Brief Summary of Minutes of Annual Meeting

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.

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. https://crops.extension.iastate.edu/cropnews/2018/10/crop-quality-hurt-rains

Hurburgh, C. 2018. Management of flood submerged grain. https://crops.extension.iastate.edu/cropnews/2018/09/management-flood-submerged-grain

Imboden, I., Afton D., Trail, F. 2017. Surface interactions of Fusarium graminearum on barley. Molecular Plant Pathology. DOI: 10.1111/mop.12616.

Franco LT, Petta T, Rottinghaus GE, Bordin K, Gomes GA, Oliveira C. 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.

Kenyon SL, Roberts CA, Kallenbach RL, Lory JA, Kerley MS, Rottinghaus GE, and Hill NS, Ellersieck MR. 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. https://doi.org/10.1094/PDIS-01-17-0103-RE

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. https://crops.extension.iastate.edu/blog/ethan-stoetzer/mycotoxins-app-available

Weatherly ME, Pate RT, Rottinghaus GE, de Oliveira Roberti Filho F, Cardoso FC. 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. https://doi.org/10.1094/PDIS-04-18-0696-PDN

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