As virus diseases cannot be controlled by traditional plant protection methods, the risk of their spread have to be minimized on vegetatively propagated plants, such as grapevine. Metagenomic approaches used for virus diagnostics offer a unique opportunity to reveal the presence of all viral pathogens in the investigated plant, which is why their application can reduce the risk of using infected material for a new plantation. Here we used a special branch, deep sequencing of virus-derived small RNAs, of this high-throughput method for virus diagnostics, and determined viromes of vineyards in Hungary. With NGS of virus-derived small RNAs we could detect not only the viruses tested routinely, but also new ones, which had never been described in Hungary before. Virus presence did not correlate with the age of the plantation, moreover phylogenetic analysis of the identified virus isolates suggests that infections are mostly caused by the use of infected propagating material. Our results, validated by other molecular methods, raised further questions to be answered before this method can be introduced as a routine, reliable test for grapevine virus diagnostics.

Grapevine can be infected by several viruses and viroids, the presence of which can lead to yield losses and vineyard decline. Our previous survey of vineyards in Hungary suggested that viral infection originates from infected propagation material. To investigate whether rootstocks can be a source of virus infections, we surveyed seventeen rootstock vineyards and two rootstock collections in Hungary to determine the virome by high-throughput sequencing of small RNAs. The presence of the viruses was also tested by RT-PCR. The results showed that viruses whose presence is routinely checked were almost absent in rootstock vineyards but were present in rootstock genotype collections. Moreover, first the time in Hungary, we detected the presence of Australian grapevine viroid in the rootstock genotype collection at Pecs. In contrast, viruses that are not regulated or not routinely tested, namely, grapevine rupestris stempitting-associated virus, grapevine Syrah virus-1 and grapevine Pinot gris virus, were detected in almost all locations in most of the varieties. The presence and absence of infected rootstock genotypes in the same vineyard together with phylogenetic analysis suggested that viral infections originated from infected propagation material. Moreover, we found the symptomatic variant of grapevine Pinot gris virus in several rootstock vineyards without symptoms, suggesting the possibility for leaf mottling and deformation disease symptoms to manifest on susceptible cultivars following grafting onto these rootstocks.

Woody perennial plants like grapevine and fruit trees can be infected by several viruses even as multiple infections. Since they are propagated vegetatively, the phytosanitary status of the propagation material (both the rootstock and the variety) can have a profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Viral derived small RNAs produced by the host immune system during viral infection can be sequenced by next-generation techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample. This method is based on Illumina sequencing of short RNAs and bioinformatics analysis of virus-derived small RNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, in order to ensure success for every user.