Simultaneous detection of grapevine viruses, viroids, and satellite RNAs by high-throughput and targeted assays

Abstract

Grapevine (Vitis vinifera L.) is considered to be among the most widely grown and major fruit crops worldwide with high economic importance. Viruses and virus-like organisms cause considerable economic losses in vitiviniculture, and considering that they cannot be controlled by conventional plant protection methods, the use of healthy vegetative propagation material is crucial. Metagenomic approaches for virus diagnostics enable rapid and accurate detection of viral pathogens in plants. To investigate the presence of viruses and virus-like organisms infecting grapevine from the Ampelographic Collection Kromberk in Slovenia, Ion Torrent small RNAsequencing (sRNA-seq) was performed, and the resulting data were analyzed using the VirusDetect pipeline. This approach revealed the presence of grapevine leafroll- associated virus 1 (GLRaV-1), grapevine leafroll-associated virus 2 (GLRaV-2), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine fanleaf virus (GFLV) and its satellite RNA(satGFLV), grapevine fleck virus (GFkV), grapevine rupestris vein feathering virus (GRVFV), grapevine Pinot gris virus (GPGV), grapevine satellite virus (GV-Sat), hop stunt viroid (HSVd), and grapevine yellow speckle viroid 1 (GYSVd-1). To validate the infections predicted by sRNA-seq, multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assays were developed, allowing simultaneous detection of different combinations of viruses, viroids, and satellite RNAs. The mRT-PCR assays represent a rapid and cost-effective tool for routine molecular diagnostics and could be applied for the detection of widespread, emerging, and rarely tested grapevine viruses, as well as viroids, which are often overlooked in standard diagnostic testing.