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.

In the present study, we conducted field surveys to detect the population density of the most important invasive weed species and their associated virus vectoring aphids in crops grown under high input field (HIF) vs. low-input field (LIF) conditions, with and without fertilizers and pesticides. The most frequent invasive weed species were annual fleabane, Erigeron annua (L.), Canadian horseweed, Erigeron canadensis (L.) and Canadian goldenrod, Solidago canadensis (L.). These species were predominantly hosts of the aphids Brachycaudus helichrysi and Aulacorthum solani under both management systems. The 13% higher coverage of E. annua under LIF conditions resulted in a 30% higher B. helichrysi abundance and ∼85% higher A. solani abundance compared with HIF conditions. To reveal the incidence of virus infection in crop plants and invasive weeds, high-throughput sequencing of small RNAs was performed. Bioinformatics analysis combined with independent validation methods revealed the presence of six viruses, but with strikingly different patterns under LIF and HIF conditions. Their presence without symptoms in invasive weeds and crop plants supports the necessity of employing new approaches to those currently employed in invasive weed management. These findings also suggest that invasive weeds could serve as hosts for local aphid species and reservoirs for plant pathogenic viruses, both under low and high input management systems. In this light, as here demonstrated, viruses transmitted by local aphid species were found to differ between the management systems; hence, the importance of B. helichrysi and A. solani as virus vectors in particular clearly needs to be re-evaluated. Altogether, we accept that the present study is a pilot one and individual virus vectoring of aphids still needs to be directly tested. Even so, it represents one of the first contributions to this particular area, and thereby paves the way for further similar applied research in the future.

Grapevine virus T (GVT) is a recently described foveavirus, which was identified from a transcriptome of a Teroldego grapevine cultivar in 2017. Recently, we surveyed vineyards and rootstock plantations in Hungary using small RNA (sRNA) high-throughput sequencing (HTS), at a time when GVT had not yet been described. A re-analysis of our sRNA HTS datasets and a survey of grapevines by RT-PCR revealed the presence of GVT in most of the vineyards tested, while at rootstock fields its presence was very rare. The presence and high variability of the virus in the country was confirmed by sequence analysis of strains originating from different vineyards. In this study, we demonstrate the presence of GVT in Hungary and show its high diversity, suggesting that GVT presence may not seriously affect grapevine health and that it could have been present in European vineyards for a long time as a latent infection.

Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT‒(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus.