Location: Mycotoxin Prevention and Applied Microbiology Research
2019 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 prepared and screened libraries of genes from two trichothecene-producing fungi, to identify and isolate genes that confer resistance to the toxins. We identified four genes in the fungus Trichothecium roseum that provide a moderate level of resistance to trichothecin, the trichothecene mycotoxin produced by this fungus. In addition, a gene that is ubiquitous in fungi that produce trichothecenes was tested in yeast as a possible resistance gene.
Other research under Objective 1 is designed to determine whether natural communities of microorganisms (microbiome) that occur in/on wheat 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 excellent ability to spread within wheat stems and found that wheat with Sarocladium consistently reduced FHB and DON contamination.
We also looked for correlations between Fusarium growth or DON content and characteristics of the microbial communities inhabiting wheat grain. In collaboration with USDA-ARS scientists in St. Paul, Minnesota, seeds were collected from a mist irrigated nursery in St. Paul, Minnesota. We compared microbiome diversity, and the relative abundances of specific microbes, in seeds with high or low levels of DON, seeds for which the amount of Fusarium was a good predictor of DON content, and seeds with large deviations from the expected relationship between the amount of Fusarium and DON content. We found bacterial communities associated with wheat seeds that substantially impact the development of FHB and the accumulation of DON in grain.
Fusarium species can form close associations (symbioses) with groups of bacteria that 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. Additionally, 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 evaluated the ability of the fungus with and without its bacterial symbiont to 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. This year, virulence and toxin assays were completed on a susceptible and a moderately resistant spring wheat variety that had been acclimated to ambient or elevated carbon dioxide levels (Objective 2). These experiments demonstrated that elevated carbon dioxide is a significant contributing factor to the severity of FHB in wheat. We also found that the moderately resistant wheat variety grown under elevated carbon dioxide produced more carbohydrates and had significantly greater decreases in protein and mineral compositions than the susceptible wheat variety at 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.
We completed a two-year study comparing the natural microbial communities in/on wheat grown at ambient carbon dioxide and in free air carbon dioxide enrichment fields. We found several changes in the microbiome of bacteria and fungi on wheat grown at elevated carbon dioxide. Fusarium was present in significantly greater abundance in plots with elevated carbon dioxide.
Research under Objective 3 is designed to provide novel targets and strategies for disease and mycotoxin control, detection of introduced Fusarium species, modeling of pathogen and toxin contamination levels, and improved understanding of the potential effect of changes in climate or crop distributions on mycotoxin contamination. This year, we made significant progress in elucidating the genomic diversity of the Fusarium species that cause FHB. We identified differences in genome sequences, interactions with crops, mycotoxin production, and fitness that occur among or within the species of Fusarium.
We substantially improved understanding of diversity, distribution, and mycotoxin potential of pathogens causing FHB by completing analyses of genetic diversity and mycotoxin production profiles for more than 3,000 Fusarium isolates collected from infected wheat and barley in the United States, Brazil and Uruguay. In addition, we completed collection, assembly, and preliminary analyses of draft genomes for 250 isolates of the most significant FHB pathogen, F. graminearum. This diversity was placed into a broader context by collecting and analyzing genetic markers based on DNA sequence data for more than 2,500 F. graminearum isolates from across the globe and will be used to advance development of plant germplasm with broad resistance to diverse FHB pathogens.
Effective disease and mycotoxin control strategies need to account for differences in the F. graminearum isolates that cause FHB. Other 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 a hard red spring wheat variety that is rated as moderately resistant 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 began testing whether the three populations are differentially adapted to winter or spring wheat varieties.
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 twelve candidate effectors that were highly expressed during wheat infection and tested them in the model plant Nicotiana benthamiana to determine if/how they suppressed or induced immunity. Several of the highly induced effectors suppressed the production reactive oxygen species, which is an important type of plant defense. We also tested the efficacy of an antimicrobial peptide to shore up plant defenses against F. graminearum. We expressed the gene for this peptide in the model plant Arabidopsis.thaliana. Transformed plants had reduced Fusarium disease development but no overall changes in the diversity of their microbiome.
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. Our initial tests indicated that low concentrations of isothiocyanates from garden cress meal completely inhibited the growth of F. graminearum without adversely affecting seed germination of wheat or barley.
Accomplishments
1. Reliable markers for predicting wheat resistance to fusarium head blight. Fusarium head blight (FHB) is a devastating fungal disease of wheat which can reduce yield and contaminate grain with harmful mycotoxins. Because FHB outbreaks are strongly associated with weather, wheat varieties are evaluated for resistance to FHB under temperature and moisture conditions that favor disease spread. Although atmospheric carbon dioxide levels are rising, wheat varieties are not currently being screened under higher carbon dioxide levels. To determine if wheat resistance ratings are accurate under elevated carbon dioxide levels, ARS researchers in Peoria, Illinois, in collaboration with scientists at the National Research Council Canada, Ottawa, Canada, examined the amount of infection, mycotoxin contamination, and natural plant defense metabolites in susceptible and moderately resistance wheat grown under current or elevated amounts of carbon dioxide. Elevated carbon dioxide led to changes in plant defenses, disease severity, and mycotoxin contamination. Overall elevated carbon dioxide increased mycotoxin contamination. This research demonstrated that resistance ratings developed for wheat grown at current carbon dioxide levels may not apply under future conditions. Although the levels of several plant metabolites associated with disease resistance changed when the wheat cultivars were grown under elevated carbon dioxide, this research identified a set of metabolic markers that can be reliably used by breeders to select for FHB resistance even under increased atmospheric carbon dioxide.
2. Global and regional contributors to mycotoxin contamination of wheat and barley. Fusarium head blight (FHB) is a destructive disease of cereals crops worldwide and a major food safety concern because FHB pathogens can contaminate grain with vomitoxin and other fungal toxins (mycotoxins). FHB is caused by a diverse set of fungal species that make different mycotoxins. Understanding which FHB species and toxin types are present in an area is key to disease and mycotoxin control programs. In this study, ARS scientists in Peoria, Illinois, worked in collaboration with scientists in Uruguay and Brazil to identify and characterize FHB pathogens from their countries. The most common FHB pathogens of wheat and barley in Uruguay and Brazil, as well as the United States, are Fusarium graminearum with the ability to make a form of vomitoxin. However, a new species, Fusarium subtropicale, was found in Brazil that produces a related mycotoxin with greater toxicity for humans and animals. Analyses of genetic diversity revealed that wheat and barley share a common FHB pathogen population that moves back and forth between these two hosts. The FHB pathogens in this study exhibited different levels of aggressiveness toward barley and different levels of resistance to two commonly used fungicides. These results provide new information on FHB pathogen and mycotoxin prevalence, host distributions, aggressiveness, and fungicide sensitivity that can be used to develop globally applicable and regionally targeted disease and mycotoxin control programs that improve crop production and food safety.
3. Plant disease resistance is weakened by fungal enzyme. Fusarium graminearum is a fungal pathogen that causes Fusarium head blight (FHB) of wheat and other cereals and reduces crop yields and quality by producing mycotoxins. Salicylic acid is an important plant signaling molecule that regulates how the plant responds to fungal pathogens. To reduce the incidence of FHB and mycotoxin contamination of grain, we need to understand how Fusarium overcomes plant defenses to cause disease. ARS scientists in Peoria, Illinois, used genome sequence data to identify a F. graminearum protein that degrades salicylic acid and found that it weakens coordinated plant defenses against FHB. The protein is a new target for disease control and mycotoxin reduction programs that improve crop production and food safety.
4. Fungal protein helps disarm plant defenses to fungal disease. Fusarium head blight (FHB) caused by the fungus Fusarium graminearum is one of the most devastating diseases of wheat and other cereals. FHB results in contamination of grain with fungal toxins, known as mycotoxins, that can be a serious threat to food safety and animal health. A better understanding of how the fungus interacts with the plant and plant defenses is needed to reduce the incidence of FHB and mycotoxin contamination of grain. ARS scientists in Peoria, Illinois, discovered a Fusarium arabinanase, a protein that can degrade plant cell walls during infection in wheat. In addition, they demonstrated that arabinanase reduced the plant immune response to fungal invasion. This protein is a new target for improving resistance to FHB and mycotoxin contamination.
5. Genetic control of trichothecene toxins in a biocontrol fungus. Trichothecenes are a group of mycotoxins that when present in crops pose health risks to people, pets, and livestock. The fungus Fusarium graminearum causes head blight of cereal crops and produces the trichothecene vomitoxin, which is toxic to plants (phytotoxic) and contributes to the ability of the fungus to cause head blight. In contrast, the biocontrol fungus Trichoderma arundinaceum inhibits the growth of many plant disease-causing fungi and produces a trichothecene that is highly toxic to other fungi but harmless to plants. In collaboration with scientists at the University of León, Spain, ARS scientists in Peoria, Illinois, identified a group of genes in T. arundinaceum that control production of trichothecenes that are not phytotoxic. This study provided targets to aid development of methods to counteract the phytotoxic effects of trichothecenes and, thereby, the plant-disease-promoting effects of the toxins.
Review Publications
Whitaker, B.K., Bakker, M.G. 2018. Bacterial endophyte antagonism toward a fungal pathogen in vitro does not predict protection in live plant tissue. FEMS Microbiology Ecology. https://doi.org/10.1093/femsec/fiy237.
Garmendia, G., Pattarino, L., Negrin, C., Martinez-Silveira, A., Pereyra, S., Ward, T.J., Vero, S. 2018. Species composition, toxigenic potential and aggressiveness of Fusarium isolates causing Head Blight of barley in Uruguay. Food Microbiology. 76:426-433. https://doi.org/10.1016/j.fm.2018.07.005.
Cuperlovic-Culf, M., Vaughan, M., Vermillion, K., Surendra, A., Teresi, J., McCormick, S. 2018. Effects of atmospheric CO2 level on the metabolic response of resistant and susceptible wheat to Fusarium graminearum infection. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-06-18-0161-R.
Pereira, C.B., Ward, T.J., Tessmann, D.J., Del Ponte, E.M., Laraba, I., Vaughan, M.M., McCormick, S.P., Busman, M., Kelly, A., Proctor, R.H., O'Donnell, K. 2018. Fusarium subtropicale sp. nov., a novel nivalenol mycotoxin-producing species isolated from barley (Hordeum vulgare) in Brazil and sister to F. praegraminearum. Mycologia. 110(5):860-871. https://doi.org/10.1080/00275514.2018.1512296.
O'Donnell, K., McCormick, S.P., Busman, M., Proctor, R.H., Ward, T.J., Doehring, G., Geiser, D.M., Alberts, J.F., Rheeder, J.P. 2018. Marasas et al. 1984 "toxigenic Fusarium species: identity and mycotoxicology" revisited. Mycologia. 110(6):1058-1080. https://doi.org/10.1080/00275514.2018.1519773.
Lindo, L., McCormick, S.P., Cardoza, R.E., Kim, H.-S., Brown, D.W., Alexander, N.J., Proctor, R.H., Gutierrez, S. 2018. Role of Trichoderma arundinaceum tri10 in regulation of terpene biosynthetic genes and in control of metabolic flux. Applied and Environmental Microbiology. 122:31-46. https://doi.org/10.1016/j.fgb.2018.11.001.
Lindo, L., McCormick, S.P., Cardoza, R.E., Busman, M., Alexander, N.J., Proctor, R.H., Gutierrez, S. 2018. Requirement of two acyltransferases for 4-O-acylation during biosynthesis of Harzianum A, an antifungal trichothecene produced by Trichoderma arundinaceum. Journal of Agricultural and Food Chemistry. 67(2):723-734. http://dx.doi.org/10.1021/acs.jafc.8b05564.
Bakker, M.G., McCormick, S.P. 2019. Microbial correlates of Fusarium load and deoxynivalenol content in individual wheat kernels. Phytopathology. https://doi.org/10.1094/PHYTO-08-18-0310-R.
Jacobs-Venter, A., Laraba, I., Geiser, D.M., Busman, M., Vaughan, M.M., Proctor, R.H., McCormick, S.P., O'Donnell, K. 2018. Molecular systematics of two sister clades, the Fusarium concolor and F. babinda species complexes, and the discovery of a novel microcycle macroconidium–producing species from South Africa. Mycologia. 110:1189-1204. https://doi.org/10.1080/00275514.2018.1526619.
Hao, G., McCormick, S., Vaughan, M.M., Naumann, T.A, Kim, H.-S., Proctor, R., Kelly, A., Ward, T.J. 2019. Fusarium graminearum arabinanase (Arb93B) enhances wheat head blight susceptibility by suppressing plant immunity. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-06-18-0170-R.
Hao, G., Naumann, T.A., Vaughan, M.M., McCormick, S.P., Usgaard, T., Kelly, A., Ward, T.J. 2019. Characterization of a Fusarium graminearum salicylate hydroxylase. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2018.03219.
Hartman, G.L., McCormick, S.P., O'Donnell, K. 2019. Trichothecene production and pathogenicity of Fusaria isolated from symptomatic soybean roots in Ethiopia and Ghana. Plant Disease. https://doi.org/10.1094/PDIS-12-18-2286-RE.
Cardoza, R.E., McCormick, S.P., Lindo, L., Kim, H.-S., Olivera, E.R., Nelson, D.R., Proctor, R.H., Gutierrez, S. 2019. A cytochrome P450 monooxygenase gene required for biosynthesis of the trichothecene toxin harzianum A in Trichoderma. Applied Microbiology and Biotechnology. 103(19):8087-8103. https://doi.org/10.1007/s00253-019-10047-2.
Lippolis, V., Porricelli, A.C.R., Mancini, E., Ciasca, B., Lattanzio, V.M.T., De Girolamo, A., Maragos, C.M., McCormick, S., Li, P., Logrieco, A.F., Pascale, M. 2019. Fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins and their glucosides in wheat. Toxins. 11(7):380. https://doi.org/10.3390/toxins11070380.
