Introduction

Plant tumorigenic bacteria belonging to the family Rhizobiaceae are associated with crown gall and cane gall diseases that can affect various plants1,2,3. 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. Lochness4. 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 disease13. 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 Hildebrand13, 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.

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 1078T/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 1078T 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 leguminosarum14 or Rhizobium aegyptiacum, Rhizobium bangladeshense and Rhizobium binae15. 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 85046T located on a neighbouring branch (Fig. 2).

Figure 1
figure 1

Maximum likelihood tree based on partial sequence of 16S rRNA gene (1273 bp) indicates the phylogenetic position of Rhizobium tumorigenes sp. nov. strains 932, 1019, 1078T and 1081 (marked in bold) and their relationship with related members of the Rhizobiaceae family. The tree was constructed using a general time reversible substitution model with a gamma distribution and invariant sites (GTR + G + I). Bootstrap values (expressed as a percentage of 1000 replications) equal to or higher than 60% are shown at nodes. Bradyrhizobium japonicum USDA 6T 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.

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Figure 2
figure 2

Maximum likelihood trees based on partial sequences of atpD – 496 bp (A), recA – 541 bp (B) and rpoB – 585 bp (C) housekeeping genes indicate the phylogenetic position of Rhizobium tumorigenes sp. nov. strains 932, 1019, 1078T and 1081 (marked in bold) and their relationship with related members of the Rhizobiaceae family. The trees were constructed using a general time reversible substitution model with a gamma distribution and invariant sites (GTR + G + I). Bootstrap values (expressed as a percentage of 1000 replications) equal to or higher than 60% are shown at nodes. Bradyrhizobium japonicum USDA 6T was used as the outgroup organism. DDBJ/EMBL/GenBank accession numbers are shown in Table S3. The scale bar represents the estimated number of nucleotide substitutions per site.

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The draft genome sequence of R. tumorigenes 1078T consisted of 5,899,412 bp (129 contigs) with an average coverage of 127.6x. For R. tubonense CCBAU 85046T, the assembly generated 85 contigs comprising of 6,540,512 bp with an average coverage of 131.8x. R. tumorigenes 1078T and R. tubonense CCBAU 85046T had similar average GC contents of 60.0% and 59.3%, respectively, which was generally in accordance with other related Rhizobium spp., e.g. R. rhizogenes ATCC 11325T (59.9%), Rhizobium tropici CIAT 899T (59.9%) or Rhizobium freirei PRF 81T (59.9%).

Genome-wide phylogeny based on 385 conserved proteins further supported distinctiveness of representative strain 1078T and its phylogenetic relationship to R. tubonense CCBAU 85046T (Fig. 3). Furthermore, whole-genome sequences of strain 1078T 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 ANI16 or is 70% for DDH17,18,19, confirming the authenticity of the novel species (Table 1). The ANIb, 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. 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 1078T was compared with R. tubonense CCBAU 85046T, which is a borderline for reliable alignment. Although evidently distantly related, R. tubonense CCBAU 85046T 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.

Figure 3
figure 3

Maximum likelihood phylogenomic tree based on concatenated sequences of 385 conserved proteins extracted from whole-genome sequences showing the evolutionary relationships between Rhizobium tumorigenes sp. nov. 1078T (marked in bold) and related Rhizobiaceae members. Branch support values equal to or higher than 60% are shown at nodes. Bradyrhizobium japonicum USDA 6T was used as the outgroup organism. DDBJ/EMBL/GenBank whole-genome accession numbers are shown in Table S3. The scale bar represents the estimated number of amino acid substitutions per site.

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Table 1 Average nucleotide identity (ANI) and in silico DNA–DNA hybridization (DDH) comparisons between Rhizobium tumorigenes sp. nov.
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The results of phenotypic characterization of novel strains are summarized in Table 2. Unlike R. tubonense CCBAU 85046T, the novel strains from blackberry were able to catabolize L-Alanine and D-Gluconic acid. On the other hand, R. tubonense CCBAU 85046T 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 question20. Moreover, biochemical tests were of limited value for classification and differentiation of some Rhizobiaceae species as indicated by Puławska, et al.21.

Table 2 Protologue for Rhizobium tumorigenes sp. nov.
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The major cellular fatty acids of the four novel strains were: 18:1 w7c (66.11–70.93%), 19:0 cyclo w8c (8.71–12.40%), Summed feature 2 (12:0 aldehyde and/or an unknown fatty acid of equivalent chain length 10.928, and 14:0 3OH/16:1 iso I; 5.88–6.23%) and 16:0 (4.07–5.63%) (Table S2). Comparing to four strains studied, R. tubonense CCBAU 85046T possessed a lower content of fatty acid 18:1 w7c (55.11%), and a higher one of 16:0 (10.65%) and 11 methyl 18:1 w7c (6.72%) (Table S2).

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 1078T 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 1078T (=DSM 104880T = CFBP 8567T) as the type strain. R. tumorigenes sp. nov. is a new plant tumorigenic species containing the Ti plasmid and the second tumorigenic species within the genus Rhizobium. Tumor-inducing ability has been limited so far to Agrobacterium spp., A. vitis and R. rhizogenes.

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

Four strains 932 (=DSM 104878 = CFBP 8566), 1019 (=DSM 104919), 1078T (=DSM 104880T = CFBP 8567T) and 1081 (=DSM 104920) recovered from tumor tissue on thornless blackberry (Rubus sp.), cultivar ‘Čačak Thornless’ were characterized in this study. They were isolated on yeast mannitol agar (YMA)12 from plant samples originating from two localities, Lučani (932 and 1019) and Arilje (1078T and 1081), in western Serbia during 2015–2016. In addition, we also included type strain of R. tubonense CCBAU 85046T for some tests. For molecular methods, total genomic DNA of the strains was extracted from bacteria grown on YMA at 22 °C for 48 h according to the procedure described by Aljanabi and Martinez22.

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 primers24, as described by Kuzmanović, et al.12. Primer sets atpD-273F/771R25, and rpoB-456F/1061R26 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 MgCl2, 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/F289927, as described before12.

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 MUSCLE28 at EMBL-EBI29.

Pairwise nucleotide identities were calculated using the p-distance model with MEGA 7.0.21 software package30. Maximum likelihood (ML) trees were generated with PhyML 3.031 by using 1000 bootstrap replicates. The most suitable substitution models were determined by the Smart Model Selection (SMS) tool32 and jModelTest 2.1.1033, according to the Akaike information criterion (AIC)34.

Genome sequencing

DNA fragmentation was performed on Covaris E210 and libraries were made with NEBNext DNA Library Prep Master Mix Set for Illumina® (NEB, USA). Sequencing was performed on Illumina MiSeq platform using MiSeq Reagent Kit v2 (500-cycles) in PE250 mode generating 3,336,198 (1078T) and 3,784,696 (R. tubonense CCBAU 85046T) sequences in pairs (Genomed SA, Poland). Sequence processing and assembly were performed using CLC Genomics Workbench 7.5.

Whole-genome sequence comparisons and phylogenomic analysis

Genome sequence of strain 1078T was compared with genome sequences of related Rhizobium spp., by calculating average nucleotide identity (ANI) values using the JSpecies Web Service35. In silico DNA–DNA hybridizations (DDH) values by the Genome-to-GenomeDistance Calculator (GGDC 2.1; http://ggdc.dsmz.de/distcalc2.php) using the recommended BLAST + alignment and formula 2 (identities/HSP length)17 were also obtained.

Genome-wide phylogeny based on 385 conserved protein sequences extracted from genome sequences of 1078T and strains of related Rhizobiaceae strains was reconstructed by using PhyloPhlAn pipeline, version 0.9936.

Phenotypic characterization

Novel strains isolated from blackberry, including R. tubonense CCBAU 85046T were phenotypically characterized by using API and Biolog tests. The API 20NE kit was used according to manufacturer’s instructions (bioMérieux) and addition of MgSO4 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 85046T 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.

Detection of tumor-inducing (Ti) plasmid and pathogenicity assay

Bacterial strains isolated from blackberry were subjected to PCR analysis using primers specific for tumor-inducing (Ti) plasmid genes: virC (VCF3/VCR3)37, virD2 (A/C’) and ipt (CYT/CYT’)38, and tms2 (tms2F1/tms2R2)39, as described before12.

Pathogenicity of the novel strains originating from Serbia was studied by inoculating stem internodes of young tomato (Solanum lycopersicum) and kalanchoe (Kalanchoe daigremontiana) plants, and hypocotyls of sunflower (Helianthus annuus) seedlings, as described before40.

Accession numbers

The DDBJ/EMBL/GenBank accession numbers for the partial 16S rRNA gene sequences of the strains 1081, 1078T, 1019 and 932 are MG018988-MG018991, respectively. Accession numbers for the partial atpD gene sequences of the strains R. tubonense CCBAU 85046T, 1019, 1078T, 1081 and 932 are MG007662-MG007666, respectively. Accession numbers for the partial recA gene sequences of the strains of the strains R. tubonense CCBAU 85046T, 1019, 1078T, 1081 and 932 are MG007667-MG007671, respectively. Accession numbers for the partial rpoB gene sequences of the strains R. tubonense CCBAU 85046T, 1019, 1078T, 1081 and 932 are MG007672-MG007676, respectively.

The draft genome sequences of R. tumorigenes 1078T and R. tubonense CCBAU 85046T have been deposited at DDBJ/EMBL/GenBank under the accession numbers PCDQ00000000 and PCDP00000000, respectively.