Improved Double-Stranded RNA Extraction and Hyphal Tip-Touch Techniques for the Detection of Mycoviruses

Abstract

Mycoviruses infect a wide range of fungal species, and many of them have double-stranded RNA (dsRNA) genomes. Using a dsRNA technique makes it easier to detect unknown mycoviruses in fungi. Previously, a few studies reported dsRNA extraction methods from fungal tissues, but the procedures required a significant amount of starting material and time, particularly in growing fungal cultures. In this study, we report an efficient and quick dsRNA extraction method that is reproducible to extract quality dsRNA of mycoviruses from a small amount of fungal culture in a short time. Extracted dsRNA was subsequently used in Illumina sequencing and reverse transcription-polymerase chain reaction assays, followed by Sanger sequencing, to confirm the identity of the mycovirus. Additionally, a rapid and efficient technique named hyphal tip-touch (HTT) was developed to identify known mycoviruses without the need for dsRNA extraction. The HTT technique uses the edge of mycelia as a template for preparing complementary DNA in the reverse transcription reaction, which was used in the polymerase chain reaction assay followed by sequencing. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Keywords

• dsRNA extraction method
• dsRNA viruses
• hyphal tip-touch
• known mycoviruses
• unknown mycoviruses