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

2020 Annual Report

Objectives
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.

Approach
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
Trichothecenes are a group of mycotoxins, toxic chemicals produced by fungi, that can contaminate grain crops, such as barley, corn, and wheat. These toxins pose health hazards to humans, livestock, and pets. Production of vomitoxin (deoxynivalenol or DON) or other trichothecene mycotoxins contributes to the ability of the fungus Fusarium graminearum to cause the disease Fusarium Head Blight (FHB) of wheat. As a result, increasing resistance of wheat to trichothecenes should increase resistance to FHB. Research under Objective 1 is designed to identify microorganisms or genes that provide resistance to trichothecenes and/or resistance to Fusarium, and thereby reduce DON contamination of grain. Because fungi that produce toxins have mechanisms to protect themselves from their own toxins, trichothecene-producing fungi are a potential source of trichothecene resistance. This year, we demonstrated that a gene (TRI14) that is present in all trichothecene-producing fungi confers resistance to trichothecenes when transferred to yeast. We also found evidence that the effect of TRI14 on resistance in trichothecene-producing species of Fusarium is masked by a second more effective resistance gene (TRI101). Further, we identified a third gene (TRG1) from the trichothecene-producing fungus Spicellum roseum that provides a moderate level of resistance to trichothecenes. Together, these findings indicate that trichothecene-producing fungi have at least 2 – 3 genes that confer resistance to the toxins. Other research under Objective 1 is designed to determine whether natural communities of microorganisms (microbiome) that occur in/on soil, plant stubble, wheat plants, seeds and heads can reduce Fusarium mycotoxins in grain. The microbiome of wheat heads frequently includes the fungus Sarocladium. This year, we identified an isolate of Sarocladium that has an excellent ability to spread throughout the entire wheat plant and found that wheat plants inhabited with Sarocladium consistently have reduced FHB and DON contamination. Fusarium species can form close associations (symbioses) with groups of bacteria than influence the ability of the fungus to cause crop diseases. This year we evaluated the potential of several bacteria that have symbiotic associations with F. graminearum to reduce the ability of the fungus to cause FHB. We identified a F. graminearum strain that is naturally associated with a species of the bacterium Paenibacillus. We cured the fungus of this bacterium and determined that the bacterial association significantly compromised the ability of F. graminearum to produce trichothecenes and cause FHB. Furthermore, we determined that the bacterial association was on the outside of the fungal hyphae (ectohyphal association) and could be transferred with the spores. Fusarium graminearum produce spores in reproductive structures call perithecia. Blocking perithecia formation and/or spore production should block the infection of crops. Research under Objective 1 is designed to determine if bacterial communities can infect F. graminearum perithecia and reduce production of spores. This year we evaluated the soil microbiomes from research plots and local field plots that had used a combination of cover crops or were left fallow overwinter. Bacterial communities from soil that had cover crops were most effective at delaying perithecia formation. We identified a bacterial Stenotrophomonas rhizophila strain that readily colonized F. graminearum and reduced perithecia formation by approximately half. We also identified a F. graminearum strain that is naturally associated with a Stenotrophomonas bacterial strain. This association appeared to be within the fungal hyphae but did not transfer with the spores. We cured the fungus of this bacterium and determined that the Stenotrophomonas association significantly reduced perithecia formation but did not affect the ability of F. graminearum to produce trichothecenes or cause FHB. The severity of FHB epidemics and contamination of grain with DON are strongly linked to weather conditions. Research under Objective 2 is designed to identify how future climate conditions and environmental stress change the expression of genes and plant defense responses of wheat plants and which changes make them more vulnerable to Fusarium infection and DON. We completed virulence and toxin assays on susceptible and moderately resistant spring wheat variety that had been acclimated to ambient or elevated carbon dioxide levels (Objective 2) and found that elevated carbon dioxide is a significant contributing factor to the severity of FHB in wheat. This year, 15 FHB moderately resistant and susceptible cultivars were grown under ambient or elevated carbon dioxide conditions, and the grain was harvested and evaluated for nutritional value. We found that the moderately resistant wheat cultivars produced more carbohydrates but significantly less protein and mineral content than susceptible wheat varieties when grown under elevated carbon dioxide. In addition to dramatically altering the nutritional quality of grain, these shifts in carbohydrate, protein, and mineral composition impact the ability of F. graminearum to produce DON, which is highly dependent on the nutrient composition of its growing medium. Therefore, changes in the nutritional composition of wheat seeds when grown in elevated carbon dioxide can have a significant impact on the susceptibility or resistance of wheat to disease spread. In addition, we found that moderately resistant cultivars currently being used in breeding programs have a greater reduction in nutritional value then susceptible cultivars. Therefore, in order to determine whether specific genetic backgrounds contribute to the observed loss in grain nutritional content, we obtained additional wheat cultivars containing genetic backgrounds with varying grain protein concentrations and six near isogenic lines containing the FHB1 loci, the genetic loci primarily responsible for FHB resistance in wheat. Effective disease and mycotoxin control strategies need to account for differences in the F. graminearum isolates that cause FHB. Research under Objective 3 is designed to determine how diversity within F. graminearum influences disease development and mycotoxin contamination. This year, we evaluated the ability of representative strains from each of three genetically distinct groups (populations) within F. graminearum to cause FHB and produce mycotoxins in two hard red spring wheat varieties, one that is rated as moderately resistant and another that is considered susceptible to the disease. We also evaluated how the populations differed in their abilities to induce plant defense responses, the biochemical changes that help wheat to combat disease. We found population-specific differences in the ability of F. graminearum to cause FHB that were only partially linked to the specific type of trichothecene mycotoxin produced. Differences in host defense responses and in the relationship between the amounts of fungal growth and the amounts of trichothecenes were also observed between pathogen populations. This year, we also tested whether the three F. graminearum populations were differentially adapted to winter or spring wheat varieties or to environmental conditions. We compared disease progression and toxin production of representative strains from each of the three populations on winter and spring wheat varieties and on spring wheat varieties experiencing flooding or mild drought stress conditions. In addition, we compared perithecia formation on wheat straw of representative strains from each of the three F. graminearum populations. In addition to trichothecene mycotoxins that help the fungus spread within wheat heads, F. graminearum secretes proteins called effectors that can trigger or suppress the plant defense response to the infection. This year, we identified three candidate effector genes that were highly induced during initial wheat infection and generated deletion mutants to determine the role of the effectors in FHB pathogenesis. Fusarium head blight virulence assays showed that loss of one of the effectors significantly reduced F. graminearum initial infection, however, none of the effectors had a significant effect on FHB spread. After harvest, Fusarium-contaminated grain can continue to produce and accumulate mycotoxins if conditions are warm and humid. Glucosinolates are a group of chemicals produced by mustard, horse radish, cress, and related plants that help defend against attacks by insects and disease-causing microorganisms. When leaves of these plants are damaged, glucosinolates are converted to and released as volatile chemicals called isothiocyanates. This year, we tested the efficacy of isothiocyanates from mustard family seed meals, low value agricultural byproducts, to reduce growth of F. graminearum and mycotoxin production during storage, processing, and malting of grain. Initial tests indicated that low concentrations of isothiocyanates from brown mustard or pennycress seed meal inhibited the growth of F. graminearum without adversely affecting seed germination of wheat or barley.

Accomplishments
1. Elevated carbon dioxide impacts nutritional quality of wheat. Rising carbon dioxide can significantly increase the severity of Fusarium Head Blight (FHB), a devastating fungal disease of wheat that reduces yield and contaminates grain with vomitoxin and other harmful mycotoxins. ARS scientists in Peoria, Illinois, discovered that some wheat cultivars with moderate resistance to FHB at current carbon dioxide levels have poorer nutritional quality when grown in higher carbon dioxide. Since changes in nutritional quality can increase the ability of Fusarium to produce vomitoxin and to spread in the plant, the resistance to FHB may also be compromised. This study showed that breeding wheat lines that maintain nutritional quality at higher carbon dioxide will complement FHB control strategies to reduce disease and mycotoxin contamination of grain.

2. Culmorin increases toxic effect of vomitoxin. Fusarium graminearum infects cereal crops and contaminates grain with vomitoxin and other harmful trichothecene mycotoxins. The role of vomitoxin in plant disease development is well characterized, but less is known about other chemicals produced by the fungus and if they aid the fungus during plant infection or contribute to the toxicity of contaminated grain. ARS scientists in Peoria, Illinois, tested culmorin, another chemical produced by F. graminearum, and mixtures of culmorin and vomitoxin on plant roots. Although culmorin did not affect plant growth, addition of culmorin to vomitoxin increased its toxicity to wheat, barley, and corn roots. In addition, the severity of Fusarium Head Blight disease was correlated with the sum of culmorin and vomitoxin produced by Fusarium. This study showed that culmorin production provides an advantage to the pathogen and that culmorin production can be targeted to aid in the control of disease development and vomitoxin contamination of grain.

3. Corn phytoalexins for disease resistance. Losses in corn production due to microbial pathogens can result in billions of dollars in lost revenue annually. Despite these great economic losses, only a limited amount of research has demonstrated how corn can defend itself against pathogens using innate antibiotics. In collaboration with scientists at University of California, San Diego, California, ARS scientists in Gainesville, Florida, and Peoria, Illinois, identified several new corn-produced antibiotics called zealexins. Genetic testing demonstrated that the antibiotics and the genes that produce them play a significant role in disease resistance. Breeding corn to enhance production of these naturally-occurring antibiotics will provide greater disease protection and alleviate economic losses from microbial-based threats to corn production.

4. Biocontrol of wheat head scab and vomitoxin with a beneficial fungus. Fusarium head blight (FHB) is a devastating fungal disease of wheat and barley worldwide. Grain harvested from infected crops can be contaminated with vomitoxin and other harmful mycotoxins produced by the fungal pathogen. These toxins reduce the value of the grain and make it unsafe to eat. A potential method to control FHB is to use beneficial fungi that naturally live inside plant tissues and increase plant immunity or produce compounds that inhibit the growth of other fungi. ARS scientists in Peoria, Illinois, tested a fungus isolated from corn that is known to produce antifungal chemicals, Sarocladium zeae, for its ability to colonize wheat. Strains of S. zeae successfully colonized internal wheat organs and survived within the plant through its life cycle. Wheat plants with S. zeae were more resistant to FHB. Sarocladium is a promising candidate for environmentally friendly control of FHB.

Review Publications
Moreira, G.M., Machado, F.J., Pereira, C.B., Neves, D.L., Tessman, D.J., Ward, T.J., Del Ponte, E.M. 2019. First report of the Fusarium tricinctum species complex causing Fusarium head blight of wheat in Brazil. Plant Disease. 104(2):586. https://doi.org/10.1094/PDIS-03-19-0552-PDN.
Wipfler, R., McCormick, S.P., Proctor, R., Teresi, J., Hao, G., Ward, T., Alexander, N., Vaughan, M.M. 2019. Synergistic phytotoxic effects of culmorin and trichothecene mycotoxins. Toxins. 11(10):555. https://doi.org/10.3390/toxins11100555.
Hao, G., Bakker, M.G., Kim, H.-S. 2020. Enhanced resistance to Fusarium graminearum in transgenic Arabidopsis plants expressing a modified plant thionin. Phytopathology. 110(5):1056-1066. https://doi.org/10.1094/PHYTO-12-19-0447-R.
Brown, D.W., Villani, A., Susca, A., Moretti,A., Hao, G., Kim, H.-S., Proctor, R.H., McCormick, S.P., 2019. Gain and loss of a transcription factor that regulates late trichothecene biosynthetic pathway genes in Fusarium. Fungal Genetics and Biology. 136:103317. https://doi.org/10.1016/j.fgb.2019.103317.
Cowger, C., Ward, T.J., Nilsson, K., Arellano, C., McCormick, S.P., Busman, M. 2020. Regional and field-specific differences in Fusarium species and mycotoxins associated with blighted North Carolina wheat. International Journal of Food Microbiology. https://doi.org/10.1016/j.ijfoodmicro.2020.108594.
De Collli, L., Elliot, C., Finnan, J., Grant, J., Arendt, E.K., McCormick, S.P., Danaher, M. 2020. Determination of 42 mycotoxins in oats using a mechanically assisted QuEChERS sample preparation and UHPLC-MS/MS detection. The Journal of Chromatography B: Biomedical Applications. 1150:122187. https://doi.org/10.1016/j.jchromb.2020.122187.
Vaughan, M.M., Ward, T.J., McCormick, S.P., Orwig, N., Hay, W.T., Proctor, R., Palmquist, D. 2020. Intrapopulation antagonism can reduce the growth and aggressiveness of the wheat head blight pathogen Fusarium graminearum. Phytopathology. 110(4):916-926. https://doi.org/10.1094/PHYTO-09-19-0341-R.
Hay, W.T., McCormick, S.P., Hojilla-Evangelista, M.P., Bowman, M.J., Dunn, R.O., Teresi, J.M., Berhow, M.A., Vaughan, M.M. 2020. Changes in wheat nutritional content at elevated [CO2] alter Fusarium graminearum growth and mycotoxin production on grain. Journal of Agricultural and Food Chemistry. 68(23):6297-6307. https://doi.org/10.1021/acs.jafc.0c01308.
Kemp, N., Vaughan, M.M., McCormick, S.P., Brown, J.A., Bakker, M.G. 2020. Sarocladium zeae is a systemic endophyte of wheat and an effective biocontrol agent against Fusarium head blight. Biological Control. 149:104329. https://doi.org/10.1016/j.biocontrol.2020.104329.
Laraba, I., Kim, H.-S., Proctor, R.H., Busman, M., O'Donnell, K., Felker, F.C., Aime, M.C., Koch, R.A., Wurdack, K.J. 2019. Fusarium xyrophilum, sp. nov., a member of the Fusarium fujikuroi species complex recovered from pseudoflowers on yellow-eyed grass (Xyris spp.) from Guyana. Mycologia. 112(1):39-51. https://doi.org/10.1080/00275514.2019.1668991.
Proctor, R.H., McCormick, S.P., Gutierrez, S. 2020. Genetic bases for variation in structure and biological activity of trichothecene toxins produced by diverse fungi. Applied Microbiology and Biotechnology. 104:5185–5199. https://doi.org/10.1007/s00253-020-10612-0.