Grapevine Pinot gris virus (GPGV) was described in Italy using a metagenomic approach: next-generation sequencing of the virus-derived small RNAs. Since that time, it has been reported all over the world. The presence of GPGV is associated with grapevine disease, but most of the time, the disease is asymptomatic. Although the host range of this virus has not been investigated, it has been found in the non-Vitis hosts, Silene latifolia and Chenopodium album. We investigated the presence of GPGV in grapevine and other plant species growing as weeds in the vineyard. Using RT-PCR, we identified GPGV in seven non-Vitis hosts: Ailanthus, Asclepias, Crataegus, Fraxinus, Rosa, Rubus, and Sambucus. In the case of Rosa and Rubus, this finding was supported by Northern blot detection of the virus. GPGV strains in non-Vitis hosts belong to the asymptomatic clade, and are clustered according to their original geographic locations. The presence of GPGV in species other than grapevine shows that besides well-known vector and propagating material-based infections, other possible entry sites for the virus can exist, which have to be taken into consideration when developing reliable regulation strategies.

Peach trees can be infected with viruses and viroids. As we do not have efficient plant protection methods against these pathogens, the prevention of infection is crucial. Fruit trees are maintained by vegetative propagation. Planting material such as certified mother trees and rootstocks should be free from viruses and viroids, and this status has to be regularly checked to prevent infections. We surveyed certified peach trees for the presence of viruses and viroids using small RNA high-throughput sequencing (HTS), an unbiased virus diagnostic method. The results of the bioinformatic analysis of HTS were validated by other molecular methods including RT-PCR, Northern blot hybridization and loop-mediated isothermal amplification (LAMP). We found the presence of plum pox virus and peach latent mosaic viroid (PLMVd) in the vector-free isolator houses, whose presence should be regularly tested. Moreover, we detected frequent infection with recently described viruses such as nectarine stem pitting-associated virus and peach-associated luteovirus (PaLV). During the survey, PLMVd and PaLV were detected for the first time in Hungary. The analysis of the presenting virus variants and possible sources of infection suggests that the source of the viral infection could be the infected propagating material. Our study emphasizes the importance of using sensitive and trustworthy diagnostic techniques to be able to detect viral infections and successfully prevent their spread by propagation material.

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.

Maintaining grape cultivation requires the continuous production of healthy propagating material and the control of economically important viruses. For more effective virus eradication, it is beneficial to try chemotherapeutic agents that have not yet been used on grapevine. We therefore tested two chemotherapeutic agents with different mechanisms of action (2-thiouracil and zidovudine) in addition to ribavirin, which is already used on grapevine. Viruses and viroids were identified in the mother plants of different grapevine cultivars by small RNA HTS (High-Throughput Sequencing), and RT-PCR verified the results. After chemotherapy, the regenerated plants were tested using RT-PCR and the efficacy of the three chemotherapeutic compounds was evaluated. Among the tested agents, ribavirin had the broadest elimination effect (the virus was not detectable by RT-PCR after 8 months) on most viruses. It proved to be particularly effective (the virus was not detectable by RT-PCR in most of the tested plants) against GFkV, GLRaV-4, GPGV and GRSPaV. The use of 2-thiouracil caused high phytotoxicity and was effective against GLRaV-4 among the viruses tested, having no effect on the other viruses. Zidovudine alone failed to eliminate any of the viruses we tested. 2-thiouracil, ribavirin and zidovudine were unable to eliminate HSVd and GYSVd-1 viroids.

Using small RNA high-throughput sequencing (HTS), we previously demonstrated the widespread distribution of grapevine Pinot gris virus (GPGV) in Hungarian vineyards. This trichovirus has been associated with a disease known as grapevine leaf mottling and deformation (GLMD). However, since GPGV has been detected in both symptomatic and asymptomatic plants, its exact role in GLMD disease is not well-characterised. Studies addressing this question suggested that differences in the GPGV susceptibility of the grapevine cultivars and the presence of variants of the virus could affect symptom development. Being able to suppress various steps of the RNA interference-based defence reactions, the viral suppressor of RNAi (VSR), encoded by the ORF3 of GPGV, can also alter the symptom development. In the present study, we compared the VSR activity of the ORF3-encoded coat protein of symptomatic and asymptomatic GPGV variants and found that both possess VSR activity. Testing the VSR activity of the ORF2-encoded movement proteins from the two variants, using a GFP-based transient gene expression assay, we found that the GPGV-MP has weak systemic VSR activity. Moreover, we found that the transient expression of the MP variants induced necrosis in the infiltrated leaves, which was stronger in the case of the symptomatic variant. To functionally characterise the crucial sequence elements of MP responsible for this difference in the necrosis between symptomatic and asymptomatic variants, the necrosis-inducing activity of GPGV-MP encoded by different natural and recombinant variants was tested. Differences in the GPGV-MP necrosis-inducing activity suggested that, besides the previously described C/T polymorphism, different phosphorylation patterns of the GPGV-MP may contribute to symptom development.