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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

2021 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
This is the final report for Project 5010-42000-048-00D, which has been replaced by Project 5010-11420-001-00D, “Improving Food Safety by Controlling Mycotoxin Contamination and Enhancing Climate Resilience of Wheat and Barley.” For additional information, please see the report for the replacement project. Mycotoxins are toxic chemicals produced by fungi that infect crops and contaminate food and feed making them unsafe to eat. The trichothecene mycotoxins produced by the fungus Fusarium graminearum can act as weapons that help the fungus to overcome wheat plant defenses and cause a disease known as Fusarium head blight (FHB). Agricultural losses due to FHB and grain trichothecene contamination can total billions of dollars every year. The overall goal of the project was to enhance food safety by developing methods to reduce levels of trichothecenes and other mycotoxins that occur in grain infected with trichothecene-producing species of Fusarium. Research under Objective 1 was designed to identify microorganisms or genes that could provide protection against trichothecenes and/or Fusarium, and thereby reduce contamination of grain. Over the course of the project we identified candidate microbes that might be used to control mycotoxin contamination by determining which types of microbes were usually found on wheat seeds with less contamination. Furthermore, we determined which microbes were consistently capable of protecting the plant from disease under variable climate conditions. We also discovered that the fungus Sarocladium was able to grow throughout the wheat plant and when present in the head provided protection against FHB and mycotoxin contamination of grain. This year we investigated the differences between two Sarocladium isolates that had varying efficacy as biocontrol agents by sequencing and comparing their genomes. We also examined the Sarocladium genomes to determine if they had the genetic blueprints to produce any known mycotoxins. Furthermore, chemical extracts of the Sarocladium isolates were evaluated to ensure that the beneficial fungus was not producing any unwanted toxins that would contaminate grain. We also conducted two experiments to determine if Sarocladium introduced into wheat as a seed treatment could effectively control FHB. This information and additional studies are needed to fully evaluate the suitability of these isolates for field application. Additionally, we discovered that the volatile chemical trichodiene suppresses mycotoxin production and engineered a beneficial fungus Trichoderma harzianum to produce trichodiene. The engineered fungus could more effectively control FHB and trichothecene contamination of wheat. Taking advantage of the knowledge that mycotoxin producing fungi have developed mechanisms to protect themselves from their own toxins, we discovered two genes (TRI14 and TRG1) that provide resistance to trichothecenes. These genes can be used to produce transgenic crops or enzymes capable of modifying trichothecenes to be less toxic. Climate change is predicted to increase the frequency and severity of FHB and mycotoxin contamination of wheat. Research under Objective 2 was designed to identify how rising atmospheric carbon dioxide will affect wheat vulnerability to FHB. Over the course of the project we showed that wheat plants grown at elevated carbon dioxide have a compromised defense response and are more prone to disease and mycotoxin contamination. We found that this effect was dependent on both the wheat cultivar and the F. graminearum isolate. We identified metabolic biomarkers that can be used by wheat breeders to predict FHB resistance level of wheat cultivars at both current and elevated carbon dioxide. This year, we evaluated the grain nutritional quality of 15 spring wheat cultivars with varying levels of FHB resistance, grown at elevated carbon dioxide. We found that some moderately FHB resistant cultivars currently used in breeding programs to enhance plant resistance to FHB had a greater loss in grain nutritional value than FHB susceptible cultivars. In addition, we evaluated how different F. graminearum isolates respond to these changes in grain nutritional composition. We found that some isolates produced more mycotoxins on grain of lower nutritional quality. Effective disease and mycotoxin control strategies need to account for the variability among F. graminearum isolates that cause FHB. Research under Objective 3 was designed to determine how diversity within F. graminearum influences disease development and mycotoxin contamination. Over the course of the project we published the first pangenome of F. graminearum and described three genetically distinct populations (NA1, NA2, NA3) among F. graminearum isolates in North America, and evaluated FHB disease caused by isolates of the three populations. On average, isolates from the NA3 population caused less disease than isolates from NA1 or NA2 on a moderately resistant wheat cultivar. Additionally, we discovered that mixtures of isolates from the same population compete with one another resulting in overall less disease and mycotoxin contamination of wheat. These findings reveal that understanding regional or field-specific pathogen population diversity may be important for the development of effective disease and mycotoxin control strategies. This year, we evaluated differences in the ability of representative strains from each of three populations to cause FHB and produce mycotoxins on wheat plants grown under different climatic conditions including drought, high temperature and elevated carbon dioxide. Climate conditions significantly contributed to differences in disease, but the observed effects were not specifically correlated with a particular population. For example, warmer temperatures and modest drought stress reduced FHB and mycotoxin accumulation. However, elevated carbon dioxide alone or in combination with warmer temperatures resulted in increased disease development. Reactive oxygen species (ROS) is one of the earliest defense responses during plant and pathogen interactions. Prior studies have shown that ROS play complex roles during F. graminearum infection and vomitoxin production. This year, we investigated ROS responses induced in wheat and barley tissues treated with chitin, a major component of fungal cell walls. Interestingly, the ROS burst typically observed following chitin-treatment was induced in barley leaves but not wheat leaves. However, we observed ROS bursts in wheat rachis nodes that are critical barriers for FHB spread in the wheat head. Furthermore, we demonstrated a positive correlation between ROS levels in wheat rachis nodes and disease spread. In addition, we determined that several defense marker genes were induced in wheat heads and rachis nodes treated with chitin. In contrast, several defense marker genes were suppressed in F. graminearum infected wheat heads. The genes identified in chitin-mediated signaling may serve as novel targets to enhance disease resistance. In addition, this research provides a new approach to prime plants with chitin to boost plant immunity and increase disease resistance. Trichothecene mycotoxins are produced by diverse fungi and pose health hazards to humans, livestock animals, and pets. All of the over 200 known trichothecenes have the same basic chemical structure but differ by the presence and absence of chemical groups attached to the basic structure. Such structural differences are of concern because they can result in marked differences in toxicity. In collaboration with scientists in Leon, Spain, we investigated whether and how direct transfer of genes between fungal species affects trichothecene production. We found evidence of horizontal transfer of genes responsible for trichothecene production from a Fusarium species to a distantly related fungus and that the function of one transferred gene changed. As a result, this fungus acquired the ability to produce an unusual trichothecene. We also found evidence that some species of the fungus Trichoderma inherited some of their trichothecene genes from an ancestral Trichoderma species but acquired other trichothecene genes by direct transfer from a distantly related fungus. As a result, the Trichoderma species produces a highly toxic subgroup of trichothecenes that are not produced by any of its close relatives. Together, these findings reveal genetic mechanisms that contribute to variation in toxin production in fungi and show how the risks that the fungi pose to food and feed safety can change.

1. Silencing mycotoxin genes with RNA interference reduces mycotoxin production in wheat. Fusarium graminearum causes Fusarium head blight (FHB) on wheat and barley. This disease not only reduces crop yield but also contaminates grains with mycotoxins, including deoxynivalenol (DON). Control of F. graminearum infection and mycotoxin contamination remains a challenge due to a lack of resistant plant varieties. DON is critical for FHB development, thus, reducing DON production will decrease yield losses to FHB and enhance food safety. A new technology known as RNA interference has shown promising results in its ability to silence gene expression. ARS researchers at Peoria, Illinois, used this technology to silence a gene critical for DON production and applied it on wheat heads under controlled conditions. The application was effective and reduced DON production, but high relative humidity altered the effectiveness. For RNA interference to be deployed for FHB and mycotoxin control in an agricultural setting, further investigations are needed to understand how environmental conditions affect RNA interference efficacy.

2. Gene for initial infection of wheat identified in Fusarium graminearum. The fungal pathogen Fusarium graminearum causes Fusarium head blight (FHB) on wheat and barley in the U.S. and worldwide. The disease results in severe yield loss and, contaminates grains with mycotoxins that are harmful to humans and animals. Control of F. graminearum infection and mycotoxin contamination remains a challenge due to a lack of resistant plant varieties and emergence of fungicide resistant strains. To develop novel and effective control methods, it is important to understand how F. graminearum causes FHB. The goal of this study was to discover factors critical for the initial infection. ARS researchers at Peoria, Illinois, found multiple fungal genes that are expressed during the initial stages of infection and investigated the role of three of these genes by generating gene knockout mutants. Although the genes had only minor effects on disease spread, experiments with knockout mutants showed that loss of one gene limited the ability of F. graminearum strains to cause initial infection. These data provide valuable information on FHB development and a new molecular target for disease and mycotoxin control.

3. Beneficial fungus engineered to reduce wheat disease and mycotoxin contamination. Fusarium head blight (FHB) is among the most devastating diseases of wheat and other small cereal crops worldwide. The disease is caused by Fusarium graminearum, a fungus that produces vomitoxin and other harmful toxins that contaminate grain and make it unsafe to eat. ARS researchers at Peoria, Illinois, discovered that a volatile chemical signals Fusarium to make less vomitoxin. In collaboration with scientists in Leon, Spain, a beneficial fungus that usually controls disease was engineered to produce this volatile signal. The engineered beneficial fungus was even more effective at controlling FHB and can be used as a new method to reduce FHB and mycotoxin contamination thereby enhancing food safety.

4. Identification of genes controlling corn defense chemicals. Plants produce specialized antifungal chemicals that help to defend against pathogens. Corn produces a large group of defense chemicals called zealexins. To effectively use these natural defense chemicals to fight fungal pathogens, it is important to understand how they are made by the plant. Therefore, ARS researchers at Peoria, Illinois, and Gainesville, Florida, identified the corn genes involved in making zealexins and found that some of the genes are redundant. Redundancy ensures that zealexins are produced even if one copy of a gene is lost. The new understanding of zealexin production in corn has provided breeders with the gene targets needed to enhance crop natural resistance to pathogens.

Review Publications
Pereira, C.B., Ward, T.J., Del Ponte, E.M., Moreira, G.M., Busman, M., McCormick, S.P., Feksa, H.R., De Almeida, J.L., Tessmann, D.J. 2020. Five-year survey uncovers extensive diversity and temporal fluctuations among fusarium head blight pathogens of wheat and barley in Brazil. Plant Pathology. 70(2):426-435.
Taylor, L., Gutierrez, S., McCormick, S.P., Bakker, M.G., Proctor, R.H., Teresi, J., Kurtzman, B., Hao, G., Vaughan, M.M. 2021. Use of the volatile trichodiene to reduce Fusarium head blight and trichothecene contamination in wheat. Microbial Biotechnology.
Hao, G., McCormick, S., Tiley, H., Usgaard, T. 2021. Detoxification and excretion of trichothecenes in transgenic Arabidopsis thaliana expressing Fusarium graminearum trichothecene 3-O-acetyltransferase. Toxins. 13(5).
Gutierrez, S., McCormick, S.P., Cardoza, R.E., Lindo, L., Alexander, N.J., Proctor, R.H. 2020. Trichoderma trichothecenes: beyond their toxic effect. In: Gupta, V.K., Zeilinger, S., Singh, H.B., Druzhinina, I. New and Future Developments in Microbial Biotechnology and Bioengineering. Amsterdam, Netherlands: Elsevier B.V. p. 281-301.
McLaughlin, J.E., Darwish, N.I., Garcia-Sanchez, J., Tyagi, N., Trick, H.N., McCormick, S., Dill-Macky, R., Tumer, N.E. 2021. A lipid transfer protein has antifungal and antioxidant activity and suppresses Fusarium Head Blight disease and DON accumulation in transgenic wheat. Phytopathology. 111(4):671-683.
Hao, G., McCormick, S., Usgaard, T., Tiley, H., Vaughan, M.M. 2020. Characterization of three Fusarium graminearum effectors and their roles during Fusarium head blight. Frontiers in Plant Science. 11. Article 579553.
Ding, Y., Weckwerth, P.R., Poretsky, E., Murphy, K.M., Sims, J., Saldivar, E., Christensen, S.A., Char, S., Yang, B., Tong, A., Shen, Z., Kremling, K.A., Buckler IV, E.S., Kono, T., Nelson, D.R., Bohlmann, J., Bakker, M.G., Vaughan, M.M., Khalil, A.S., Betsiashvili, M., Briggs, S.P., Zerbe, P., Schmelz, E.A., Huffaker, A. 2020. Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity. Nature Plants. (6):1375-1388.
Laraba, I., McCormick, S.P., Vaughan, M.M., Geiser, D.M., O'Donnell, K. 2021. Correction: Phylogenetic diversity, trichothecene potential, and pathogenicity within Fusarium sambucinum species complex. PLoS ONE. 16(4). Article e0250812.
Hao, G., McCormick, S., Vaughan, M. 2021. Effects of double-stranded RNAs targeting Fusarium graminearum TRI6 on Fusarium head blight and mycotoxins. Phytopathology.