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

Emiliya Navrotskaya,Eugeniya Yurchenko,Elena Porotikova,Eva Varallyay,Zsuzsanna Nagyne Galbacs,Svetlana Vinogradova

doi:10.3390/v13122432

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.

Highlights

The viticulture and viniculture industries are important economic constituents in many countries [1]
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 small RNAs (sRNAs) high-throughput sequencing (HTS) and the molecular method RT-PCR, which made it possible to identify 13 viruses and 4 viroids

Summary

Introduction

The viticulture and viniculture industries are important economic constituents in many countries [1]. Grapevines are the host for the largest number of viruses among cultivated species [4]. More than 85 grapevine-infecting viruses are currently known [5]. About 30 of them are caused by the 4 disease complexes [3]. Five viruses are associated with grapevine leafroll disease and cause the reddening or yellowing of leaves with veins remaining green and the down-rolling of leaves [7,8,9]. Six viruses among them are vitiviruses and Grapevine rupestris stem pitting-associated virus (GRSPaV), which affect the woody cylinder, cambium tissues, bark and associated with rugose wood complex [10]. Four viruses of the fleck complex cause clearing of the small veins, with the appearance of translucent spots, wrinkling, twisting and curling upward of the leaves [3]

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