Wu, X.; Xi, H.; Ryder, M.; Searle, I. R.

Grapevine crown gall is a globally significant disease typically attributed to Agrobacterium species carrying tumour-inducing (Ti) plasmids. In this study, we identify Neorhizobium vitis as a previously unrecognised crown gall-associated pathogen. Four modern isolates from symptomatic grapevines in South Australia and four historical strains collected in New South Wales in 1989 induced weak but consistent neoplastic growths on sunflower hypocotyls and carrot discs. Long-read genome sequencing revealed that all strains lack canonical Ti plasmids and the oncogenes iaaM and ipt , which are essential for classical auxin- and cytokinin-mediated tumorigenesis. Phylogenomic analyses reassigned all strains to the Neorhizobium clade, correcting the original classification of historical isolates as Agrobacterium vitis . The persistence of tumorigenic activity despite the absence of known virulence genes suggests the involvement of alternative, yet uncharacterized pathogenic mechanisms. These findings revise the non-pathogenic status of Neorhizobium , establish N. vitis as a novel tumorigenic lineage, and underscore the value of re-evaluating legacy isolates with modern genomic tools.