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

Location: Mycotoxin Prevention and Applied Microbiology Research

2022 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
Research conducted under Objective 1 focuses on identifying Fusarium graminearum virulence factors that can be targeted to reduce grain mycotoxin contamination. Plant pathogens secrete effector proteins to overcome plant defenses and cause disease. Effectors essential for pathogen infection are ideal targets to control disease. This year, we developed a computational analysis pipeline to identify effector proteins that make F. graminearum such a successful pathogen. We used effector prediction-programs to evaluate genomes of 199 representative Fusarium species. We evaluated the identified candidate effectors with comprehensive bioinformatics tools for the functional annotation (e.g., domain searches and potential homologs with other known effectors of known function) and used an artificial intelligence program to predict if the 3D structure of candidates identified resembled the structure of known effectors. Based on these analyses, we selected 21 candidate-effector genes and found that seven were highly expressed during initial wheat infection. Investigations are underway to generate mutants for these seven effectors and to test their virulence in plants. Our prior studies showed that the genetic background or population identity of F. graminearum (NA1, NA2, NA3) contributed to disease aggressiveness (how rapidly disease spreads on wheat heads). NA1 and NA2 strains were more aggressive in comparison to NA3 strains. NA1 and NA2 population strains primarily produce the toxin deoxynivalenol (DON), while NA3 population strains produce a chemical variant known as NX. This year, we compared initial infection of wheat heads caused by five strains from each of the three populations. Two of the three experimental replicates indicated that NA3 strains were more successful at causing initial infection on wheat, but one experiment indicated there were no population specific differences. Interestingly, during the first week of infection a higher mycotoxin level was detected in wheat heads inoculated with NA3 strains than those inoculated with NA1 or NA2 strains. Collectively, our results suggest that NA3 strains may have an advantage at causing initial disease infection of wheat. Objective 2 aims to identify germplasm that can be used by breeders to simultaneously target climate resilient disease resistance and high grain quality traits. Rising atmospheric carbon dioxide (CO2) has been shown to increase wheat susceptibility to Fusarium head blight (FHB). Thus, we are evaluating the influence of elevated CO2 on FHB disease resistance traits. This year, we evaluated the reliability of a genetic region, called quantitative trait loci (QTL) Fhb5, that is associated with wheat Type I resistance (or resistance to initial infection) of F. graminearum. We conducted three fully replicated experiments with four wheat cultivars with or without Fhb5 at ambient CO2 (450 ppm) or elevated CO2 (1000 ppm). Within seven days of inoculation, cultivars with Fhb5 showed significantly more disease at elevated CO2. DON contamination was greater at elevated CO2 in all the cultivars evaluated, especially those with Fhb5. These results suggest that disease resistance provided by Fhb5 is compromised at elevated CO2. Further research is underway to confirm these results and determine the underlying cause. Additionally, we evaluated the reliability of Fhb1, the predominant FHB resistance QTL utilized in wheat breeding. We evaluated 12 near isogenic wheat lines, with or without Fhb1. We found that Fhb1 did not impact wheat agronomic performance and provided a consistent reduction in disease spread, despite the increase in pathogen aggressiveness at elevated CO2. This information provides plant breeders with confidence in the continued utilization of Fhb1 as a marker for resistance, as its presence consistently provides resistance regardless of rising CO2. This year, we also evaluated wheat cultivars of varying genetic backgrounds and grain protein contents to identify traits associated with maintained grain protein content at elevated CO2. We demonstrated that the Fhb1 QTL was not associated with grain protein content loss in wheat grown at elevated CO2. However, the genetic background from which most moderately resistant cultivars are derived contributes to a more severe grain protein loss at elevated CO2. We analyzed F. graminearum metabolite production on wheat grain with high and low protein content. F. graminearum strains from NA1 and NA2 populations were inoculated on grain previously grown at ambient CO2 or elevated CO2, and thus varied in protein content. The amount of DON per unit fungal biomass increased for strains of both F. graminearum populations; however, the increase was on average 3-fold for NA1 strains and only 0.5-fold for NA2 strains. Furthermore, 15 F. graminearum strains were used to inoculate grain of two wheat cultivars of naturally high and low protein content and allowed to colonize the grain at ambient CO2 or elevated CO2. Analyses of mycotoxin production are currently underway. Objective 3 is targeted to manipulating microbial populations or metabolites to control trichothecene contamination of grain and malting barley. Use of beneficial microbes to reduce FHB is an effective sustainable and ecofriendly approach. We previously showed that under controlled growth chamber conditions wheat plants harboring the beneficial fungus Sarocladium zeae were less susceptible to FHB and mycotoxin contamination. This year we evaluated a seed soak introduction method of S. zeae on wheat. Since seed coats are commonly used and do not increase farmer input costs, this method will likely be most easily incorporated. Thus, wheat seed of two independent FHB susceptible wheat cultivars were soaked in a S. zeae solution prior to planting. The beneficial fungus colonized both wheat cultivars and effectively provided control of FHB spread and DON accumulation. We tested if the seed soak application method of S. zeae would reduce symptoms of Fusarium crown rot, an allied disease of F. graminearum impacting wheat stem tissue; however, the treatment did not provide any significant control of crown rot. Furthermore, we tested if S. zeae would control FHB and mycotoxin accumulation in barley, but no significant difference was detected in S. zeae seed treated barley plants in comparison to the untreated controls. However, S. zeae was not recovered from barley tissues suggesting that it may not efficiently colonize barley when inoculated using a seed coat method. Thus, alternative introduction methods are being explored for barley. Additionally, we evaluated the ability of nine diverse bacterial endophytes including Paenibacillus to control FHB by spraying the flowering wheat heads of a susceptible and moderately resistant wheat cultivar with a dilute bacterial suspension. Contrary to expectations, utilization of this application method of biocontrol bacteria resulted in enhanced wheat susceptibility to FHB for all the bacteria evaluated. Thus, the seed soak method was used in subsequent experiments. The seed soak method provided a vast improvement over the head spray method under greenhouse conditions. The results of the seed soak experiments showed that the bacterial biocontrol effect was dependent on the host cultivar. Overall, host genetic resistance was most effective at controlling disease spread. Integrated control strategies that attempt to couple wheat genetic resistance with biocontrol agents will require detailed validation experiments as many of the bacteria tested here were only capable of providing disease control in one of the wheat cultivars. Controlling the spread of F. graminearum spores has great potential for disease control. Cereal crops are infected by F. graminearum spores that are produced in small, darkly pigmented fruiting bodies known as perithecia. A build-up of pressure inside perithecia causes the spores to be forcibly ejected into the air where they are caught up in air currents that carry them to crops. To examine the role of perithecial pigmentation in spore production and ejection, we are generating mutants of F. graminearum with colorless perithecia by deleting a gene required for pigment production. For comparison purposes, we are also generating mutants of F. neocosmosporiellum that produces lightly pigmented perithecia that exude rather than forcibly eject spores. We also discovered that altering lighting conditions can result in colorless perithecia production. We developed a new methodology to evaluate differences in perithecia structural strength by comparing physical compression characteristics on a highly sensitive rheometer. Pigmented and colorless perithecia were individually harvested and slowly compressed. The colorless perithecia were far more pliable and appeared to lack the rigidity of the fully pigmented structures. Furthermore, we determined that the perithecia substantially accumulate numerous aromatic compounds during their maturation. This allowed us to identify how the crosslinking of fungal pigments may be involved in the strengthening of fungal perithecia prior to spore ejection. Other research under Objective 3 was designed to develop a plant derived biofumigant treatment to prevent toxin contamination in malting small grains. This year we tested the use of mustard seed meal derived volatiles to reduce Fusarium contamination and mycotoxin production during malting. Treatments during malting stages inhibited mycotoxin production without damaging wheat or barley germination. Furthermore, the volatiles were almost entirely driven off during the final heat treatment step of the malting process, dramatically reducing the likelihood of off flavors in the final product. This research provides maltsters with an organic biofumigation treatment method for wheat and barley grain that reduces Fusarium contamination and mycotoxin accumulation during the malting process.

Accomplishments
1. Discovered Fusarium head blight resistant wheat becomes less nutritious at elevated carbon dioxide. Higher atmospheric carbon dioxide can cause some cereal crops to produce more starch resulting in a lower grain protein and mineral content, making it less nutritious. In addition, these crops may become more susceptible to disease. Fusarium head blight is a fungal disease of wheat, barley, and other cereal crops that causes billions of dollars in annual yield losses and contamination with mycotoxins which makes the grain unsafe to eat. ARS researchers in Peoria, Illinois, in collaboration with wheat breeders from the University of Minnesota, compared wheat varieties that were susceptible or moderately resistant to the disease and measured the nutritional quality of the grain from plants grown with current or elevated concentrations of carbon dioxide. They discovered that wheat varieties with moderate resistance to Fusarium Head blight were more likely to produce grain with poorer nutritional quality when they were grown at elevated carbon dioxide. Furthermore, some F. graminearum strains responded to the reduced nutritional content in moderately resistant wheat by producing more mycotoxins. This study demonstrated the importance of identifying wheat cultivars that maintain nutritional quality and disease resistance with rising atmospheric carbon dioxide.

2. Demonstrated atmospheric carbon dioxide, temperature and Fusarium strain differentially influence mycotoxin contamination of corn and wheat. Fusarium fungi are devastating pathogens that infect cereal crops causing billions of dollars in annual yield losses and poison grains with mycotoxins making it unsafe to eat. Climate change is predicted to increase the frequency and severity of Fusarium disease and mycotoxin contamination of cereal grains. However, it has been unclear how rising atmospheric carbon dioxide and temperature will specifically impact Fusarium graminearum ear rot of corn and head blight of wheat. ARS researchers in Peoria, Illinois, showed that both economically important crops were more susceptible to mycotoxin contamination when grown at elevated carbon dioxide, but warmer temperatures reduced mycotoxin contamination. Additionally, the effects of carbon dioxide and temperature were dependent on the F. graminearum strain, and under the combined stress conditions a strain that produced the highest amount of mycotoxin in corn, produced the least in wheat. This study provides valuable information needed to determine the risk of Fusarium disease outbreaks and mycotoxin contamination in the future and will be of interest to farmers and regulatory agencies.

3. Revealed that genetic analyses reveal that gene organization can distinguish friend from foe fungi. Trichothecene mycotoxins are poisonous compounds produced by the fungus Fusarium and ten other kinds of fungi. These mycotoxins help Fusarium cause infection of crop plants such as wheat and potatoes. In Fusarium, and most of the other fungi that produce trichothecenes, the eight to fifteen genes that control their production are grouped together. However, gene number and organization vary in Trichoderma species, which can be beneficial fungi that protect crops or harmful fungal pathogens. ARS researchers in Peoria, Illinois, in collaboration with researchers in Leon, Spain, analyzed 35 Trichoderma species to see if they produced trichothecenes and to determine how their toxin genes were organized. They found that the tri5 gene, which controls the critical first step in trichothecene production, is widely distributed in Trichoderma, but other toxin genes and trichothecene production are less common. Trichoderma species that have only tri5 were able to inhibit trichothecene production by other fungi. These results indicate that Trichoderma species with only tri5 have the potential to control crop diseases caused by trichothecene producing species of Fusarium and reduce contamination of crops with the toxins

4. Determined controlling pathogen load on neighboring plants may be key to reducing mycotoxin contamination. The plant microbiome, including pathogens and beneficial microbes, can have direct impacts on plant health and productivity, but where precisely plants get their microbiomes remains unclear. Using next generation sequencing of fungal communities, ARS researchers in Peoria, Illinois, in collaboration with North Carolina State University and Indiana University researchers, discovered that soil fungi only marginally contributed to the fungal communities on plant leaves. Other surrounding plants were the primary source of fungi to the leaf microbiome, and the extent to which other plants contributed to leaf fungal microbiomes was dependent on precipitation with more rain leading to more plant-to-plant fungal spread. This knowledge can be applied to understanding how pathogens or biocontrol microorganism spread within natural and agricultural communities.

5. Updated Fusarium species identification database. Fusarium is a devastating group of diverse toxin-producing plant pathogens responsible for multibillion U.S. dollar losses each year to the world’s agricultural economy. Accurate species-level identification of these pathogens is crucial for disease diagnosis and management. However, the Fusarium genus is comprised of more than 400 genetically distinct species that cannot be accurately identified by morphology alone. To address the critical need for species identification, ARS researchers in Peoria, Illinois, in collaboration with researchers at Pennsylvania State University, improved identification of Fusarium pathogens by updating FUSARIUM-ID (https://github.com/fusariumid/fusariumid), a web-accessible DNA sequence database for the identification of Fusarium, by adding new DNA sequence data that help distinguish between species. This more comprehensive database will enable scientists worldwide to accurately identify Fusarium species using DNA sequence data. This database is being used by a wide range of scientists and quarantine officials charged with minimizing the threat these pathogens pose to global agricultural biosecurity and food safety.

6. Discovered tissue-specific wheat defense responses correlate with susceptibility to Fusarium infection. The fungus Fusarium graminearum causes Fusarium head blight (FHB), a devastating disease of wheat. FHB not only reduces crop yield but also contaminates grain with a fungal toxin called vomitoxin, that make the grain unsafe to eat. One way that plants react to fungal infection is by releasing reactive oxygen species (ROS). ARS researchers in Peoria, Illinois, evaluated ROS responses in different parts of the wheat plant that had been treated with chitin, a polysaccharide that is a major component of fungal cell walls, insect exoskeletons, and crustacean shells. While there was no ROS increase in wheat leaves treated with chitin, typical ROS responses were found in the central part of the wheat heads, the route by which FHB spreads. The discovery that the chitin induced ROS response is correlated with tissue susceptibility suggests that the ROS response may assist F. graminearum infection, and comparisons between tissue responses may aid in identifying methods of resistance. Further, this study identified defense genes that were turned on in wheat heads treated with chitin. These genes may serve as novel targets to improve disease resistance.

Review Publications
Zaret, M.M., Bauer, J.T., Clay, K., Whitaker, B.K. 2021. Conspecific leaf litter induces negative feedbacks in Asteraceae seedlings. Ecology. 102(12). Article e03557. https://doi.org/10.1002/ecy.3557.
Hao, G., Tiley, H., McCormick, S. 2022. Chitin triggers tissue-specific immunity in wheat associated with Fusarium head blight. Frontiers in Plant Science. 13. Article 832502. https://doi.org/10.3389/fpls.2022.832502.
Hay, W.T., McCormick, S.P., Vaughan, M.M. 2021. Effects of atmospheric CO2 and temperature on wheat and corn susceptibility to Fusarium graminearum and deoxynivalenol contamination. Plants. 10(12). Article 2582. https://doi.org/10.3390/plants10122582.
Whitaker, B.K., Giauque, H., Timmerman, C., Birk, N., Hawkes, C.V. 2021. Local plants, not soils, are the primary source of foliar fungal community assembly in a C4 grass. Microbial Ecology. 84:122–130. https://doi.org/10.1007/s00248-021-01836-2.
Selling, G.W., Hojilla-Evangelista, M.P., Hay, W.T., Utt, K.D., Grose, G.D. 2022. Preparation and properties of solution cast films from pilot scale cottonseed protein isolate. Industrial Crops and Products. 178. Article 114615. https://doi.org/10.1016/j.indcrop.2022.114615.
Hay, W.T., Anderson, J.A., McCormick, S.P., Hojilla-Evangelista, M.P., Selling, G.W., Utt, K.D., Bowman, M.J., Doll, K.M., Ascherl, K.L., Berhow, M.A., Vaughan, M.M. 2022. Fusarium head blight resistance exacerbates nutritional loss of wheat grain at elevated CO2. Scientific Reports. 12. Article 15. https://doi.org/10.1038/s41598-021-03890-9.
Guttierrez, S., McCormick, S.P., Cardoza, R.E., Kim, H.-S., Yugueros, L.L., Vaughan, M.M., Carro-Huerga, G., Busman, M., Saenz de Miera, L.E., Jaklitsch, W.M., Zhuang, W.-Y., Wang, C., Casquero, P.A., Proctor, R.H. 2022. Distribution, function, and evolution of a gene essential for trichothecene toxin biosynthesis in Trichoderma. Frontiers in Microbiology. 12. Article 791641. https://doi.org/10.3389/fmicb.2021.791641.
O'Donnell, K., Whitaker, B.K., Laraba, I., Proctor, R.H., Brown, D.W., Broders, K., Kim, H.-S., McCormick, S.P., Busman, M., Aoki, T., Torres-Cruz, T.J., Geiser, D.M. 2022. DNA sequence-based identification of Fusarium: A work in progress. Plant Disease. 106(6):1597-1609. https://doi.org/10.1094/PDIS-09-21-2035-SR.
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.
Naumann, T.A., Sollenberger, K.G., Hao, G. 2022. Production of selenomethionine labeled polyglycine hydrolases in Pichia pastoris. Protein Expression and Purification. 194. Article 106076. https://doi.org/10.1016/j.pep.2022.106076.
Kang, S., Lumactud, R., Li, N., Bell, T.H., Kim, H.-S., Park, S.-Y., Lee, Y.-H. 2021. Harnessing chemical ecology for environment-friendly crop protection. Phytopathology. 111(10):1697-1710. https://doi.org/10.1094/PHYTO-01-21-0035-RVW.
Kim, H.-S., Park, S.-Y., Kang, S., Czymmek, K.L. 2022. Time-lapse imaging of root pathogenesis and fungal proliferation without physically disrupting roots. In: Coleman, J., editor. Fusarium wilt. Methods in Molecular Biology, vol 2391. New York, NY: Humana. p. 153-170. https://doi.org/10.1007/978-1-0716-1795-3_13.
Nichea, M.J., Proctor, R., Probyn, C.E., Palacios, S.A., Cendoya, E., Sulyok, M., Chulze, S.N., Torres, A.M., Ramirez, M.L. 2021. Fusarium chaquense,sp. nov, a novel type A trichothecene-producing species from native grasses in a wetland ecosystem in Argentina. Mycologia. 114(1):46-62. https://doi.org/10.1080/00275514.2021.1987102.