Within VirFree the optimization of HTS for the identification of known and new fruit tree and grapevine viruses or viroids was addressed. Pipelines using the software CLC Genomics Workbench 10, Geneious Prime® 2022.2.1 and MacVector, Inc. were developed and optimized for the detection of plant viruses in woody plants. These pipelines were used for screening of samples resulting in the detection of more than 60 viruses/viroids, including five new virus species and a new viroid-like RNA in fruit trees as well as several divergent strains of known viral pathogens of woody plants. On several occasions the viruses and viroids identified were reported for the first time in a country, thus expanding the list of endemic pathogens, and in new hosts which directly affect their epidemiology. The data obtained was used to update detection assays and determine the previously unknown, etiological agents of diseases. The high number of new sequences from different isolates obtained from woody plants were incorporated in alignments to assess the specificity of the existing RT-PCR assays and redesign the primers where needed. RT-PCR assays were developed for the detection of CiVA, PrVI and ctaHVd-LR1 to establish their spread and assist further studies. RT-PCR and RT-qPCR assays were also developed for several viral pathogens (CVTR, GVE, GVF, GVH, GVT, GPGV, GRLDaV and PeSV), and they will become useful tools in the production of high-quality plant material. LAMP assays were designed for the sensitive and rapid detection of grapevine viruses (GVA, GPGV, GRLDaV) and a fruit tree viroid (ASBVd) however further experimentation will be needed before their use for diagnosis. In addition, nanobodies were raised against a grapevine infecting pathogen (RpRSV) and incorporated in an ELISA assay thus improving its diagnostic efficiency. The method is being exploited by the involved consortium participants. Moreover, the phytosanitary improvement of nursery material and optimization of the micropropagation methods for fruit trees through training and exchange of knowledge on virus elimination and micropropagation techniques was attempted. Fruit tree species of interest (Prunus species, pomegranate etc.) were micropropagated and, in some cases, attempts were made for sanitation. Also, the stage of acclimatization/hardening of young plants was optimized and a best practices protocol was prepared, to produce high-quality plant material of fruit trees. The proposed protocol is currently being used by the Greek nursery that participated in VirFree. Finally, the results of VirFree were disseminated to the scientific community, stakeholders and the general public through national and international conferences, press releases, presentations in open public events, social media and the project’s website –www.virfree.eu. Moreover, 18 scientific articles related to VirFree activities, detection methods and new virus species were published, and the sequences obtained in this project have or will be submitted to public databases, making the results readily available to everyone.
