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

Location: Mycotoxin Prevention and Applied Microbiology Research

2023 Annual Report

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.

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 focuses on identifying Fusarium graminearum virulence factors that can be used as targets to reduce grain contamination with trichothecene mycotoxins. Plant pathogens secrete effectors to suppress plant defenses and promote disease. Effectors critical for pathogen infection are ideal targets to control disease. This year, we developed a computational analysis pipeline to identify effectors by using a combination of genome sequence analyses and artificial intelligence programs. With this pipeline, we identified 2,916 putative effectors from 23 Fusarium species complexes. Of these 50 candidates were selected for evaluation and 11 were highly expressed during F. graminearum wheat infection. Deletion mutants were generated for five candidates, and the effect of these mutants on Fusarium head blight (FHB) initial infection was evaluated. Results showed that four of the effectors play a role in F. graminearum initial infection. To support community research and share resulting data, we developed a Fusarium protein structure database which compares three-dimensional structures of target proteins among species. Previous results showed that F. graminearum North American populations (NA1, NA2, NA3) differed in disease spread in wheat. NA1 and NA2 strains typically spread faster than NA3 strains. Previously, we showed that NA3 strains were more successful at causing initial infection on wheat and produced more trichothecenes per fungal biomass than NA1 and NA2 strains. This year we compared initial infection of F graminearum populations on barley. NA1 strains had a higher infection rate than NA2 and NA3 strains, but more toxin per fungal biomass was detected in barley inoculated with NA3 strains than NA1 and NA2 strains. Our results suggest that NA3 strains can cause greater trichothecene contamination on wheat and barley which poses a food safety concern. Fusarium population genomics analyses identified population specific differences in a circadian clock gene, so we also tested how light duration influenced F. graminearum growth and mycotoxin production. Transcriptomic analyses are in progress to identify genes associated with toxin biosynthesis that are regulated by light. We previously showed that FHB resistant wheat cultivars become more susceptible at elevated carbon dioxide (CO2). As part of Objective 2, we tested if a genetic region called Fhb5 that increases wheat resistance to initial infection of F. graminearum, was less effective at elevated CO2. While trichothecene contamination at elevated CO2 was significantly greater in all the cultivars evaluated, the difference was greatest in cultivars with Fhb5. Since Fhb5 is thought to contribute to wheat anther extrusion as a mechanism of enhanced resistance, we investigated the effect of elevated CO2 on anther extrusion. Metabolomic analyses of the different parts of the wheat flower from different cultivars under variable conditions are currently underway. To confirm that Fhb5 directly contributes to compromised FHB resistance and grain quality at elevated CO2, we obtained near isogenic lines (plants with nearly identical genetic makeup except for a specific genetic locus) with and without Fhb5. Additionally, we tested if elevated CO2 compromised the ability of the genetic region Fhb1 that is associated with resistance to disease spread of F. graminearum. Fhb1 is the most widely used FHB resistance locus in breeding programs. We evaluated the disease resistance of 12 near isogenic lines with and without Fhb1. We found that Fhb1 was not associated with increased disease severity at elevated CO2 and maintained a consistent disease resistance efficacy. This information provides plant breeders with confidence in the continued utilization of Fhb1 as a marker for disease resistance. To determine how elevated CO2 effects F. graminearum trichothecene production in different host species or cultivars, we inoculated wheat and barley grain of high and low protein content with F. graminearum strains from each North American population and incubated them at ambient or elevated CO2. Results revealed that grain protein content and F. graminearum population contributed to differences in trichothecene accumulation. CO2 did not have a direct effect. Lower protein content was associated with higher levels of trichothecene accumulation in wheat and barley. NA3 strains accumulated higher amounts of trichothecenes in comparison to other populations in both crops. As part of this work, we identified for the first time a glycosylated form of a relatively new trichothecene type called NX. Fungi, yeast and plants can have glucosyltransferases that detoxify trichothecene mycotoxins by attaching a sugar moiety to the toxin. Trichothecene glucosides have been considered masked mycotoxins because they may not be detected with standard analytical methods. Since attempts by other researchers to enzymatically remove the glucoside from trichothecenes have been unsuccessful, we used Blastobotrys yeast to produce trichothecene glucosides standards to be used with analytical methods to accurately measure the total amount of trichothecenes being produced. Objective 3 aims to manipulate microbial populations or metabolites to control trichothecene contamination. This year, we continued efforts to identify agronomically feasible strategies to introduce Sarocladium zeae into wheat, barley and corn. We tested a low dosage seed soak and foliar spray of S. zeae, into wheat and barley. Neither introduction method provided FHB control comparable to seed soaking with a high dosage of S. zeae. Thus, experiments to identify the minimum effective dosage are currently underway. Additionally, a material transfer research agreement was established with a seed coating company to explore the use of commercial seed coat methods with S. zeae. To understand the mechanisms by which S. zeae provides control against FHB, we performed genome sequencing, comparative genomic and metabolomic analyses. Interestingly, pyrrocidine, an S. zeae metabolite previously shown to inhibit F. verticillioides fumonisin production, did not inhibit F. graminearum production or the predominant trichothecene, deoxynivalenol (DON). Lastly, the ability of S. zeae to control F. graminearum trichothecene production on corn was evaluated by soaking yellow dent corn in different concentrations of S. zeae prior to inoculation with F. graminearum. Two different dosages of S. zeae eliminated production of DON. Field experiments evaluating the efficacy of S. zeae to control Fusarium in corn are underway. To understand how microbiome associations can improve barley breeding efforts to enhance FHB resistance, we evaluated the microbiome of 10 barley varieties across four U.S. states. Results showed that certain genetic backgrounds associate with more beneficial taxa than others. Thus, selection of barley genotypes that develop associations with beneficial taxa may provide breeders with new targets for resistance. FHB can be caused by a complex of diverse Fusarium species, each of which can produce different mycotoxins, and compete for residence on host plants. Thus, it is important to know which species are causing disease and how they interact to target control methods accordingly. We collected symptomatic wheat heads from 19 Illinois farms, then we isolated and sequenced 1200 Fusarium strains. Most isolates belonged to the Fusarium sambucinum species complex. Identification of the mycotoxin chemical type for each isolate is in progress. We also evaluated the effect of co-inoculating wheat with F. graminearum and F. poae or F. avenaceum, which can be found to co-exist in agricultural fields. Our results showed that when F. poae colonizes wheat and barley first, it reduces deoxynivalenol (DON) accumulation. This year we also started characterizing the genomic diversity and disease phenotypes of 91 FHB causing isolates used by six different barley screening programs. Genomes have been sequenced for 30 strains and disease assays have been conducted for 10 strains in two barley varieties. F. graminearum spores are produced in pigmented fruiting bodies known as perithecia. Spores are forcibly expelled from perithecia and initiate FHB epidemics by infecting wheat and barley flowers. To identify ways to block the spread of spores, we are investigating whether perithecial pigmentation affects spore formation/expulsion. Using a mutagenesis approach, we determined that pigmented and nonpigmented perithecia produced similar numbers of spores and expelled them similar distances. However, after exposure to ultraviolet light, nonpigmented perithecia expelled fewer spores and expelled them shorter distances than pigmented perithecia. These results indicate that perithecial pigmentation has a protective role during development of F. graminearum spores and that blocking pigmentation could reduce the ability to cause infection. Other Objective 3 aims were to develop a plant derived biofumigant treatment to prevent fungal toxin contamination in malting small grains. Mycotoxin accumulation during malting causes millions of dollars in annual losses for the U.S. malting and brewing industry. This year, we evaluated the use of mustard seed meal derived volatiles to reduce Fusarium contamination and mycotoxin production during the malting of commercial barley samples. The biofumigation treatments were able to inhibit Fusarium growth and prevent additional mycotoxin contamination of the grain. The seed meal treatments did not affect barley germination. This research provides maltsters with an organic biofumigation treatment method that prevents Fusarium growth and reduces mycotoxin accumulation during the malting process. The volatile hydrocarbon precursor of trichothecenes tichodiene can also be used as a biofumigant to reduce trichothecene production. So, we developed a pilot-scale production method for of trichodiene by fungal fermentation.

1. Safely fighting fungi with fungi depends on the chemical weapons the friendly fungus brings to battle. Fungal pathogens claim approximately 20% of global crop yield estimated at $200 billion each year and can further reduce grain value by contaminating crops with mycotoxins. The beneficial fungus Trichoderma can inhibit growth of fungal pathogens and mycotoxin contamination and can be deployed to reduce these agricultural losses. ARS researchers in Peoria, Illinois, in collaboration with researchers at the University of Leon, Leon, Spain, determined that the chemicals farnesol, aspinolide, and harzianum A all help Trichoderma successfully control fungal pathogens. This research will help identify Trichoderma strains that are most effective and safe to fight crop diseases.

2. Discovered a previously unknown toxin gene transfer mechanism among Fusarium fungi. Trichothecene mycotoxins can cause billion-dollar losses yearly to the U.S. wheat and barley industry. Trichothecenes were thought to only be produced by two groups of species of the fungus Fusarium. ARS researchers in Peoria, Illinois, discovered trichothecene production in a new distantly related Fusarium species. Additionally, they found that this new species acquired the ability to produce trichothecenes through direct transfer of genes instead of through the normal process of inheritance. This study demonstrated that trichothecene toxin production occurs more widely among Fusarium species than was previously known. Insight into the genetic processes by which fungi can gain the ability to produce toxins alerted public health agencies, such as the Food and Drug Administration and the Food Safety and Inspection Service, of the potential risk, and provides plant pathogen researchers with knowledge needed to ensure that fungi capable of acquiring the ability to produce toxins do not evade detection and pose a threat to human and animal health.

3. Created treatment capable of chemically changing a fungal toxin. Grain contaminated with fungal toxins called mycotoxins can cause billions of dollars in annual yield losses. The mycotoxin deoxynivalenol (DON) is produced by Fusarium graminearum, a fungus that causes Fusarium head blight of wheat and barley and ear rot of corn. DON is a food safety concern because if consumed it is harmful to human and livestock health. The chemical structure of DON lends to its toxicity. ARS researchers in Peoria, Illinois, discovered that combining a fungal enzyme with a chemical mediator called TEMPO can chemically alter the structure of DON making it less toxic. This work serves as a potential treatment to chemically modify DON and reduce its toxicity.

4. A newly discovered form of Fusarium toxin has a unique function in initial infection. Fusarium graminearum is among the most destructive fungal pathogens of wheat and barley because it poisons grain with harmful toxins called trichothecenes which reduce cereal crop yield and contaminate grain. Most F. graminearum strains in the U.S. produce the trichothecene mycotoxin called deoxynivalenol (DON) which promotes disease spread within the infected plant. A recently identified emerging F. graminearum population produces a trichothecene called NX with a slightly different chemical structure than DON. ARS researchers in Peoria, Illinois, discovered that NX not only plays a similar role as DON but also has a unique function in enhancing pathogen initial infection. This study provides the knowledge needed to develop a F. graminearum population specific control strategy that has the potential to eliminate infection and not just reduce disease spread after infection.

5. Modified wheat to avert Fusarium attack. Fusarium head blight (FHB) is one of the most devastating diseases of wheat and other cereals resulting in billions of dollars in economic losses. The disease is predominantly caused by the fungus Fusarium graminearum and reduces crop yield and contaminates grain with mycotoxins that are a serious threat to food safety and animal health. ARS researchers in Peoria, Illinois, and Manhattan, Kansas, in collaboration with researchers at Kansas State University and the University of Nebraska, discovered a Fusarium protein (FgN1s1) suppresses plant immunity allowing the fungus to cause disease. Transgenic wheat plants capable of silencing FgN1s1 were created and shown to be two times more resistant to FHB. This study improves our understanding of crop-fungal pathogen interactions and provides a targeted approach to enhance wheat resistance against FHB.

6. Manipulating the wheat microbiome by the application of beneficial bacteria can indirectly control mycotoxin contamination by enhancing plant health and resilience to pathogen stress. Fusarium head blight (FHB) is a devastating fungal disease of wheat causing contamination of grain with mycotoxins that are harmful if consumed resulting in billions of dollars in yield losses. While breeding programs have developed some wheat varieties that are moderately resistant to FHB, farmers currently control the disease using expensive fungicide applications, which can lead to the proliferation of resistant fungal strains. As an alternative to fungicide, ARS researchers in Peoria, Illinois, discovered that some bacteria indirectly mediated resistance to FHB. Specifically, the bacteria did not directly compete with or kill the Fusarium fungal pathogen. However, some bacterial strains boosted overall plant health and resilience to the Fusarium pathogen. The most effective strain at providing control was dependent on the wheat variety. The greatest reduction of mycotoxin contamination which was 73% was achieved by a Paenibacillus strain in one variety but in another variety the most effective strain was a Serratia isolate which reduced mycotoxin production by 43%. This research identified new and more environmentally safe approaches to control FHB and ensure food safety.

7. Confirmed that the Fhb1 genomic marker for wheat resistance against Fusarium head blight will maintain efficacy at elevated atmospheric carbon dioxide (CO2) concentrations. The fungus Fusarium graminearum causes Fusarium head blight (FHB), a disease of cereal crops that contaminates grain with hazardous mycotoxins making it unfit for consumption and results in significant yield losses. Rising atmospheric CO2 is predicted to increase FHB outbreaks and mycotoxin contamination. Concerningly, wheat varieties resistant to FHB suffer greater grain protein loss at elevated CO2. ARS researchers in Peoria, Illinois, in collaboration with wheat breeders from the University of Minnesota, showed that Fhb1 did not negatively affect wheat growth, development, yield, or grain protein content at elevated CO2. This report provided wheat breeders with confidence in the use of Fhb1 as a genomic marker to breed for FHB resistance and climate resilience. This work is helping wheat breeders develop wheat lines that maintain resistance to FHB and nutritional value regardless of rising atmospheric CO2.

8. Modeled mycotoxin contamination to guide management of Illinois corn. Mycotoxin contamination and associated fungal diseases of U.S. corn reduces grain quality and safety and causes significant economic losses. ARS researchers in New Orleans, Louisiana, and Peoria, Illinois, in collaboration with the Illinois Department of Agriculture, used historical mycotoxin data from the state of Illinois to develop a model that can predict mycotoxin contamination with 94% accuracy. Validation was done by using mycotoxin data from 2021 which was not included in the model. Analyses for aflatoxin and fumonisin mycotoxins showed that in addition to climate conditions during crop growth, climate conditions and the amount of plant growth during the month of March prior to corn planting can influence grain contamination at the end of the growing season. This research provides extension agencies, corn farmers, and stakeholders with new methods and a much larger window of opportunity to protect corn from mycotoxin contamination before and during the growing season.

Review Publications
Cardoza, R.E., Mayo-Prieto, S., Martinez-Reyes, N., McCormick, S.P., Carro-Huerga, G., Campelo, M.P., Rodriguez-Gonzalez, A., Lorenzana, A., Proctor, R.H., Casquero, P.A., Gutierriez, S. 2022. Effects of trichothecene production by Trichoderma arundinaceum isolates from bean-field soils on the defense response, growth and development of bean plants (Phaseolus vulgaris). Frontiers in Plant Science. 13. Article 1005906.
Proctor, R.H., Hao, G., Kim, H.-S., Whitaker, B.K., Laraba, I., Vaughan, M.M., McCormick, S.P. 2022. A novel trichothecene toxin phenotype associated with horizontal gene transfer and a change in gene function in Fusarium. Toxins. 15(1). Article 12.
Cardoza, R.E., McCormick, S.P., Lindo, L., Mayo-Prieto, S., Gonzalez-Caron, D., Martinez-Reyes, N., Carro-Huerga, G., Rodríguez-González, Á., Proctor, R.H., Casquero, P.A., Guttierrez, S. 2022. Effect of farnesol on Trichoderma physiology and fungal-plant interaction. The Journal of Fungi. 8(12). Article 1266.
Shanakhat, H., McCormick, S.P., Busman, M., Rich, J.O., Bakker, M.G. 2022. Modification of deoxynivalenol by a fungal laccase paired with redox mediator TEMPO. Toxins. 14(8). Article 548.
Cardoza, R.E., McCormick, S.P., Izquierdo-Bueno, I., Martínez-Reyes, N., Lindo, L., Brown, D.W., Collado, I.G., Proctor, R.H., Gutierrez, S. 2022. Identification of polyketide synthase genes required for aspinolides biosynthesis in Trichoderma arundinaceum. Applied Microbiology and Biotechnology. 106:7153–7171.
Whitaker, B.K., Vaughan, M.M., McCormick, S.P. 2023. Biocontrol impacts on wheat physiology and Fusarium head blight outcomes are bacterial endophyte strain and cultivar specific. Phytobiomes Journal. 7(1):55-64.
Chen, H., Su, Z., Tian, B., Hao, G., Trick, H., Bai, G. 2022. TaHRC suppresses the calcium-mediated immune response and triggers wheat Fusarium head blight susceptibility. Plant Physiology. 190(3):1566-1569.
Castano-Duque, L., Vaughan, M., Lindsay, J., Barnett, K., Rajasekaran, K. 2022. Gradient boosting and bayesian network machine learning models predict aflatoxin and fumonisin contamination of maize in Illinois – First USA case study. Frontiers in Microbiology. 13. Article 1039947.
Flor-Weiler, L., Behle, R.W., Berhow, M.A., McCormick, S.P., Vaughn, S.F., Muturi, E.J., Hay, W.T. 2023. Bioactivity of brassica seed meals and its compounds as ecofriendly larvicides against mosquitoes. Scientific Reports. 13. Article 3936.
Wilson, V.C., McCormick, S.P., Kerr, B.J. 2023. Feeding thermally processed spray-dried egg whites, singly or in combination with 15-acetyldeoxynivalenol or peroxidized soybean oil on growth performance, digestibility, intestinal morphology, and oxidative status in nursery pigs. Journal of Animal Science. 101. Article skac429.
Hao, G., McCormick, S., Tiley, H., Gutierrez, S., Yulfo-Soto, G., Vaughan, M.M., Ward, T.J. 2023. NX trichothecenes are required for Fusarium graminearum infection of wheat. Molecular Plant-Microbe Interactions. 36(5):294-304.
Hay, W.T., Anderson, J.A., Garvin, D.F., McCormick, S.P., Vaughan, M.M. 2022. Fhb1 disease resistance QTL does not exacerbate wheat grain protein loss at elevated CO2. Frontiers in Plant Science. 13. Article 1034406.