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Optimization of mycovirus detection methods obtained from NGS data analysis
PÍCHALOVÁ, Barbora
One well-known pathogenic fungus Armillaria causing Armillaria root disease which is characterised by infecting the root system of woody plants, resulting in basal decay and the development of a white syrrocium under the bark of trees. Several genome sequences of mycoviruses have already been published in research on this disease, including viruses from the Mymonoviridae, Botourmiaviridae, Partitiviridae, Virgaviridae as well as a newly described group of viruses known as ambi-like viruses. In this study, alongside ambi-like viruses and partitiviruses, by in silico analysis of virus sequences obtained via NGS methods unveiles the identification of previously unknown beny-like viruses within Armillaria spp. from the Czech Republic. To confirm the presence of beny-like viruses in vitro, reverse transcription with PCR was used. Total RNA was isolated using a commercial kit and its presence in the samples was confirmed electrophoretically. The sequence obtained using the Illumina system's NovaSeq 6000 sequencing platform was used to design the triple pairs of primers Beny F1 and Beny R1, Beny F2 and Beny R2, and BV1 F and BV1 R, in which potential ORFs were designed. The primers were designed to target the longest ORF encoding RdRp, the most conserved gene in RNA viruses. The DNA fragment obtained by reverse transcription was amplified by PCR followed by reamplification. Finally the samples were evaluated electrophoretically for the presence of viral single-stranded RNA.
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