High-Throughput Sequencing of Small RNAs for Diagnostics of Grapevine Viruses and Viroids in Russia

Abstract

The use of high-throughput sequencing (HTS) technology has led to significant progress in the identification of many viruses and their genetic variants. In this study, we used the HTS platform to sequence small RNAs (sRNAs) of grapevine to study the virome. Isolation of RNA was performed using symptomatic grapevines collected from commercial vineyards in Krasnodar Krai in 2017–2018. To determine the viromes of vineyards, we used an integrated approach that included a bioinformatic analysis of the results of sRNA HTS and the molecular method RT-PCR, which made it possible to identify 13 viruses and 4 viroids. Grapevine leafroll-associated virus 4 (GLRaV-4), Grapevine Syrah Virus-1 (GSyV-1), Raspberry bushy dwarf virus (RBDV), Australian grapevine viroid (AGVd), and Grapevine yellow speckle viroid 2 (GYSVd-2) were identified for the first time in Russia. Out of 38 samples analyzed, 37 had mixed infections with 4–11 viruses, indicating a high viral load. Analysis of the obtained sequences of fragments of virus genomes made it possible to identify recombination events in GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, GVT, GPGV, GRSPaV, GVA, and GFLV. The obtained results indicate a wide spread of the viruses and a high genetic diversity in the vineyards of Krasnodar Krai and emphasize the urgent need to develop and implement long-term strategies for the control of viral grapevine diseases.

Virome of Grapevine Germplasm from the Anapa Ampelographic Collection (Russia)

“…Validation of 47 libraries confirmed the presence of 20 viruses and 3 viroids in the analyzed samples (Figure 1). The most common (found in more than 50% of libraries) were GRSPaV, GPGV, GFkV, GSyV-1, as well as HSVd and GYSVd-1 viroids, which confirms our previous observations on the wide distribution of these pathogens in Russia [34,35,49]. Not a single virus-free plant was found.…”

“…Molecular groups for GLRaV-2 were determined in silico by RFLP analysis according to the method described by us previously [34,35,42].…”

Elimination of Eight Viruses and Two Viroids from Preclonal Candidates of Six Grapevine Varieties (Vitis vinifera L.) through In Vivo Thermotherapy and In Vitro Meristem Tip Micrografting

“…The first report of natural infection of vines with RBDV was published in Slovenia in 2003 [56]. Outside Slovenia, there have been few reports of grapevine infections by this virus [57][58][59][60]. Although several studies have reported RBDV elimination from raspberries using different methods [27,[61][62][63], to the best of our knowledge, there are no reports of RBDV elimination from grapevines.…”

Small RNA Sequencing and Multiplex RT-PCR for Diagnostics of Grapevine Viruses and Virus-like Organisms

“…High-throughput sequencing technology (HTS), which targets all nucleic acid types, enables rapid and accurate detection, including previously described and novel viruses and virus-like organisms [ 14 , 15 , 16 , 17 ]. An approach that enables virus discovery through HTS technology and assembly of small RNAs (small RNA sequencing, sRNA-seq) has proven to be highly efficient in the detection of new RNA and DNA grapevine viruses [ 4 , 7 , 18 , 19 ], virome studies [ 20 , 21 , 22 , 23 ], and to evaluate the efficacy of different elimination methods such as chemotherapy, somatic embryogenesis, and meristem tissue culture [ 24 , 25 ]. All in silico predicted grapevine viral infections are most commonly validated using RT-PCR [ 21 , 22 , 23 , 24 , 25 , 26 ].…”

“…An approach that enables virus discovery through HTS technology and assembly of small RNAs (small RNA sequencing, sRNA-seq) has proven to be highly efficient in the detection of new RNA and DNA grapevine viruses [ 4 , 7 , 18 , 19 ], virome studies [ 20 , 21 , 22 , 23 ], and to evaluate the efficacy of different elimination methods such as chemotherapy, somatic embryogenesis, and meristem tissue culture [ 24 , 25 ]. All in silico predicted grapevine viral infections are most commonly validated using RT-PCR [ 21 , 22 , 23 , 24 , 25 , 26 ]. Several other molecular diagnostic methods as well as immunological detection methods, and biological indexing are used in plant virology [ 27 ].…”