Rhizobium tumorigenes sp. nov., a novel plant tumorigenic bacterium isolated from cane gall tumors on thornless blackberry

Four plant tumorigenic strains 932, 1019, 1078T and 1081 isolated from cane gall tumors on thornless blackberry (Rubus sp.) were characterized. They shared low sequence identity with related Rhizobium spp. based on comparisons of 16S rRNA gene (≤98%) and housekeeping genes atpD, recA and rpoB (<90%). Phylogenetic analysis indicated that the strains studied represent a novel species within the genus Rhizobium, with Rhizobium tubonense CCBAU 85046T as their closest relative. Furthermore, obtained average nucleotide identity (ANI) and in silico DNA–DNA hybridization (DDH) values calculated for whole-genome sequences of strain 1078T and related Rhizobium spp. confirmed the authenticity of the novel species. The ANI-Blast (ANIb), ANI-MUMmer (ANIm) and in silico DDH values between strain 1078T and most closely related R. tubonense CCBAU 85046T were 76.17%, 84.11% and 21.3%, respectively. The novel species can be distinguished from R. tubonense based on phenotypic and chemotaxonomic properties. Here, we demonstrated that four strains studied represent a novel species of the genus Rhizobium, for which the name Rhizobium tumorigenes sp. nov. is proposed (type strain 1078T = DSM 104880T = CFBP 8567T). R. tumorigenes is a new plant tumorigenic species carrying the tumor-inducing (Ti) plasmid.

Plant tumorigenic bacteria belonging to the family Rhizobiaceae are associated with crown gall and cane gall diseases that can affect various plants [1][2][3] . The presence of a large conjugal tumor-inducing (Ti) plasmid in the genome of the host strain is essential for pathogenicity. So far, tumorigenic strains have been identified within multiple species of the genus Agrobacterium, as well as within species Allorhizobium vitis (i.e. Agrobacterium biovar 3/Agrobacterium vitis) and Rhizobium rhizogenes (i.e. Agrobacterium biovar 2/Agrobacterium rhizogenes).
Rubus spp. have been identified as natural hosts of tumorigenic Rhizobiaceae strains. Crown gall disease that was mostly associated with tumorigenic strains of R. rhizogenes and A. tumefaciens species complex (i.e. Agrobacterium biovar 1/Agrobactrium tumefaciens), including recently described species Agrobacterium arsenijevicii has been frequently reported on Rubus spp. [4][5][6][7][8][9][10][11][12] . In general, crown gall disease symptoms include formation of tumors on roots and crowns of infected plants. In addition, tumorigenic R. rhizogenes strains were also isolated from aerial tumors formed at pruning wounds of blackberry-raspberry (Rubus occidentalis-Rubus idaeus) hybrid of cv. Lochness 4 . On the other hand, cane gall disease is characterized by formation of tumors on the cane surface that may increase in size and number and completely girdle affected cane sections in advanced stages of disease 13 . Although Agrobacterium rubi was initially recognized as a causal agent of cane gall disease of Rubus spp. 13 , later reports on this disease are limited or entirely lacking.
In this study, we observed plants of thornless blackberry (Rubus sp.) showing cane gall symptoms corresponding to those described before by Hildebrand 13 , that originated from two plantations in western Serbia. Although disease developed repeatedly every year, it was not lethal for infected blackberry plants nor caused significant losses in yield. Here, we characterized atypical tumorigenic strains isolated from cane gall tumors by using a polyphasic taxonomic approach and demonstrated that they represent a novel tumorigenic species within the genus Rhizobium. Bootstrap values (expressed as a percentage of 1000 replications) equal to or higher than 60% are shown at nodes. Bradyrhizobium japonicum USDA 6 T was used as the outgroup organism. DDBJ/EMBL/GenBank accession numbers are given in Table S3. The scale bar represents the estimated number of nucleotide substitutions per site.

Results and Discussion
Four atypical strains isolated from thornless blackberry showing cane gall symptoms, originating from two localities in western Serbia, were characterized by using polyphasic taxonomic methods. The strains studied possessed identical 16S rRNA gene sequences (calculated for the length of 1309 bp). Furthermore, strains originating from the same locality (932/1019 and 1078 T /1081) possessed identical sequences of atpD, recA and rpoB housekeeping genes. On the other hand, strains 932 and 1019 had high sequence identities (>97.5%) with strains 1078 T and 1081 based on analysis of partial sequences of atpD (496 bp), recA (541 bp) and rpoB (585 bp) housekeeping genes (Table S1), suggesting that they are closely related and belong to the same species. The strains exhibited different PCR MP fingerprints (Fig. S1), which excluded the possibility of their clonal origin. However, strains originating from the same locality showed similar fingerprints, differing by several bands (Fig. S1).
The strains studied shared 16S rRNA gene sequence identity ≤98% with related Rhizobium spp. (Table S1). It is notably low value, taking into account 16S rRNA gene sequence identities between related Rhizobium species being above 99%, and in some cases even 100%, as it was shown, for example, for Rhizobium laguerreae and Rhizobium leguminosarum 14 or Rhizobium aegyptiacum, Rhizobium bangladeshense and Rhizobium binae 15 . Moreover, nucleotide identity values were remarkably low (<90%) when comparing atpD, recA and rpoB gene sequences of novel strains and related species (Table S1).
Based on 16S rRNA gene phylogeny, strains studied were grouped within the genus Rhizobium, however, they formed a separate cluster, with Rhizobium tubonense as their closest relative (Fig. 1). For further phylogenetic analysis, we selected species closely related to novel strains and included representative members of the Rhizobiaceae family. Phylogenetic trees generated by using partial sequences of atpD, recA and rpoB genes confirmed independent clustering of the novel strains with R. tubonense CCBAU 85046 T located on a neighbouring branch (Fig. 2).
Genome-wide phylogeny based on 385 conserved proteins further supported distinctiveness of representative strain 1078 T and its phylogenetic relationship to R. tubonense CCBAU 85046 T (Fig. 3). Furthermore, whole-genome sequences of strain 1078 T and related Rhizobium spp. were compared by using ANI-Blast (ANIb), ANI-MUMmer (ANIm) and in silico DDH methods. Obtained values were far below the proposed threshold for species delineation, which ranges between 95-96% for ANI 16 or is 70% for DDH [17][18][19] , confirming the authenticity of the novel species (Table 1). The ANIb, ANIm and in silico DDH values between strain 1078 T and most closely related R. tubonense CCBAU 85046 T were 76.17%, 84.11% and 21.3%, respectively. In case of ANIm, less than 20% of the genome was aligned for all strains used for comparison, and the alignment was assigned by the software as suspicious. However, besides other strains when it was below 15%, almost 20% (19.11%) of the genome was aligned when strain 1078 T was compared with R. tubonense CCBAU 85046 T , which is a borderline for reliable alignment. Although evidently distantly related, R. tubonense CCBAU 85046 T was considered as a closest known relative of novel strains isolated from blackberry, with respect to their phylogenetic, phylogenomic and genomic relatedness. Therefore, phenotypic and chemotaxonomic characterization was performed in order to determine additional traits distinguishing these two species.
The results of phenotypic characterization of novel strains are summarized in Table 2. Unlike R. tubonense CCBAU 85046 T , the novel strains from blackberry were able to catabolize L-Alanine and D-Gluconic acid. On the other hand, R. tubonense CCBAU 85046 T utilized L-Lactic acid, contrary to the novel strains studied. However, many genes encoding transport and catabolism of carbon and nitrogen compounds can be plasmid-borne, and therefore, the role of phenotypic tests in taxonomy of Rhizobium spp. has been recently called into question 20 . Moreover, biochemical tests were of limited value for classification and differentiation of some Rhizobiaceae species as indicated by Puławska, et al. 21 .
By using PCR, presence of virC, virD2, ipt and tms2 genes was detected in all four strains studied, indicating that they carry the Ti plasmid required for plant tumorigenic ability. In pathogenicity assay, all strains caused tumors on inoculated sunflower seedlings and kalanchoe plants. In contrast to strains 1078 T and 1081 which clearly induced tumors on kalanchoe stems, tumors induced by strains 932 and 1019 were smaller, which could suggest differences in the virulence of the strains. In case of tomato, the reaction of plants was variable, since strains caused either very small and inconspicuous tumors, or symptom development was absent.
Overall, based on the polyphasic characterization of the four strains isolated from cane gall tumors on thornless blackberry, we propose that they represent a novel species, Rhizobium tumorigenes sp. nov., with 1078 T The new species is registered at Digital Protologue website the (http://imedea.uib-csic.es/dprotologue/) under the taxonumber TA00285. The description of the new species is given in Table 2.

Materials and Methods
Bacterial strains and DNA extraction. PCR melting profile (PCR MP) fingerprinting. Genetic diversity among four novel strains was investigated by a method of PCR melting profile (PCR MP) with two sets of restriction enzymes, adaptors and primers: ApaI and HindIII as described by Puławska, et al. 23 . Denaturation temperatures 91 °C and 89 °C were used for PCR MP with ApaI and HindIII enzymes, respectively. PCR amplification and sequencing of 16S rRNA and housekeeping genes. The amplification and sequencing of nearly complete 16S rRNA gene was performed by using fD1 and rP2 primers 24 , as described by Kuzmanović,et al. 12 . Primer sets atpD-273F/771R 25 , and rpoB-456F/1061R 26 were used for amplification and sequencing of atpD and rpoB gene fragments, respectively. PCR reactions were performed in a 25 µl volume with master mix containing 1 × Colourless GoTaq Flexi buffer (Promega Corp., USA), 1.5 mmol l −1 MgCl 2 , 0.2 mmol l −1 of each dNTP, 0.2 µmol l −1 of each primer, 0.5 U of GoTaq Flexi DNA polymerase (Promega Corp., USA) and 40-60 ng of DNA template. The thermal profile for amplification of atpD gene fragment was as described by Gaunt,et al. 25 , except that total of 35 cycles with annealing temperature of 60 °C, followed by final extension at 72 °C for 5 min were used. For amplification of rpoB gene fragment, the PCR conditions were as follows: initial denaturation at 95 °C for 5 min; 35 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min and extension at 72 °C for 1 min. A final extension at 72 °C for 5 min was conducted. The amplification and sequencing of recA gene fragment was performed by using primers F2898/F2899 27 , as described before 12 . Gene sequence comparison and phylogenetic analysis. The phylogenetic analysis and sequence comparisons were conducted on 16S rRNA gene sequence, and sequences of atpD, recA and rpoB housekeeping genes. Sequences of related Rhizobiaceae strains were retrieved from NCBI GenBank and included into the analysis (Table S3). The obtained sequences were aligned using MUSCLE 28 at EMBL-EBI 29 .
Pairwise nucleotide identities were calculated using the p-distance model with MEGA 7.0.21 software package 30 . Maximum likelihood (ML) trees were generated with PhyML 3.0 31 by using 1000 bootstrap replicates. The most suitable substitution models were determined by the Smart Model Selection (SMS) tool 32 and jModelTest 2.1.10 33 , according to the Akaike information criterion (AIC) 34 .    Phenotypic characterization. Novel strains isolated from blackberry, including R. tubonense CCBAU 85046 T were phenotypically characterized by using API and Biolog tests. The API 20NE kit was used according to manufacturer's instructions (bioMérieux) and addition of MgSO 4 in order to improve bacterial growth as described before by Saidi, et al. 14 . Utilization of sole carbon sources was tested with Biolog GEN III microplates by using protocol C2 according to the instructions of the manufacturer (Biolog, Inc., Hayward, CA, USA). Measurements were taken after incubation of API strips and Biolog microplates at 20 °C for 72 h.
Chemotaxonomic analysis. Analysis of cellular fatty acid composition of the novel strains isolated from blackberry, including R. tubonense CCBAU 85046 T was performed by the Microbial Identification System (Sherlock version 6.1, TSBA40 method), as recommended by the manufacturer. Since the bacteria did not grow well on standard trypticase soy agar (TSA) medium, they were cultured on YMA at 22 °C for 36 h.