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.

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.

Among other pathogens, more than 80 viruses infect grapevine. The aim of this work was to study the virome diversity of grapevine viruses and mycoviruses of a vineyard using high-throughput sequencing technologies. The grapevine virome was studied in symptomatic vines of the Rkatsiteli cultivar (V. vinifera) collected at the vineyards of the Krasnodar Krai in Russia. Ribosomal-depleted total RNA and isolated small RNAs were used for library preparation and high-throughput sequencing. Six grapevine-infecting viruses and two viroids were validated by RT-PCR and analyzed phylogenetically. We identified the presence of grapevine leafroll-associated virus 3, grapevine Pinot gris virus, grapevine virus T, grapevine rupestris stem-pitting-associated virus, grapevine fleck virus, and grapevine rupestris vein feathering virus, as well as two viroids, grapevine yellow speckle viroid 1 and hop stunt viroid. We also studied the mycovirome of the vineyard and identified nine viruses with single-stranded positive-sense RNA genomes: alternaria arborescens mitovirus 1, botrytis cinerea mitovirus 1, botrytis cinerea mitovirus 2, botrytis cinerea mitovirus 3, botrytis cinerea mitovirus 4, sclerotinia sclerotiorum mitovirus 3, botrytis cinerea hypovirus 1, grapevine-associated narnavirus 1, and botrytis virus F. In addition, we identified botrytis cinerea hypovirus 1 satellite-like RNA and two single-stranded negative-sense RNA viruses. This is the first study of grapevine mycoviruses in Russia. The obtained result will contribute to the development of biocontrol strategies 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.

In 2019, random samples of Panicum miliaceum growing as a weed were surveyed to uncover their virus infections at two locations in Hungary. This pilot study revealed infection with three viruses, two appearing for the first time in the country. As follow-up research, in the summer of 2021, we collected symptomatic leaves of several monocotyledonous plants in the same locations and determined their viromes using small RNA high-throughput sequencing (HTS). As a result, we have identified the presence of wheat streak mosaic virus (WSMV), barley yellow striate mosaic virus (BYSMV), barley virus G (BVG), and two additional viruses, namely Aphis glycines virus 1 (ApGlV1) and Ljubljana dicistrovirus 1 (LDV1), which are described for the first time in Hungary. New hosts of the viruses were identified: Cynodon dactylon is a new host of BYSMV and LDV1, Echinocloa crus-galli is a new host of BVG, ApGlV1 and LDV1, Sorghum halepense is a new host of ApGlV1, and Panicum miliaceum is a new host of LDV1. At the same time, Zea mays is a new host of ApGlV1 and LDV1. Small RNA HTS diagnosed acute infections but failed to detect persistent ones, which could be revealed using RT-PCR. The infection rates at the different locations and plant species were different. The phylogenetic analyses of the sequenced virus variants suggest that the tested monocotyledonous weeds can host different viruses and play a virus reservoir role. Viral spread from the reservoir species relies on the activity of insect vectors, which is why their management requires an active role in plant protection strategies, which need careful planning in the changing environment.

Micropropagated Catharantus roseus plants infected with ‘Candidatus Phytoplasma asteris’ showed virescence symptoms, witches’ broom symptoms, or became asymptomatic after their planting in pots. Nine plants were grouped into three categories according to these symptoms, which were then employed for investigation. The phytoplasma concentration, as determined by qPCR, correlated well with the severity of symptoms. To reveal the changes in the small RNA profiles in these plants, small RNA high-throughput sequencing (HTS) was carried out. The bioinformatics comparison of the micro (mi) RNA and small interfering (si) RNA profiles of the symptomatic and asymptomatic plants showed changes, which could be correlated to some of the observed symptoms. These results complement previous studies on phytoplasmas and serve as a starting point for small RNA-omic studies in phytoplasma research.