Grapevines are propagated by grafting, but the rootstocks used in commercial plantations are susceptible to several diseases. In this study, we focused on a novel root and trunk rot disease of grapevine plantlets that show symptoms during cold storage, before field establishment. Our objectives were to study the aetiology, symptomatology, plant resistance responses, and mode of action of the pathogen that was initially identified as Fusarium. The characterisation of this pathosystem was performed by isolation, pathogenicity assays, genetic diversity studies with BOX-PCR, and identification by sequencing a fragment of the tef1 gene. Scanning electron microscopy and X-ray spectroscopy were used to study the mode of action and plant resistance responses. The results showed that 12 species of Fusarium, initially isolated from both healthy and diseased plantlets, and classified into 4 species complexes, were pathogenic to grapevines. Comparative analyses between diseased and healthy roots showed typical resistance responses in diseased plantlets, including tyloses formation, translocation of Ca, and accumulation of Si. Field experiments confirmed that 100% of the diseased plantlets died within 90 days of transplantation. This study contributes to a better understanding of root and trunk rot disease under cold storage and provides insights for the development of management strategies.