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Research Project: Novel Methods for Controlling Trichothecene Contamination of Grain and Improving the Climate Resilience of Food Safety and Security Programs

Location: Mycotoxin Prevention and Applied Microbiology Research

2017 Annual Report

1a. Objectives (from AD-416):
Objective 1: Identify and characterize microorganisms and microbial genes that can reduce trichothecene contamination of grain-based food and feed. Sub-objective 1.1: Determine the role of natural microbial populations in reducing Fusarium mycotoxins in wheat. Sub-objective 1.2: Identify trichothecene resistance mechanisms in a diversity of trichothecene-producing fungi. Objective 2: Determine the effects of climate change on susceptibility of wheat and corn to contamination by trichothecenes and other Fusarium mycotoxins. Sub-objective 2.1: Evaluate the effects of environmental conditions associated with climate change on susceptibility of wheat and corn to Fusarium infection and trichothecene contamination. Sub-objective 2.2: Identify stress-induced changes in plant metabolism and transcription associated with Fusarium infection and deoxynivalenol under projected future climate conditions. Objective 3: Determine the genomic diversity of Fusarium Head Blight pathogens and identify species or population-specific differences in host-pathogen interactions, mycotoxin production, or pathogen fitness under different climatic conditions. Sub-objective 3.1: Determine the genomic diversity and population affinities of NX-2 strains in relation to other FHB pathogens in North America, and utilize comparative genomics to identify regions of the genome influenced by adaptive evolution. Sub-objective 3.2: Characterize competitive interactions of Fusarium graminearum populations on spring and winter wheat. Sub-objective 3.3: Characterize changes in the aggressiveness and mycotoxin production of FHB species, chemotype groups, and populations in response to different climatic conditions.

1b. Approach (from AD-416):
In recent years, the world has experienced an increase in mycotoxin contamination of grains due to climatic and agronomic changes that encourage fungal growth during cultivation. We will isolate and characterize major contributors (yeasts, filamentous fungi, and bacteria) to the microbial community associated with wheat cultivation. Microorganisms isolated from the wheat phyllosphere and rhizosphere will be evaluated both for their efficacy as biocontrol agents of mycotoxigenic Fusarium, and for their ability to detoxify or degrade mycotoxins. We will identify and characterize trichothecene detoxification genes from microbes capable of surviving mycotoxin exposure. As a parallel approach to trichothecene detoxification we will identify resistance mechanisms from diverse fungi that produce trichothecenes and have naturally developed strategies to cope with exposure to these toxins. Plants have evolved complex signaling mechanisms to respond to stress; however, simultaneous challenges by abiotic and biotic stress factors results in the activation of diverse signals that can have synergistic and antagonistic effects on each other. Additive abiotic stress can alter plant health and susceptibility to mycotoxins. We will evaluate the effects of environmental conditions associated with climate change on susceptibility of wheat and corn to Fusarium infection and trichothecene contamination and identify changes in plant physiology or defense that influence mycotoxin contamination. Climate induced physiological changes that occur in the host and influence mycotoxins and/or Fusarium infection will be useful as markers in plant breeding programs aimed at developing climate resilient fungal resistance strategies. Fusarium graminearum and other members of the F. graminearum species complex (FGSC) are the primary cause of Fusarium Head Blight (FHB) and trichothecene contamination of wheat worldwide. Understanding diversity at the level of species, genetic populations, and trichothecene chemotypes is critical to the development of effective disease control and mycotoxin reduction strategies. We will determine the extent, distribution, and significance of genomic diversity among FHB pathogen populations, species, and chemotype groups. Finally, we will test hypotheses regarding species, population, or chemotype-specific differences, in host-pathogen interactions, mycotoxin production, or pathogen fitness under different climatic conditions in order to understand the influence of host and climatic variables on pathogen composition and trichothecene contamination.

3. Progress Report:
Production of vomitoxin (deoxynivalenol or DON) or other trichothecene mycotoxins contributes to the ability of Fusarium graminearum to cause Fusarium Head Blight (FHB). As a result, increasing trichothecene resistance in wheat should increase resistance to FHB. Research under Objective 1 is designed to identify microorganisms or genes that provide detoxification of trichothecenes or resistance to Fusarium and thereby reduce vomitoxin contamination of grain. Soil samples were collected from experimental fields, microorganisms were then isolated and tested for their ability to grow in the presence of vomitoxin. Microorganisms were also isolated from both FHB infected and non-infected heads of winter and spring wheat. Over 30 candidates were sequenced to determine species identification and tested for ability to detoxify trichothecenes. Wheat head, stalk, and rhizosphere samples were also collected from fields growing two winter wheat varieties and we initiated isolation and identification of microorganisms from the samples. (Objective 1) Bacteria and fungi were isolated from surface disinfested wheat tissues collected from Illinois, Minnesota, and Kansas. Microbiome profiling of wheat plants was used to discover the suite of microbial associates colonizing wheat in a way that avoids the biases associated with ease of cultivation. This method assesses microbial community composition via analysis of DNA signatures rather than requiring the growth of individual organisms. Three potential bacteria antagonists of Fusarium were selected to test if they can limit FHB under field conditions. In addition, microorganisms from the NRRL (ARS) culture collection have been tested for the ability to transform or have resistance to vomitoxin or other trichothecenes. Aerobic enrichment cultures with extended exposure to trichothecenes were grown in order to isolate organisms capable of metabolizing, resisting, or biotransforming trichothecenes. Methods were also developed for looking at the relationships between microbiome composition of single wheat seeds, F. graminearum density and vomitoxin contamination. (Objective 1) Some fungi and yeasts can detoxify trichothecenes by linking the toxins to sugars, forming trichothecene-glycosides. Plants can also detoxify trichothecenes by forming glycosides. Trichothecene glycosides are considered masked mycotoxins and are a food safety problem because they may escape detection with the analytical methods that have been used to keep vomitoxin, T-2 toxin, and other trichothecenes out of food and feed. Yeast species that were found to detoxify trichothecenes by adding a sugar were used to prepare diacetoxyscirpenol glucoside. In collaboration with scientists at University of Wisconsin, HT-2 toxin glucoside was synthesized with a glucosyltransferase from rice. These glucosides are being used to develop analytical detection methods and complete risk assessments of the masked mycotoxins. Toxin-producing fungi generally have a mechanism to protect themselves from the harmful effects of the toxin(s). In order to identify genes responsible for trichothecene self-protection in eight genera of trichothecene-producing fungi, we generated genome sequences for the fungi and identified the region of chromosomes with genes responsible for trichothecene biosynthesis, and for possible self-protection. We developed an assay to transfer genes from these fungi into the yeast Saccharomyces cerevisiae and determine whether the transferred genes protect the yeast from the harmful effects of trichothecenes. In addition, we completed production of trichothecenes needed for the yeast selection assays. (Objective 1) The severity of FHB epidemics and the accumulation of associated trichothecene mycotoxins in wheat kernels are strongly driven by climatic factors. In order to evaluate the effects of atmospheric carbon dioxide concentrations on wheat susceptibility to FHB and mycotoxin contamination, we conducted virulence assays at both ambient and elevated carbon dioxide levels with two F. graminearum toxin producing strains and a non-toxin producing strain on spring wheat varieties that had been acclimated to corresponding carbon dioxide levels. This year we experimentally replicated these experiments to assess reproducibility both in growth chambers and in the Free Air Concentration Enrichment (FACE) field at University of Illinois at Champaign-Urbana. To further determine the mechanisms by which carbon dioxide levels influence wheat defenses against Fusarium species, we measured phytohormone response levels and examined the expression of defense related genes. In collaboration with scientists at the National Research Council of Canada, metabolic profiles of control and infected plants at ambient and elevated carbon dioxide were analyzed using nuclear magnetic resonance (NMR). This research addresses the effects of climate change on plant susceptibility to disease and stress-induced changes in plant metabolism associated with FHB (Objective 2). Research under Objective 3 is designed to provide novel targets and strategies for disease and mycotoxin control, information needed for detection of introduced pathogens and modeling of pathogen and toxin contamination levels, and improved understanding of the potential effect of changes in climate or crop distributions on mycotoxin contamination. To address milestones under Objective 3, genomic diversity and global population structure was assessed via whole genome sequencing (50X coverage) of more than 270 isolates selected to represent global diversity within F. graminearum, generating a dataset consisting of 1.1 million single nucleotide polymorphisms (individual genetic differences). Analyses of these data demonstrated that F. graminearum harbors extensive genetic diversity that is strongly connected to geographic differences, such that Europe, Asia, North America, and Oceania harbor distinct sets of F. graminearum populations. Our results also indicated that one of the three genetic populations found to be rapidly increasing in some parts of North America was likely introduced from Asia, demonstrating the potential for transcontinental movement of pathogen populations to exacerbate food production and food safety issues. Using information on population-level diversity derived from genomic analyses, we conducted comparative analyses of aggressiveness in moderately resistant wheat, identifying significant differences in aggressiveness between the three major genetic populations of F. graminearum found in northern U.S. and Canada (NA1, NA2, NA3). Analyses of toxin production and host defense response are underway, but results to date suggest that successful deployment of specific host resistance genes may depend on the pathogen populations found in a given region. In addition, analyses of pathogen composition and toxin potential were conducted in collaboration with scientists from Mexico, Brazil, and Uruguay to develop a better understanding of the global significance of different FHB pathogen species, their toxin types, and their potential to cause FHB in the U.S. under current and predicted future climate conditions. A high-throughput assay for assessing FHB species or population-specific differences in optimum temperatures for growth was developed and will be used to complement in planta analyses of pathogen adaptation to different climate conditions. In addition to trichothecenes mycotoxins, F. graminearum produces and secretes proteins called effectors that can help the fungus initiate or spread infection in wheat heads or suppress host plant defenses. We used bioinformatics to identify 136 effector candidates from F. graminearum and measured the expression of 26 of the proteins at different stages of infection. Some effectors were only expressed at an early infection stage while others were highly induced at later stages of infection. Four effector genes with homologues in other plant pathogenic fungi were selected and knockout mutants were made to test the effects of the proteins on virulence (FHB) and toxin production. Wheat inoculated with mutants of one of these effectors had significantly reduced FHB symptoms and toxin contamination. We also found that 70 of the 136 candidate effectors were specifically enriched in North American populations NA2 and/or NA3. Knockout mutants of one of this group of effectors, salicylate hydroxylase, were tested for virulence and toxin production. In order to reduce vomitoxin production and thereby FHB, we used double stranded RNA (dsRNA) to target a key regulator of vomitoxin biosynthesis. The expression was significantly reduced in wheat heads treated with the dsRNA. Treatment with dsRNA reduced disease spread (FHB) and the amount of vomitoxin produced levels in wheat grown under low relative humidity.

4. Accomplishments
1. New tools to detect Fusarium head blight pathogens and determine their toxin potential. Fusarium head blight (FHB) is a major disease of wheat, barley, and other cereals that reduces yield and contaminates grain with mycotoxins that can be a significant problem for public health and animal production. A number of different fungi can cause FHB and contaminate grain with a variety of toxins. However, understanding of FHB pathogen diversity, distributions, and toxin potential is incomplete and traditional methods of pathogen identification and toxin analyses are either ineffective or expensive and time consuming. In research with international collaborators, ARS scientists in Peoria, Illinois, identified and described a novel species (Fusarium praegraminearum) capable of causing FHB in wheat and developed new genetic tools to identify strains of this FHB pathogen. A similar set of genetic tools were used to document for the first time that the FHB pathogen F. meridionale is present in wheat fields in North America, and to determine the extent and distribution of different toxin types within a widespread FHB pathogen, F. culmorum. The ability to detect, accurately identify, and determine the distribution and toxin potential of FHB pathogens will enable scientists and producers to develop and deploy FHB and mycotoxin control strategies effective against the FHB pathogens and toxin types prevalent in a given region.

2. Elevated carbon dioxide reduces the emission of defense related volatiles from corn. Insect damage allows mycotoxigenic fungi to establish and grow on corn, leading to reduced yields and corn that may be unsafe for humans and other animals. Plants release airborne chemicals (volatiles) in response to infection or attack by the caterpillars of insect pests. These chemicals are used in direct and indirect plant defense. To determine if these key defense signals are altered by changes in climatic conditions, ARS scientists in Peoria, Illinois, and in Gainesville, Florida, compared the amounts of caterpillar induced volatiles produced by sweet corn grown at current and elevated carbon dioxide. Corn grown at high carbon dioxide levels released significantly lower concentrations of volatiles than corn grown at ambient carbon dioxide levels. These results suggest corn may become more susceptible to insect pests as atmospheric carbon dioxide levels increase, and identified a trait that can be used in plant breeding programs to improve the natural defense response of corn, which would result in increased yields, reduced mycotoxin contamination of grain, and limited use of pesticides.

3. Barley sugar transfer gene helps to control Fusarium head blight. Fusarium head blight (FHB) is a devastating disease of small grain cereal crops that causes yield reductions and contamination of grain with the trichothecene mycotoxins nivalenol (NIV) or deoxynivalenol (DON). These toxins are harmful to the health of humans and livestock because of their ability to block protein synthesis. They are also important virulence factors for FHB, therefore plants that detoxify DON and NIV have improved resistance to the disease. Plants infected with mycotoxin-producing fungi can detoxify DON by attaching the toxin to a sugar. In collaboration with scientists at the University of Minnesota, ARS scientists in Peoria, Illinois, a gene from barley that produces a sugar transfer enzyme that efficiently detoxifies DON and NIV was introduced into an FHB susceptible wheat variety. Wheat that expressed this barley gene was significantly more resistant to both the toxins and to FHB. This research represents a critical step toward development of wheat with increased resistance to DON and NIV and serves as a guide for transgenic and traditional breeding approaches to increase resistance to DON in cereal crops and thereby improve food safety and crop production.

4. Key structural features identified in mycotoxin detoxifying enzyme. Fusarium Head Blight (FHB) is a devastating disease of small grain cereal crops that results in both yield reductions and contamination of grain with the mycotoxin deoxynivalenol (DON). DON is harmful to the health of humans and livestock and is also an important virulence factor for FHB, helping the disease to spread in the plant. Plants that can detoxify DON have improved resistance to the disease. In collaboration with scientists at the University of Wisconsin, ARS scientists in Peoria, Illinois, characterized a plant enzyme that rice plants use to disable DON, and used X-ray crystallography to identify key structural features of the enzyme. This research will allow researchers to engineer plant enzymes with improved ability to detoxify DON, provide a route to increasing resistance to FHB, and thereby improve both food safety and crop production of cereal crops.

5. Microorganisms isolated from soil detoxify vomitoxin. Vomitoxin (deoxynivalenol or DON) is a trichothecene mycotoxin produced when the fungus Fusarium infects small grains including wheat and barley. Ingestion of DON contaminated grain can cause diarrhea, hemorrhaging, and feed refusal. DON helps the fungus to spread into the grain. In collaboration with researchers at Virginia Polytechnic Institute and State University, ARS scientists in Peoria, Illinois, found that bacteria in soil collected from agricultural and landscape fields were able to detoxify DON both in culture and in contaminated grain. This research demonstrates the potential use of microorganisms in the environment to remediate mycotoxin contaminated materials such as dried distillers grains derived from fuel ethanol production and used as feed for livestock. These microbes also serve as a source of new detoxification genes that can be expressed in plants to improve resistance to Fusarium disease and thereby improve food safety and crop production.

6. Biocontrol tools to fight plant disease. Both the biocontrol fungus Trichoderma arundinaceum and the plant pathogen Botrytis cinerea have an arsenal of chemical weapons that they use to interact with each other and plants. T. arundinaceum produces the trichothecene sesquiterpene harzianum A (HA) which is toxic to other fungi but not toxic to plants and helps turn on genes for natural defenses that plants use to fight fungal pathogens. B. cinerea is an airborne fungus that causes pre-harvest soft rotting of field and greenhouse grown horticultural crops and post-harvest rotting of vegetable, fruits, and flowers. In collaboration with scientists at University of Leon, Spain, ARS scientists in Peoria, Illinois, found that HA and other T. arundinaceum chemical weapons affect the genes for production of botrydial, a toxin produced by B. cinerea that helps it invade and kill plant tissues, as well as fight back against the biocontrol fungus. This research provides scientists with new tools to develop effective pathogen control strategies that promote crop protection and food safety.

Review Publications
Wetterhorn, K.M., Newmister, S.A., Caniza, R.K., Busman, M., McCormick, S.P., Berthiller, F., Adam, G., Rayment, I. 2016. Crystal structure of Os79 (Os04g0206600) from Oryza sativa: a UDP-glucosyltransferase involved in the detoxification of deoxynivalenol. Journal of Biochemistry. 55(44):6175-6186.

Malmierca, M.G., Izquierdo-Bueno, I., McCormick, S.P., Cardoza, R.E., Alexander, N.J., Barua, J., Lindo, L., Casquero, P.A., Collado, I.G., Monte, E., Gutierrez, S. 2016. Trichothecenes and aspinolides produced by Trichoderma arundinaceum regulate expression of Botrytis cinerea genes involved in virulence and growth. Environmental Microbiology. 18(11):3991-4004.

Laraba, I., Boureghda, H., Abdallah, N., Bouaicha, O., Obanor, F., Moretti, A., Geiser, D.M., Kim, H.-S., McCormick, S.P., Proctor, R.H., Kelly, A.C., Ward, T.J., O'Donnell, K. 2017. Population genetic structure and mycotoxin potential of the wheat crown rot and head blight pathogen Fusarium culmorum in Algeria. Fungal Genetics and Biology. 103:34-41.

Li, X., Michlmayr, H., Schweiger, W., Malachova, A., Shin, S., Huang, Y., Dong, Y., Wiesenberger, G., McCormick, S., Lemmens, M., Fruhmann, P., et al. 2017. A barley UDP-glucosyltransferase inactivates nivalenol and provides Fusarium Head Blight resistance in transgenic wheat. Journal of Experimental Botany. 68(9):2187-2197.

Grafenhan, T., Johnston, P.R., Vaughan, M.M., McCormick, S.P., Proctor, R.H., Busman, M., Ward, T.J., O'Donnell, K. 2017. Fusarium praegraminearum sp. nov. is a novel nivalenol mycotoxin-producing pathogen from New Zealand can induce head blight on wheat. Mycologia. 108(6):1229-1239.

Acharya, J., Bakker, M.G., Moorman, T.B., Kaspar, T.C., Lenssen, A.W., Robertson, A.E. 2017. Time interval between cover crop termination and planting influences corn seedling disease, plant growth, and yield. Plant Disease. 101(4):591-600. doi: 10.1094/PDIS-07-16-0975-RE.

Block, A.K., Vaughan, M.M., Christensen, S.A., Alborn, H.T., Tumlinson, J.H. 2017. Elevated carbon dioxide reduces emission of herbivore induced volatiles in Zea mays. Plant Cell and Environment. doi:10.1111/pce.12976.

Vaughan, M.M., Block, A., Christensen, S., Allen, L.H., Schmelz, E.A. 2017. The effects of climate change associated abiotic stresses on maize phytochemical defenses. Phytochemistry Reviews. doi: 10.1007/s11101-017-9508-2.

Wilson, N.M., McMaster, N., Gantulga, D., Soyars, C., McCormick, S.P., Knott, K., Senger, R.S., Schmale, D.G. 2017. Modification of the mycotoxin deoxynivalenol using microorganisms isolated from environmental samples. Toxins. 9(4):1-11.

Proctor, R.H., Vaughan, M.M. 2017. Targeting fumonisin biosynthetic genes. In: Moretti, A., Susca, A., editors. Mycotoxigenic Fungi. Vol 1542. New York, NY: Springer. p. 201-214.