Location: Cereal Disease Lab
2025 Annual Report
Objectives
Objective 1: Investigate the biology of FHB infection, mycotoxin accumulation, and pathogenesis in the barley-Fusarium and related pathosystems. (NP303, C2, PS2A). This will include screening wheat lines for infection by Fusarium graminearum and accumulation of mycotoxins as well as detailed analysis of pathogen infection and process of toxin accumulation.
• Sub-objective 1.A. Determine the protein content, spatial architecture, and functional significance of the toxin biosynthetic apparatus in Fusarium graminearum.
• Sub-objective 1.B. Test for differentiation of the endoplasmic reticulum for specific primary and secondary terpenoid metabolite pathways upon trichothecene induction.
Objective 2: Characterize pathogen diversity by studying natural fungal populations.
• Sub-objective 2.A. Test for population subdivision among strains of F. graminearum isolated from native grasses versus those collected as pathogens on wheat or barley.
Objective 3: Develop novel strategies for disease resistance in durum wheat and barley.
• Sub-objective 3.A. Characterize the gene expression pattern changes in FHB resistant durum cultivars produced by removal of CG methylation.
• Sub-objective 3.B. Characterize the genetic transmission of mutated loci and develop molecular markers for use in cultivar improvement.
Approach
Understanding how pathogens produce toxins and cause disease on different hosts can lead to improved management strategies for disease control. Specific approaches include: 1) Protein tagging, advanced microscopy, and protein-protein interaction techniques will be used to characterize multi-enzyme complexes involved in toxin biosynthesis and fungal pathogenesis; 2) FHB levels, strain diversity, and the nature of associated fungal communities, will be monitored by population genetic and metagenomic approaches improving the ability to forecast the economic impact and the design of effective management strategies; and 3) Novel sources of FHB resistance and mycotoxin tolerance will be developed and characterized for crop plants.
Progress Report
Understanding how pathogens produce toxins and cause disease can improve management strategies for controlling disease outbreaks and protect the food supply. In support of Objective 1, research continued investigating the biology of Fusarium head blight (FHB) infection, mycotoxin accumulation, and pathogenesis on barley-FHB and related pathosystems. For Sub-objective 1.A. investigating protein-protein interactions that regulate mycotoxin biosynthesis, conditions for proximity-dependent biotinylation in Fusarium graminearum were optimized, including the amount of exogenous biotin added to fungal cultures, incubation time, protein extraction conditions, desalting methods to remove excess biotin, and biotinylated protein enrichment using streptavidin. Initial TRI4 protein samples were processed and analyzed via mass spectrometry but only a few proteins were identified. Work is ongoing to troubleshoot the protein enrichment step and increase the amount of sample for mass spectrometry analysis. In parallel, F. graminearum was transformed with constructs to create biotin ligase fusions at the native gene locus for additional TRI proteins. Fungal transformants are being validated via multiple genetic and biochemical approaches. The transformed strains will be added to our proximity-dependent biotinylation pipeline once those experiments are completed. These experiments will generate new insights into how the Fusarium mycotoxin biosynthetic pathway is regulated and could lead to new strategies to inhibit toxin production, thus increasing food safety. For Sub-objective 1.B., the goal is to reconstruct predictive network models of host and pathogen signaling during infection and use the networks to identify candidate genes that control the interaction between barley and Fusarium graminearum. We collected global proteomics and phospho-proteomics data from a time course corresponding to different stages of infection including the transition from biotrophic to necrotrophic pathogen growth. Proteins and phospho-peptides that respond dynamically to pathogen infection were identified via statistical analyses. The proteomics data were integrated with RNA-seq data to reconstruct gene regulatory networks (focused on transcription factors that control the expression of target genes). Kinase signaling networks (focused on activated kinases that modulate the activity of target proteins) were inferred using phospho-proteomics data. Transcription factors and kinases have large influences on cellular responses to stress and typically drive the host response to pathogens. Using these network models, we predicted the genes that have the largest impact on disease resistance and susceptibility . These genes are being prioritized for functional analyses to test their contribution to FHB disease outcomes. Identifying new genetic targets for breeders to use for the development of cereal crops with enhanced disease resistance will improve crop yields and food safety.
In support of Objective 2, research continued characterizing FHB pathogen diversity by studying natural pathogen populations. The goal of this work is to improve our ability to forecast damage caused by different FHB strains to develop effective management strategies. Our successful screening of FHB pathogens against different hosts and host tissues resulted in the development and validation of a high-throughput coleoptile assay that is predictive of head-blight symptoms. This new method has tremendously increased our ability to screen isolates (reducing growth periods and space required by up to 80%), enabling population-level insights into host range. These efforts have been bolstered by our concurrent genomic analysis of 513 Fusarium genomes from diverse hosts. This genomic analysis has revealed that wild grasses in Minnesota are more likely to harbor only a subset of Fusarium populations. This result may suggest host-specific adaptation, but must be differentiated from geographic patterns where some strains are present only in certain areas, an ongoing effort that requires sampling in additional states. We are also incorporating samples from emerging FHB threats observed in Africa. While state-level differentiation of Fusarium populations has been observed before, these patterns tend to shift between years, making it difficult to decide what isolates to screen with. To help inform wheat and barley screening efforts, we have been collaborating with the USDA-ARS Mycotoxin Prevention and Applied Microbiology Research Uniton a U.S. Wheat and Barley Scab Initiative (USWBSI) -funded project to determine the underlying genetics of common Fusarium used in nursery screenings. Our results reveal that while these isolates are widespread across the diversity of the fungus, some geographically widespread clades are not represented, leaving U.S. wheat vulnerable to existing threats. Together, our efforts have offered new tools to screen Fusarium head blight pathogens that could be implemented by other scientists or private agricultural companies and offer new insights into the host specificity of different pathogenic lineages that may be useful to scientists and private industry in developing novel disease control strategies that are targeted to specific lineages.
In support of Objective 3, research continued developing novel strategies for disease resistance in durum Wheat, which is generally highly susceptible to Fusarium head blight. For Sub-objective 3.A., RNA-seq analysis was performed on mutant and parental durum wheat lines at 12- and 48- hours post-inoculation with F. graminearum. Comparison of the mutants with the respective parental lines provided significant information on the gene expression changes underlying the enhanced resistance. Interestingly, the three mutant lines showed distinct mechanisms of resistance based on their transcript profiles. This observation suggests that the epigenetic modifications conferring disease resistance were unique in each line. Upon analysis, we found several regulatory genes such as WRKY, PHD, C3H, CAMTA, bHLH, C2H2, NAC, and MYB were differentially expressed and might be contributing to resistance. For Sub-objective 3.B., five FHB-resistant mutant lines were crossed with two advanced durum varieties lines ('ND Grano', and 'ND Stanley'). These F1 lines were then backcrossed again to the advanced durum varieties to generate BC1F1 lines. We selected 10 backcross-derived seeds per population to advance to the next generation. One plant (based on agronomic characteristics and seed yield) per cross was selected and ~200 of its progeny to the next generation by selfing. We have advanced over 200 lines per population (over 2,000 total lines) to the BC1F3 generation. These lines will be further advanced to BC1F4 and seed increased. With adequate seed, phenotypic characterization of FHB disease response will start in the field during the summer of 2026. These new lines can be used in breeding programs to enhance Fusarium disease resistance in durum wheat.
Accomplishments
1. Ensuring the safety of U.S. grain and food supplies with better diagnostic tools. Some fungal pathogens that infect crop plants produce mycotoxins that are dangerous for humans and livestock to eat. Vomitoxin is a mycotoxin produced by Fusarium head blight (FHB) pathogens that is a major problem on grain crops like wheat and barley. When a farmer delivers their harvest to a grain elevator, it is tested for vomitoxin. If the levels are high enough, the shipment could be discounted (the farmer must sell it at a lower price) or even outright rejected, which can have significant impacts on farmer profits and rural communities. Different strains of FHB fungi can produce variant types of vomitoxin and are present in different parts of the country, but their distributions are changing. So, tracking fungal populations and the mycotoxins they produce is critical to understanding mycotoxin risks in grain crops and ensuring a safe food supply. USDA-ARS researchers in Saint Paul, Minnesota and Peoria, Illinois collaborated with researchers at the University of Minnesota to develop a new tool that can determine the type of vomitoxin a fungal strain produces based on short regions of its DNA sequence. This is the first assay that can diagnose all four of the vomitoxin variants in a single tube, making it a high-throughput alternative to existing assays that scientists use to characterize fungal strains. The tool is being applied to pathogen surveillance and field surveys to track pathogen populations in different wheat and barley growing regions. Together, these efforts will inform mycotoxin risk assessments in grain crops to ensure U.S. food safety and the continued profitability of U.S. grain farmers and processors.
2. Discovery of a new Fusarium head blight (FHB) causing pathogen in the Fusarium graminearum species complex (FGSC). FHB is one of the most devastating diseases of wheat, barley and other cereals, causing devastating losses in the U.S.; the disease also causes mycotoxin contamination, together threatening the nation’s food safety and food security. Species in the FGSC are the most widespread and the most pathogenic species to cause FHB, making them some of the most important plant pathogens in the world. In recent years FHB has emerged as a new problem in Eastern Africa. USDA-ARS researchers at the Cereal Disease Laboratory in Saint Paul, Minnesota studied the origin of this new emergence and discovered a new FGSC species that they have now formally described as Fusarium kistleri. The researchers performed genome sequencing, creating a high-quality reference to define the genetics of this new threat to U.S. and global agriculture. The discovery of this new pathogen enables efforts to screen for F. kistleri to make sure it does not emerge in the U.S. and is being used to screen existing U.S. cereal germplasm to cement disease- control strategies to protect U.S. food security and food safety.
3. Development of high-throughput methodology enables germplasm development and disease screening. Fusarium head blight (FHB), caused by a complex of Fusarium species, is one of the most devastating cereal diseases, leading to significant economic losses and contamination of grain with harmful mycotoxins that threaten global cereal production and human health. Fusarium infections are highly variable depending on what species are involved or what weather conditions are present; this variation has presented a significant limitation in the face of large-scale virulence testing that is currently labor-intensive and time-consuming. This year, researchers at the USDA-ARS Cereal Disease Laboratory in Saint Paul, MN identified a new high-throughput assay that offers robust insights into pathogen virulence and host resistance in a fraction of the time. Classical head-infection assays require plants to be grown for several months and require expensive growth chamber or greenhouse space. Assays pioneered by the ARS scientists can be performed on seedlings, reducing space and time commitments by up to 80%. While the assays dramatically shortened the time and cost of assays, the researchers confirmed that they are still predictive of head blight disease. The researchers demonstrated the high-throughput assays offer robust insights into pathogen virulence and host resistance, differentiating FHB-causing species and demonstrating the power of known host-resistances against common threats. These assays promise to shorten screening times needed to identify new and promising cereal germplasm, identify the threat of emerging diseases, and will allow for larger experiments to determine the interaction of variables that may contribute to FHB progression, offering a new line of defense for America’s crops and enabling new approaches through optimized research.
Review Publications
Singh, L., Drott, M.T., Elmore, J.M. 2025. Identification and differentiation of the Fusarium graminearum NX-2 chemotype using High-Resolution Melting (HRM). Plant Disease. 109(2):435-444. https://doi.org/10.1094/PDIS-09-23-1972-RE.
Singh, L., Drott, M.T., Kim, H., Proctor, R., Mccormick, S.P., Elmore, J.M. 2024. A Multiplex High-Resolution Melting (HRM) assay to differentiate Fusarium graminearum chemotypes. Scientific Reports. https://doi.org/10.1038/s41598-024-81131-5.
Rafiei, V., DeGenring, L., Schwister, E.M., Elmore, J.M., Dubey, M., Karlsson, M., Drott, M.T. 2025. Comparative analysis of distinct phenotyping methods for assessing wheat resistance and pathogen virulence among Fusarium species causing head blight disease. Plant Methods. 21. Article 85. https://doi.org/10.1186/s13007-025-01402-8.