Agrobacterium cucumeris sp. nov. isolated from crazy roots on cucumber (Cucumis sativus)

Agrobacteria are a group of microorganisms naturally occurring in soil and they can be either pathogenic or non-pathogenic. Their pathogenic properties result from the presence of the tumor-inducing (Ti) or the root-inducing (Ri) plasmid. As a result of integration of the fragment of Ti and Ri plasmids in host plant cells, crown gall disease or crazy (i.e. hairy) root disease may appear, respectively, in many plant species (Bosmans et al., 2015, Pulawska, 2010). Both diseases lead to significant yield losses, and therefore reduce the profitability of crop production (Campillo et al., 2012).

The taxonomy of the genus Agrobacterium has been constantly changing since Conn (Conn, 1942) created it and classified it in the family Rhizobiaceae (Conn, 1938). The differentiation of the newly discovered species was initially based on the comparison of their phenotypic features (Allen et al., 1974) and pathogenic properties, however, it has been proven that the pathogenicity is plasmid-borne (Genetello et al., 1977) which means that this feature can be lost or transferred to other bacteria (Kerr, 1969). The process of discovering more agrobacteria from various sources led to the separation of biotypes (biovars) based on biochemical and physiological tests (Keane et al., 1970, Kerr and Panagopoulos, 1977, Kersters and De Ley, 1984). Molecular methods based on DNA sequence analysis began to be increasingly used and allowed for more precise determination of the taxonomic affiliation of microorganisms. In this context, particular attention was paid to the usefulness of the 16S rRNA sequences (Woese, 1987), nonetheless pointing to some limitations, especially at the level of discrimination among species (Puławska and Kałużna, 2012, Stackebrandt and Goebel, 1994).

In 2001, Young et. al. proposed to amalgamate Agrobacterium, Allorhizobium and Rhizobium into genus Rhizobium, based on the results of 16S rDNA sequence similarity (Sawada et al., 1993, Young et al., 2001), fatty acid profiles and biochemical features among members of the mentioned genera (Young et al., 2001). Nevertheless, the taxonomic status of these genera was reconsidered when their classification, based mainly on phenotypic traits, DNA-DNA hybridization (DDH) and 16S rRNA sequence analysis, was supplemented by the results of MLSA analysis of both individual housekeeping genes and concatenated sequence analyses of rrs, atpD, recA and rpoB genes (Mousavi et al., 2015). Currently (October 2022), the genus Agrobacterium includes 14 validly described species and 8 species with not validly published names yet (LPSN, https://www.bacterio.net).

In our previous study (Warabieda et al., 2021), from the diseased cucumber plants with symptoms of crazy roots, strains belonging to genera Agrobacterium, Rhizobium and Pararhizobium.were isolated. Among them, 8 rhizogenic Agrobacterium strains were isolated on MG + Te (Mougel et al., 2001) and 1A + 2E (Puławska and Sobiczewski, 2005) media. All of them caused crazy roots in pathogenicity tests on sunflowers. In order to determine the presence of the rol and vir genes on the pathogenicity-related plasmid (Ri plasmid), PCR amplification tests were performed and their results showed that all Agrobacterium strains were positive towards their possession of these genes (Warabieda et al., 2021). Vir genes are involved in the transfer of the T-DNA (transferred DNA) to the plant cell, which is essential for infection and next disease symptoms development (Otten, 2018), whereas rol genes are responsible for increase of the sensitivity of plants to auxins that participate in development and growth processes in plant life cycles (Shen et al., 1988). All obtained Agrobacterium strains possessed identical recA sequences and the phylogenetic analysis based on the sequence of this gene indicated their separate phylogenetic lineage, which suggested that they could belong to a new species (Warabieda et al., 2021).

In this work, we studied 3 non-clonal Agrobacterium strains: O132T, O115 and O34 and our aim was to determine and characterize their phylogenetic status and describe them as a new Agrobacterium species.

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