Vanja Miljanić1*, Jernej Jakše1, Sebastjan Radišek2 and Nataša Štajner1
1Biotechnical Faculty, University of Ljubljana, Slovenia
2Slovenian Institute of Hop Research and Brewing
vanja.miljanic [at] bf.uni-lj.si
Abstract
Verticillium dahliae is a soil-borne phytopathogenic fungus and the causal agent of Verticillium wilt, a destructive vascular disease affecting hundreds of economically important plant species worldwide. Its broad host range, long-term survival and persistence in soil make disease control particularly challenging. Mycoviruses, or fungal viruses, have garnered increasing attention due to their potential role in altering fungal pathogenicity and their suitability for biological control.
In our study, 33 isolates of V. dahliae were obtained from the culture collection of the Slovenian Institute of Hop Research and Brewing to test for the presence of mycoviruses. The first step in the search for viral-infected fungi is to look for the presence of dsRNAs, as all RNA viruses have a dsRNA stage during their infection cycle. To isolate dsRNA, fungi were cultured on cellophane membranes overlaying CDA plates. The dsRNA was isolated by the cellulose chromatography method. After digestion with DNase I (New England BioLabs) and S1 nuclease (Thermo Fisher Scientific), the dsRNA segments were analyzed by electrophoresis on a 1% agarose gel. Two out of the 33 tested fungal isolates were found to be virus-infected. Libraries of dsRNAs were constructed using the Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) and were barcoded using the Xpress™ RNA-Seq Barcode 1–16 Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The yield and size distribution of the amplified cDNA libraries were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed on the Ion GeneStudio S5 Prime System (Thermo Fisher Scientific). The obtained reads were processed with bioinformatics tools CLC Genomic Workbench and Genomics Server (Qiagen). The applied method revealed the presence of bisegmented Verticillium albo-atrum partitivirus 1 (VaaPV1; family Partitiviridae) and Verticillium dahliae RNA virus 1 (VdRV1; unassigned non-segmented +RNA virus). Given that some mycoviruses influence the pathogenicity of their fungal hosts, ongoing research focuses on assessing the impact of VaaPV1 and VdRNA1 on host virulence.
Keywords: Verticillium wilt, VaaPV1, VdRNA1
Acknowledgement: This research was funded by the Slovenian Research and Innovation Agency (ARIS): research project J4-70163 awarded to Vanja Miljanić and research program P4-0077 Genetics and Modern Technologies of Crops.