Grapevine latent viroid and Grapevine hammerhead viroid-like identified by next generation sequencing in an ancient grapevine germplasm collection in Italy

"Up to now, nearly 70 different viruses are known to infect grapevines, about half of which are associated with major diseases. By contrast, only six grapevine-infecting viroids and a viroid- like RNA, which is likely a novel viroid, have been identified. Although most of the viroids generally do not elicit severe symptoms in grapevines, some of them are the agent of diseases in certain environmental conditions or in combination with certain viruses. Moreover, some of grapevine-infecting viroids may cause severe diseases in other crops. While the identification of the five grapevine viroids dates back to the end of the 900s, Grapevine latent viroid (GLVd) and Grapevine hammerhead viroid-like RNA (GHVd) were identified only recently thanks to the application of next-generation sequencing (NGS) technologies. NGS technologies have, indeed, revolutionized the biodiversity studies and are going to deeply impact on the diagnostic protocols. NGS-based metagenomics approaches are particularly useful to investigate the plant associated viromes, offering a unique opportunity to reveal the presence of all viral agents in the investigated plants, including still unknown entities. In this work, we used an NGS-based approach in order to characterize the virome associated to a grapevine germplasm collection located in Grinzane-Cavour (Piedmont, Northern Italy), comprehending a wide diversity of current and ancient grapevine varieties. The collection includes around 400 grape cultivars from North-Western Italy, as well as regional, national and international varieties used as references, and it is among the largest and richest in materials neglected, endangered to disappear. NGS of cDNA libraries of small RNAs from about one third of the accessions in the germplasm collection was performed, and the virome was reconstructed using an ad hoc bioinformatics pipeline. Results were then validated by other independent molecular methods. Thanks to this approach, viruses routinely present in the grapevines, along with recently discovered viruses and viroids, and viral entities never described before in Italy were identified. In particular, we detected for the first time in Italy the two recently discovered viroids, GLVd and GHVd. According to our data, GLVd presence is limited to few plants in the collection, originally from extra-European territories, i.e. Armenia, Georgia, Uzbekistan and North America, whereas GHVd is more widely distributed among the grapevine accessions, including extra-European, European and Italian grapevines."