Location: Cereal Disease Lab
2022 Annual Report
Objectives
Objective 1: Discover specific factors involved in pathogenicity, sporulation and toxin synthesis for the FHB pathogen and related fungi by applying genomic and functional approaches.
Sub-objective 1.A. Functionally characterize cellular processes and structures that determine plant pathogenesis.
Sub-objective 1.B. Identify genes uniquely or differentially expressed during development that defines pathogen structure and function.
Objective 2: Relate fungal genotypes to mycotoxin production in fungal strains in field production environments to aid in developing enhanced methods of control.
Sub-objective 2.A. Monitor genetic changes in critical pathogen populations by pathogen surveys.
Sub-objective 2.B. Characterize populations of Fusarium from native North American grasses that may be sources of novel pathogen genotypes and/or host resistance.
Objective 3: Optimize metagenomic and functional approaches to define the phytobiome of healthy and diseased plants naturally infested with the FHB fungus.
Sub-objective 3.A. Characterize phytobiome and soil carbon composition.
Sub-objective 3.B. Determine the relative abundance of competitive phenotypes and impacts on plant productivity.
Objective 4: Identify novel sources of plant disease resistance to FHB and mycotoxins produced by FHB fungi to improve breeding for resistance.
Sub-objective 4.A. Characterize the epigenetic changes of FHB resistant durum cultivars produced by altering the DNA methylation pattern.
Sub-objective 4.B. Characterize durum lines missing a portion of chromosome 2A region that may contain the FHB suppressor locus.
Objective 5: Introgress new genes of scab resistance into barley and wheat germplasm.
Approach
Improved management strategies are needed to maintain adequate plant disease control. Specific approaches include: 1) Genetic information obtained from the fungal pathogen, Fusarium, will be used to identify genes factors responsible for fungal pathogenesis, possibly leading to novel approaches to control FHB disease and reduce toxin levels in grain; 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; 3) Novel sources of FHB resistance and mycotoxin tolerance will be developed for plants.
Progress Report
This is the final progress report for this project which terminated March 2022. See the report for the replacement project, 5062-21220-024-000D, "Plant-Fungal Interactions and Host Resistance in Fusarium Head Blight of Barley and Wheat" for additional information.
Substantial progress was made on all objectives. However, Year 4 and Year 5 Milestones in Objectives 1 and 2 had considerably less progress than initially projected due to the Covid-19 travel and lab restrictions through March 2022. The turnover of project personnel, due to the retirement of the former lead SY and the onboarding of the new SY in the Fall of 2021, contributed to slower progress in completing Milestones.
Objective 1: Discover specific factors involved in pathogenicity, sporulation and toxin synthesis for the Fusarium head blight pathogen and related fungi by applying genomic and functional approaches.
A combination of approaches was used to dissect the molecular basis for pathogenicity and toxin biosynthesis in Fusarium graminearum, the causal agent of Fusarium head blight (FHB). Proteomics analysis of fluorescence-activated cell sorting (FACS)-enriched Tri4-RFP cell lysates under toxin-inducing conditions identified known and previously unidentified candidate proteins involved in tricothecene toxin biosynthesis. Tri14p, a protein of unknown function but required for toxin biosynthesis, was enriched in the toxisome proteome and shown to co-localize with Tri4p in toxisome structures. Candidate proteins and negative controls were genetically tagged with fluorescent proteins and subjected to preliminary fluorescence resonance energy transfer (FRET) to test for physical protein-protein interactions with known toxisome components. Deletion mutants were created for the Fusarium Tri14 gene and introduced into several genetic backgrounds that allow for fluorescent examination of fungal cells to test for alterations in toxisome formation. Gene constructs for creating mutants and misdirecting proteins were prepared and are available for future use.
To complement the proteomics and microscopy analysis of toxisome components, a detailed RNA-seq time course of toxin induction in Fusarium graminearum was performed. Three replicates of fungal cultures growing in toxin-inducing and non-inducing media were collected at 24-, 48-, and 72-hours post-transfer. Differential gene expression analysis identified the transcripts that are up- and down-regulated at each timepoint of toxin induction. Biological process enrichment analysis was performed to identify the high-level cellular changes at each timepoint, and the results suggest a dynamic coordination of primary and secondary metabolism during toxin induction. Additionally, the RNA-seq data was integrated with available protein-protein interaction data to identify protein complexes that are coordinately activated during toxin biosynthesis. Together, these analyses were used to predict gene candidates that regulate or participate in toxisome formation and/or toxin production. Mutant strains with deletions in candidate genes were created and are awaiting phenotyping for alterations in toxin production.
Objective 2: Relate fungal genotypes to mycotoxin production in fungal strains in field production environments to aid in developing enhanced methods of control.
In 2017-2019, FHB pathogen isolates were collected through surveys of wheat production fields and native grasses around Minnesota. Diseased and symptomless plant samples were cultured on media that inhibits non-Fusarium species and isolates were purified via single-spore descent. For a subset of isolates, Koch’s postulates were fulfilled by inoculating healthy plants in the lab, monitoring disease symptoms, and re-isolating the causal pathogen. In 2020-2021, due to Covid-19 restrictions the progress on this objective was halted.
Objective 3: Optimize metagenomic and functional approaches to define the phytobiome of healthy and diseased plants naturally infested with the FHB fungus.
Field sampling and culture collection was performed for 2 seasons at the Cedar Creek Ecosystem Science Reserve. Individual fungal (Fusarium) and bacterial (Streptomyces) isolates were purified from field samples. Reciprocal in vitro antibiotic inhibition assays were performed to characterize potential antagonistic interactions between Fusarium and Streptomyces isolates. Inhibitory activity of Streptomyces communities in each soil sample was evaluated against every single sympatric (from that soil sample) and allopatric (from samples taken from different individuals of the same plant species and plant community richness). For each soil sample, the proportion of Streptomyces that exhibit antagonistic activities against each sympatric or allopatric Fusarium isolate was calculated, and the size of the inhibition zone surrounding every inhibitory Streptomyces was determined. Fusarium inhibition of Streptomyces was evaluated for all possible sympatric and allopatric Fusarium–Streptomyces isolate pairwise combinations using the agar-disc method. Additionally, nutrient use profiles were evaluated for each Streptomyces (n =120) and Fusarium (n = 84) isolate for 95 single carbon sources. Culture collection data was posted online as part of a collaborative project. Together, these findings suggest a central role of antibiotic inhibition and nutrient competition in mediating Streptomyces–Fusarium interactions and determining their functional capacities and coevolutionary dynamics in soil. Such antagonistic interplays can offer novel opportunities for the fight against plant diseases caused by pathogenic Fusarium using antagonistic Streptomyces (e.g., Fusarium wilt and root rots) and vice versa (e.g., Streptomyces scabs).
Objective 4: Identify novel sources of plant disease resistance to FHB and mycotoxins produced by FHB fungi to improve breeding for resistance.
We have created populations of durum wheat segregating for FHB disease resistance and developed a radiation hybrid breeding population. Resistance was determined to be stable in subsequent generations. Transcriptome sequencing of plant samples collected from various lines and different infection period was completed. Bioinformatics analysis of the transcriptome data from modified resistant durum lines was completed and differentially expressed genes (elevated and suppressed >5 fold; log2 value) were identified for further characterization. This resulted in a list of 25 candidate genes that are in common between the samples. We are now confirming this finding by quantitative PCR amplification at various time points and additional lines. The narrowed list of candidate genes was used to identify a number of mutant durum lines from a collection of EMS mutants. These lines are now being characterized for their response to FHB as well as their gene expression patterns.
To characterize durum lines missing a portion of chromosome 2A region that may contain the FHB suppressor locus, populations were created and advanced. These lines were genotyped for deletion breaks along the chromosome. Select lines were further advanced and phenotyped. Initial testing indicated that some of these lines showed greatly improved resistance to FHB. Populations were advanced and phenotyped to confirm the initial results and assure resistance is true and not false positives. Promising lines have been identified and are being further characterized to confirm the enhanced disease phenotype and we have introgressed critical deletions into adapted germplasm for further evaluation.
Accomplishments
1. Halting fungal "toxin factories" may make small grains safer. Halting fungal "toxin factories" may make small grains safer. Harmful byproducts of fungi, called mycotoxins, threaten food safety and cause losses in wheat and barley yield and grain quality. Little is known about structures within fungal cells that make high-level production of mycotoxins possible. ARS researchers located in Saint Paul, Minnesota, have discovered that the fungal products vomitoxin and culmorin that contaminate wheat and barley grain are produced within specialized portions of fungal cells called toxisomes. The formation of toxisomes converts normal fungal cells into proverbial "toxin factories". Moreover, treatments such as phenamacril that prevent toxisome formation greatly reduce the ability of the fungus to produce mycotoxins. Phenamacril binds to and inhibits Fusarium myosin I, which is required for toxisome formation. Such treatments, and new "designed" chemicals with a similar mode of action, may supplement fungicide applications and be important for developing novel strategies for preventing the contamination of grain with vomitoxin and other mycotoxins. An expanded toolbox of safe and environmentally-friendly chemicals will directly benefit farmers and consumers.
2. Genes identified in resistant durum wheat are involved in plant defense. The plant disease Fusarium head blight (FHB) is an important constraint to profitable production of durum wheat in the upper Midwest. Currently planted durum cultivars lack consistently high levels of resistance to the disease. To identify new genes for disease resistance, ARS researchers located in Saint Paul, Minnesota studied differences in the genes expressed by disease susceptible lines and a newly developed M4 line that has moderately high FHB resistance. During infection, the resistant M4 line activated many categories of genes associated with an active plant disease defense response. These genes are being further characterized to better define the mechanism of resistance and assist in breeding better cultivars. These new native sources of FHB resistance in durum wheat with the gene markers will be a great boon to the effort in breeding for resistant cultivars and the growers in the Upper Great Plains of the United States.
