Introduction

Tumorigenic agrobacteria affect various fruit species and are responsible for crown gall disease that may cause significant economic losses in orchards and nurseries (Puławska 2010). Tumor formation on crown and roots of the host plant is a typical symptom of the disease. Pathogenicity of the strains is mainly determined by the presence of conjugative tumor-inducing (Ti) plasmid in their genome (Van Larebeke et al. 1974; Kerr et al. 1977).

Taxonomy of the genus Agrobacterium is debatable and still not fully resolved. Although Young et al. (2001) proposed inclusion of all Agrobacterium species into the genus Rhizobium, this taxonomy revision was disputed and not widely accepted (Farrand et al. 2003). The transfer of Agrobacterium rhizogenes (biovar 2) to genus Rhizobium was subsequently supported, while the rest of the members remained within Agrobacterium genus (Lindström and Young 2011). However, a formal proposal with a definition or the new borders of genus Agrobacterium has not yet been published. Since Agrobacterium tumefaciens (biovar 1) is not a homogenous species but composed of at least 11 genomic species (G1 to G9, G13 and G14), it was proposed that they should be collectively called the A. tumefaciens species complex until being formally named (Costechareyre et al. 2010; Lindström and Young 2011). So far, only genomic species G2, G4 and G14 were formally named as Rhizobium pusense (Panday et al. 2011), Agrobacterium radiobacter (Conn 1942) and Rhizobium nepotum (Puławska et al. 2012a), respectively. Despite the proposal of name “Agrobacterium fabrum” for genomic species G8 (Lassalle et al. 2011), its standing in nomenclature still pending (http://www.bacterio.net/).

A. tumefaciens genomic species can be clearly delineated by genotypic-based methods: DNA-DNA hybridizations (De Ley 1974; De Ley et al. 1973; Popoff et al. 1984), amplified fragment length polymorphism (AFLP) (Portier et al. 2006; Mougel et al. 2002), sequence analysis of housekeeping recA gene (Costechareyre et al. 2010) and recA-based PCR approach (Shams et al. 2013). Although 16S rRNA gene sequencing may be suitable for identification of Agrobacterium species, it lacks the resolution power to discriminate among genomic species of A. tumefaciens complex (Mougel et al. 2002).

Raspberry is a natural host of tumorigenic Agrobacterium and Rhizobium strains. The disease may be particularly serious on certain cultivars (Burr et al. 1993). While the Agrobacterium rubi is recognized as causal agent of cane gall disease of Rubus spp. (Hildebrand 1940), tumorigenic strains of A. tumefaciens species complex (Alippi et al. 2012; Milijašević et al. 2007) and Rhizobium rhizogenes (Weller et al. 2004; Burr et al. 1993; Hobolth 1973; Peluso et al. 2003; Süle 1978) were predominantly isolated from raspberry showing crown gall symptoms. Moreover, nonpathogenic strain belonging to recently described species R. nepotum (Puławska et al. 2012a) was also recovered from galled raspberry.

Serbia is one of the world’s leading raspberry producers (96,078 t in 2012 [http://faostat3.fao.org/]) and exporters. Raspberry production and export is of a strategic importance for agriculture in this country. During the last 3 years, high incidence of crown gall disease was recorded in some young raspberry plantations throughout Serbia. Infected plants showed stunted growth and significantly decreased yield. Newly introduced cultivars Polka and Tulameen were associated with disease.

Therefore, in this study we isolated and identified tumorigenic bacteria from diseased plants originating from six localities throughout Serbia and investigated their pathogenic properties. Furthermore, we evaluated genetic diversity of isolated strains and their Ti plasmids by using PCR, PCR-RFLP and ERIC-PCR methods. Phylogenetic position of representative strains was elucidated by sequence analysis of 16S rDNA and recA housekeeping gene. Although Milijašević et al. (2007) identified A. tumefaciens as causal agent of the disease in Western Serbia, assessment of genotypic variation among tumorigenic strains occurring on raspberry in Serbia has not been performed so far.

Materials and methods

Bacterial strains

A total of 16 Agrobacterium strains isolated from raspberry, including 14 from Serbia and two from Poland, were used in this study (Table 1). Strains from Serbia were isolated from tumor tissue of diseased plants collected in six localities during 2011–2013. Nonselective yeast mannitol agar (YMA; 10 g l−1 mannitol, 1 g l−1 yeast extract, 1 g l−1 CaCO3, 0.1 g l−1 NaCl, 0.5 g l−1 K2HPO4, 0.2 g l−1 MgSO4 · 7H2O, 18 g l−1 agar; pH 7.2) and/or selective MG agar medium amended with tellurite (K2TeO3; 70 μg ml−1) (Mougel et al. 2001) were used for isolations. Plates were incubated at 27 °C for 3–5 (isolation on YMA) or 4–7 days (isolation on MG agar medium amended with tellurite).

Table 1 Strains of Agrobacterium and Rhizobium spp. used in this study
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Bacterial colonies resembling Agrobacterium spp. phenotype were purified and maintained on YMA medium for further testing. Two strains from Poland were previously identified as tumorigenic A. tumefaciens and R. rhizogenes (J. Puławska, unpublished data) (Table 1). In addition to Agrobacterium strains isolated from raspberry, ten reference strains of Agrobacterium spp. and Rhizobium spp. were also used in this study (Table 1). For DNA extraction, bacteria were grown on King’s medium B (King et al. 1954) at 27 °C for 24–48 h, to reduce production of polysaccharides.

Pathogenicity tests

Pathogenicity of the strains was determined by inoculating carrot root discs, young tomato (Lycopersicon esculentum L.) plants and sunflower (Helianthus annuum L.) seedlings. One-year old raspberry plants (cv. Meeker), as a natural host, were inoculated with five representative strains. Four carrot root discs were inoculated with each strain by pipetting 100 μl of bacterial suspension (approx. 106 CFU/ml) on the abaxial side of the disc (Moore et al. 2001). Tomato and sunflower plants were inoculated as described before (Kuzmanović et al. 2014). Potted raspberry plants were inoculated in stem internodes following the same procedure as for tomato and sunflower. Three plants were inoculated per strain. Inoculated plants were maintained in a greenhouse at 24 ± 3 °C. Tumor formation was scored during 3–4 weeks (carrot, tomato, and sunflower) or 2 months (raspberry) after inoculation.

Physiological and biochemical properties

The strains were characterized using following physiological and biochemical tests: oxidase reaction, growth in 2 % NaCl and at 35 °C, 3-ketolactose production, acid clearing on PDA amended with CaCO3, ferric ammonium citrate test, motility at pH 7.0, citrate utilization, production of acid from meso-erythritol (erythritol) and D-(+)-melezitose monohydrate (melezitose), and alkali from L-(+)-tartaric acid disodium salt (tartrate) (Moore et al. 2001).

DNA extraction

Genomic DNA was isolated from bacterial suspensions (approx. 108 CFU/ml) using the DNeasy plant mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA quality was checked by agarose gel electrophoresis. The DNA samples were diluted to 10–20 ng/μl and stored at −20 °C for further analysis.

PCR analysis

Bacterial strains isolated from raspberry were analyzed by PCR using primers specific for plasmid virC (VCF3/VCR3) (Suzaki et al. 2004), virD2 (A/C’) and ipt (CYT/CYT’) (Haas et al. 1995), and tms2 (tms2F1/tms2R2) (Puławska and Sobiczewski 2005) genes, as well as chromosomal 23S rRNA gene (UF/B1R/B2R/AvR/ArR) (Puławska et al. 2006). The primers specific for virD2 and ipt genes, and 23S rRNA gene-specific primers were used in duplex (Haas et al. 1995) and multiplex PCR (Puławska et al. 2006) reactions, respectively.

PCR amplifications was also conducted with primer pair F749 (Mougel et al. 2001) and F14 (aka vir-14 or FGPvirG15’) (Picard et al. 1992) designed to amplify the intergenic region between virB11 and virG genes of Ti plasmid. This primer pair amplifies 432 bp product from nopaline-type (e.g. pTiC58 and pTi-SAKURA), or 384 bp product from octopine-type (e.g. pTiAch5 and pTi15955) and agropine/mannopine-type (e.g. pTiBo542) Ti plasmids, as determined by NCBI Primer-Blast tool (Ye et al. 2012). The opine-type of pathogenic plasmid was further examined with primers derived from octopine-type (ocsF/ocsR) and nopaline-type (RBF/RBR) Ti plasmids (Tan et al. 2003), and virF gene of octopine-type Ti plasmid (virFF1/virFR2) (Bini et al. 2008) used in separate PCR reactions.

PCR amplifications were carried out using a 2720 Thermal Cycler (Applied Biosystem, Foster City, CA, USA). The reactions were performed in 15 μl mixtures and contained 1× DreamTaq Green Buffer (Thermo Scientific, Vilnius, Lithuania), 200 μM of each dNTP, 0.5 μM of each primer, 0.3 U of DreamTaq DNA polymerase (Thermo Scientific, Vilnius, Lithuania) or Taq DNA polymerase (Invitrogen, Sao Paulo, Brazil), and 2 μl of template DNA. The thermal profiles were as described in the original publications, except for PCR amplifications with primers VCF3/VCR3 and F749/F14. The thermal profile used for PCR with primer pair VCF3/VCR3 was as follows: initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and elongation at 72 °C for 1 min, and final extension at 72 °C for 7 min. With primer pair F749/F14 thermal cycling conditions were programmed as described by Rhouma et al. (2006). The PCR products were resolved by electrophoresis in 1.5 % (w/v) agarose gel in 1× TAE buffer. The gels were stained in ethidium bromide solution (1 μg/ml) and the amplicons were visualized under UV light.

PCR-RFLP analysis of Ti plasmid virA-virB2 region

The vir region (virA-virB2 genes) of Ti plasmids was further analyzed by PCR-RFLP method. PCR was carried out by using primer pair FGPvirA2275/FGPvirB2164 amplifying 1673 bp fragment from nopaline-type Ti plasmids (Ponsonnet and Nesme 1994). The reaction mixtures (25 μl) contained 1× DreamTaq Buffer (Thermo Scientific, Vilnius, Lithuania), 200 μM of each dNTP, 0.4 μM of each primer, 10 % of dimethyl sulfoxide (DMSO), 0.6 U of DreamTaq DNA polymerase (Thermo Scientific, Vilnius, Lithuania) and 2 μl of template DNA. The thermal profile was as follows: initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 1 min, annealing at 62 °C for 1 min, and elongation at 72 °C for 1.5 min, and final extension at 72 °C for 5 min.

PCR products were digested with restriction endonuclease HhaI (CfoI) (Thermo Scientific, Vilnius, Lithuania), as recommended by the manufacturer. Restriction fragments obtained after 4 h of digestion were separated in 2.5 % agarose gel at 80 V for 5 h, stained with ethidium bromide and visualized under UV light.

ERIC-PCR

ERIC-PCR fingerprinting (Versalovic et al. 1991) was used to asses genetic diversity among strains studied. The PCR mixtures (25 μl) contained 1× PCR buffer (Invitrogen, Sao Paulo, Brazil), 1.5 mM MgCl2, 200 μM of each dNTP, 2 μM of each primer (ERIC1R and ERIC2), 0.16 mg/ml of bovine serum albumin, 10 % of DMSO, 2 U of Taq DNA polymerase (Invitrogen, Sao Paulo, Brazil) and 2 μl of template DNA. The thermal profile was as described previously by Rademaker and De Bruijn (Rademaker and De Bruijn 1997). Electrophoresis was performed in a 1.5 % (w/v) agarose gel in 1× TAE buffer at 60 V for 30 min and 75 V for 5 h. After the run, gels were stained in ethidium bromide solution and photographed under UV light.

Sequence analysis 16S rRNA and recA genes

Representative strains were selected for phylogenetic analysis of the 16S rRNA and recA housekeeping gene sequences. Region of 16S rRNA gene was amplified with primers fD1 and rP2 (Weisburg et al. 1991). The PCR reactions (50 μl) contained 1× DreamTaq Buffer (Thermo Scientific, Vilnius, Lithuania), 200 μM of each dNTP, 0.2 μM of each primer, 1.5 U of DreamTaq DNA polymerase (Thermo Scientific, Vilnius, Lithuania) and 3 μl of template DNA. The thermal profile was as described above for amplifying virA-virB2 region, except that an annealing temperature of 55 °C was used. The recA gene sequences were amplified by using primers F2898 and F2899 (Costechareyre et al. 2010; Shams et al. 2013). The reaction mixtures (50 μl) contained 1× DreamTaq Buffer (Thermo Scientific, Vilnius, Lithuania), 200 μM of each dNTP, 0.5 μM of each primer, 5 % of DMSO, 1 U of DreamTaq DNA polymerase (Thermo Scientific, Vilnius, Lithuania) and 3 μl of template DNA. The thermal profile was as described by Costechareyre et al. (2010).

The sequencing of PCR-amplified partial 16S rRNA gene fragments was performed using the same primers as for amplifications, while the recA PCR products were sequenced with standard T7 and T3 primers by Macrogen Europe (The Netherlands). The obtained sequences were compared with those in the NCBI database (October 2014) by using the BLASTn algorithm (Altschul et al. 1997). The sequence alignment was conducted using ClustalW algorithm implemented in MEGA version 6 software package (Tamura et al. 2013). Reference sequences of related Agrobacterium and Rhizobium species were also included. Phylogenetic analysis was carried out by maximum likelihood (ML) method using the raxmlGUI 1.3 (Silvestro and Michalak 2012) and the GTR + G model (general time-reversible model with gamma-distributed rates) of evolution with 100 thorough bootstraps.

Results

Identification of the isolated strains

Bacterial colonies phenotypically resembling Agrobacterium spp. were isolated on YMA and MG agar medium amended with tellurite. In order to differentiate pathogenic ones, isolated strains were subsequently subjected to PCR analysis using VCF3/VCR3 primers specific for Ti/Ri plasmid virC gene. Out of 46 tested strains, 36 were determined to be phytopathogenic. A total of 14 strains were selected for further study (Table 1). In duplex PCR using virD2 and ipt gene-specific primers, two specific DNA fragments were amplified in all tested strains from raspberry. Likewise, strains were also positive in PCR with primers specific for tms2 gene and intergenic region between virB11 and virG genes of Ti plasmid. Overall, based on PCR analysis, Ti plasmid was detected in 14 tested strains originating from raspberry.

In order to differentiate strains to the species level, we performed multiplex PCR assay targeting 23S rRNA gene sequences. Twelve strains isolated in Serbia were identified as R. rhizogenes, one strain as A. tumefaciens, whereas the remaining one gave two amplification products specific for both these species. The strains identified by PCR as A. tumefaciens and R. rhizogenes exhibited bacteriological properties typical for these species, while the atypical strain KFB 330 showed features of A. tumefaciens, except for positive reaction in citrate utilization test (Table 2).

Table 2 Bacteriological characteristics of strains isolated from raspberry compared to reference strains of Agrobacterium and Rhizobium spp
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Pathogenic properties of the strains

All strains studied induced typical tumors on carrot root discs, young tomato plants and sunflower seedlings. Five representative strains KFB 323, KFB 330, KFB 337, MAL 1.1.2 and MAL 1.1.4 that were tested for tumorigenicity by inoculation of raspberry plants caused characteristic symptoms at the inoculation sites. Control strains of A. radiobacter (B6) and A. vitis (K309T) were tumorigenic on carrot root discs, young tomato plants and sunflower seedlings, while the strains of A. tumefaciens genomic species G8 (C58) and A. rubi (ATCC 13335T) induced crown gall symptoms at stem internodes of raspberry. The plants inoculated with nonpathogenic strain of R. nepotum (39/7T) and SDW developed no symptoms.

Characterization of Ti plasmid

The Ti plasmid of the strains originating from raspberry was characterized by PCR analysis targeting sequences specific for nopaline-type, octopine-type and agropine/mannopine-type Ti plasmids. In PCR using primer pair F749/F14 the product of 432 bp was amplified in all tested strains from raspberry, suggesting they carry nopaline-type of Ti plasmid. Furthermore, out of 16 strains, specific PCR products were amplified from 15 strains when primers specific for nopaline-type Ti plasmid (RBF/RBR) were used. The remaining strain KFB 324 gave a weak positive PCR signal with coamplification of multiple nonspecific fragments. Specific amplification products were not obtained when primers specific for octopine-type Ti plasmid (ocsF/ocsR) were used. Overall, Ti plasmid of all strains studied from raspberry was classified as nopaline-type (Table 1).

Based on PCR-RFLP analysis of Ti plasmid virA-virB2 region two different restriction profiles (N1 and N2) were found (Table 1). The genetic group N1 included 11 strains belonging to R. rhizogenes and A. tumefaciens species complex. The remaining five R. rhizogenes strains were characterized by N2 PCR-RFLP profile (Table 1). The PCR-RFLP groups were not associated with geographical origin of the strains or raspberry cultivars from which they were isolated.

ERIC-PCR genotyping

The genetic profiles generated by ERIC-PCR allowed differentiation of the strains and provided insight into their genetic diversity (Fig. 1). A total of nine unique ERIC-PCR patterns were identified among the strains studied (Table 1, Fig. 1). Although strains of R. rhizogenes exhibited similar genetic profiles, they were differentiated into six distinct genetic groups (Table 1, Fig. 1). On the other hand, A. tumefaciens strains KFB 337 and MAL 1.1.4, and atypical strain KFB 330 exhibited distinguishably different genetic profiles (Fig. 1). Associations between the strains’ ERIC-PCR profile and geographic origin or raspberry cultivars from which they were isolated were not found. Strains belonging to genetic groups III, V and VI were isolated from different geographic areas (Table 1).

Fig. 1
figure 1

ERIC-PCR patterns of the strains isolated from raspberry. The different ERIC-PCR patterns are indicated by Roman numerals above the lanes. Strain designations are indicated above the respective lanes. Lanes M, molecular size marker GeneRuler DNA Ladder Mix (#SM0333; Thermo Scientific, Vilnius, Lithuania)

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16S rRNA and recA phylogeny

The representative strains KFB 323, KFB 330, KFB 337, MAL 1.1.2 and MAL 1.1.4 were further characterized by partial sequencing of their 16S rDNA and recA housekeeping gene. Phylogenetic analysis of the 16S rRNA gene sequences (1304 bp for R. rhizogenes and 1308 bp for A. tumefaciens species complex) confirmed identity of strains KFB 323 and KFB 337 assigned as R. rhizogenes and A. tumefaciens, respectively (Fig. 2). Atypical strain KFB 330 was closely related to species belonging to A. tumefaciens complex (Fig. 2). Its closest relatives were type strains of R. nepotum (99.8 % sequence identity, two SNPs) and A. radiobacter (99.3 %, nine SNPs).

Fig. 2
figure 2

Maximum likelihood tree based on 16S rRNA gene sequence indicating phylogenetic relationships of strains studied (marked in bold) and related members of the Rhizobiaceae family. Bootstrap values ≥ 70 are shown at nodes. The scale bar represents the number of substitutions per site. DDBJ/EMBL/GenBank accession numbers are shown in parentheses. Ochrobactrum anthropi (strain LMG 3331T) is used as the outgroup organism

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In the phylogenetic tree based on recA gene sequences (869 bp) the strains of R. rhizogenes (KFB 323 and MAL 1.1.2) were grouped with type strain of this species (Fig. 3). Furthermore, the recA sequence analysis revealed that strains MAL 1.1.4 and KFB 337 belong to A. tumefaciens genomic species G1 and G8, respectively. A total of 21 different recA alleles have been so far described among genomic species G1, whereas six unique alleles have been found within genomic species G8 (Costechareyre et al. 2010; Shams et al. 2013; Lamovšek et al. 2014). The strain MAL 1.1.4 possessed a recA-G1-15 allele, which was found among nonpathogenic strains isolated from agricultural soils in Slovenia (Lamovšek et al. 2014). On the other hand, novel allele named recA-G8-7 was identified in strain KFB 337. BLASTn searches on NCBI database failed to find sequences identical to one of strain KFB 337.

Fig. 3
figure 3

Maximum likelihood tree based on recA gene sequence indicating phylogenetic relationships of strains studied (marked in bold) and related members of Agrobacterium sp. and Rhizobium sp. Bootstrap values ≥ 70 are shown at nodes. The scale bar represents the number of substitutions per site. Allele codes for particular species/genomic species of A. tumefaciens complex are given. DDBJ/EMBL/GenBank accession numbers are shown in parentheses. Bradyrhizobium japonicum (strain USDA 6T) is used as the outgroup organism

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Atypical strain KFB 330 clustered within a clade of A. tumefaciens species complex (Fig. 3). However, this strain formed separate phylogenetic lineage, different from all known genomic species (Fig 3). This strain was closely related to genomic species G1 and had the highest level of identity (96.9 %) to the sequence of nonpathogenic strain CFBP 5622 isolated from rhizosphere of Solanum nigrum in France. Moreover, based on BLASTn searches, we were unable to find sequences more closely related than 96.9 %.

Discussion

Insights into the genetic diversity of phytopathogenic Agrobacterium spp. and Rhizobium spp. may be particularly important for better understanding of the crown gall epidemiology and ecology of the disease causal agent. The aim of this study was to characterize and differentiate tumorigenic bacteria from raspberry in Serbia. The crown gall has been already recorded on raspberry in this country (Milijašević et al. 2007), but also on other Rubus sp. such as blackberry (Arsenijević 1989). However, during the last 3 years, crown gall disease was also observed in some young raspberry plantations suffering considerable losses.

Our results revealed extensive genetic diversity of tumorigenic bacteria associated with crown gall of raspberry in Serbia, although a limited number of strains were analyzed. At least two different species were identified as causal agents of the disease. Out of 14 strains isolated from the tumors, 12 were identified as R. rhizogenes, whereas the remaining two (KFB 330 and KFB 337) belonged to A. tumefaciens species complex (Table 1).

The high diversity of agrobacteria was observed within the one locality in western Serbia where strains of R. rhizogenes (KFB 328, KFB 329 and KFB 334), A. tumefaciens genomic species G8 (KFB 337) and atypical strain belonging to A. tumefaciens species complex (KFB 330) were isolated from the same raspberry plantation. Similarly, strains of A. tumefaciens and R. rhizogenes were also isolated from the same forest or stone fruit nursery, but also from a single tumor (Nesme et al. 1987; Nesme et al. 1992; Kuzmanović et al. 2013).

On the other hand, strains studied were generally homogenous with respect to opine-type of Ti plasmid, since they all harbored nopaline-type of Ti plasmid. Additionally, all strains showed identical pathogenic properties in pathogenicity test on several test plants. Nevertheless, they were differentiated into two genetic groups based on PCR-RFLP analysis of Ti plasmid virA-virB2 region. The fact that strains belonging to different species or genomic species originating from same plantation (KFB 328, KFB 329, KFB 330 and KFB 337) share the same Ti plasmid genotype suggests that these Ti plasmids are transferable between distantly related Agrobacterium and Rhizobium strains in nature, confirming the previous findings (Nesme et al. 1992; Michel et al. 1990).

The strains belonging to different taxonomic entities of Agrobacterium sp. and Rhizobium sp. exhibited substantially different ERIC-PCR profiles (Fig. 1). However, when analyzing intraspecies diversity of ERIC-PCR patterns, strains of R. rhizogenes showed similar genetic profiles (Fig. 1). They were divided into six genetic groups (Table 1, Fig. 1). Since genetic groups III, V and VI were composed of strains originating from different geographic areas, it can be assumed that they have a common origin and were probably disseminated by movement of infected plant material. It is likely that the pathogen systemically colonizes the raspberry plants and that may be latently present within propagation material. Systemic nature has been already proven for different agrobacteria in various plant species (Tarbah and Goodman 1987; Jones and Raju 1988; Cubero et al. 2006; Yakabe et al. 2012; Zoina et al. 2001).

By recA gene sequence analysis we confirmed identity of strains studied and differentiated those belonging to A. tumefaciens species complex (Table 1, Fig. 3). In addition, we also found novel allele (recA-G8-7) within genomic species G8, which increases the known diversity of this genomic species. Until now, total of 70 different alleles were found within A. tumefaciens species complex (Costechareyre et al. 2010; Lamovšek et al. 2014; Shams et al. 2013). Further assessment of allelic diversity among agrobacteria will contribute to the more efficient epidemiological surveillance and crown gall outbreak investigations.

Although strain KFB 330 was identified as a member of A. tumefaciens species complex, it had some atypical properties and therefore was not fully identified. In multiplex PCR targeting 23S rRNA gene sequences, it gave two amplification products specific for both A. tumefaciens and R. rhizogenes. This feature was also recorded for strains 39/7, 7/1 and 0 (Puławska et al. 2006), later described as novel species within A. tumefaciens species complex ‐ R. nepotum (Puławska et al. 2012a). However, biochemical and physiological tests as well as sequence analysis of recA housekeeping gene showed that KFB 330 is not a member of R. nepotum, although this strain was clustered with type strain of this species by phylogenetic analysis of 16S rRNA gene sequences (Fig. 2). The phylogenetic position of atypical strain remained unclear since it formed separate phylogenetic lineage, different from all known genomic species of A. tumefaciens species complex (Fig 3).

Morphological, physiological and biochemical methods are the oldest tools for studying prokaryotes, but still essential for the characterization and classification of bacteria (Tindall et al. 2010). When we performed set of differential bacteriological tests, atypical strain KFB 330 generally exhibited characteristics of A. tumefaciens, apart from positive reaction in citrate utilization test (Table 2). Phenotypic variations among strains of A. tumefaciens have been already recorded in literature (Bouzar and Moore 1987; du Plessis et al. 1984; Holmes and Roberts 1981; Süle 1978). Presently, most of the genomic species of A. tumefaciens complex are not formally named since they still cannot be differentiated by clear and stable discriminative phenotypic traits. However, R. pusense (genomic species G2), R. radiobacter (G4) and R. nepotum (G14) are phenotypically distinguishable by physiological and biochemical tests (Puławska et al. 2012a; Panday et al. 2011). In addition, ability to degrade ferulic acid and caffeic acid is reported as specific characteristic feature of genomic species G8 (“A. fabrum”) (Shams et al. 2012; Lassalle et al. 2011).

In summary, our results revealed the existence of a high degree of genetic variation among pathogenic Agrobacterium and Rhizobium strains isolated from tumor tissue. The data presented in this paper highlight the importance of crown gall bacteria on raspberry and contribute to taxonomic studies of agrobacteria.