Inhibition of Hop Stunt Viroid by Exogenous Double-Stranded RNA in Micropropagated Grapevine Plantlets

Hop stunt viroid (HSVd) infection reduces grapevine (Vitis vinifera) yield and quality, leading to significant economic losses. Conventional methods for producing virus-free plantlets often fail to completely eliminate viroids, necessitating alternative strategies. In this study, RNA interference (RNAi) was induced by applying HSVd-specific double-stranded RNA (dsRNA) to infected grapevine plantlets. Exogenous dsRNA treatment significantly reduced HSVd levels, as confirmed by reverse transcription polymerase chain reaction and digital PCR. Fluorescently labeled (Cy3) dsRNA uptake was detected in plant tissues, while small RNA sequencing revealed an accumulation of HSVd-derived small interfering RNA, indicating RNAi activation. Notably, the inhibitory effect persisted through three successive generations without additional treatment, and similar suppression was observed in HSVd-infected cucumber plants. These findings highlight the efficacy and durability of exogenous dsRNA applications as a sustainable and non-transgenic approach for viroid control in grapevine cultivation.