Research Project: Improving Food Safety by Controlling Mycotoxin Contamination and Enhancing Climate Resilience of Wheat and Barley

Location: Mycotoxin Prevention and Applied Microbiology Research

2024 Annual Report

Objectives
Objective 1: Identify Fusarium graminearum (Fg) virulence factors and/or fitness traits that can be targeted to reduce grain mycotoxin contamination. [C1, PS2] Sub-objective 1.A: Identify and characterize core effectors of Fg that can be targeted to reduce initial infection of wheat and barley. Sub-objective 1.B: Identify and characterize Fg population-specific factors that contribute to differences in virulence and mycotoxin contamination of wheat and barley. Objective 2: Identify germplasm that can be used by breeders to simultaneously target climate resilient mycotoxin resistance and high grain quality traits. [C1, PS2] Sub-objective 2.A: Evaluate the resilience of FHB resistance to e[CO2] in MR and S wheat cultivars. Sub-objective 2.B: Determine the impact of e[CO2] on the production of Fg mycotoxins and other secondary metabolites during growth on wheat and barley grains. Sub-objective 2.C: Determine the impact of e[CO2] on the nutritional quality of FHB MR parent wheat lines and identify potential breeding strategies to maintain grain quality. Objective 3: Manipulate microbial populations or metabolites to control trichothecene contamination of grain and malting barley. [C1, PS2, PS5] Sub-objective 3.A: Evaluate the efficacy of Sarocladium and Paenibacillus as biocontrol agents to control FHB and mycotoxin contamination of grains. Sub-objective 3.B: Determine how perithecial pigmentation affects production, discharge, and germination of Fusarium ascospores so that these metabolites can be manipulated to reduce Fg inoculum in the field. Sub-objective 3.C: Develop a biofumigant from plant derived metabolites to inhibit fungal growth and mycotoxin production during barley malting.

Approach
Mycotoxins are poisonous fungal metabolites that contaminate cereals, making them unsafe for human or livestock consumption. Contamination originates in the field during grain development when crops become infected by mycotoxigenic fungal pathogens that can result in diseases with significant economic losses. Fusarium head blight (FHB), is a devastating disease of wheat and barley that is caused primarily by the fungal pathogen Fusarium graminearum (Fg) which produces mycotoxins, including trichothecenes and zearalenone. FHB is a complex ecological problem that has been difficult to eradicate because infection is dependent on multiple interacting factors. The severity of FHB is contingent on the prevalence, virulence, and aggressiveness of the pathogen, the genetic potential of the host plant’s resistance, as well as other abiotic and biotic environmental factors that influence the outcome of the plant-pathogen interactions. Previous efforts by scientists in the FHB community have laid a foundation of information on pathogen virulence and host resistance, but we need to further understand how environmental factors shape the outcome and impact of interactions. Using a holistic approach that tackles the problem from multiple angles, we propose to target factors that can be manipulated to impose mycotoxin control: 1) Fg pathogenic fitness, 2) resilience of crop resistance, and 3) beneficial microorganisms and microbial or plant metabolites. Knowledge obtained from this approach will aid in the development of integrated climate-resilient control strategies for FHB and mycotoxin contamination of grains, thereby reducing the impact of mycotoxins on our food supply. These studies will ultimately benefit growers; small grain breeders; stakeholders in the food and feed industry; other research scientists; regulatory agencies (United States Food and Drug Administration, USDA Federal Grain Inspection Service, and Animal Plant Health Inspection Service); and most importantly the consumer.

Progress Report
Objective 1: The devastating fungal pathogen Fusarium graminearum, uses proteins and toxins to promote disease in crops. The identification of genes involved in the production of these molecular factors can be targeted by strategies, such as gene silencing, to reduce disease and toxin contamination. This year, we continued to search for such molecular factors including effector genes. Eleven effector candidate genes that were highly expressed during F. graminearum infection of wheat were evaluated. Deletion mutants were generated for five candidates, and the effect of these mutants on F. graminearum initial infection was tested. In total, we found five effectors involved in F. graminearum infection of wheat. We were also able to restore the function of an effector by introducing the wildtype gene back into the mutant, thus confirming its role in disease development. In collaboration with scientists in Ponferrada, Spain, we identified a gene required for construction of the macrocyclic trichothecenes produced by a fungal pathogen of vegetable crops. F. graminearum North American populations (NA1, NA2, NA3) differ in disease spread in wheat. Previously, we showed that NA3 strains were more successful at causing initial infection on wheat, and they produced and accumulated more trichothecenes than NA1 and NA2 strains. In contrast, NA1 strains were more infectious in barley than NA2 and NA3 strains, but NA3 produced more toxin per fungal biomass than NA1 strains. However, NX toxins were not essential for barley infection. Our data suggest that NA3 strains can cause more trichothecene contamination. To support community research and share resulting data, we developed Fusarium Protein Toolkit, a web-based utility (https://fusarium.maizegdb.org/) which contains AlphaFold 3D structures of effectors and other proteins found in F. graminearum. At this website, users can explore 3D protein structures for similarities to help identify their potential function. Users can also compare structural differences in proteins across diverse Fusarium species to predict the potential impact of genetic diversity on the biology of these fungi. The toolkit leverages protein language models to predict how genetic variations might affect protein function. Objective 2: This year, we confirmed that the Fhb1 genetic region used by wheat breeders worldwide to select for Fusarium head blight (FHB) resistance was not associated with the observed disproportionate decline in grain protein and mineral nutrient content of resistant wheat lines at elevated carbon dioxide (CO2). Furthermore, we confirmed that the Fhb1 genetic region was a reliable marker for resistance regardless of rising CO2. Results with Fhb1 near isogenic lines (NIL) suggested that other genetic factors in the background of resistant lines were responsible for the observed decline in grain nutritional quality and FHB resistance. Therefore, we investigated the effects of elevated CO2 on NIL possessing Fhb1 and/or Fhb5 in a European wheat background. However, wheat from the European background displayed increased FHB susceptibility and mycotoxin contamination at elevated CO2, as observed in U.S. wheat varieties. At current CO2 conditions, wheat lines possessing both Fhb1 and Fhb5 had the least disease and mycotoxin contamination, but at elevated CO2 this resistance was lost, and the plants exhibited disease and toxin levels equivalent to susceptible control wheat varieties. Also, we tested if the genetic region GPC-B1, which is associated with increased protein and mineral nutrient content in wheat grain, could be used to ameliorate nutritional losses at elevated CO2. Experiments investigating how elevated CO2 influenced F. graminearum trichothecene production revealed that grain protein content and F. graminearum strain were significant contributing factors. We analyzed mycotoxin production of F. graminearum strains from each North American population on grain from nine different wheat varieties that had been grown at ambient and elevated CO2. The protein content of the grain, trichothecene contamination levels and fungal biomass were estimated and the relationship between these variables was determined. The impact of CO2 induced changes in grain protein content on trichothecene production was strain dependent and usually more toxin was produced on grain with lower protein content. This would suggest that grain with less protein may be more susceptible to mycotoxin contamination. We inoculated grain of high and low protein content with Aspergillus flavus strains that produce aflatoxin and showed that protein content was only a contributing factor in barley. Objective 3: A field experiment was performed to determine the ability of Sarocladium zeae to protect field corn from Fusarium verticillioides infection. Prior to pollination, ear silk channels of corn were inoculated with S. zeae. Then, the ears were challenged with F. verticillioides. Pyrrocidine B production was used to evaluate the success of S. zeae colonization, while visual fungal damage and fumonisin contamination of kernels was used to evaluate the biocontrol capacity of S. zeae. Results suggest that S. zeae can inhibit fumonisin contamination in corn. To investigate the molecular mechanisms by which S. zeae can control Fusarium, 25 Sarocladium strains from the ARS Culture Collection and recently isolated strains from Illinois fields were screened for their ability to block F. graminearum trichothecene production. Metabolomic analyses revealed that S. zeae strains produce both antifungal pyranones and volatile sesquiterpenes that may enhance their biocontrol activity. Exposure of F. graminearum to S. zeae volatiles inhibited trichothecene production. Studies to determine optimal application methods of the biocontrol fungus and/or its metabolites to control trichothecene contamination are underway. S. strictum was isolated from asymptomatic, field-grown barley. In vitro studies demonstrated that this S. strictum isolate could reduce F. graminearum production of the trichothecene, deoxynivalenol (DON). To evaluate the efficacy of this isolate to control mycotoxin contamination in planta, a gnotobiotic system was developed using sterile growth boxes, soil, and fertilizer for a rapidly-maturing barley variety. Fifty-two wheat and barley farms across North Dakota and Illinois were sampled for mycotoxin contamination in 2022-2024. Analysis of 2022 samples revealed unexpectedly high nivalenol (an emerging trichothecene toxin of concern) contamination across 19 winter wheat farms in Illinois. Samples of winter wheat and spring barley collected during 2023 and 2024 are being analyzed to determine 1) whether nivalenol contamination is of rising concern in the midwestern United States and 2) whether nivalenol contamination is present during a high scab risk year. Analysis of microbiome datasets from North Dakota and Illinois revealed important insights on the ability of the microbiome to modulate trichothecene contamination in barley and winter wheat, respectively. The fungal spike microbiome was more responsive to disease pressure from F. graminearum than the bacterial spike microbiome – thus, manipulations of the fungal microbiome may hold greater promise for intervention strategies than the bacterial microbiome. An unsupervised machine learning approach revealed specific microbial colonizers associated with reductions in mycotoxin content in wheat. Cryptococcus, Hanaella, and Epicoccum colonizers to wheat heads were associated with reduced mycotoxin concentrations, while Sporidiobolus and Kabatiella colonizers to corn debris early in the growing season were associated with reductions in disease – potentially indicating an early intervention strategy for the control of disease and mycotoxins by targeting the microbiome of the previous crop’s debris. We also investigated pigmentation of sexual fruiting bodies (perithecia) of F. graminearum to identify targets that block initiation of FHB epidemics. Spores produced within perithecia are forcibly expelled, carried by air currents and rain to cereal crops, where they initiate FHB. Previously, we generated mutant strains of F. graminearum that produced nonpigmented perithecia. This year, we used the sexual cycle of the fungus to remove nontarget mutations from the mutant strains. Subsequent experiments with the resulting strains confirmed that pigmentation protects the sexual spores from radiation. We also found that growing mixtures of multiple F. graminearum strains together suppresses perithecia and spore production compared to individual strains. Post-harvest food losses due to fungal and insect pests pose a significant threat to food security. Insect damage often coincides with fungal diseases that reduce yields, lower end use quality, and endanger food safety from mycotoxins. The fungal pathogen F. graminearum can produce mycotoxins in infected plant tissues, making them unsafe for use as food or feed due to significant health hazards. Infected grains can continue to accumulate mycotoxins during storage, especially in poor conditions which are often exacerbated by insect pests. We developed biofumigant treatments from plant derived sources to control major pests of cereal crops during storage. Plants from the mustard family produce highly volatile isothiocyanates when damaged by pests. These plant defense compounds were utilized to fumigate cereals in simulated storage experiments. The biofumigation treatments inhibited F. graminearum growth and prevented mycotoxin contamination of stored cereal grains. Furthermore, the treatments were highly effective against insect pests which is known to be a vector of mycotoxigenic fungi. This research provides producers with biofumigation technology that can reduce fungal and insect pest damage during grain storage.

Accomplishments
1. Risk assessment and control strategies for an emerging mycotoxin in cereal crops. Fusarium graminearum causes significant food safety concerns because it infects cereal crops and contaminates grain with harmful mycotoxins. Most F. graminearum strains in the United States produce deoxynivalenol (DON), a mycotoxin regulated by the Food and Drug Administration, but an emerging population of F. graminearum strains produces the mycotoxin NX. Since little was known about the aggressiveness of these F. graminearum strains or how NX functions during plant disease, ARS researchers in Peoria, Illinois, compared disease development of NX-producing strains with strains in other North American populations. While NX producing strains typically spread more slowly in wheat, they produced more mycotoxins during infection. Both NX and DON promote disease spread, but NX also has a unique function in enhancing initial infection. This work informs stakeholders of the potential risk of the emerging NX mycotoxin, but also identifies population specific control strategies.

2. Climate resilient markers for Fusarium head blight resistance. Wheat is the primary food grain produced in the United States and is a key staple in many other countries. Wheat production and food safety are challenged by a devastating fungal disease called Fusarium head blight (FHB) which reduces yield and contaminates grain with mycotoxins. Concerningly, some wheat varieties are more susceptible to disease at higher atmospheric carbon dioxide. Because carbon dioxide levels are increasing, ARS researchers in Peoria, Illinois, investigated how traits and genetic backgrounds that have provided some resistance to FHB under current atmospheric conditions will perform under higher levels of carbon dioxide. Some wheat varieties with more FHB resistance had poorer nutritional quality when grown at elevated atmospheric carbon dioxide. These effects were not associated with the primary genetic marker (Fhb1) globally used by wheat breeders in selecting for FHB resistance. This research demonstrated the importance of breeding for climate resilient disease resistance. Furthermore, it provided wheat breeders with confidence in the use of Fhb1 as a resilient marker of FHB resistance and high nutritional value regardless of rising carbon dioxide.

3. Using a fungal self-defense gene to make crops that are resistant to toxins. The fungal pathogen Fusarium graminearum causes a cereal disease called Fusarium head blight (FHB), which contaminates grain with deoxynivalenol (DON) and related mycotoxins that pose a threat to human and livestock health. Despite breeding efforts, control of FHB and mycotoxin contamination remains a challenge due to inconsistent host resistance and the emergence of fungicide resistant strains. F. graminearum that produces DON has a gene that gives it built-in resistance to the toxin. Therefore, ARS researchers in Peoria, Illinois, and Manhattan, Kansas, in collaboration with researchers at Kansas State University, tested a strategy to enhance crop resistance to DON by making transgenic wheat with the Fusarium self-defense gene. The transgenic wheat plants were more resistant to FHB and had 50% less DON. These transgenic plants provide an effective way to reduce agricultural losses and food safety concerns associated with DON contamination in wheat and other cereal grains.

4. Stimulating plant immunological memory reduces toxic fungal contaminants. Fungal toxins frequently contaminate cereal grains resulting in critical international food safety and food trade issues. Enhanced crop resistance to the fungi that produce the toxins would be the most economical and ecofriendly control strategy, but completely resistant wheat and barley varieties have not been identified. Therefore, ARS researchers in Peoria, Illinois, tested individual and combinations of different fungal cell wall components as a way to boost plant immunity to infection and reduce the amount of toxin in infected grains. Results showed that plants treated with two fungal cell wall components, laminarin and chitin, stimulated the greatest defense response and reduced the amount of toxin contaminating the grain. This study identified a way to boost plant resistance to toxin producing fungi that will improve food safety.

5. Changes in the wheat microbiome at elevated atmospheric carbon dioxide influences grain risk to Fusarium mycotoxins. Fusarium fungi can cause Fusarium head blight (FHB) of wheat and other cereal crops. FHB results in billions of dollars in annual yield losses and results in grain contamination with a toxin that makes it unsafe to eat. Communities of microbes that live on crops play a significant role in overall plant health, productivity, and resistance to pathogens. However, it is unclear how rising atmospheric carbon dioxide will affect the wheat microbiome – including its pathogens such as Fusarium. ARS researchers in Peoria, Illinois, and Urbana, Illinois, in collaboration with a scientist at the University of Manitoba, Canada, discovered that elevated carbon dioxide changes the makeup of the bacterial and fungal microbiome of wheat. Interestingly, the abundance of Fusarium was also altered by atmospheric carbon dioxide, but the effect was strain-specific with certain Fusarium strains showing increased abundance while others displayed reduced abundance at elevated carbon dioxide. These results suggest that certain Fusarium strains will gain an advantage, become more abundant and pose greater risk as atmospheric carbon dioxide increases.

6. Designing mycotoxin detoxification enzymes. Trichothecenes are a group of fungal toxins that frequently occur in cereal crops and cause approximately $1.5 billion in annual losses to agricultural economies of the United States and Canada. Enzymes can be used to detoxify trichothecenes and mitigate losses. However, one barrier to this approach is the inability of enzymes to detoxify all trichothecenes because of differences in their chemical structures. To overcome this barrier, ARS researchers in Peoria, Illinois, in collaboration with scientists at the Ponferrada Campus of the University of Leon, Spain, identified components of the trichothecene structures that limit the ability of enzymes to modify the toxins. These results provide information needed to design general detoxification enzymes that can target diverse trichothecenes and thereby reduce economic losses caused by the toxins.

7. Regional dominance of crops in landscape reduces microbiome diversity on local farms. Communities of microbes living on crops can greatly contribute to overall plant health, and productivity, but it remains unclear how intensive agricultural practices have shaped the crop microbiome. Researchers at North Carolina State University, in collaboration with ARS researchers in Peoria, Illinois, discovered that the dominance of crops, at the cost of natural vegetation, across the landscape can lead to reduced fungal biodiversity in leaves of corn, soy, switchgrass, and wheat crops. Additionally, researchers identified major differences in the composition of the microbiome according to crop type, largely due to the differences among crops in their growth yield traits and growing season. These results indicate that intensive agricultural practices may have broad consequences for the role of microbiomes in crop health. Manipulating the communities will increase crop quality, safety and productivity.

8. Insights into the evolution of Fusarium graminearum and the secret to its success. The fungus Fusarium graminearum causes head blight of wheat and barley and contaminates grain with toxins that pose serious food safety concerns and economic losses. Development of control strategies has been hindered by the diversity of F. graminearum strains and their multifaceted methods of successfully causing disease. To understand if changes in agricultural practices and environmental conditions influence the evolution of F. graminearum, ARS researchers in Peoria, Illinois, and collaborators at Kansas State University compared the DNA sequences of F. graminearum strains in North America and discovered marked genomic differences among the strains. These differences include gain and loss of many genes, including genes that help the fungus infect plants, provide resistance to pesticides, and produce toxins. Knowledge of these genomic changes in fungi that adapt to environmental changes will aid in the development of resilient control strategies.

Review Publications
Hao, G., Rhoades, N.A., McCormick, S. 2023. Chitin and laminarin additively trigger wheat reactive oxygen species but not resistance to Fusarium head blight. Plant Direct. 7(10). Article e538. https://doi.org/10.1002/pld3.538.
Bakker, M.G., Whitaker, B.K., McCormick, S.P., Ainsworth, E.A., Vaughan, M.M. 2023. Manipulating atmospheric CO2 concentration induces shifts in wheat leaf and spike microbiomes and in Fusarium pathogen communities. Frontiers in Microbiology. 14. Article 1271219. https://doi.org/10.3389/fmicb.2023.1271219.
Hay, W.T., Anderson, J.A., Garvin, D.F., McCormick, S.P., Busman, M., Vaughan, M.M. 2023. Elevated CO2 can worsen Fusarium Head Blight disease severity in wheat, but the Fhb1 QTL provides reliable disease resistance. Plants. 12(20). https://doi.org/10.3390/plants12203527.
Yulfo-Soto, G., McCormick, S., Hui, C., Bai, G., Trick, H.N., Hao, G. 2024. Reduction of Fusarium head blight and trichothecene contamination in transgenic wheat expressing Fusarium graminearum trichothecene 3-O-acetyltransferase. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2024.1389605.
Whitaker, B.K., Heiniger, R.W., Hawkes, C.V. 2023. Foliar fungal communities in agroecosystems depend on crop identity and neighboring vegetation. Frontiers in Microbiomes. 2. Article 1216462. https://doi.org/10.3389/frmbi.2023.1216462.
Hao, G., Proctor, R.H., Brown, D.W., Rhoades, N.A., Naumann, T.A., Kim, H.-S., Gutierrez, S., McCormick, S.P. 2024. TRI14 is critical for Fusarium graminearum infection and spread in wheat. Applied Microbiology. https://doi.org/10.3390/applmicrobiol4020058.
Whitaker, B.K. 2024. Diversity in the phyllosphere – greater than the sum of its parts?. New Phytologist. https://doi.org/10.1111/nph.19907.
Bethke, G., Huang, Y., Hensel, G., Heinen, S., Liu, C., Wyant, S.R., Li, X., Quin, M.B., McCormick, S.P., Morrell, P.L., Dong, Y., Kumlehn, J., Salvi, S., Berthiller, F., Muehlbauer, G.J. 2023. UDP-glucosyltransferase HvUGT13248 confers type II resistance to Fusarium graminearum in barley. Plant Physiology. https://doi.org/10.1093/plphys/kiad467.
Cardoza, R.E., McCormick, S.P., Martinez-Reyes, N., Rodríguez-Fernández, J., Busman, M., Proctor, R.H., Gutiérrez, S. 2024. Analysis of substrate specificity of cytochrome P450 monooxygenases involved in trichothecene toxin biosynthesis. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-023-12950-1.
Dhakal, U., Kim, H.-S., Toomajian, C. 2024. The landscape and predicted roles of structural variants in Fusarium graminearum genomes. Genes, Genomes, and Genomics. https://doi.org/10.1093/g3journal/jkae065.
Andorf, C.M., Haley, O., Hayford, R.K., Portwood II, J.L., Harding, S.F., Sen, S., Cannon, E.K., Gardiner, J.M., Kim, H., Woodhouse, M.R. 2024. PanEffect: a pan-genome visualization tool for variant effects in maize. Bioinformatics. 40(2). Article btae073. https://doi.org/10.1093/bioinformatics/btae073.
Laraba, I., Ward, T.J., Cuperlovic-Culf, M., Azimi, H., Xi, P., McCormick, S.P., Hay, W.T., Hao, G., Vaughan, M.M. 2023. Insights into the aggressiveness of the emerging North American population 3 (NA3) of Fusarium graminearum. Plant Disease. https://doi.org/10.1094/PDIS-11-22-2698-RE.
Jeong, E., Lim, J.Y., Proctor, R.H., Lee, Y.-W., Xu, J., Shi, J., Liu, X., Seo, J.-A. 2023. Genome sequence resource of the head blight pathogens Fusarium asiaticum and F. graminearum isolated from cereal crops and gramineous weeds in Korea and China. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-10-22-0120-A.
Hao, G., Naumann, T.A., Chen, H., Bai, G., McCormick, S., Kim, H.-S., Tian, B., Trick, H.N., Naldrett, M.J., Proctor, R. 2023. Fusarium graminearum effector FgNls1 targets plant nuclei to induce wheat head blight. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-12-22-0254-R.