The genus Agrobacterium was initially proposed by Conn (Conn, 1942) for three bacterial species that had rhizogenic or tumorigenic properties toward plants or were non-pathogenic. Over the years, the genus steadily grew to comprise more species. It was later shown to be a polyphyletic group based on 16S rRNA gene phylogenies and Genome BLAST Distance Phylogeny, GBDP (Hördt et al., 2020). The taxonomic status of Agrobacterium as a separate genus from its sister clade Rhizobium has been the subject of intense debate (Farrand et al., 2003), but nowadays, Agrobacterium is broadly accepted as a separate genus based on genomic and phenotypic features (Farrand et al., 2003, Lassalle et al., 2017, Ramírez-Bahena et al., 2014). While in the early years, plant pathogenicity was considered a key phenotypic trait for this genus, this point of view evolved to include non-pathogenic species as well, as the genus was redefined based on molecular taxonomy (Flores-Félix et al., 2020).

Members of this group possess genomes with a chromosome, besides one or several circular and linear chromids, and often the presence of plasmids. The presence of plasmids is not always indicative of pathogenicity, but in some cases, they carry conjugative elements that act as oncogenic tumour-inducing (Ti) or root-inducing (Ri) plasmids, causing crown gall and hairy root disease, respectively (Schierstaedt et al., 2019, Weisberg et al., 2020).

The presence of a linear chromid and a gene coding for a protelomerase (telA), an enzyme that protects genomic DNA against nuclease activity, are important features identified by Ramírez-Bahena et al. (Ramírez-Bahena et al., 2014) in the genomes of most members of Agrobacterium genus. These features were proposed as key to identifying members of Agrobacterium (de Lajudie et al., 2019); but the availability of current genomic information and the use of Overall Genome Relatedness Indexes (OGRI) bring new perspectives for establishing a robust framework not only for species delimitation but also for genus assignment (Kuzmanović et al., 2022, Wirth and Whitman, 2018).

Isolated mostly from soils and as plant-associated bacteria, phenotypically, this group includes motile, aerobic and non-pigmented rod-shaped bacteria with gram-negative cell wall type. At the time of writing (January 25, 2023), the genus Agrobacterium comprises seventeen validated non-synonymous species and six non-validated species in the List of Prokaryotic names with Standing in Nomenclature, LPSN (Parte et al., 2020).

In the present work, we characterise two strains belonging to a novel species of the genus Agrobacterium. The first strain, LMG 31532 (R-72433), was isolated from roots of wild-grown Alkanna tinctoria (L.) plants collected in Northern Greece (Rat et al., 2021). Later, we found a related strain based on 16S rRNA gene similarity, LMG 31531T (R-31762), previously isolated in our laboratory from a soil sample in Melle, Belgium (Verbaendert et al., 2011). In this study, we used a polyphasic and comparative genomic approach to characterise the aforementioned strains taxonomically. We concluded that they represent a novel species for which the name Agrobacterium divergens sp. nov. is proposed.