H 7650 instrument

The EV pellets collected at the bottom of tubes were directly fixed with 100 μL of modified Karnovsky’s fixative solution⁸⁰ (2.0% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2) for 1 h at 4 °C. After fixation, the pellets were carefully recovered with a spatula and embedded in LR White resin (Agar Scientific, Stansted, Essex, LDN, UK). Ultra-thin sections were prepared with an Ultracut UCT microtome (S9329, Leica, S9329, Wetzlar, Germany) and the specimens were stained with 6% uranyl acetate (Wako, Tokyo, Japan) and 3% lead citrate (Wako) on formvar-coated (Nissin EM, Tokyo, Japan) nickel grids (S-300 square mash, Gilder, Grantham, UK). Transmission electron microscopy (TEM) images were obtained with a H-7650 instrument (Hitachi, Co., Tokyo, Japan).

To obtain viral particles, 30 g of mycelia was ground and mixed with 100 mM phosphate buffer (pH 7.4). After the removal of the cellular debris, the lysate was subjected to ultracentrifugation at 4 °C for 2 h to obtain the sediment, which was suspended in 100 mM phosphate buffer. The extract was further subjected to ultracentrifugation in sucrose density gradients (100 to 500 mg/mL with intervals of 100 mg/mL) [16 (link)]. The fraction containing the virus particles was carefully collected and dialyzed overnight. The dialyzed fractions were collected through ultracentrifugation for 2 h and suspended in 50 μL of 0.05 M phosphate buffer for further analysis. The structure of the virus-like particles was visualized using a transmission electron microscope (TEM) on an H-7650 instrument installed at the Center for University-Wide Research Facilities at Chonbuk National University (Hitachi, Tokyo, Japan) after negative staining with 2% uranyl acetate. The viral dsRNA elements from the crude extract were extracted with phenol, chloroform, and isoamyl alcohol, precipitated with ethanol, and visualized through agarose gel electrophoresis. Viral proteins were detected through 10 % SDS-PAGE analysis.