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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

2018 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
Production of vomitoxin (deoxynivalenol or DON) or other trichothecene mycotoxins contributes to the ability of Fusarium graminearum to cause Fusarium Head Blight (FHB) of wheat. As a result, increasing resistance to trichothecenes should increase wheat 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 DON contamination of grain. Fungi that produce toxins generally have mechanisms to protect themselves from their own toxins. The microbiology and genetic tools necessary to identify genes that confer resistance to trichothecene in Fusarium and other fungi that produce the toxins were developed. Improved understanding of interactions between Fusarium and other members of crop microbiomes is also expected to produce novel strategies for limiting disease and mycotoxin accumulation. Collection of microbes from wheat-associated environments was expanded, with several hundred new bacterial isolates collected from surface-sterilized wheat tissues (Objective 1). These isolates were tested for ability to antagonize Fusarium graminearum and limit FHB under field conditions. In addition, isolates and consortia of microbes were screened for the ability to biotransform or detoxify DON. Wheat kernels from a disease-conductive field environment were used to observe relationships between F. graminearum load, mycotoxin content, and microbiome characteristics. 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 DON, T-2 toxin, and other trichothecenes out of food and feed. This year, in collaboration with scientists at University of Wisconsin, ARS scientists in Peoria, Illinois synthesized DON glucoside using an enzyme from rice. Trichothecene glucosides are being used to develop analytical detection methods and complete risk assessments of the masked mycotoxins. Trichothecene production in Fusarium is generally found in two groups of species called the Fusarium equiseti-incarnatum complex (FIESC) and Fusarium sambucinum complex (FSSC). This year, a trichothecene gene cluster was discovered outside of these two groups, in Fusarium buharicum. Trichothecenes produced by this species were identified, and significant differences were found between FIESC/FSSC and F. buharicum in the function of one of the trichothecene genes. Toxin production by fungi can affect agriculture in two different ways. Production by fungi that cause crop diseases can lead to contamination of crops with toxins that are harmful to human and animal health. In contrast, toxin production by fungi that are used to control crop diseases can contribute to the control. Understanding the genetic control of toxin production in fungi is critical for reducing toxin contamination in crops and for manipulating toxin production in fungi used to control crop diseases. In collaboration with scientists at the University of León, Spain, researchers investigated the genetic control of trichothecene production in Trichoderma, a fungus that is used for biocontrol of crop diseases caused by other fungi. This year ARS scientists in Peoria, Illinois, investigated how two genes control trichothecene toxin production in Trichoderma and Fusarium and found that there were important differences between the two groups of fungi. Inactivation of either gene in Fusarium completely blocked trichothecene production, but inactivation of either gene in Trichoderma reduced, but did not completely block, trichothecene production, and induced production of other antifungal chemicals. In Trichoderma, these genes affect regulation of genes that is not restricted to trichothecene pathways. In wheat, the severity of FHB epidemics and contamination of grain with DON are strongly driven by climatic factors. This year, virulence and toxin assays were conducted on susceptible and resistant spring wheat varieties that had been acclimated to ambient or elevated carbon dioxide levels, and then inoculated with either F. graminearum strains that produce DON or with a non-toxin producing strain (Objective 2). Experiments were completed in growth chambers and at the Free Air Concentration Enrichment (FACE) field at University of Illinois with a moderately resistant spring wheat variety. In addition to virulence assays, changes in plant hormones were measured and the expression of defense related genes was examined. In collaboration with scientists at the National Research Council of Canada, nuclear magnetic resonance (NMR) was used to characterize and compare the metabolic fingerprints (metabolomics) of control and infected plants grown at ambient or elevated carbon dioxide levels. New markers were identified that will predict resistance or susceptibility to FHB regardless of atmospheric carbon dioxide levels. This will be useful in selecting varieties that maintain resistance even as atmospheric carbon dioxide levels rise. These metabolic markers can be used by breeders to improve FHB resistant varieties that are resilient to rising carbon dioxide concentrations. This year, researchers evaluated the effects of atmospheric carbon dioxide concentrations and temperature on wheat and corn susceptibility to FHB and mycotoxin contamination after inoculation with F. graminearum strains from the two predominant North American populations (NA1 and NA2). This research will aid in forecast modeling of FHB as well as the development of climate resilient management strategies. Research under Objective 3 is designed to provide novel targets and strategies for disease and mycotoxin control, detection of introduced pathogens, 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 assess species or population-specific differences in host-pathogen interactions, researchers conducted comparative analyses of aggressiveness and toxin production in moderately resistant wheat, and identified significant differences between the three major genetic populations of F. graminearum found in the northern U.S. and Canada (NA1, NA2, and NA3). Metabolomic and transcriptomic analyses of the wheat defense responses to the three different populations are in progress. The growth, pathogenicity, and toxin production of individual isolates and mixtures of isolates from NA1 and NA2 populations were compared (Objective 3). Researchers found that most NA1 and NA2 isolates produced more mycelia in liquid culture when grown individually than when they were grown in a mixture of isolates from the same population. Disease generally spread faster in wheat heads inoculated with an individual isolate rather than a mixture of isolates. However, the effect of intrapopulation interactions was much less pronounced in NA2 than in NA1 and significant differences in the amount of disease were infrequently observed among treatments and primarily limited to early stages of infection. Toxin levels were not consistently affected by intrapopulation interactions. Results suggested that pathogen population dynamics influence FHB, and understanding regional pathogen composition could improve disease forecasting and local management practices. Plants mount defenses against fungal invasion. The plant chemical salicylic acid (SA) plays an important role in regulating plant defense response against pathogens. Pathogens can circumvent plant SA-mediated defense pathways by degrading SA with enzymes called hydroxylases. This year, as part of a study on the genetic differences between F. graminearum populations (Objective 2), an SA hydroxylase gene, which was unique to F. graminearum strains in the NA2 population, was identified and characterized. Researchers also characterized another SA hydroxylase gene, present in both populations, that was highly induced in wheat tissue and by the addition of SA to liquid cultures. In addition to producing trichothecene mycotoxins, F. graminearum secretes proteins called effectors that can trigger or suppress plant defense response. This year, researchers studied a Fusarium effector gene which is expressed during early stages of infection. This effector, which suppressed plant defence response, encodes an enzyme that breaks down a component of plant cell walls. Deletion of this gene significantly reduced FHB severity and DON production in mutants.

1. An improved plant enzyme to reduce mycotoxin contamination and Fusarium Head Blight in cereal crops. Deoxynivalenol (DON) is a toxin produced by Fusarium graminearum, a fungus that causes Fusarium Head Blight (FHB) of wheat, barley, and other cereals. DON promotes the spread of FHB within a plant; therefore plants that can detoxify DON have improved resistance to the disease. There is a need for detoxification enzymes with broader or altered specificity because DON is part of a family of toxins known as trichothecenes that can be produced by Fusarium species that cause FHB worldwide. ARS scientists in Peoria, Illinois, in collaboration with scientists at University of Wisconsin, used X-ray crystallography to guide modification of a DON detoxification enzyme from rice plants in order to produce an improved enzyme that can disable a wide variety of trichothecenes as well as DON. The new enzyme provides a means to control FHB across a broad spectrum of Fusarium species and is a good candidate for incorporation in crop improvement programs.

2. Discovery of genes responsible for mycotoxin diversity improves mycotoxin monitoring and risk assessment. Trichothecenes are fungal toxins that frequently contaminate food and feed crops and, as a result, pose health risks to humans and domestic animals. Collectively fungi produce over 150 different trichothecenes, each with a similar but distinct chemical structure that can vary markedly in toxicity and in the risks they pose to human and animal health. ARS scientists in Peoria, Illinois, used a combination of chemical, molecular, genetic, and genome sequencing technologies to determine the origins and the genetic basis for this toxin diversity. The researchers identified specific genes that were acquired, lost, or changed function resulting in different trichothecene structures. These findings can be used to rapidly determine toxin production potential and assess the potential risks that different fungi pose to food and feed safety.

3. Identification of Fusarium Head Blight (FHB) pathogens in Mexico facilitates disease and mycotoxin control programs across North America. FHB is a disease of cereal crops worldwide and a major food safety concern because FHB pathogens can contaminate grain with trichothecenes and other mycotoxins. Information on FHB pathogen and mycotoxin diversity in Mexico has been extremely limited, but is needed to improve disease and mycotoxin control efforts across North America. In collaboration with scientists at multiple universities in Mexico, ARS scientists in Peoria, Illinois, used DNA sequencing and toxin analyses to characterize FHB isolates collected from wheat in Mexico. They identified nine previously described species and 30 isolates representing potentially novel species. Significant regional differences in pathogen composition were found, with Fusarium boothii being predominant in the Mixteca region of Southern Mexico and Fusarium avenaceum and related fungi being predominant in the Highlands region of Central Mexico. Fusarium graminearum, which is the dominant FHB pathogen in other parts of North America, was not present among the isolates collected in Mexico. None of the Fusarium avenaceum related isolates produced trichothecenes, but many were able to produce other mycotoxins including chlamydosporol and enniatin. These results provide new information on FHB pathogen and mycotoxin prevalence that can be used to develop regionally targeted risk assessments as well as disease and mycotoxin control programs that improve crop production and food safety.

4. Genomics reveals novel targets to control Fusarium Head Blight (FHB) of wheat and barley. Fusarium graminearum is the major cause of FHB, a significant disease of wheat, barley, and other cereal crops worldwide. In addition, the fungus contaminates grain with mycotoxins that pose a significant threat to food safety and animal health. The recent appearance of novel pathogen populations and toxin types in North America is of concern because this diversity may include novel adaptations that enable the fungus to rapidly respond to FHB control measures. ARS scientists in Peoria, Illinois, sequenced the genomes of 60 isolates of F. graminearum from across North America to identify genes that help this important pathogen adapt to agricultural environments. They demonstrated that there are at least three distinct pathogen populations in North America and identified 121 genes that distinguish these populations. In addition, they identified 14 regions of the genome that harbor population-specific adaptations. The findings suggest that each population uses a unique set of tools to invade and obtain nutrients from their hosts, compete with other microbes, and adapt to different climatic conditions. The genes identified in this study will be of use to scientists, disease control specialists, and producers working to develop and deploy more effective disease and mycotoxin control measures that counter adaptations within all of the major FHB pathogen populations.

5. Fungal mock community developed to improve microbiome research. Plants, soils, and many other environments host diverse communities of microorganisms called microbiomes. Microbiomes can have substantial impacts on plant health and food safety. For instance, plant disease may be reduced when the plant microbiome is able to prevent pathogen colonization. Similarly, food safety may be protected by microbiomes that are able to limit the accumulation of mycotoxins in grain. Current research methods for studying microbiomes are typically based on analysis of deoxyribonucleic acid (DNA), which offers several advantages over older methods. However, there are still limitations to DNA-based methods and these can lead to erroneous conclusions. In order to address this problem, ARS scientists in Peoria, Illinois, developed artificial mixtures of fungal DNA that can be used as an experimental control to identify sources of error in microbiome studies, so that methods can be improved and limitations can be considered when interpreting results. The resources developed in this research will be shared with other scientists to promote the adoption of best practices in microbiome studies. As a consequence, the understanding of the role of microbiomes in plant health and food safety will advance more quickly, leading to new strategies for supporting the production of a safe and abundant food supply.

6. New method developed to improve identification of fungi that cause Fusarium Head Blight (FHB) in wheat and barley. FHB is a destructive disease of cereal crops worldwide and a major food safety concern due to grain contamination with trichothecenes and other mycotoxins. Fusarium graminearum, a member of the Fusarium graminearum species complex (FGSC), is the dominant FHB pathogen in many parts of the world. However, a number of other Fusarium species, including other members of the FGSC, may also be present in some regions or in other crops. To facilitate identification of the major causative agent of FHB, ARS scientists in Peoria, Illinois, worked with scientists in Uruguay to develop an easy and inexpensive method to differentiate F. graminearum from other species within the FGSC. This will aid in proper species identification that is critical to research aimed at improving disease and mycotoxin control programs.

7. Differences in fungicide sensitivity discovered among Fusarium Head Blight (FHB) pathogens of wheat and barley. Fungi within the Fusarium graminearum species complex (FGSC) are responsible for FHB of wheat and barley. These fungi also contaminate grain with trichothecene mycotoxins that pose a significant threat to food safety and animal health. Fungicides are often used to control disease and mycotoxin contamination. ARS scientists in Peoria, Illinois, collaborated with scientists in Brazil to determine the sensitivity of FGSC isolates to two triazole fungicides (tebuconazole and metconazole). Tests performed on three different species as well as isolates with different toxin types revealed that all isolates were sensitive to both triazole fungicides at concentrations used in field applications. However, these fungi were significantly less sensitive to tebuconazole as compared with metconazole. The results reported here facilitate monitoring of mycotoxins in barley and the deployment of effective management strategies to reduce FHB and mycotoxin contamination of grain.

Review Publications
Wetterhorn, K.M., Gabardi, K., Michlmayr, H., Malachova, A., Busman, M., McCormick, S.P., Berthiller, F., Adam, G., Rayment, I. 2017. Determinants and expansion of specificity in a trichothecene UDP-glucosyltransferase from Oryza sativa. Journal of Biochemistry. 56(50):6585-6596.
Kelly, A.C., Ward, T.J. 2018. Population genomics of Fusarium graminearum reveals signatures of divergent evolution within a major cereal pathogen. PLoS One. 13(3):e0194616.
Ceron-Bustamante, M., Ward, T.J., Kelly, A.C., Vaughan, M.M., McCormick, S.P., Cowger, C., Leyva-Mir, S.G., Villasenor-Mir, H.E., Ayala-Escobar, V., Nava-Diaz, C. 2018. Regional differences in the composition of Fusarium head blight pathogens and mycotoxins associated with wheat in Mexico. International Journal of Food Microbiology. 273:11-19.
Garmendia, G., Umpierrez-Failache, M., Ward, T.J., Vero, S. 2017. Development of a PCR-RFLP method based on the transcription elongation factor 1-a gene to differentiate Fusarium graminearum from other species within the Fusarium graminearum species complex. Food Microbiology. 70:28-32.
Machado, F.J., Nicolli, C.P., Moller, P.A., Arruda, R., Ward, T.J., Del Ponte, E.M. 2017. Differential triazole sensitivity among members of the Fusarium graminearum species complex infecting barley grains in Brazil. Tropical Plant Pathology. 42(3):197-202.
Bakker, M.G. 2018. A fungal mock community control for amplicon sequencing experiments. Molecular Ecology Resources. 18(3):541-556.
Bakker, M.G., Brown, D.W., Kelly, A.C., Kim, H.-S., Kurtzman, C.P., McCormick, S.P., O'Donnell, K.L., Proctor, R.H., Vaughan, M.M., Ward, T.J. 2018. Fusarium mycotoxins: a trans-disciplinary overview. Canadian Journal of Plant Pathology. 40(2):161-171.
Hossain, M.Z, McCormick, S.P., Maragos, C.M., 2018. An imaging surface plasmon resonance biosensor assay for the detection of T-2 toxin and masked T-2 toxin-3-glucoside in wheat. Toxins. 10(3)119.
Lee, S., Chen, Y., Gorski, L.A., Ward, T.J., Osborne, J., Kathariou, S. 2018. Listeria monocytogenes source distribution analysis indicates regional heterogeneity and ecological niche preference among serotype 4b clones. mBio. 9(2):1-12.
Vaughn, S.F., Dinelli, F.D., Jackson, M.A., Vaughan, M.M., Peterson, S.C. 2018. Biochar-organic amendment mixtures added to simulated golf greens under reduced chemical fertilization increase creeping bentgrass growth. Industrial Crops and Products. 111:667-672.
Proctor, R.H., McCormick, S.P., Kim, H.-S., Cardoza, R.E., Stanley, A.M., Lindo, L., Kelly, A., Brown, D.W., Lee, T., Vaughan, M.M., Alexander, N.J., Busman, M., Gutierrez, S. 2018. Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi. PLoS Pathogens. 14(4): e1006946.
Vaughan, M., Backhouse, D., Del Ponte, E.M. 2016. Climate change impacts on the ecology of Fusarium graminearum species complex and susceptibility of wheat to Fusarium head blight: a review. World Mycotoxin Journal. 9(5):685-700.
Christensen, S.A., Sims, J., Vaughan, M.M., Hunter III, C.T., Block, A.K., Willett, D.S., Alborn, H.T., Huffaker, A., Schmelz, E.A. 2018. Commercial hybrids and mutant genotypes reveal complex protective roles for inducible terpenoid defenses. Journal of Experimental Botany. doi:10.1093/jxb/erx495.
Laraba, I., Keddad, A., Boureghda, H., Abdallah, N., Vaughan, M.M., Proctor, R.H., Busman, M., O'Donnell, K. 2018. Fusarium algeriense, sp. nov., a novel toxigenic crown rot pathogen of durum wheat from Algeria is nested within the Fusarium burgessii species complex. Mycologia. 106(6):935-950.
Lindo, L., McCormick, S.P., Cardoza, R.E., Brown, D.W., Kim, H.-S., Alexander, N.J., Proctor, R.H., Gutierrez, S. 2018. Effect of deletion of a trichothecene toxin regulatory gene on the secondary metabolism transcriptome of the saprophytic fungus Trichoderma arundinaceum. Fungal Genetics and Biology. 119:29-46.