Botryosphaeria dieback is a grapevine trunk disease with a worldwide distribution associated with Diplodia seriata and Neofusicoccum parvum, among several other Botryosphaeriaceae species. The aforementioned xylem-inhabiting fungi cause wood lesions and leaf and berry symptoms, and eventually lead to the death of the plant. The aim of this work was to develop a simple model system to reproduce the foliar symptoms caused by D. seriata and N. parvum to better characterize fungal pathogenicity and determine the mechanisms involved in symptom development. Green stems of grafted 'Aragonez' grapevine cuttings were inoculated with three isolates of N. parvum and two isolates of D. seriata with different degrees of virulence and the experiment was repeated four times from 2011 to 2014. Three months after inoculation, the lesions associated with N. parvum were larger than those associated with D. seriata. Similarly, 8 months after inoculation, the percentage of plants showing foliar symptoms was greater in the N. parvum treatments than in the D. seriata treatments. During the emergence of foliar symptoms, plant stress-related responses were modulated in green stems and leaves, especially a downregulation of superoxide dismutase (SOD) and fasciclin-like arabinogalactan protein (fascAGP) and an upregulation of stilbene synthase (STS) genes with an accumulation of phenolics. In conclusion, the simple model system developed allowed a good characterization of isolate pathogenicity and correlation with foliar symptoms of Botryosphaeria dieback, namely spots on leaf margin and blade.