Location: Sunflower Improvement Research
Project Number: 3060-21220-034-038-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2025
End Date: Dec 31, 2026
Objective:
The objectives of this project are: 1) Characterize the prevalence and effect of the Mitovirus on S. sclerotiorum isolates in North Dakota, 2) characterize the transcriptional dynamics of S. sclerotiorum infected with Mitovirus, and 3) Characterize the small RNA profiles of S. sclerotiorum isolates infected and non-infected with Mitovirus.
Approach:
The aim of this project is to evaluate the Mitovirus prevalence among North Dakota S. sclerotiorum isolates and evaluate its potential for use as a component of the gene silencing construct previously generated. Specifically, 1) Characterize the prevalence and effect of the Mitovirus on S. sclerotiorum isolates in North Dakota. Isolates of S. sclerotiorum will be collected from commercial fields and evaluated for the presence of Mitovirus along with samples from isolates collected in previous years. Infected isolates will be cultured. Following fungal growth, the new cultures will be checked for mitovirus infection using reverse transcription PCR (RT-PCR). The Effect of Mitovirus infection on S. sclerotiorum virulence will be assessed in canola. Canola plants at the flowering stage will be inoculated with S. sclerotiorum isolates infected with the Mitovirus and a similar number will be inoculated with its Mitovirus-free version. After inoculation, lesion size and plant mortality will be monitored. The production of pathogenicity factors will be compared between mitovirus-infected and mitovirus-free S. sclerotiorum mycelia. The production of sclerotia by mitovirus-infected and mitovirus-free isolates will be evaluated in canola plants. The number and weight of sclerotia produced will be recorded as indicators of fungal growth and development, which may be influenced by the presence of the mitovirus. 2) Objective 2. Characterize the transcriptional dynamics of S. sclerotiorum infected with Mitovirus. To sequence the complete genome of the Mitovirus, total RNA will be extracted from two Mitovirus-infected S. sclerotiorum isolates. The RNA sequences will be uploaded to the VirFind website to identify mitovirus sequences. After identifying the majority of the mitovirus genome sequence, any missing regions will be amplified via PCR and subsequently sequenced. Upon obtaining the complete genomic sequence of S. sclerotiorum Mitovirus, phylogenetic analysis will be conducted to determine the virus strain and its relatedness to other Mitoviruses. Transcriptome analysis will be conducted in virus-infected and virus-free S. sclerotiorum isolates. Differentially expressed genes (DEGs) will be classified using the PANTHER classification system to identify functional categories. Genes showing significant upregulation or downregulation in the transcriptomic analysis will be validated using quantitative PCR (qPCR); and 3) Characterize the small RNA profiles of S. sclerotiorum isolates infected and non-infected with Mitovirus. Total small RNA will be extracted from the samples using the MagMAX™ mirVana™ Total RNA Isolation Kit. The extracted RNA will be sent to be sequenced with Illumina sequencing to generate high-quality small RNA sequence data. The small RNA profiles will be analyzed using appropriate bioinformatics tools and software. Differentially expressed small RNAs will be classified into coding and non-coding regions. Small RNA sequences with fewer than 20 reads or those identified as ribosomal or transfer RNAs (tRNAs) will be excluded from the analysis.