP than are WT plants.  Barley plants containing similarly edited genes are under construction using a modified tissue culture protocol for barley (Hordeum vulgare, Hv; cv. Conlon). 

PA Station: The Kuldau lab is isolating and characterizing microbes with the ability to inhibit the growth of the Fusarium Head Scab pathogen, Fusarium graminearum, as well microbes capable of transforming the mycotoxin DON produced by this fungus.  To date the group has isolated nine bacteria able to inhibit growth of F. graminearum in plate assays.  These organisms belong to the genera Bacillus, Brevibacillus, and Burkholderia based on 16S rDNA sequencing.  Eight of these isolates inhibit germination of F. graminearum macroconidia when co-cultured.  After 24 hours, some isolates inhibit all macroconidia germination.  Eleven bacterial isolates putatively transform DON based on an initial selection process.  Studies using purified DON and analysis by gas chromatography confirm the DON transforming status of three of these isolates.  Field studies conducted to assess the ability of two of the bacterial isolates to reduce Fusarium Head Blight Disease in the field did not show any effects with respect to disease incidence or severity. Effects of these bacteria on DON content of harvested grain is ongoing.

Objective 3: Better Understand the Biology and Ecology of Mycotoxigenic Fungi.

KY/OK Station: Collaborators at the University at Kentucky (Schardl) and the Noble Research Institute, LLC (Young) conducted DNA sequencing and assembly of draft genomes for Epichloë species symbiotic with tall fescue, including three strains of Epichloë coenophiala, and isolates of three other species as yet unnamed, but designated FaTG-2, -3 and -4. The three newly sequenced strains of E. coenophiala were from Iberia and Morocco (Mediterranean). These, as well as FaTG-3, lacked the genes for ergot alkaloid biosynthesis, bur had genes for indole-diterpene, peramine and loline alkaloid biosynthesis. Strains of FaTG-2 and FaTG-4 had genes for indole-diterpene, peramine and ergot alkaloid biosynthesis. The presence of alkaloids of these three classes in plants with these species of endophytes was confirmed by chemical analysis. They conclude that the Mediterranean strains of E. coenophiala, as well as FaTG-3, have alkaloid profiles that are probably desirable for endophytes of tall fescue forage cultivars if the indole-diterpenes that they produce are non-toxic to livestock.

MI Station: The Trail lab has performed a microbiome experiment on wheat across four management strategies (organic, conventional, no till, low nitrogen input).  The microbiome does not vary significantly with treatment.  They collected fungal and bacterial isolates from field grown wheat and tested them in culture for antagonism to F. graminearum. Four isolates had positive effects in reducing damping off in seedlings by F. graminearum. The Trail lab has also investigated how the surface interaction of Fusarium graminearum on barley promotes life cycle stages, such as perithecia production, conidiation and infection. Trichomes are important points of interactions with germinating conidia, as has been shown in wheat and Brachypodium. Interestingly, F. graminearum interacts with cells high in silica content for all of the important stages of its life cycle. 

NE Station: Wheat was sampled in the field throughout the state of Nebraska during the 2017 growing season and assayed for Fusarium head blight. Collections from the previous year were also analyzed to determine: Fusarium species infecting wheat and mycotoxin chemotype. Three Fusarium species - F. boothii, F. poae, and F. acuminatum - were isolated in addition to the expected F. graminearum. The predominant toxin chemotype remains DON+15-ADON, as it has been in previous years; <5% of samples give conflicting results on molecular chemotype tests, and these samples do not produce toxins in planta under standard infection conditions.

OK Station: The Young lab at the Noble Research Institute, LLC, has been using a surveillance pipeline to discover grass endophytes (Epichloë species) with the potential to produce bioactive alkaloids such as ergot alkaloids, indole-diterpenes, lolines and peramine.  In 2017, they screened all available Festuca pratensis (meadow fescue) in the The United States Department of Agriculture National Plant Germplasm System. Of the 324 available F. pratensis PI lines, 26% were considered to have some level of endophyte infection. Of the infected PI lines 79% were considered to contain Epichloe uncinata.  The remaining 21% of samples will be further evaluated as some were associated with the likely ability to produce ergot alkaloids. The surveillance pipeline has also been used to evaluate other grass-endophyte associations that have been recently published.

KY Station: With Dr. Trail of MI, L. Vaillancourt developed and investigated a set of F. graminearum mutants that were deleted in various mating type genes (the entire MAT1 locus, or the MAT1-1-1 or MAT1-2-1 genes alone).  The MAT1 KO strains had normal pathogenicity on both spring wheat (cultivar Norm) and winter wheat (cultivar Pioneer 2555).  However, both of the individual MAT gene KO strains were significantly reduced in pathogenicity on both types of wheat.  The latter result was not consistent with a recent publication that showed no effect of knocking out these genes on pathogenicity to a Chinese wheat cultivar Trail and Vaillancourt wrote a pre-proposal together to support further studies, and will be continuing to work with these mutants together in the future

VA Station: Schmale, Trail, and colleagues 'squeezed' perithecia of Fusarium graminearum to provide insights into the strength of the perithecial wall and the quantity of ascospores. A mechanical compression testing instrument was used to determine the structural failure rate of perithecial walls from three different strains of F. graminearum (two wild-type strains, and a mutant strain unable to produce asci). The MPCC increased as perithecia matured, and the highest number of ascospores was found in older perithecia. The results indicated that for every additional day of perithecial aging, the perithecia become more resilient to compression forces. In the future, compression testing may provide a unique method of determining perithecial age in the field, which could extend to management practices that are informed by knowledge of spore release and dispersal. This work was published in Fungal Genetics and Biology in September 2016.

WI Station: Keller and Yu continued their studies of development and mycotoxin production in the widely distributed Aspergillus flavus, an opportunistic pathogen of plants and humans. Aspergillus flavus can produce the mycotoxin aflatoxin B1 (AFB1), the most potent carcinogen found in nature. The main means of dissemination of this fungus is producing a massive number of asexual spores (conidia), which are dispersed in the soil and air. In agricultural fields, these spores are carried to corn ears by insects or the wind where they grow in maize kernels and produce AFB1. Aspergillus fungi’s conidia formation and maturation is governed by the central genetic regulatory circuit BrlAàAbaAàWetA. In the current year, the group reported that WetA is a multi-functional regulator that couples spore differentiation and survival, and governs proper AFB1 production in A. flavus. The deletion of wetA results in the formation of conidia with defective cell walls and no intra-cellular trehalose, leading to reduced stress tolerance, a rapid loss of viability, and disintegration of spores. WetA is also required for normal vegetative growth, hyphal branching, and production of aflatoxins. Targeted and genome-wide expression analyses reveal that WetA exerts feedback control of brlA and influences expression of 5,700 genes in conidia. Functional category analyses of differentially expressed genes between wild type and wetA null mutant conidia RNA-seq data indicate that WetA contributes to spore integrity and maturity by properly regulating the metabolic pathways of trehalose, chitin, β -(1,3)-glucan, β-(1,3)-glucan, melanin, hydrophobins, and secondary metabolism more generally. Moreover, 160 genes predicted to encode transcription factors are differentially expressed by the absence of wetA, suggesting that WetA may play a global regulatory role in conidial development. Collectively, we have revealed that the evolutionarily conserved WetA protein plays a global regulatory role in governing growth, development, bridging spore differentiation and survival, and aflatoxin biosynthesis in A. flavus.

Synergistic Activities

• The Schmale and Trail labs collaborated to study factors that influence the strength of the Fusarium graminearum perithecial wall.
• The Vaillancourt and Trail labs are collaborating on the interaction between mating type locus and pathogenicity in  Graminearum.  They have performed pathogenicity trials on knockouts of the MAT loci and demonstrated that the degree of pathogenicity on wheat varies and appears to associate with the MAT loci present in the strain. 
• The Keller and Trail labs communicate regularly about the development of novel means of controlling aflatoxin.  They regularly share information and approaches, assisting each other in moving forward towards development of controls to aflatoxin contamination.
• The Young and Schardl labs at the University at Kentucky (Schardl) and the Noble Research Institute, LLC (Young) collaborated to sequecne and assemble draft genomes for Epichloë species symbiotic with tall fescue.
• The Young and Schardl labs collaborated to understand the relationship between the ecology of Epichloë spp and mycotoxin production.
• The University of Kentucky (Schardl), Noble Research Institute, LLC (Young), West Virginia University (Daniel G. Panaccione) and others collaborated on a grant proposal to the NSF Dimensions of Biodiversity program, Though the application was unsuccessful, plans are to revise and resubmit the application in 2018.
• The Brown and Schardl groups have collaborated on a number of projects to develop aflatoxin extraction methods, examine the factors that influence aflatoxin accumulation and fungal biomass and investigate the use of biochar for the remediation of aflatoxin M1 from contaminated milk.
• The NJ Station collaborated with the Schardl group at KY to use the elegans system to analyze the cytotoxicity of N-formylloline (NFL) isolated from the endophyte Epichloe.
• The MO Station’s work on mycotoxin additives is funded by commercial companies that supply mineral and feed supplements.Impacts
• In collaboration with Dr. Christopher Schardl at University of Kentucky, the NJ group used the elegans system to analyze the cytotoxicity of N-formylloline (NFL) isolated from the endophyte Epichloë. These studies revealed that NFL was far more toxic to worms than was DON, and also severely reduced fecundity. These results shows that C. elegans can be used to study the cytotoxicity of fungal toxins other than DON and that this system may be of general use for understanding the mode of action of emerging mycotoxins.
• The MO Station has developed assays to test the efficacy of various sorbants and additives for their ability to reduce the exposure of animal tissues to mycotoxins. Information from this project in combination with in vivo mycotoxin detoxification studies of the most successful candidates by collaborating researchers can be used to advise producers, extension personnel, and regulatory agencies on how to safely manage the utilization of mycotoxin contaminated grains in animal feeds.
• The NJ group have developed dedicated CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 nuclease) gene-editing platforms to disrupt genes that condition susceptibility to Fusarium graminearum (Fg) infection to engineer FHB resistance in the model plant Arabidopsis. Disease susceptibility assays as well as Fusarium growth assays show that the gene-edited AtHSK and At2OGO Arabidopsis plants are more resistant to infection by graminearum-GFP than are WT plants. This suggests the possibility to use this new gene-editing approach to produce resistant crop plants.
• The PA and MI groups have identified novel microbes and microbial metabolites that inhibit the growth of mycotoxigenic fungi and detoxify mycotoxins, opening the possibility for sustainable biologically-based management in the future.