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Summary Grapevine (Vitis spp.) is a widespread fruit tree hosting many viral entities that interact with the plant modifying its responses to the environment. The production of virus‐free plants is becoming increasingly crucial for the use of grapevine as a model species in different studies. Using high‐throughput RNA sequencing, the viromes of seven mother plants grown in a germplasm collection vineyard were sequenced. In addition to the viruses and viroids already detected in grapevine, we identified 13 putative new mycoviruses. The different spread among grapevine tissues collected in vineyard, greenhouse and in vitro conditions suggested a clear distinction between viruses/viroids and mycoviruses that can successfully be exploited for their identification. Mycoviruses were absent in in vitro cultures, while plant viruses and viroids were particularly accumulated in these plantlets. Somatic embryogenesis applied to the seven mother plants was effective in the elimination of the complete virome, including mycoviruses. However, different sanitization efficiencies for viroids and grapevine pinot gris virus were observed among genotypes. The absence of mycoviruses in in vitro plantlets, associated with the absence of all viral entities in somaclones, suggested that this regeneration technique is also effective to eradicate endophytic/epiphytic fungi, resulting in gnotobiotic or pseudo‐gnotobiotic plants.

Grapevine is one of the most cultivated fruit plant among economically relevant species in the world. It is vegetatively propagated and can be attacked by more than 80 viruses with possible detrimental effects on crop yield and wine quality. Preventive measures relying on extensive and robust diagnosis are fundamental to guarantee the use of virus-free grapevine plants and to manage its diseases. New phenotyping techniques for non-invasive identification of biochemical changes occurring during virus infection can be used for rapid diagnostic purposes. Here, we have investigated the potential of Raman spectroscopy (RS) to identify the presence of two different viruses, grapevine fan leaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in Vitis vinifera cv. Chardonnay. We showed that RS can discriminate healthy plants from those infected by each of the two viruses, even in the absence of visible symptoms, with accuracy up to 100% and 80% for GFLV and GRSPaV, respectively. Chemometric analyses of the Raman spectra followed by chemical measurements showed that RS could probe a decrease in the carotenoid content in infected leaves, more profoundly altered by GFLV infection. Transcriptional analysis of genes involved in the carotenoid pathway confirmed that this biosynthetic process is altered during infection. These results indicate that RS is a cutting-edge alternative for a real-time dynamic monitoring of pathogens in grapevine plants and can be useful for studying the metabolic changes ensuing from plant stresses.