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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

2016 Annual Report

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.

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.

Progress Report
Project 5010-42000-048-00D replaces Project 5010-42000-042-00D and has a start date of 01/19/2016. Scientists have begun planning and conducting research to fulfill the 12-month milestones. The production of trichothecene mycotoxins contributes to the ability of the Fusarium graminearum to cause Fusarium Head Blight (FHB). As a result, increasing trichothecene resistance in wheat should increase resistance to FHB. Toxin-producing fungi generally have a mechanism to protect themselves from the harmful effects of the toxin(s). Therefore, in order to identify novel mechanisms of trichothecene resistance, we are examining eight genera of trichothecene-producing fungi to identify the genes responsible for their self-protection. As a first step, we generated genome sequences for these fungi and identified the region of chromosomes with genes responsible for trichothecene biosynthesis. Microorganisms that survive in the same environment as toxin-producing fungi also require toxin-resistance mechanisms. Wheat head, stalk, and rhizosphere samples have been collected from two different winter wheat varieties and we are isolating microorganisms from these substrates. Thirty candidate microorganisms have been isolated that can grow in the presence of the trichothecenes deoxynivalenol (DON) and T-2 toxin and they have been sequenced to identify species. We have also initiated enrichment cultures of field soil microorganisms and have identified candidates that convert DON to the non-toxic products, 3-keto DON and epi-DON. In addition, we completed production of DON required to complete large scale screening of fungi and other microorganisms for detoxification genes. This research addresses identification and characterization of microorganisms and microbial genes that can reduce trichothecene contamination of grain (Objective 1). The severity of FHB epidemics and accumulation of associated trichothecene mycotoxins in wheat kernels is strongly driven by climatic factors. In order to evaluate the effects of carbon dioxide levels on susceptibility of wheat to FHB, and toxin production and virulence of F. graminearum, we conducted virulence assays at both ambient and elevated carbon dioxide using F. graminearum and spring wheat varieties that we had acclimated to higher carbon dioxide levels. In order to determine the mechanisms by which abiotic stress influences wheat defenses against Fusarium species, we examined the expression of eight genes involved in plant defense. 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). In order to determine the genomic diversity and population structure of Fusarium graminearum, and to identify genes responsible for adaptive traits in these mycotoxigenic pathogens (Objective 3), we performed whole-genome sequencing (50X coverage) on 60 F. graminearum strains isolated from the U.S. and Canada. Analyses based on 505,748 single nucleotide polymorphisms identified three genetic populations of F. graminearum that were strongly associated with the type of trichothecene toxin (15ADON, 3ADON, and NX-2) produced. However, we found evidence of genetic exchange between populations indicating the potential for exchange of toxin types and other adaptations. Comparative genomic analyses resulted in the identification of genes that likely contribute to population-specific adaptations. These included toxin biosynthetic genes and genes that contribute to temperature adaptation and UV radiation tolerance in other fungi. We identified 13,669 core genes that were present in all isolates and more than 80 genes that were population-enriched (found exclusively, or at significantly higher frequency, in one of the three genetic populations examined). Population-enriched genes included plant effectors, fungal-specific toxins, secondary metabolite enzymes, and self-recognition proteins. We also determined the distribution and prevalence of the novel NX-2 toxin type from a global collection of more than 5,000 F. graminearum isolates. The results indicated that NX-2 originated in, and is potentially restricted to, southern Canada and the northern U.S. These data also demonstrated that the NX-2 toxin type occurs on a broader set of cereal hosts (wheat, oat, and barley) than was previously recognized. In addition, the NX-2 chemotype was demonstrated to have evolved in response to a significant change in selective pressure, suggesting this novel toxin type may provide an adaptive advantage to FHB pathogens in some environments or on some hosts. These analyses suggest that F. graminearum populations have been subjected to distinct selective pressures that have contributed to differences in how they exploit the agricultural landscapes of North America. Accounting for these population-specific adaptations will be critical to the development of robust disease and toxin control programs. The relative prevalence of different hosts can have a major impact on FHB species and trichothecene chemotype composition. Research on the relative competitive ability of genetic and geographic populations of F. graminearum on spring and winter wheat will include direct competition assays between pairs of pathogen populations using two spring (Barlow, Norm) and winter (AgriMax 413, AgriMax 438) wheat lines (Objective 3). Since the winter wheat varieties require a three month vernalization period, they must be planted prior to the spring wheat varieties so that host anthesis can be approximately synchronized. Seed for winter and spring wheat varieties have been obtained and plants have been grown to maturity two or more times. Differences between spring wheat flowering dates have been determined and methods have been developed to synchronize maturity. We are also developing methods to enhance uniform flowering in winter wheat varieties.

1. The distribution and prevalence of the novel NX-2 mycotoxin revealed through global molecular surveillance of Fusarium head blight pathogens. Fusarium graminearum and related fungi are responsible for Fusarium head blight (FHB) and other economically destructive diseases of cereal crops world-wide. In addition, these fungi contaminate grain with mycotoxins that pose a significant threat to food safety and animal health. Strains of F. graminearum with a previously unknown mycotoxin type, termed NX-2, were recently identified from FHB infected wheat. However, the origin, distribution, and global prevalence of this novel toxin type is unknown. ARS scientists in Peoria, Illinois, in collaboration with an international team of scientists, determined the distribution, prevalence, and evolutionary history of NX-2 strains from a global collection of more than 2,500 F. graminearum isolates. The results expanded the known geographic distribution of NX-2 strains to include the northeastern U.S., expanded the known host range of NX-2 strains to include oat and barley, and indicated that the NX-2 toxin type may be restricted to southern Canada and the northern US where it occurs at low frequency on cereal crops. In addition, we identified nine genetic changes in a toxin production gene that are specific to NX-2 strains and demonstrated that this novel toxin has a unique evolutionary history indicating it may provide an advantage to FHB pathogens in some environments. These results promote food safety and cereal production by providing information and molecular tools needed for efficient and effective monitoring of mycotoxin contamination and for the development of cereals that are broadly resistant to FHB infection.


Review Publications
Li, N., Alfiky, A., Vaughan, M.M., Kang, S. 2016. Stop and smell the fungi: Fungal volatile metabolites are overlooked signals involved in fungal interaction with plants. Fungal Biology Reviews. 30(3):134-144.
Kelly, A., Proctor, R.H., Belzile, F., Chulze, S.N., Clear, R.M., Cowger, C., Elmer, W., Lee, T., Obanor, F., Waalwijk, C., Ward, T.J. 2016. The geographic distribution and complex evolutionary history of the NX-2 trichothecene chemotype from Fusarium graminearum. Fungal Genetics and Biology. 95:39-48.