Progress 07/01/24 to 06/30/25
Outputs
Target Audience:The target audience for our project includes other researchers, extension educators, and producers of wheat and maize. This project will generate valuable new protocols and resources (e.g. the Monsterplex genotyping protocol, strains, genome sequences, the Fast Flowring Mini Maize ear rot system, mapping populations), for the research community, as well as strengthening a highly beneficial international collaboration. This will move us closer to our goal of improving our ability to predict epidemics of GER and FHB by tracking specific markers associated with selective pathogen population shifts and incorporating this information with disease forecasting models so that growers can make better, more profitable decisions. Farmers in the U.S. are acutely aware of the potential damage in wheat from FHB and in maize from GER, but they may not always link the two diseases together, especially from a management perspective. As part of this project, we willdevelop outputs to increase awareness of the link between FHB and GER and translate our research results into resources that will have a broad impact in the agricultural community. Since this is only the first year of the project, we have not made any reports to our target audience as of yet. Changes/Problems:There were no major changes or problems encountered during the first year of the project. However, there was one minor modification to Objective 2: while we had originally planned to evaluate selection in Fast-Flowering Mini Maize (FFMM) under two temperature conditions, budget reductions from NIFA necessitated a revision. As a result, we will now conduct evaluations under a single temperature condition. This change has been reviewed and approved by NIFA. What opportunities for training and professional development has the project provided?During the first year of the project, we successfully onboarded a postdoctoral researcher and a graduate student at the University of Kentucky. In addition, two graduate students, one based in Kansas and another in Brazil, have contributed significantly to the project, along with one undergraduate researcher in Brazil. All trainees have received hands-on training in plant pathology, genetics, and genomics through their active involvement in project-related research. The graduate students and postdoctoral researcher have also participated in professional development activities offered through their respective institutions, including opportunities in teaching, outreach, and grantsmanship. As integral members of the research team, each trainee has been responsible for regular progress reporting and has undergone ongoing performance assessments to support their continued growth and development. How have the results been disseminated to communities of interest?In this first year of the project, we have not made any presentations or produced any refereed publications yet. The University of Kentucky Martin-Gatton College of Food, Agriculture, and Environmentpublicized the project through an article in their online news venue. This article can be accessed at the following URL:https://news.ca.uky.edu/article/university-kentucky-leads-international-effort-combat-debilitating-crop-fungus The article was picked up by the industry magazine"FarmWorld", whichpublished a follow up article that can be accessed at the following URL:https://www.farmworldonline.com/News/NewsArticle.asp?newsid=35212 What do you plan to do during the next reporting period to accomplish the goals?During the first year of the project, our primary focus was on assembling essential materials and optimizing experimental protocols and analytical tools. These foundational efforts were successful and have positioned us to move forward confidently in the second year. Upcoming activities will include continuation of our field experiments according to the planned crop rotation schedules, as well as processing samples collected during the first round of experiments. This will involve genomic and toxicity assessments to characterize pathogen traits and potential host associations. We also plan to generate additional sequencing data from Brazilian strains, which will be analyzed upon completion to further enhance our understanding of population structure and host adaptation across geographic regions. For Objective 3, we will conduct replicated inoculation experiments using the 70 recombinant progeny strains on Fast-Flowering Mini Maize to assess their aggressiveness and mycotoxin production. In parallel, all 70 progeny will be subjected to MonsterPlex genotyping, enabling us to evaluate potential associations between genetic markers and high aggressiveness on maize, wheat, or both hosts.
Impacts
What was accomplished under these goals?
To identify loci in the Fusarium graminearum species complex (FGSC) that may be experiencing host-specific selection, we are expanding genomic resources by sequencing a geographically and host-diverse collection of isolates, with particular emphasis on improving maize isolate representation. Previous comparative studies focused heavily on wheat-infecting isolates from the Upper Midwest and Canada. To address this imbalance, we assembled a collection of over 400 FGSC isolates from wheat, maize, and a small number from hemp. The collection includes 179 isolates from wheat, 99 from maize, and 10 from hemp, primarily from the southeastern and mid-Atlantic U.S.; 11 wheat and 24 maize isolates from Canada; and 32 wheat and 11 maize isolates from Brazil. We also included 37 isolates representing other FGSC species common in Brazil, including F. meridionale (24 wheat, 13 maize), F. asiaticum (2 wheat), F. austroamericanum (2 wheat), and F. cortaderiae (2 wheat). All isolates are preserved on silica and/or in glycerol for long-term storage. Whole-genome sequencing was completed for 24 isolates (all from Kentucky and Indiana), all of which belonged to the NA1, 15ADON chemotype. Five clonal isolates were removed following clone correction. This pilot study demonstrated that our sequencing pipeline performed reliably, validating its use for larger batches. An additional set of 96 isolates, mostly maize-derived and sampled from the Upper Midwest and Southeastern U.S., was recently submitted for sequencing, with data expected soon. To inform future comparisons between maize- and wheat-associated populations, we have reanalyzed existing phylogenomic datasets, which primarily include wheat isolates with a few from other cereals. Surprisingly, our analyses revealed substantial introgressions from related species, particularly F. boothii and F. gerlachii, into the F. graminearum genome. These introgressed regions are discrete, recombinogenic, and limited in scope, whereas the rest of the genome appears largely clonal. These findings suggest that previous reports of high recombination in F. graminearum populations likely reflect ancestral rather than ongoing gene flow. Among 225 strains analyzed, evidence of recent recombination was found in only 18, with most of those occurring outside the U.S. and a notable proportion derived from barley. These patterns of historical introgression and limited contemporary recombination are essential to consider when conducting genome-wide association studies to identify loci linked to aggressiveness on maize versus wheat. To examine how different cropping regimes shape FGSC population dynamics and selection pressures, we initiated field experiments in both Kentucky and Brazil. The Kentucky field study began in summer 2025 at the University of Kentucky Research and Education Center (UKREC) in Princeton. Treatment 1 (continuous maize) and Treatment 3 (a maize-wheat-soybean rotation) were inoculated with a representative mixture of 11 genetically diverse F. graminearum strains (nine wheat, one maize, and one hemp isolate). Inoculations were performed by injecting 2 × 104 spores/mL into the silk channel of maize ears. Ears were harvested in late August, and GER incidence was assessed. In Treatment 1, incidence among 185 sampled ears was 90%, with 58% showing more than 50% kernel infestation. In Treatment 3, incidence among 148 sampled ears was 88%, with 48% exhibiting more than 50% infection. No GER symptoms were observed on uninoculated ears, confirming successful establishment of the pathogen from experimental inoculations. In Brazil, the field study can takeadvantage of the climate that allows continuous cropping. Treatments 1 (continuous maize) and 3 (maize-wheat rotation) were sown in March 2025. Inoculations used a mixture of 10 genetically diverse strains representing both F. graminearum and F. meridionale, isolated from maize and wheat. Maize inoculation was conducted in June via silk injection of 1 × 105 macroconidia/mL, and ears were harvested in late August. GER incidence was evaluated: a low incidence of Fusarium Ear Rot, and no other ear rot pathogens, were also observed. Treatment 2 (continuous wheat) was sown in May 2025 and inoculated by spraying a 1 × 104 spores/mL suspension at early anthesis in August. This lower concentration was used to balance disease pressure and grain yield. FHB incidence was assessed on 50 heads per plot at the soft dough stage on September 15. Wheat harvest is scheduled for early October 2025. To complement field studies, we established a controlled-environment infection system using Fast-Flowering Mini Maize (FFMM). Plants were grown from seed and hand-pollinated. Inoculation occurred five days after pollination using various concentrations of spore suspension. We found that 1 × 104 spores/mL was optimal for inducing infection while still permitting kernel development, as higher concentrations led to premature ear death. To support host-association studies and future field trials, we developed a genotyping platform targeting genetically informative loci. We analyzed global variation across 225 F. graminearum genomes and selected 253 loci meeting two criteria: each contained at least two SNPs with minor allele frequencies >0.1, and the flanking sequences were invariant across strains. These loci are spaced approximately every 150 kb across the four chromosomes. In a pilot study using DNA from 96 strains, 82 yielded complete data, and all 253 loci were successfully amplified and genotyped, a 100% recovery rate, which exceeds the typical ~90%. We attribute this high success to our phylogenetically informed primer design pipeline. A second, larger run with 292 strains confirmed the robustness of the platform and provided strong genetic resolution for identifying recombinants and clones. To streamline analysis of this genotyping data, we developed an automated MonsterPlex analysis pipeline as a ShinyApp. This application allows users to upload raw MonsterPlex data directly, and the app performs sequence alignment, generates read metrics, calls SNPs, creates FASTA alignments, builds phylogenetic trees, and exports tree images within the dashboard. The pipeline is fully functional and currently undergoing user interface improvements in preparation for web deployment. To dissect the genetic basis of host-specific aggressiveness, we performed a controlled cross between a heterothallic tester strain derived from the maize isolate PH-1 and a homothallic wheat isolate from North Dakota. We recovered 70 recombinant progeny, all confirmed by marker analysis. These have been preserved for long-term use. The progeny were evaluated for aggressiveness and toxin production on susceptible spring wheat ('Wheaton') in three replicated trials. No transgressive segregants more aggressive than the parents were observed; however, several progeny were significantly less aggressive or less toxic. The phenotypic extremes were validated in further replicated experiments. Whole-genome sequencing was performed on selected high- and low-aggressiveness strains, and preliminary analysis revealed a region on chromosome 2 associated with increased aggressiveness to wheat.
Publications
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