NC_old1183: Mycotoxins: Biosecurity, Food Safety and Biofuels Byproducts (NC129, NC1025)

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[10/20/2015] [11/27/2017] [10/05/2018] [10/20/2019] [02/03/2021]

Date of Annual Report: 10/20/2015

Report Information

Annual Meeting Dates: 10/09/2015 - 10/09/2015
Period the Report Covers: 10/01/2010 - 10/01/2015

Participants

Brief Summary of Minutes

Please see attached for NC_old1183's termination report, as well as the NC1183 2015 report.

Minutes Attachment:

NC OLD1183 termination report (2).pdf

Accomplishments

Publications

Impact Statements

Date of Annual Report: 11/27/2017

Report Information

Annual Meeting Dates: 09/26/2017 - 09/26/2017
Period the Report Covers: 10/01/2016 - 09/30/2017

Participants

Participants Attachment:

NC1183 Participants.pdf

Brief Summary of Minutes

Minutes Attachment:

NC1183 minutes of annual meeting.pdf

Accomplishments

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 <ul> <li>The Schmale and Trail labs collaborated to study factors that influence the strength of the Fusarium graminearum perithecial wall.</li> <li>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. </li> <li>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.</li> <li>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.</li> <li>The Young and Schardl labs collaborated to understand the relationship between the ecology of Epichloë spp and mycotoxin production.</li> <li>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.</li> <li>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.</li> <li>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.</li> <li>The MO Station’s work on mycotoxin additives is funded by commercial companies that supply mineral and feed supplements.Impacts </li> <li>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.</li> <li>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.</li> <li>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.</li> <li>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.</li> </ul>

Publications

Impact Statements

Information and Technology Transfer: A new Project Web site is now active and can be found at https://www.mycotoxins-nc1183.com/ . We expect this site will serve our efforts to better communicate with stakeholders and assist in promoting cooperation among project members and between project members and other researchers and stakeholders.

Date of Annual Report: 10/05/2018

Report Information

Annual Meeting Dates: 05/18/2018 - 05/18/2018
Period the Report Covers: 10/01/2017 - 09/30/2018

Participants

Participants Attachment:

NC1183_May_18_participants_list.pdf

Brief Summary of Minutes

Minutes Attachment:

Minutes_NC1183_May_18_2018.pdf

Accomplishments

Objective 1: Develop data for use in risk assessment of mycotoxins in human and animal health. VA: The Schmale lab is conducting experiments to track the mycotoxin zearalenone in swine reproductive tissues. IA Station: Field and laboratory studies were conducted with Fusarium temperatum, a cryptic species within F. subglutinans. Mycotoxin production was measured in vitro and in the field. Strains of F. temperatum produced beauvericin and moniliformin, but strains of F. subglutinans sensu stricto did not. Neither produced trichothecenes. Silk channel inoculations of maize plants in the field resulted in elevated levels of fusaric acid, fusaproliferin, beauvericin, and moniliformin. Only non-inoculated treatments had significant fumonisin contamination. Occurrence of F. temperatum in maize fields appears to alter the mycotoxin profile of contaminated grain, which also changes mycotoxin risk for humans and other animals consuming maize grain. In a USAID-funded project, a survey of mycotoxins in dairy and poultry feed (and ingredients) was conducted across the country of Rwanda in 2017.  Over 3,300 samples were collected from farmers, feed vendors and processors from all 30 districts in Rwanda in six rounds over a period of 6-7 months. Approximately 170 milk samples were also collected from dairy producers in one of the rounds of sample collection and analyzed using fluorometry. Fumonisin contamination was low (mean 1-1.5 ppm when grouped by type of producer—either poultry farmer, dairy farmer, feed vendor, or feed processor) aflatoxin was high (mean 89-109 ppb for the same groupings).  Maize bran tended to be most highly contaminated ingredient. There was a trend for increasing contamination over time in storage. MO Station: The Fusarium/Poultry Research Laboratory continues to evaluate 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.   Objective 2: Establish integrated strategies to manage and reduce mycotoxin contamination in cereals and in forages. IA: Field studies with Fusarium temperatum are being conducted to assess the value of insect management with transgenic insect resistance in order to reduce the risk of mycotoxins associated with this fungal species. VA: The Schmale lab has developed a variety of different detoxification strategies for DON. The lab continues to provide mycotoxin testing services for wheat and barley researchers associated with the USWBSI. A former graduate student in the Schmale Lab, Dr. Nina Wilson, developed and delivered a unit on mycotoxins for high school students in Virginia. This resulted in a peer-reviewed publication in the Science Teacher. MO:  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: Combined field and lab studies show consistent increase of DON in stored grains of a susceptible and a moderately resistant variety of wheat treated with strobilurins (increase equal to or greater than in untreated wheat; manuscript in preparation). PA: 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 four bacteria able to inhibit growth of F. graminearum in plate assays and remove DON from culture media.  Studies using purified DON and analysis by gas chromatography confirm the DON transforming status of the four isolates. These organisms belong to the genera Brevibacillus, and Burkholderia based on 16S rDNA sequencing.  Eight additional isolates inhibit growth of F. graminearum, and four reduce DON in culture media.  Eight isolates inhibited germination of F. graminearum macroconidia.  After 24 hours, some isolates inhibit all macroconidia germination.  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. In some cases, application of bacteria in the field resulted in higher DON levels than in control plots with no bacterial application.   Objective 3: Better Understand the Biology and Ecology of Mycotoxigenic Fungi NE Station:  Field surveys of wheat for FHB causal organism and toxin genotype continued in 2017 and 2018; effectively no head blight in 2017, and FHB geographically circumscribed in 2018 (little to none in eastern, but high incidence in central, Nebraska). Analysis of previous years’ samples confirmed Fusarium boothii in geographically distinct samples of Nebraska wheat (published as disease note in Plant Disease, 2018). KY: In collaboration with Dr. Trail of MI, L. Vaillancourt is investigating a set of Fusarium 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 have normal pathogenicity on both spring wheat (cultivar Norm) and winter wheat (cultivar Pioneer 2555).  However, both of the individual MAT gene KO strains are significantly reduced in pathogenicity on both types of wheat. Trail and Vaillancourt wrote a joint proposal, and received two years of funding from the USWBSI in the fall of 2018 to continue to work with these mutants together.  The proposed work will include a cytological analysis and a transcriptome study to compare the mutants with the wild type in wheat heads. Vaillancourt is also collaborating with Dr. Emerson Del Ponte of Universidade Federal de Viçosa to investigate F. graminearum and F. meridionale strains causing ear and stalk rot diseases in corn in Brazil versus in the U.S. A dual degree student has characterized a collection of Brazilian strains representing both species isolated from both corn and wheat.  He has found that a majority of the strains from both species cluster together into a single group, based on various phenotypic characteristics in vitro and in planta (corn ears and stalks).  Current work is focused on evaluating the potential for cross-fertility among members of these two species in vitro and in the field. WI: 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 BrlA-->AbaA-->WetA. 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.  The Keller lab also works with two other mycotoxigenic species, Fusarium spp. and Pencillium expansum.  Her lab is currently focused on polymicrobial interactions of phytobacteria with Fusarium and Aspergillus species best summarized in publications (ACS Chemical Biology 13(1):171-179 ; ISME J.  10(9):2317-30 ; MBio 9:3 May/June; doe:10.1123/mBio.00820-18 ).  Her lab has identified two P. expansum genes, LaeA and CreA, that regulate virulence on apple and patulin synthesis (Mol Plant Pathol 18:1150-1163 ; Mole Plant Pathology.  doi: 10.1111/mpp.12734. [Epub ahead of print]).   Synergistic Activities The Vaillancourt and Trail labs are collaborating on the interaction between mating type locus and pathogenicity in F. 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.  They have received funding for a joint project to continue these studies. The MO Station’s work on mycotoxin additives is funded by commercial companies that supply mineral and feed supplements. 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.

Publications

Di, R.; Zhang, H.; Lawton, M.A. Transcriptome Analysis of C. elegans Reveals Novel Targets for DON Cytotoxicity. Toxins 2018, 10, 262.  doi:10.3390/toxins10070262 Gdanetz K. and trail F. 2017. The wheat microbiome under four management strategies, and the potential for endoohytes in disease protection. Phytobiomes 1: 158-168. Hurburgh, C. and Robertson, A.  2018. Crop Quality hurt by rains. <a href="https://crops.extension.iastate.edu/cropnews/2018/10/crop-quality-hurt-rains">https://crops.extension.iastate.edu/cropnews/2018/10/crop-quality-hurt-rains</a> Hurburgh, C. 2018. Management of flood submerged grain. <a href="https://crops.extension.iastate.edu/cropnews/2018/09/management-flood-submerged-grain">https://crops.extension.iastate.edu/cropnews/2018/09/management-flood-submerged-grain</a> Imboden, I., Afton D., Trail, F. 2017. Surface interactions of Fusarium graminearum on barley. Molecular Plant Pathology. DOI: 10.1111/mop.12616. <a href="https://link.springer.com/journal/12550/onlineFirst/page/1">Franco LT, Petta T, Rottinghaus GE, Bordin K, Gomes GA, Oliveira C</a>. Co-occurrence of mycotoxins in maize food and maize-based feed from small-scale farms in Brazil: a pilot study.  Mycotoxin Research https://doi.org/10.1007/s12550-018-0331-4, 2018. <a href="https://dl.sciencesocieties.org/publications/cs/abstracts/58/2/925">Kenyon SL, Roberts CA, Kallenbach RL, Lory JA, Kerley MS, Rottinghaus GE, and Hill NS, Ellersieck MR</a>. Vertical distribution of ergot alkaloids in the vegetative canopy of tall fescue. Crop Sci 58(2):925-931, 2018. Leslie, J.F., Lattanzio, V., Audenaert, K., Battilani, P., Cary, J., Chulze, S.N., De Saeger, S., Gerardino, A., Karlovsky, P., Liao, Y., Maragos, C.M., Meca, G., Medina, A., Moretti, A., Munkvold, G., Mulè, G., Njobeh, P., Pecorelli, I., Perrone, G., Pietri, A., Palazzini, J.M., Proctor, R.H., Rahayu, E.S., Ramírez, M.L., Samson, R., Stroka, J., Sulyok, M., Sumarah, M., Waalwijk, C., Zhang, Q., Zhang, H., and Logrieco, A.F. 2018. MycoKey round table discussions of future directions in research on chemical detection methods, genetics and biodiversity of mycotoxins. Toxins Vol. 10, 109. doi:10.3390/toxins10030109 Munkvold, G.P., Weieneth, L., Proctor, R., Busman, M., Blandino, M., Susca, A., Logrieco, A., and Moretti, A. 2018. Pathogenicity of fumonisin-producing and nonproducing strains of Aspergillus species in section Nigri to maize ears and seedlings. Plant Dis. 102: 282-291.<a href="https://doi.org/10.1094/PDIS-01-17-0103-RE"> https://doi.org/10.1094/PDIS-01-17-0103-RE</a> Munkvold, G.P., Arias, S.L., Taschl, I., and Gruber-Dorninger, C. 2018. Mycotoxins in Corn – Occurrence, Impacts, and Management. Pp. in Corn Chemistry and Technology, 3rd Edition. Eds.  Am. Assoc. Cereal Chemists, St. Paul, MN. Munkvold, G.P. 2017. Fusarium species and their associated mycotoxins. Ch 4 (pp. 51-106) In: Methods Molecular Biology, Vol. 1542: Mycotoxigenic Fungi. Antonio Moretti and Antonia Susca (Eds). Springer. Shi C, An S, Yao Z, Young CA, Panaccione DG, Lee ST, Schardl CL, Li C. (2018) Toxin-producing Epichloë bromicola symbiotic with the forage grass, Elymus dahuricus, in China. Mycologia 109:847-859. DOI: 10.1080/00275514.2018.1426941 Stoetzer, E.  2018. Mycotoxin App Available. <a href="https://crops.extension.iastate.edu/blog/ethan-stoetzer/mycotoxins-app-available">https://crops.extension.iastate.edu/blog/ethan-stoetzer/mycotoxins-app-available</a> <a href="https://ac.els-cdn.com/S0377840117307137/1-s2.0-S0377840117307137-main.pdf?_tid=689b1c26-d447-11e7-bf35-00000aab0f6c&acdnat=1511879062_16e20fbdebefde6fab647bfc4e5c6029">Weatherly ME, Pate RT, Rottinghaus GE, de Oliveira Roberti Filho F, Cardoso FC</a>. Physiological responses to a yeast and clay-based adsorbent during an aflatoxin challenge in Holstein cows. Animal Feed Science and Technology 235147-157, 2018. Wegulo, S.N., Valverde-Bogantes, E.,  Bolanos-Carriel, C., Hallen-Adams, H.,  Bianchini, A., McMaster, N., and Schmale, D.G. 2018. First Report of Fusarium boothii Causing Head Blight of Wheat in the United States. Plant Disease. <a href="https://doi.org/10.1094/PDIS-04-18-0696-PDN">https://doi.org/10.1094/PDIS-04-18-0696-PDN</a> Wilson, N., Dashiell, S., McMaster, N., Bohland, C., and Schmale, D. 2018. Could Your Food be Contaminated with Toxins? Educating High School Students about Mycotoxins in Feed and Food Products. The Science Teacher 86 (1): 46-52. Yi M, Hendricks WQ*, Kaste J, Charlton ND*, Nagabhyru P, Panaccione DG, Young CA. (2018) Molecular identification and characterization of endophytes from uncultivated barley. Mycologia 110:453-472 DOI:10.1080/00275514.2018.1464818

Impact Statements

KY initiated a dual degree program with the Universidade Federal de Viçosa in Brazil, with the first Ph.D. student who will be conducting research on the genetic diversity of Fusarium species causing stalk and ear rot diseases in maize in Brazil and in the United States.

Date of Annual Report: 10/20/2019

Report Information

Annual Meeting Dates: 09/12/2019 - 09/12/2019
Period the Report Covers: 10/01/2018 - 09/30/2019

Participants

Attendance in person in Blacksburg, Virginia
David Schmale, Virginia Tech
Nicole McMaster, Virginia Tech
Celia Jimenez-Sanchez, Virginia Tech
Erica Pack, Virginia Tech
Heather Hallen-Adams, University of Nebraska Lincoln
David Jackson, University of Nebraska, Lincoln
Gretchen Kuldau, Penn State University
Di Rong, Rutgers University
John Leslie, Kansas State University
Lisa Vaillancourt, University of Kentucky
Aline Vieira de Barros, University of Kentucky
Gabdiel Yulfo-Soto, University of Kentucky

Virtual attendance via Zoom:
Dan Panaccione, West Virginia University
Gary Munkvold, Iowa State University
David Jackson, University of Nebraska
Nancy Keller, University of Wisconsin Madison
Steve Ensley, Kansas State University
Zach Noel, Michigan State University

Brief Summary of Minutes

Minutes Attachment:

Minutes_NC1183_Hallen_Adams_Sept_13_2019.pdf

Accomplishments

ACTIVITIES   Objective 1: Develop data for use in risk assessment of mycotoxins in human and animal health. KS: K-State and the University of Nebraska-Lincoln are the two institutions primarily responsible for mycotoxin work in the USAID-sponsored Feed the Future (FTF) Innovation Lab for the Reduction of Post-Harvest Losses.  These efforts have resulted in buy-ins to the project from USAID missions in Kabul, Afghanistan ($1.2 million), Khatmandu, Nepal ($1.2 million), and Tegucigalpa, Honduras ($600,000). Health concerns were of critical importance in all three countries, with economic concerns about exports also important in Afghanistan.      The work in Afghanistan is finished, and joint publications are in preparation.  Work on raisins and other dried fruits has been in collaboration with BOKU at the University of Vienna (Austria) and with the CNR Institute for the Science of Food Production in Bari, Italy.      The work in Nepal was designed to overlap with and extend the work of the FTF Nutrition Innovation Lab to look for correlations between agriculture and the health of pregnant women and infants.  Approximately 95% of the women in the Banke district in Nepal are positive for aflatoxin in the blood. Stunting amongst children in this district exceeds 25%. A preliminary survey suggests a potential problem with aflatoxin in chilies and soy nuggets in addition to expected problems in peanuts and maize.  A lab was established at the Nepal Academy of Sciences and staff for the lab have been to Nebraska for training. Initial sample analysis is complete with peanuts and maize having the highest levels of aflatoxin contamination, followed by intermediate levels of contamination for dried chilies and soy nuggets, and relatively little aflatoxin contamination in either rice or wheat-based weaning foods.      In Honduras the focus was on maize in the western highlands where childhood stunting again exceeded 25% of the population.  A testing lab was established at Zamorano University. Maize was the only crop surveyed. Samples were assayed for both aflatoxin and fumonisin, with fumonisin appearing to be the more frequent contaminant.      Dr. Steve Ensley is in the process of establishing a mycotoxin analysis lab at K-State.  The lab will focus initially on animal feeds. The mycotoxin analysis will be performed by LC/MS/MS and a panel of mycotoxins will be analyzed at one time.  In preliminary studies this year, multiple samples had 10 ppm deoxynivalenol, and others had fumonisins at > 100 ppm. Plans for the coming year are to increase the capacity of the lab and its analytic capabilities.      As part of the European Horizon 2020 Research program, a series of roundtable discussion focused on future research areas in mycotoxicology were held at a meeting in Ghent, Belgium.  Gary Munkvold from Iowa State also participated in the discussions and is one of 31 co-authors of the resulting paper. A second joint effort was the development and publication of a mycotoxin charter with 17 co-authors to encourage more equitable regulation and increased efforts to address mycotoxin contamination in less-developed countries.   VA: The Schmale lab conducted experiments to track the mycotoxin zearalenone in swine reproductive tissues. A feeding study was conducted to track ZON and the metabolite α-zearalenol (α-ZOL) in swine reproductive tissues. Thirty pubertal gilts, approximately six months old, were randomly assigned to one of three treatments, with 10 pigs in each treatment group: (Treatment 1) base feed (solvent-only) for 21 days, (Treatment 2) ZON-spiked feed for 7 days followed by base feed for 14 days, or (Treatment 3) ZON-spiked feed for 21 days. Reproductive tracts were collected from each pig and dissected into the anterior vagina, posterior vagina, cervix, uterus, ovaries, and broad ligament. ZON was found in the anterior vagina, posterior vagina, cervix, and ovaries, with significantly higher concentrations in the cervix. α-ZOL was not found in any of the tissue types studied.     Objective 2: Establish integrated strategies to manage and reduce mycotoxin contamination in cereals and in forages.   VA: The Schmale lab has developed a variety of different strategies for the detoxification and transport of DON. The lab continues to provide mycotoxin testing services for wheat and barley researchers associated with the USWBSI. A new method was developed to quantify mycotoxins in sorghum using GC/MS.   NE: Data on field and lab studies showing consistent increase of DON in stored grains of a susceptible and a moderately resistant variety of wheat treated with strobilurins (increase equal to or greater than in untreated wheat) is accepted for publication and will be published late 2019 or early 2020.   PA: Fumonisin mycotoxins are regularly found in corn grain and silage, and their levels are regulated in Europe and subject to guidelines in the United States.  Ingestion is known to cause fatal livestock diseases, and to be associated with cancer and neural tube defects in humans. The use of Bt-maize engineered to resist feeding by lepidopteran caterpillars has in some systems reduced the occurrence of fumonisins but this technology cannot be applied for organic production.  As such other management approaches are needed for these systems. Additionally, there are gaps in knowledge as to the role of maize genotype in acquisition of fumonisin producers from the environment. Fusarium verticillioides is the most commonly found fumonisin producer on maize in Pennsylvania and in most of the United States.  F. proliferatum is another species found associated with maize in north America.      Six maize genotypes with varying in kernel resistance to fumonisin were assessed for Fusarium endophyte colonization after growth at the Penn State Russell E. Larson Agricultural Research Center at Rock Springs.  Shoot tissues including leaf, stem, reproductive tissues and also prop roots were sampled, subject to surface sterilization and plated to Fusarium selective media.  Over 420 Fusarium isolates were obtained and identified based on partial sequence of the elongation factor 1-alpha.  Of these 27% are either F. verticillioides (25%) or F. proliferatum (2%).  F. verticillioides was found in all plant tissue sampled with the highest incidence in the prop root (22%) and the kernel (16%).  Of the six maize genotypes used in this study, four reached maturity. Of those that reached maturity there were differences in terms of the percentage of fumonisin producers out of the total number of Fusarium isolated.  The two most sensitive lines Lancaster Surecrop and Country Gentleman had 14% and 21% fumonisin producers respectively, while the moderately resistant line Gaspe Flint had 30% and the resistant line Tama Flint had 45.5% fumonisin producers.  Compared to the aggregate data for all lines, 27% fumonisin producers, the susceptible lines had a lower frequency of fumonisin producers while the resistant line had a higher frequency. These data suggest that maize genotype and potentially kernel resistance to fumonisin may be factors in colonization by fumonisin producing fusaria.    MS: The Brown lab at MSU is designing and implementing a more proactive integrated food safety system for MS.  Food-borne diseases are a major cause of illness and death, the Center for Disease Control estimates that 48 million people get sick, 128 thousand are hospitalized and 3 thousand die from food-borne diseases each year in the United States and most of these cases are caused from unspecified agents.  The Food Safety Modernization Act (FSMA) was prompted and signed into law to enable the FDA to better protect public health by strengthening the food safety system. Maintaining a healthy, safe food supply is critical to the health of our communities, and requires a system of oversight for how food is grown, transported, processed, prepared and consumed.  This requires a commitment to inter-agency coordination for a food safety system that integrates activities to prevent, as well as response to food-borne illness when outbreaks occur. The food manufacturing industry has continued to grow in Mississippi and now has more than 250-food processing companies that employ almost 30,000 workers. Mississippi has a strong sense of food culture and locally produced foods are cherished as strong connection points to tradition.  The MSDH and the MSCL are tasked with protecting this industry, its consumers and ensuring safe food products. However, this protection web extends beyond the State and requires collaboration from all stakeholders. These stakeholders can be divided into government (FDA, USDA, CDC, DHS, EPA, State and local regulators); the food industry (farmers, ingredient supplies, processors/manufactures, distributors, food service, retailers); academia (education, research, extension); and consumers.   When food safety problems occur time is the enemy as public health and lives are at stake, as well as the livelihoods of industries.  Surveillance and trace-back are paramount to this mission.  Therefore, we are working with these agencies to design and implement a more proactive integrated food safety system for Mississippi to prevent food-borne illnesses. As part of this system we have tested for mycotoxin contamination in corn based manufactured food products.  To date, none of the tested products have resulted in a recall.      The Brown lab is investigating the use a modified Douglas Fir biochar to remove aflatoxin M1 from milk. Biochar, charcoal produced from plant matter, has a very high absorption capacity and can be used as an economical filter alternative. The biochar was modified using methanol has been proven to bind and remove tetracycline from aqueous solutions. Since the chemical structure of aflatoxin M1 contain similar functional groups to tetracycline, we designed a methanol-modified biochar from the removal of aflatoxin from milk and other contaminated liquids, such as beer and coffee.   MS: Shan lab, our research projects involve next generation sequencing-assisted crop breeding. Incorporating corn genetic resistance is an ideal means to reduce aflatoxin in corn. Crop breeding benefits greatly from the precise information obtained through genome sequencing. Genomic information such as gene markers, QTL region composition, and genes for introgression of important agronomic traits can be revealed from comparative whole genome sequence analysis among corn inbred lines carrying various levels of aflatoxin reduction. Next generation sequencing allows precise sequence comparisons among breeding lines, gene by gene, providing large amount of DNA markers and make precision breeding possible. Our research is focused on corn inbred line Mp313E which carries host plant resistance to Aspergillus flavus and exhibits low level of aflatoxin in mature kernels. We are working on whole genome sequence comparative analysis on near isogenic lines (QTL-NILs) derived from Mp313E, carrying major QTLs from Mp313E with enhanced host resistance observed in field trials.   NJ (Lawton and Di Labs):      Gene Editing of FHB Susceptibility Loci. We have adopted CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated endonuclease 9) gene editing technology to mutate genes involved in conditioning FHB susceptibility.  We have employed Arabidopsis as the model plant to study the feasibility of knocking-out FHB susceptibility genes to achieve FHB resistance. After generating and assaying the effects of gene editing of these loci in Arabidopsis, we have used these plants to identify their functional orthologues from barley via a complementation assay.  Once confirmed, we then employ CRISPR/Cas9 to edit these loci in barley and determine their contribution to disease susceptibility      We identified three FHB susceptibility genes that are likely involved in FHB susceptibility: ethylene insensitive 2 (EIN2), homoserine kinase (HSK) and 2-oxoglutarate Fe(II)-dependent oxygenase (2OGO).  We used the Arabidopsis (At) CRISPR vector pAt-sgRNA-Cas9 and produced AtEIN2-, AtHSK- and At2OGO-edited Arabidopsis plants with vectors, pRD182, pRD212 and pRD207 respectively.  Our vectors contain PAtU6::AtEIN2/AtHSK/At2OGO-gRNA//PAtUbi::Cas9/TAtUbi in the plant expression vector pCAMBIA1300, with the hygromycin resistance gene as the selectable marker.  Agrobacterium-mediated transformants (T1 and T2 generations) were validated for editing of target sites by restriction fragment length polymorphism (RFLP), T7 endonuclease 1 assay and sequencing of PCR-amplified gDNA fragment spanning the target sites. To-date, we have validated: 15 AtEIN2-edited plants each with different mutations at their respective target sites, 14 At2OGO-edited Arabidopsis plants, each with different mutations at their respective target sites, and we have also produced AtHSK-KO Arabidopsis plants whose characterization at the DNA sequence level is in progress.       For both AtEIN2-edited plants and At2OGO-edited plants, we have selected from the T2 generation and confirmed by RFLP analysis transgene-free homozygous plants.  This confirms the validity of the approach of using CRISPR/Cas9 gene editing to alter endogenous gene sequences in plants that are no longer considered to be GMOs.       Inoculation of staged florets from WT and edited plants with Fg tagged with green fluorescent protein (GFP) allowed us to the progression of infection, monitor symptom development, and quantitate defense responses.  We determined the levels of Fg-GFP by real-time PCR using GFP-specific primers and Arabidopsis actin gene as an endogenous control for relative quantification.  Our results show that both At2OGO and AtEIN2-KO plants displayed a markedly slower FHB disease development with a consequent reduction in the levels of Fg-GFP, compared to WT plants.       RNA-SEQ of Cultivar Conlon. To identify which barley homologs of 2OGO, EIN2 and HSK are involved in FHB susceptibility, we wished to complement the corresponding gene-edited Arabidopsis plants (At2OGO-KO and AtEIN2-KO) with barley cDNA isolated from the U.S. cultivar Conlon.  The genome of Morex barley, a US spring six-row malting cultivar, has been published.  We focused, however, on Conlon, because this cultivar is more susceptible to Fg than is Morex, and provides a better baseline for determining the effects of inactivating genes that condition susceptibility in barley. We used the Illumina HiSeq platform to perform RNAseq analysis of barley cv. Conlon, an important North American two-rowed cultivar and obtained high quality cDNA reads of 44170060, over 74% of which mapped to the Morex reference genome.   The results have been uploaded to NCBI GenBank (Project #PRJNA563590).       Complementation assay for Barley susceptibility genes. We identified and sequenced cDNAs of Conlon HvEIN2, HvHSK and Hv2OGO.  Introduction of either gene into the corresponding Arabidopsis gene edited plants provided a complementation assay for ability to revert the phenotype of these plants back to fully-susceptible.  Both HvEIN2 and Hv2OGO cDNAs were cloned into plant expression vectors with kanamycin resistance selectable marker and complement-transformed into the transgene-free, homozygous lines for the corresponding gene edited mutant plants. Our results showed that both AtEIN2-KO/HvEIN2 plants and At2OGO-KO/Hv2OGO plants recovered susceptibility to near-WT levels FHB. These results indicate that both the barley HvEIN2 Hv2OGO genes condition FHB susceptibility and marks them as rationale targets for gene editing in barley and other grains.  These findings have been incorporated into a manuscript that will be submitted for publication in the period just following this reporting period.         Development of a Barley tissue culture and regeneration system for barley cv. Conlon.  We have used CRISPR/Cas9 gene editing to produce two Hv2OGO mutants in the Conlon cultivar.  We have modified the barley tissue culture protocol to regenerate Conlon barley more efficiently, as this is a bottleneck for our studies and others in the field.  Therefore, we constructed several CRISPR-editing vectors using barley (Hv), rice (Os) and wheat (Ta) U3 or U6 promoter, which have been used effectively in other cultivars, such as Golden Promise.         Using immature or mature embryos with either gene gun or Agrobacterium transformation methods, we have regenerated four plantlets from pRD383 (targeting Hv2OGO). RFLP analysis indicates that three of the regenerated plants produced NdeI-uncut fragments of the PCR-amplified, target site-spanning gDNA. Sequence analysis of the target site from these putatively edited barley plants confirm the presence of mutations near the NdeI target site, all resulting in amino acid changes in these two plants.  These plants, together with gene-edited plants in progress for HvHSK and HvEIN2, provide the basis for determining the contribution of these genes to FHB susceptibility and to generating GMO-free, gene-edited plants with an enhanced disease performance in the field.     MI: The Trail lab has been characterizing the microbiomes of a corn/soy/wheat rotation. Leaf, stalk and root microbiomes have been performed for each rotation since 2013 and we are now adding in the seed microbiomes due to USDA-AFRI funding. Collections of isolates of bacteria and fungi from plant organs were challenged in vitro with F. graminearum.  Isolates that affected growth of F. graminearum were used to determine their ability to protect wheat seedlings infected with F. graminearum.  We identified three endophytes from this collection that have exhibited robust abilities to reduce development of seedling blight and in some cases increase seedling growth.  Testing of these for protection against head blight of wheat resulted in increased seed weight, reduced DON accumulation and less disease. We tested the activity of volatiles from these isolates and found no evidence for the response being volatile induced.  We are gearing up for field trials this coming spring.   KY: Christopher Schardl has received a grant from the Mycological Society of America to use CRISPR technology to eliminate genes for ergot alkaloid production in the fungus Epichloë coenophiala, a ubiquitous and important seed-transmitted symbiont (endophyte) of the widely planted pasture and forage grass, tall fescue. Although important for stand longevity and productivity, the most common E. coenophiala strains produce ergot alkaloids and, if livestock are grazed on pastures or fed hay with these endophyte strains they fail to thrive, produce less or no milk, and can suffer reproductive problems. Non-producers of ergot alkaloids have been identified by researchers world wide, and are deployed in tall fescue cultivars, but for various reasons their effects on fitness of the cultivars may not always emulate that of the “common toxic” strains. Therefore, we chose to eliminate ergot alkaloid genes from an established E. coenophiala strain. There were several technical problems to address in this effort: The fungus is a complex interspecific hybrid with two copies of ergot alkaloid genes, it is asexual and slow growing, and tedious to reintroduce into the host plant. Furthermore, the technique must generate a non-transgenic fungus for regulatory and public acceptance. Evidence so far indicates success in eliminating the genes required for ergot alkaloid biosynthesis without net introduction of exogenous genes in the genome.     Objective 3: Better Understand the Biology and Ecology of Mycotoxigenic Fungi. NE: Fusarium boothii (first detected in field surveys in 2018; first report in US on wheat) isolates have been subjected to greenhouse wheat infection studies. Three F. boothii isolates show low infectivity and deoxynivalenol production when compared with 13 F. graminearum isolates, but are within the range of variation shown by F. graminearum. A hybrid between F. boothii and F. graminearum was detected in field trials, as were one F. acuminatum and 2 F. poae isolates (67 F. graminearum isolates examined). Manuscript in preparation; grant proposal submitted.      We are developing a multiplex PCR for identification of species within the Fusarium sambucinum species complex, with success differentiating and identifying F. graminearum, F. culmorum, F. boothii, F. asiaticum, and F. gerlachii, and will be working to expand, optimize, and validate this protocol.   NJ (Lawton and Di Labs): We are addressing this project goal using the model multicellular animal C. elegans.  DON appears to act in humans at a number of different levels, and many of these features can also be addressed in the C. elegans model.  These include the roles of genes that condition programmed cell death, the role of endocytotic pathways in toxin cellular transport, and the role of toxin-induced protein aggregation in neurological degeneration.  We have established the worm system to study the mode of DON intoxication. We have mapped out the gene expression profile throughout the entire genome of C. elegans upon DON intoxication by genome-wide RNAseq analysis.  The interaction of several of these genes and their contribution to conditioning sensitivity to DON has now been confirmed by RNAi (RNA interference) analysis.  Our studies reveal that DON affects the expression of many genes outside of those involved in innate immunity, suggesting that this toxin may affect diverse molecular and cellular processes as well as development.  These findings have been published. Current work focuses on understanding how these cellular and molecular pathways function in ameliorating the effects of mycotoxins and of exploring the use of external agents (chemicals and natural products) to lessen the impact of these toxins on animals and humans exposed to them in their diet.   KY: In collaboration with Frances Trail of MI, L. Vaillancourt is investigating a set of Fusarium 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 have normal pathogenicity on both spring wheat (cultivar Norm) and winter wheat (cultivar Pioneer 2555). However, both of the individual MAT gene KO strains are significantly reduced in pathogenicity on both types of wheat.  Work in the past year confirmed that the original two MAT1-1-1 and MAT1-2-1 KO strains are significantly less aggressive than the WT on Norm and on Wheaton spring wheat. Additional independent KO strains were evaluated and most (but not all) are also less aggressive than the WT. Different KO strains vary quantitatively in a variety of phenotypes, including interfertility in crosses. Work over the next year will focus on additional phenotyping of a representative collection of multiple MAT1-1-1 and MAT1-2-1 KO strains, and evaluation of the hypothesis that the KO mutants have additional random mutations that modify their behavior in pathogenicity and mating tests.      Vaillancourt is also collaborating with Emerson Del Ponte of Universidade Federal de Viçosa, and David Schmale of VT, to investigate F. graminearum and F. meridionale strains causing ear and stalk rot diseases in corn in Brazil versus in the U.S. A dual degree student has characterized a collection of Brazilian strains representing both species isolated from both corn and wheat.  Results show that the two species are similarly aggressive on corn ears and stalks, although isolates of F. graminearum recovered from corn are more aggressive to corn than isolates recovered from wheat. F. graminearum is much more fertile than F. meridionale, and more pathogenic to wheat.    KY and WV: Christopher Schardl and Daniel Panaccione are collaborating on a project to assess the diversity and roles of defensive alkaloids in wild grasses. The study is in partnership with local K-12 schools. The students will be involved in a wide range of research methods including plant collection and identification, curation of herbarium specimens and associated data, chemical analysis by thin layer chromatography, genetic analysis by PCR, and bioinformatic analysis of DNA sequences. This has been incorporated in a collaborative grant proposal to NSF.    KY: Christopher Schardl and collaborators have published a study of gene expression by E. coenophiala as it colonizes different host tissues, including ovaries as an essential step in transgenerational maintenance of the symbiosis. We found that colonization of reproductive structures was accompanied by dramatically enhanced expression of genes for stress tolerance, response to reactive oxygen species, and protein chaperones and chaperonins (Nagabhyru et al. 2019). In another study we investigated tall fescue plant gene expression in response to E. coenophiala and water deficit stress, identifying endophyte-induced changes associated with enhancement of stress tolerance (Dinkins et al. 2019). These studies are important for future choices of endophyte and plant lines to use for development on nontoxic cultivars.   WI: The Keller lab focuses on molecular mechanisms controlling mycotoxin synthesis and virulence on Aspergillus and Penicillium spp.  One emphasis is how epigenetics influences pathogenesis and secondary metabolism in these genera.  Her lab has shown that the histone reader protein, SntB, is required for aflatoxin synthesis and sclerotia production by A. flavus.  Furthermore loss of sntB impairs the ability of the fungus to colonize seed.  Similarly sntB deletion reduces virulence of Penicillium expansum on apple and decreases patulin synthesis although increases citrinin production in vitro.  The work in P. expansum is in collaboration with Israeli scientists.  SntB works by silencing certain regions of the genome while allowing expression of other regions.  Deletion of SntB resulted in the identification of a new secondary metabolite with immunomodulatory properties in A. flavus.  Additional studies focus on fungal/bacterial interactions and the outcomes on mycotoxin synthesis and disease in polymicrobial communities.     WI: The Yu lab continue their studies of understanding the mechanisms governing sporulation 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 BrlA-->AbaA-->WetA. The final step in this cascade is controlled by the WetA protein, which governs not only the morphological differentiation of spores but also the production and deposition of diverse metabolites including AFB1 into spores. While WetA is conserved across the genus Aspergillus, the structure and degree of conservation of the wetA gene regulatory network (GRN) remained largely unknown. The group led by Yu and Rokas carried out comparative transcriptome analyses of comparisons between wetA null mutant and wild-type conidia in three representative species spanning the diversity of the genus Aspergillus: A. nidulans, A. flavus, and A. fumigatus. The group discovered that WetA regulates asexual sporulation in all three species via a negative-feedback loop that represses BrlA, the cascade’s first step. Furthermore, data from chromatin immunoprecipitation sequencing (ChIP-seq) experiments in A. nidulans conidia suggest that WetA is a DNA-binding protein that interacts with a novel regulatory motif. Several global regulators known to bridge spore production and the production of secondary metabolites show species-specific regulatory patterns in the reported data. These results suggested that the BrlA→AbaA→WetA cascade’s regulatory role in cellular and chemical asexual spore development is functionally conserved but that the wetA-associated GRN has diverged during Aspergillus evolution. These results shed light on how gene regulatory networks in microorganisms control important biological processes and evolve across diverse species. Moreover, these studies provided the first clear and systematic dissection of WetA, an evolutionarily and functionally conserved regulator of morphological and chemical development of filamentous fungal conidiation. Furthermore, studies have revealed the molecular mechanisms of WetA as a likely DNA-binding, multifunctional regulator governing the diverse processes of cellular differentiation, AFB1 biosynthesis, and cell survival across a genus of filamentous fungi, advancing our knowledge of spore formation and mycotoxin production in pathogenic and toxigenic fungi.   SYNERGISTIC ACTIVITIES <ul> <li>The Vaillancourt and Trail labs are collaborating on the interaction between mating type locus and pathogenicity in F. 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.  They have received funding for a joint project to continue these studies.</li> <li>The Vaillancourt, Del Ponte, and Schmale labs have developed a collaborative project to study the phenotypic variation between two phylogenetic species within the Fusarium graminearum clade that cause Gibberella ear and stalk rot and head blight of wheat in Brazil.</li> </ul> <ul> <li>The NJ Station collaborated with the KY Station to assess the impact and molecular mechanisms of Epichloë toxins on C. elegans.</li> </ul>

Publications

Publications (10/1/2018 to 9/30/2019) <ol> <li>Di, R.; Zhang, H.; Lawton, M.A. Transcriptome Analysis of C. elegans Reveals Novel Targets for DON Cytotoxicity. Toxins 2018, 10, 262.  doi:10.3390/toxins10070262</li> <li>Hurburgh, C. and Robertson, A.  2018. Crop Quality hurt by rains. <a href="https://crops.extension.iastate.edu/cropnews/2018/10/crop-quality-hurt-rains">https://crops.extension.iastate.edu/cropnews/2018/10/crop-quality-hurt-rains</a></li> <li>Hurburgh, C. 2018. Management of flood submerged grain. <a href="https://crops.extension.iastate.edu/cropnews/2018/09/management-flood-submerged-grain">https://crops.extension.iastate.edu/cropnews/2018/09/management-flood-submerged-grain</a></li> <li>Ferrara, M., M. Haidukowski, A. F. Logrieco, J. F. Leslie, & G. Mulè.  20xx. A CRISPR-Cas9 system for genome editing of Fusarium proliferatum. (submitted).</li> <li><a href="https://link.springer.com/journal/12550/onlineFirst/page/1">Franco LT, Petta T, Rottinghaus GE, Bordin K, Gomes GA, Oliveira C</a>. Co-occurrence of mycotoxins in maize food and maize-based feed from small-scale farms in Brazil: a pilot study.  Mycotoxin Research https://doi.org/10.1007/s12550-018-0331-4, 2018.</li> <li>Gdanetz K, & Trail F. The phytobiomes of a 3-crop rotation: organs as centers of microbial diversity, a role for spore dispersal in organ-specific taxa.</li> <li><a href="https://dl.sciencesocieties.org/publications/cs/abstracts/58/2/925">Kenyon SL, Roberts CA, Kallenbach RL, Lory JA, Kerley MS, Rottinghaus GE, and Hill NS, Ellersieck MR</a>. Vertical distribution of ergot alkaloids in the vegetative canopy of tall fescue. Crop Sci 58(2):925-931, 2018.</li> <li>Leslie, J.F., Lattanzio, V., Audenaert, K., Battilani, P., Cary, J., Chulze, S.N., De Saeger, S., Gerardino, A., Karlovsky, P., Liao, Y., Maragos, C.M., Meca, G., Medina, A., Moretti, A., Munkvold, G., Mulè, G., Njobeh, P., Pecorelli, I., Perrone, G., Pietri, A., Palazzini, J.M., Proctor, R.H., Rahayu, E.S., Ramírez, M.L., Samson, R., Stroka, J., Sulyok, M., Sumarah, M., Waalwijk, C., Zhang, Q., Zhang, H., and Logrieco, A.F. 2018. MycoKey round table discussions of future directions in research on chemical detection methods, genetics and biodiversity of mycotoxins. Toxins Vol. 10, 109. doi:10.3390/toxins10030109</li> <li>Leslie, J. F. & J. B. Morris. 20xx.  Talking about mycotoxins. (submitted).</li> <li>Logrieco, A. F., J. D. Miller, M. Eskola, R. Krska, A. Ayalew, R. Bandyopadhyay, P. Battilani, D. Bhatnagar, S. Chulze, S. De Saeger, P. Li, G. Perrone, A. Poapolathep, E. S. Rahayu, G. S. Shephard, F. Stepman, H. Zhang, & J. F. Leslie. 2018.  The Mycotox Charter: Increasing awareness of and concerted action for minimizing mycotoxin exposure worldwide. Toxins 10: 149. DOI: 10.3390/toxins10040149.</li> <li>McMaster, N., Acharya, B., Harich, K., Grothe, J., Mehl, H., and Schmale, D.G. 2019. Quantification of the Mycotoxin Deoxynivalenol (DON) in Sorghum using GC-MS and a Stable Isotope Dilution Assay (SIDA). Food Analytical Methods 12 (10): 2334-2343. <a href="https://doi.org/10.1007/s12161-019-01588-3">https://doi.org/10.1007/s12161-019-01588-3</a></li> <li>Mohamed Nor, N. M. I., B. Salleh & J. F. Leslie.  2019. Fusarium species from sorghum in Thailand.  The Plant Pathology Journal 35: 301-312. <ol start="10"> <li>DOI: org/10.5423/ppj.oa.03.2019.0049</li> </ol> </li> <li>Munkvold, G.P., Weieneth, L., Proctor, R., Busman, M., Blandino, M., Susca, A., Logrieco, A., and Moretti, A. 2018. Pathogenicity of fumonisin-producing and nonproducing strains of Aspergillus species in section Nigri to maize ears and seedlings. Plant Dis. 102: 282-291.<a href="https://doi.org/10.1094/PDIS-01-17-0103-RE"> https://doi.org/10.1094/PDIS-01-17-0103-RE</a></li> <li>Munkvold, G.P., Arias, S.L., Taschl, I., and Gruber-Dorninger, C. 2018. Mycotoxins in Corn – Occurrence, Impacts, and Management. Pp. in Corn Chemistry and Technology, 3rd Eds.  Am. Assoc. Cereal Chemists, St. Paul, MN</li> <li>Shi C, An S, Yao Z, Young CA, Panaccione DG, Lee ST, Schardl CL, Li C. (2018) Toxin-producing Epichloë bromicola symbiotic with the forage grass, Elymus dahuricus, in China. Mycologia 109:847-859. DOI: 10.1080/00275514.2018.1426941</li> <li>Stoetzer, E.  2018. Mycotoxin App Available. <a href="https://crops.extension.iastate.edu/blog/ethan-stoetzer/mycotoxins-app-available">https://crops.extension.iastate.edu/blog/ethan-stoetzer/mycotoxins-app-available</a></li> <li>Torres, A. M., S. A. Palacios, N. Yerkovich, J. M. Palazzini, P. Battilani, J. F. Leslie, A. F. Logrieco & S. N. Chulze.  2019. Fusarium head blight and mycotoxins in wheat: Prevention and control strategies across the food chain.  World Mycotoxin Journal 12: (in press). DOI: org/10.3920/wmj2019.2438.</li> <li>Vismer, H. F., G. S. Shephard, L. van der Westhuizen, P. Mngqawa, V. Bushula-Njah, & J. F. Leslie.  2019. Mycotoxins produced by Fusarium proliferatum and pseudonygamai on maize, sorghum and pearl millet grains in vitro. International Journal of Food Microbiology 296: 31-36. DOI: org/10.1016/j.ijfoodmicro.2019.02.016. </li> <li><a href="https://ac.els-cdn.com/S0377840117307137/1-s2.0-S0377840117307137-main.pdf?_tid=689b1c26-d447-11e7-bf35-00000aab0f6c&acdnat=1511879062_16e20fbdebefde6fab647bfc4e5c6029">Weatherly ME, Pate RT, Rottinghaus GE, de Oliveira Roberti Filho F, Cardoso FC</a>. Physiological responses to a yeast and clay-based adsorbent during an aflatoxin challenge in Holstein cows. Animal Feed Science and Technology 235147-157, 2018.</li> <li>Wegulo, S.N., Valverde-Bogantes, E.,  Bolanos-Carriel, C., Hallen-Adams, H.,  Bianchini, A., McMaster, N., and Schmale, D.G. 2018. First Report of Fusarium boothii Causing Head Blight of Wheat in the United States. Plant Disease 102:2642.</li> <li>Wilson, N., Dashiell, S., McMaster, N., Bohland, C., and Schmale, D. 2018. Could Your Food be Contaminated with Toxins? Educating High School Students about Mycotoxins in Feed and Food Products. The Science Teacher 86 (1): 46-52.</li> <li>Yi M, Hendricks WQ*, Kaste J, Charlton ND*, Nagabhyru P, Panaccione DG, Young CA. (2018) Molecular identification and characterization of endophytes from uncultivated barley. Mycologia 110:453-472 DOI:10.1080/00275514.2018.1464818</li> <li>Álvarez-Escribano I, Sasse C, Bok JW, Na H, Amirebrahimi M, Lipzen A, Schackwitz W, Martin J, Barry K, Grigoriev IV, Gutiérrez G, Cea S, Marcos AT, Grigoriev IV, Keller NP, Braus GH, Cánovas D (2019) Genome sequencing of evolved aspergilli populations reveals robust genomes, transversions in flavus, and sexual aberrancy in non-homologous end-joining mutants.  BMC Biology. 17(1):88. Doi: 10.1186/s12915-019-0702-0.</li> <li>Greco C, Pfannenstiel BT, Liu J, Keller NP (2019) Depsipeptide aspergillicins revealed by chromatin reader protein deletion.  ACS Chemical Biology 14(6):1121-1128.</li> <li>Lan H, Wu L, Sun R, Keller NP, Yang K, Ye L, He S, Zhang F, Wang S (2019).<a href="https://www.ncbi.nlm.nih.gov/pubmed/30986121"> The HosA histone deacetylase regulates aflatoxin biosynthesis through direct regulation of aflatoxin cluster genes.</a> Mol Plant Microbe Interact. 2019 Apr 15. doi: 10.1094/MPMI-01-19-0033-R. [Epub ahead of print]</li> <li>Pfannenstiel BT, Greco C, Sukowaty AT, Keller NP (2018) The epigenetic reader SntB regulates secondary metabolism, development and global histone modifications in Aspergillus flavus.  Fungal Genet Biol.  SI on epigenetics. S1087-1845(18)30170-1. </li> <li>Tannous J, Kumar D, Barad S, Dubey A, Sionov E, Prusky D, Keller NP (2018) Fungal attack and host defense pathways unveiled in near avirulent interactions of Penicillium expansum creA mutants on apples. Mole Plant Pathology.  19: 2635-2650.</li> <li>Venkatesh N, Keller NP (2019) Mycotoxins in conversation with bacteria, fungi and plants.  Frontiers Microbiology. 10:403. doi: 10.3389/fmicb.2019.00403.</li> <li>Tannous J, Keller NP (2019) Mycotoxins. Chapter 129.  In<a href="http://www.asmscience.org/content/book/10.1128/9781555817381"> Manual of Clinical Microbiology, 12th Edition</a>, ASM Press  </li> <li>Galindo-Castaneda, T., Brown, K., Kuldau, G. A., Roth, G. W., Wenner, N. G., Swayamjit, R., Schneider, H., and J. P. Lynch. Root cortical anatomy is associated with differential pathogenic and symbiotic fungal colonization in maize. Plant Cell Environ. 2019;1-16, https://doi.org/10.1111/pce.13615.</li> <li>Parish F, Williams WP, Windham GL and Shan X (2019) Differential Expression of Signaling Pathway Genes Associated With Aflatoxin Reduction Quantitative Trait Loci in Maize (Zea mays L.). Microbiol. 10:2683. doi: 10.3389/fmicb.2019.02683</li> <li>Dinkins RD, Nagabhyru P, Young CA, West CP, Schardl CL (2019) Transcriptome analysis and differential expression in tall fescue harboring different endophyte strains in response to water deficit. The Plant Genome 12-2-180071. doi 10.3835/plantgenome2018.09.0071</li> <li>Nagabhyru P, Dinkins RD, Schardl CL (2019) Transcriptomics of Epichloë-grass symbioses in host vegetative and reproductive stages. Molecular Plant-Microbe Interactions 32: 194-207. doi 10.1094/MPMI-10-17-0251-R</li> </ol>

Impact Statements

KY initiated a dual degree program with the Universidade Federal de Viçosa in Brazil, with the first Ph.D. student who will be conducting research on the genetic diversity of Fusarium species causing stalk and ear rot diseases in maize in Brazil and in the United States. The first student completed his 12 month stay in the U.S. and will defend his dissertation in the spring of 2020.

Date of Annual Report: 02/03/2021

Report Information

Annual Meeting Dates: 09/15/2020 - 09/15/2020
Period the Report Covers: 10/01/2015 - 09/30/2020

Participants

Brief Summary of Minutes

Please see attached file below for NC1183's termination report (2015-2020) and impact statement.

Minutes Attachment:

NC1183 2015 to 2020 Termination Report and Impact Statement.pdf