For the pathogen isolation, 20 samples of necrotic buds from diseased twigs were taken. Small pieces of tissue were taken from the margins of necrotic lesions and were first disinfected with 70% ethanol, then cut and crushed using a mortar and pestle in sterile phosphate-buffered saline (0.27% Na₂HPO₄, 0.04% NaH₂PO₄, 0.8% NaCl). After 20 min, the suspensions were plated on King’s medium B (King et al., 1954). After 2 days of incubation at 26°C, a total of 43 fluorescent colonies were selected and purified on Sucrose Nutrient Agar (SNA) (Difco nutrient agar, supplemented with 0.5% sucrose; Difco Laboratories, Detroit, MI, USA) and maintained at −80°C in Luria Bertani medium supplemented with 20% glycerol (Bertani, 1951). The strains CFBP 11 (P. syringae pv. syringae), CFBP 1582 (P. syringae pv. syringae), and CFBP 2119 (P. syringae pv. morsprunorum), were used as reference strains (Collection Francaise de Bacteries Phytopathogenes [CFBP], Angers, France).

Pathogenicity testing of the obtained isolates were checked by artificial inoculation of unripe green lemon (Citrus limon) fruits, bean pods (Phaseolus vulgaris) and leaves of mandarin and liliac (Syringa vulgaris) using the methods described by Lelliott and Stead (1987) and Klement (1990). Each experiment was repeated twice for all tested isolates. The bacterial cells were grown for 2 days on SNA at 25°C, and bacterial suspensions were prepared in sterile distilled water (SDW) and photometrically adjusted to the final concentration of 10⁸ cfu/ml. Control fruits and leaves were also included in tests, but treated with reference strains and SDW as positive and negative controls, respectively.

The lemons were surface sterilized by being dipped into 2% NaClO solution for 2 min and then shortly rinsed with SDW. Four punctures were made on the fruits (1 mm wide, 3 mm deep), by using a sterile hypodermic needle and per 20 μl of the bacterial suspension (10⁶ cfu/ml) was placed on each rupture. Bean pods were inoculated by injection of a bacterial suspension (10⁷ cfu/ml) into young bean pods with a hypodermic syringe. Inoculated fruits and pods were kept in plastic bags and incubated at 25°C for 7 days under conditions of high humidity (70-80%). For each isolate, 12 sites involving three fruits/pods were inoculated. Pathogenic reactions were assessed seven days after inoculation (DAI).

Lilac (Syringa vulgaris cv. Sensation) and mandarin (cv. Owari) were ruptured with a sterile hypodermic needle by pricking the leaf midrib and into areas on the wing of the petiole of fully expanded leaves, and inoculated with a 20 μl drop of bacterial suspension (10⁶ cfu/ml) deposited on a newly made rupture. Inoculated leaves and the petioles were placed in plastic bags and kept under room temperature conditions and high humidity (80%). For each tested isolate and controls, five different leaves and petioles were inoculated. Pathogenic reactions were assessed 14 DAI.

In addition, twigs of mandarin trees were longitudinally ruptured (1 cm) using a sterile scalpel, and per 100 μl of the bacterial suspension (10⁶ cfu/ml) was placed onto the cuts. Twigs were kept in clear polyethylene bags for 24 h under room temperature conditions and high humidity (70%-80%). For each tested isolate and controls, three stems were inoculated. Symptoms were assessed 28 DAI.

Bacterial isolates and reference strains were tested for LOPAT (levan production, oxidase reaction, potato soft rot, arginine dihydrolase activity, and tobacco hypersensitivity) tests (Lelliott and Stead, 1987). Levan production was estimated by growing isolates on SNA medium. For oxidase reaction test, bacteria were grown on SNA medium for 24 h at 25°C until colonies were obtained. Colonies were picked with a loop and rubbed on filter-paper impregnated with 1% (w/v) aqueous tetramethyl-p-phenylene diamine dihydrochloride solution. Production of a purple colour within 10 s was recorded as positive, its development in 10 to 60 s as delayed positive, and the absence of coloration or its still later development as negative (Kovacs, 1956). Pectinase activity was tested by adding bacterial suspension (10⁶ cfu/ml) onto the potato slices and incubated for 24 h at 25°C. Each isolate was tested for arginine dihydrolase production by the method of Thornley (Thornley, 1960), by growing in medium with L-arginine. They were then covered with a 5-mm layer of sterile liquid paraffin, tightly capped, and incubated at 25°C for 7 days. A positive test for the presence of arginine dihydrolase was indicated by a deep pink to red color, whereas a negative test was demonstrated by an absence of color change in the medium. Tobacco hypersensitivity, was tested on leaves of the tobacco. Inoculum were prepared in SDW as described for the pathogenicity test. Reaction to Gram stain, oxiditive/fermentative metabolism of glucose (O/F) and fluorescenent pigment production on the King’s medium B, were also tested (Lelliott and Stead, 1987; Schaad et al., 2001). In addition, GATT tests (Gelatine and Aesculine hydrolysis, formation of Tyrosinase activity and Tartrate metabolism) for the differentiation within the species P. syringae were carried out (Kaluzna et al., 2012). Gelatin liquefaction was done by growing bacteria in tubes containing 12% (w/v) gelatin, which were incubated up to 15 days at 25°C and then held at 4°C for 15 min before determining liquefaction. β-Glucosidase activity of tested isolates was estimated by growing isolates on 0.1% (w/v) aesculin agar medium and incubating four days at 25°C. Positive reaction is dark brown color of the medium after incubation. Tyrosinase activity of isolates were estimated on agar medium containing 0.1% (w/v) L-tyrosine by incubating 4 days at 25°C. A positive test is red dark brown color of the medium after incubation. Utilization of tartarate as a sole carbon source on Simmons basal medium (Holding and Collee, 1971) containing 0.2% (w/v) sodium tartarate. After 21 days of ingubation at 25°C if reaction is positive medium turn color from green to blue.

Total genomic DNA was extracted using a modification of the procedure of Ausubel et al. (1992). All tested 43 Montenegrian mandarin isolates and P. syringae reference strains were grown on SNA solid medium for 48 h at 25°C. Bacterial cells were washed in 1.5 ml of SDW and centrifuged at 4,000g for 10 min at 4°C. The resulting pellet was washed twice in 500 μl 0.85% NaCl, recentrifuged and washed once in 0.1 M NaPO₄ buffer, pH 6.8. Cells were treated with 10% sodium dodecyl sulfate (SDS) and with proteinase K at 37°C for 1 h. DNA was purified by adding 100 μl 5 M NaCl and 80 μl solution of 10% hexadecyltrimethyl ammonium bromide (CTAB) in 1 M NaCl at 65°C for 10 min extracted with an equal volume of chloroform and centrifuged for 10 min at 8,000g. The aqueous phase was transferred to a new tube and precipitated by adding 0.6 vol of isopropanol. The DNA was then washed with 70% ethanol and re-dissolved in 100 μl Tris-EDTA (TE; 10 mM Tris, 1 mM EDTA, pH 8.0), and quantified spectrophotometrically at 260 nm.

In order to detect the possible presence of the syrB gene in all tested isolates, the primer pair B1 (5′-CTTTCCGTGGTCTTGATGAGG-3′) and B2 (5′-TCGATTTTGCCGTGATGAGTC-3′) was used. PCR amplification procedure was described by Sorensen (1998). For negative control, template was replaced with the same volume of SDW. Additionally, reference strains of P. syringae pv. syringae (CFBP 11 and CFBP 1582) and P. syringae pv. morsprunorum (CFBP 2119) were tested as templates for PCR amplification. The PCR was programmed for 35 cycles, denaturation at 94°C for 1.5 min, primer annealing at 60°C for 1.5 min, DNA extension for 3.0 min at 72°C and additional extension at 72°C for 10 min. PCR amplifications were performed in a Mastercycler personal model (Eppendorf, Hamburg, Germany).

An enterobacterial repetitive intergenic consensus (ERIC)-PCR with primer pair ERIC1R (5′-ATGTAAGCTCCTGGGGAT TCAC-3′) and ERIC2 (5′-AAGTA AGTGACTGGGGTGAGCG-3′) have been used for differentiation among the isolates (Lupski and Weinstock, 1992). ERIC-PCR conditions were as previously described by de Bruijn (1992). Amplification of PCR was performed with a Mastercycler personal model (Eppendorf) by using the following cycles: 1 initial cycle at 95°C for 7 min; 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 65°C for 8 min with a single final extension cycle at 65°C for 15 min and a final soak at 4°C. The PCR amplifications were performed in triplicate. Amplified PCR products were separated by gel electrophoresis on 1% agarose gels in 0.5× TAE buffer for 1 h at 5 V/cm, stained with 0.05 μl/ml ethidium bromide and visualized under UV illumination. Fingerprints generated from different strains were compared visually.

Amplicons for the partial 16S rDNA sequences were generated using universal primers (5′-GAGAGTTTGATCCTGGCTCAG-3′) and P6 (5′-CTACGGCTACCTTGTTACGA-3′) (Grifoni et al., 1995) and reaction conditions described by Scortichini et al. (2005). PCR products were purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The sequencing was performed by Macrogen Inc. (Seoul, Korea) and the sequences were deposited in the GenBank database. The MEGA6 software package (Tamura et al., 2013) was used to align sequences from forward and reverse strands and generate consensus sequences. A basic local alignment search tool BLAST (Altschul et al., 1997) was used to compare unidentified sequences with sequences in public databases. Neighbor joining (NJ) analyses were performed using 16S rDNA sequences which were assembled and edited using FINCHTV v.1.4.0 (http://www.geospiza.com). Multiple alignments and comparisons with reference strains were performed using CLUSTALW integrated into MEGA6 software (Tamura et al., 2013).

MLSA was performed in order to reveal the relationship between the P. syringae pv. syringae isolates obtained from mandarin and the P. syringae reference strains. Gene fragments of three housekeeping genes such as, gyrB, rpoD, and gap1 coding for DNA gyrase B, sigma factor 70 and glyceraldehyde-3-phosphate dehydrogenase, respectively, were amplified from genomic DNA and sequenced with primers described by Sarkar and Guttman (2004). PCR products were purified with the QIAquick PCR purification kit (Qiagen) following the manufacturer’s instructions. Sequencing was performed by Macrogen Inc. and the sequences were deposited in the GenBank database. Sequences were edited, aligned, and compared with other Pseudomonas sp. sequences as is described in the study given by Sarkar and Guttman (2004).

In order to estimate the evolutionary relation between P. syringae pv. syringae isolates obtained from mandarin and the pathotype reference strains obtained from PAMDB database, phylogenetic trees were built using NJ analyses. Gene sequences were assembled and edited using FINCHTV v.1.4.0 (http://www.geospiza.com). Multiple alignments and comparisons with reference strains for each of the genes were performed using CLUSTAL W integrated into MEGA6 software (Tamura et al., 2013). Pseudomonas fluorescens was used as out-group.

Minimum spanning tree (MST) was generated, where isolates are the nodes of a tree using SplitsTree software (Huson and Bryant, 2006). The method estimates a probability of ancestry for each individual isolate from each of the groups. The individual isolates are assigned to one cluster or jointly to two or more clusters if their genotypes indicate that they were admixed.

During the spring of 2013 and 2014, severe outbreaks of citrus blast were observed in mandarin (cv. Owari) in the regions of Bar and Ulcinj in Montenegro (Fig. 1). Isolations of bacteria from necrotic buds yielded almost pure cultures of whitish, translucent bacterial colonies that were fluorescent on King’s medium B after 48 h of incubation. A total of 43 separate bacterial colonies were selected and purified on SNA for further analysis (Table 1). On SNA, isolates formed large (2-3 mm in diameter), convex (levan positive), cream-whitish, mucoid colonies 2-3 DAI.

P. syringae strains isolated from diseased mandarin tissues induced typical symptoms of P. syringae pv. syringae on an inoculated unripe lemon, appearing as deep, black necrotic pits. On dwarf bean pods, isolates produced water-soaked lesions with reddish margins 3 DAI. In syringe-inoculated lilac leaves, necrotic areas were seen 2-4 DAI. Inoculated petioles showed water-soaked or black areas, which expanded along the mid vein of the leaves, 7-10 DAI. Symptoms on mandarin leaves and petioles were observed 2 DAI in the form of a brown necrotic area at the point of inoculation. Lesions enlarged slightly on leaves until 10 DAI and brown-black necroses were recorded. On inoculated mandarin twigs there was typical and extensive tissue necrosis, while wilting and dieback were observed 4 weeks after inoculation. No symptoms were observed on SDW inoculated plants.

All isolates showed similar biochemical and physiological characteristics that were typical of P. syringae. They were Gram-negative, fluorescent on King’s medium B, showed strictly aerobic metabolism of glucose, produced levan, and none produced oxidase, arginine dehydrolase and pectinase. All bacterial isolates caused a hypersensitive reaction on the tobacco leaves. These reactions correspond to LOPAT Ia group which includes P. syringae pathovars.

All isolates were positive for gelatin and aesculin hydrolysis, but negative for tyrosinase activity and metabolism of tartrate, demonstrating typical characteristics for bacteria P. syringae pv. syringae.

PCR amplification with primers B1 and B2 amplified the specific 752 bp band for the syrB gene in all tested 43 isolates and two reference P. syringae pv. syringae strains, CFBP 11 and CFBP 1582. The strain of P. syringae pv. morsprunorum CFBP 2119 failed to show amplification and hence did not possess the syrB gene.

To determine genetic diversity of the 43 P. syringae pv. syringae isolates, ERIC-PCR genomic fingerprints were generated. DNA banding patterns were identical among tested isolates and P. syringae pv. syringae reference strains, and did not show any genetic diversity among them. Considering the fact that there was no difference among the isolates, a further genetic study was performed with a reduced number of isolates (12 in total) on the basis of the geographic origin of the isolates.

Molecular identification of isolates from mandarin was performed using 16SrDNA gene sequencing and MLSA. The sequence analysis of the conserved 16SrDNA gene deposited in NCBI GenBank database under accession numbers KP099969 to KP099980 (Table 2), confirmed that isolates originated from mandarin belong to P. syringae pv. syringae showing 100% homology with P. syringae pv. syringae strain ICMP 3023 from GenBank (accession number NR_117820).

The sequences generated with three housekeeping genes (gyrB, rpoD, and gap1), were deposited in the GenBank database under accession numbers KP099981 to KP100028 (Table 2). A NJ tree constructed for the concatenated data set showed that the mandarin isolates were grouped together with the following reference strains from the database; P. syringae pv. aceris LMG 2106, P. syringae pv. atrofaciens LMG 5095, P. syringae pv. coryli NCBI 4273, P. syringae pv. japonica MAFF 301072, P. syringae pv. lapsa LMG 2206, P. syringae pv. pisi NCPPB 2585, P. syringae pv. solidagae ICMP 16925, and P. syringae pv. syringae LMG 1247 (Fig. 2).

An MST using the MLSA data using statistical parsimony with split network revealed no ambiguous connections between pathovars and confirmed clustering of testing isolates and reference strains associated within the same genomospecies 1 (Fig. 3). The first cluster (colored in black) included all of our 43 mandarin isolates and members of genomospecies 1. The second cluster included members of genomospecies 2 (colored in blue). The other six clusters present the strains which belong in genomospecies 3, 4 and 6-9 (represented in red, green, light blue, orange, white-green, and white-red, respectively).