Genetic Diversity and Virulence of Xanthomonas Campestris Pv

Plant Pathology (2019) 68, 1448–1457 Doi: 10.1111/ppa.13064

Genetic diversity and virulence of Xanthomonas campestris pv. campestris isolates from Brassica napus and six Brassica oleracea crops in Serbia

T. Popovica* , P. Mitrovicb, A. Jelusi cc, I. Dimkicd, A. Marjanovic-Jeromela b, I. Nikolicd and S. Stankovicd aInstitute for Plant Protection and Environment, Teodora Drajzera 9,11040 Belgrade; bInstitute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad; cInstitute for Multidisciplinary Research, Kneza Viseslava 1,11000; and dUniversity of Belgrade – Faculty of Biology, Studentski Trg 16,11000 Belgrade, Serbia

The present study provides insight into the diversity of 147 Xanthomonas campestris pv. campestris (Xcc) isolates obtained from six Brassica oleracea vegetable crops (broccoli, cabbage, cauliflower, collard greens, kale, kohlrabi) and the winter oilseed rape crop Brassica napus, collected from different regions in Serbia in 2014. The XCF/XCR pathovar-specific primer set was used for fast preliminary identification. In repetitive sequence-based PCR (BOX, ERIC and REP) of all isolates, a higher level of genetic diversity was found in winter oilseed rape isolates compared to isolates from the other hosts. ERIC and REP-PCR showed the highest heterogeneity, with 10 and nine banding patterns, respectively. The REP-PCR results showed the highest correlation (70%) with those obtained with multilocus sequence analysis (MLSA), performed with 10 housekeeping genes (fusA, gap-1, gltA, gyrB1, lacF, lepA, rpoD, dnaK, fyuA and gyrB2). Three distinct phylogenetic groups of winter oilseed rape isolates were detected using MLSA. Two genes, gltA and rpoD, showed the greatest ability to identify and discriminate winter oilseed rape Xcc isolates from isolates of the other six hosts. The lepA gene exhibited specific three-nucleotide changes in sequences of some of the isolates. Results of virulence testing of 18 representative isolates showed statistically significant host–pathogen specialization for Xcc isolates from winter oilseed rape, cauliflower, kale and kohlrabi. In conclusion, oilseed rape isolates are more genetically diverse and show greater specialization to their host in comparison to the rest of the tested isolates from other brassica hosts.

Keywords: black rot, brassicas, multilocus sequence typing, repetitive element PCR

Introduction vegetables have undeniable economic significance, both around the world and in Serbia, with cabbage being the Black rot of crucifers, caused by the plant pathogenic most common representative. It should be noted that bacteria Xanthomonas campestris pv. campestris (Xcc), Xcc was first found in central Serbia during the 1960s is a serious and destructive disease which is gradually on forage kale crops (B. oleracea var. sabellica), where spreading worldwide (Popovic et al., 2013a, 2013b, its effects caused up to 80% crop loss (Perisic & Panic, 2014; Singh et al., 2016). The pathogen is known to 1964). In later years, Xcc started causing damage on affect all cultivated brassica crops in Serbia that belong other cultivated crucifers – cabbage, cauliflower and kale to two Brassica species, viz. B. oleracea: vegetable crops, (Jovanovic et al., 1997; Obradovic & Arsenijevic, 1999). with the varieties capitata (cabbage), botrytis (cauli- The first appearance on winter oilseed rape (B. napus) flower), italica (broccoli), sabauda (kale), gongylodes was reported in 2010 (Popovic et al., 2013b). (kohlrabi) and acephala (collard greens); and B. napus: So far, 11 Xcc races (1–11) have been determined on oil seed crop. Typical symptoms of this disease include the basis of interactions with differential cultivars (Cruz characteristic V-shaped necrotic lesions at the foliar mar- et al., 2017). In view of the importance of identifying gins and vein blackening, which may lead in the final this plant pathogenic bacterium at the species, pathovar stages to substantial yield and economic losses. Brassica or race level, many genotyping methods have lately been proposed that are superior to classical methods of identification. Investigators have developed Xcc-specific primer sets (Park et al., 2004; Berg et al., 2005; Singh et al., *E-mail: [email protected] 2016) and race 3-specific molecular markers (Afrin et al., 2018) for fast molecular identification. The most com- monly used techniques for accurate identification and Published online 5 July 2019 typing of Xanthomonas isolates at species and subspecies

1448 ª 2019 British Society for Plant Pathology Diversity of Xanthomonas campestris 1449

level include DNA fingerprinting techniques based on the glucose, nitrate reduction, H2S and indole production, and amplification of repetitive elements, such as REP (repeti- starch, gelatin and esculin hydrolysis (Lelliott & Stead, 1987). tive extragenic palindromic), ERIC (enterobacterial repet- Table 1 presents a list of the selected isolates, their host plants itive intergenic consensus) and BOX (Rademaker et al., and the isolation localities. The Xcc NCPPB 1144 strain isolated 1998; Singh et al., 2016), as well as multilocus sequence from B. oleracea var. capitata (cabbage) was used as a reference strain for comparison in all tests. typing (MLST) and multilocus sequence analysis (MLSA) The highly sensitive and accurate XCF/XCR pathovar-specific using two schemes, both using different housekeeping primer set (Park et al., 2004) was used for molecular identifica- genes: fusA (elongation factor 4), gap-1 (glyceraldehyde- tion of the 147 isolates obtained (Table S1). For DNA extrac- À 3-phosphate dehydrogenase A), gltA (citrate synthase), tion, bacterial suspensions (106 CFU mL 1) of isolates were gyrB1 (DNA gyrase B), lacF (PTS system lactose-specific heated to 95 °C for 10 min in a water bath and cooled on ice. EIIA component) and lepA (elongation factor 4) The debris was pelleted by centrifugation for 5 min at 7600 g. (Almeida et al., 2010), or rpoD (RNA polymerase sigma Supernatants were used for amplification and routinely stored at factor RpoD), dnaK (chaperone protein DnaK), fyuA À20 °C (Dashti et al., 2009). µ (TonB dependent receptor) and gyrB2 (Young et al., The PCR amplification was performed using 12.5 L Dream- 2008). A combination of these methods is the most effec- Taq Green PCR Master Mix (Thermo Fisher Scientific), mixed with 1 µL of sample DNA, 1 µL of each of the primers (10 µM) tive way to identify bacteria and assess genetic diversity, and 9.5 µL of ultrapure DNase/RNase-free water (Gibco), to although they all have advantages and limitations in obtain a total reaction volume of 25 lL. terms of the process of application, prices of the reagents PCRs consisted of an initial denaturation for 5 min at 94 °C; needed, reproducibility and levels of phylogenetic and then 35 cycles of 15 s at 94 °C, 15 s at 58 °C and 30 s at taxonomic resolution (Rademaker et al., 1998). 72 °C; and a final extension for 5 min at 72 °C (Park et al., Because the presence of Xcc on cultivated brassicas 2004). Amplified PCR products were electrophoresed in a 1% (broccoli, cabbage, cauliflower, collard greens, kale, agarose gel, stained with ethidium bromide and checked for the kohlrabi and winter oilseed rape) has not been studied in presence of a specific band at 535 bp. detail so far in Serbia, the main objective of this study was to isolate and characterize Xcc isolates obtained Genotyping of Xanthomonas campestris pv. campestris from crucifers from different regions on the basis of their pathogenic and genetic features. Genomic DNA extraction For this purpose, genomic DNA from all 147 tested Xcc isolates and the reference strain NCPPB 1144 was extracted using the Materials and methods CTAB extraction method of Le Marrec et al. (2000) modified by Dimkic et al. (2013). Pure cultures of all isolates were grown ° Sample collection and pathogen isolation on YDC agar at 26 C for 48 h. Single bacterial colonies were suspended in 500 µL SDW and centrifuged at 10 000 g for Brassica vegetables (broccoli, cabbage, cauliflower, collard 10 min. The pellet obtained was resuspended in TE buffer, and greens, kale, kohlrabi) and winter oilseed rape plants with visi- incubated with a mix of 10% (w/v) sodium dodecyl sulphate À ble black rot symptoms on leaves were collected in northern and (SDS) and 20 mg mL 1 proteinase K in nuclease-free water at central regions of Serbia during the period from August to Octo- 37 °C for 30 min. Samples were then treated with 100 µLof ber 2014. In total, seven localities with 15 brassica fields 5 M NaCl and heated at 65 °C for 20 min after the addition of (Table 1) were visited and 10 diseased plants per field were 3% hexadecyltrimethyl ammonium bromide (CTAB, pH 8.0). taken for pathogen isolation. Diseased leaves from each host The DNA was purified with 750 µL chloroform and centrifuged were first washed in tap water and dried at room temperature for 10 min at 10 000 g. The top (aqueous) phase was recovered on filter paper. Small leaf sections were cut from the margin of by adding isopropanol and centrifugation at 10 000 g for necrotic and healthy leaf tissue, immersed in sterile distilled 15 min. At the final phase, the obtained pellet was washed with water (SDW) and macerated. Nutrient agar (NA) was used for 1 mL of 96% ice-cold ethanol, centrifuged at 10 000 g for isolation and preliminary distinguishing of Xcc colonies from 10 min and dried at room temperature for 30 min. The pelleted saprophytes. Yellow, translucent, circular and raised colonies DNA was dissolved in 50 µL TE buffer (50 mM Tris, pH 8, that developed after 48 h of incubation at 26 °C were trans- 1mM EDTA) and stored at À20 °C. ferred to yeast extract-dextrose-calcium carbonate (YDC) agar and incubated at 26 °C for 72 h. Plates were observed for the Repetitive element PCR fingerprinting presence of characteristic pale yellowish, convex and mucoid The method of repetitive element PCR (rep-PCR) fingerprinting bacterial colonies. One hundred and forty-seven isolates with was used to determine genetic diversity among the 147 Xcc iso- the typical Xcc colony morphology were selected for further lates originating from six brassica vegetables and winter oilseed study. Isolates were kept as stock cultures in sterile Luria Ber- rape collected from seven localities in two Serbian regions. In this tani (LB) broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) study, rep-PCR fingerprinting was performed using BOX containing 20% (v/v) sterile glycerol and stored at À20 °C. Iso- (BOXA1R), ERIC (ERIC1R/ERIC2) and REP (REP1R-I/REP2-I) lates from oilseed rape will be deposited in the National Collec- oligonucleotide primers (Louws et al., 1994; Versalovic et al., tion of Plant Pathogenic Bacteria (NCPPB), UK. 1994). Primer sequences are shown in Table S1. The DNA purity and concentrations for all tested isolates were measured with a NanoDrop spectrophotometer and equalized. All PCRs were per- Preliminary identification formed according to a modified procedure of Louws et al. (1994) Isolates were tested for Gram-staining, the Kovacs oxidase reac- and Versalovic et al. (1994). A total of 25 µL PCR mix consisted tion, catalase production, oxidative/fermentative metabolism of of: 1 µL of sample total DNA, 2.5 µLof109 KAPA Taq Buffer

Plant Pathology (2019) 68, 1448–1457 1450 T. Popovic et al.

Table 1 Bacterial isolates originated from brassicas and rep-PCR groups.

Isolation Rep-PCR

Isolate code Host Cultivar Locality BOX ERIC REP

Xc6 Winter oilseed rape Banacanka Kovilj I I I Xc9, Xc10 Winter oilseed rape Banacanka Kovilj II II II Xc11-Xc13 Winter oilseed rape Banacanka Kovilj I I I Xc16-Xc18 Winter oilseed rape Banacanka Kovilj I I I Xc19 Winter oilseed rape Banacanka Kovilj III III III Xc20 Winter oilseed rape Unknown Novi Sad III IV III Xc21, Xc22 Winter oilseed rape Unknown Novi Sad III I II Xc23 Winter oilseed rape Unknown Novi Sad III V IV Xc24 Winter oilseed rape Unknown Novi Sad III IV III Xc25 Winter oilseed rape Unknown Novi Sad III I III Xc26 Winter oilseed rape Unknown Novi Sad III IV II Xc27 Winter oilseed rape Unknown Novi Sad I VI V Xc28 Winter oilseed rape Unknown Novi Sad III IV I Xc29 Winter oilseed rape Unknown Novi Sad III I III Xc30 Winter oilseed rape Unknown Novi Sad IV IV III Xc31 Winter oilseed rape Unknown Novi Sad V III VI Xc32 Winter oilseed rape Unknown Novi Sad I VI V Xc33 Winter oilseed rape Unknown Novi Sad III IV III Xc34 Winter oilseed rape Unknown Novi Sad III IV VI Xc35 Cabbage Futoski Futog VI VII VI Xc36–Xc39 Cabbage Futoski Futog VI VII VII Xc40–Xc42 Cabbage Futoski Futog VI VII VII Xc43–Xc47 Cabbage Hybrid Futog VI VII VII Xc48 Cabbage Hybrid Futog VI VIII VII Xc49, Xc50 Cabbage Hybrid Futog VI VII VII Xc51–Xc56 Cabbage Unknown Temerin VI VII VII Xc57–Xc59 Cabbage Unknown Sabac VI VII VII Xc60 Cabbage Unknown Sabac VI VIII VII Xc61, Xc62 Cabbage Unknown Sabac VI VII VII Xc63–Xc70 Cabbage Unknown Vrnjacka Banja VI VII VII Xc71–Xc74 Cabbage Unknown Cacak VI VII VII Xc75–Xc78 Cabbage Unknown Cacak VI VII VII Xc101–Xc110 Cauliflower Unknown Futog VI VII VII Xc111 Cauliflower Unknown Temerin VI VII VIII Xc112–Xc116 Cauliflower Unknown Temerin VI VII IX Xc117 Kale Unknown Temerin VI VII IX Xc118, Xc119 Kale Unknown Temerin VI VII VIII Xc120–Xc122 Kale Unknown Temerin VI VII VIII Xc123–Xc128 Kale Unknown Temerin VI VII VII Xc129 Kale Clause Futog VI VII VII Xc130 Kale Clause Futog VI VII IX Xc131 Kale Clause Futog VI VII IX Xc132, Xc133 Kale Clause Futog VI VII VII Xc134, Xc135 Kale Clause Futog VI VII IX Xc136, Xc137 Kale Clause Futog VI VII VIII Xc138, Xc140 Kale Clause Futog VI VII IX Xc141 Collard greens Unknown Temerin VI VII IX Xc142 Collard greens Unknown Temerin VI VII VIII Xc143 Collard greens Unknown Temerin VI VII IX Xc144, Xc145 Collard greens Unknown Temerin VI VII VIII Xc146-Xc149 Collard greens Unknown Temerin VI VII IX Xc150, Xc151 Collard greens Unknown Temerin VI VII IX Xc152 Collard greens Unknown Temerin VI VII VIII Xc153-Xc159 Broccoli Unknown Temerin VI VII IX Xc160, Xc161 Broccoli Unknown Temerin VI VII IX Xc162-Xc170 Broccoli Sakata Futog VI VII VII Xc171 Kohlrabi Unknown Vrnjacka Banja VI VII VII

(continued)

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Table 1 (continued)

Isolation Rep-PCR

Isolate code Host Cultivar Locality BOX ERIC REP

Xc172 Kohlrabi Unknown Vrnjacka Banja VI VII VIII Xc173 Kohlrabi Unknown Vrnjacka Banja VI II VIII Xc174, Xc175 Kohlrabi Unknown Vrnjacka Banja VI VII VIII Xc176, Xc177 Kohlrabi Unknown Vrnjacka Banja VI VII VIII Xc178 Kohlrabi Unknown Vrnjacka Banja VI IX IX NCPPB 1144a Cabbage Unknown UK VI X IX

Isolates marked in bold represent 18 isolates selected for further molecular characterization and virulence determination. aReference strain NCPPB 1144 is used as a positive control in all tests.

B and 0.5 µLof10mM dNTP mixture (KAPA Biosystems); 2 µL extension cycle at 72 °C for 10 min. The PCR products were (10 µM) of one of the three primer sets; 16.8 µL (ERIC and REP purified using the QIAquick Gel Extraction kit and QIAquick/ PCR) or 18.8 µL (BOX PCR) of ultrapure DNase/RNase-free 250 PCR Purification kit (QIAGEN) and sequenced in both À water; and 0.2 µLof(5UµL 1) KAPA Taq polymerase (KAPA directions by the Macrogen sequencing service (Netherlands) Biosystems). using the same primers that were used for the amplification. Amplified PCR products (5 µL) mixed with 1.5 µLofThermo The partial coding nucleotide sequences for all 10 genes were Scientific 6 9 DNA-loading dye were separated using gel elec- aligned using the automated CLUSTALW alignment feature of the trophoresis on 1% agarose gel with 0.5 9 TBE buffer BIOEDIT v. 7.0.5 program. All sequences were manually checked (5 9 TBE: 1.1 M Tris; 900 mM borate; 25 mM EDTA; pH 8.3) for quality and trimmed to the following lengths: fusA (663 À and ethidium bromide (0.5 lgmL 1). Gel electrophoresis was per- nt), gap1 (774 nt), gltA (547 nt), gyrB1 (477 nt), lacA (507 formed for 2.5 h under a constant voltage of 90 V and amperage nt), lepA (841 nt), gyrB2 (774 nt), rpoD (778 nt), fyuA (631 of 300 mA. The surveyed distance of DNA fingerprints was nt) and dnaK (631 nt). The trimmed sequences were compared checked in relation to a 100 bp DNA ladder (Nippon Genetics Eur- with those available in the NCBI (National Centre for Biotech- ope GmbH). Differences in band position and the level of genetic nology Information) database using the nucleotide BLAST and similarity between the obtained profiles were computed using REST- deposited with accession numbers listed in Table S2. Individual DIST and NEIGHBOUR functions of the PHYLIP 3.69 program (Felsen- phylogenetic trees were constructed in MEGA 7 for each of the stein, 2005). The unweighted pair-group method with an sequenced genes and rooted with X. campestris pv. vesicatoria arithmetic mean (UPGMA) was used to construct a phylogenetic (CP017190) from the NCBI database as an outgroup strain, tree with the NJPLOT tree-drawing program (Perriere & Gouy, using the neighbour-joining clustering method. Concatenated 1996). sequences with a total size of 6623 nt were also constructed to simultaneously compare all the selected isolates, the reference Multilocus sequence analysis strain and an outgroup strain on the basis of all 10 used genes. MLSA was performed with 18 representative Xcc isolates (Xc6, In both cases, genetic distances were computed using the Xc9, Xc20, Xc23, Xc27, Xc30, Xc31, Xc32, Xc40, Xc48, Kimura two-parameter nucleotide substitution model (Kimura, Xc75, Xc110, Xc120, Xc150, Xc160, Xc170, Xc175, Xc178) 1980). selected on the basis of rep-PCR patterns obtained, and the reference strain NCPPB 1144. Two MLSA schemes have been Pathogenicity used, with six housekeeping genes: fusA, gap-1, gltA, gyrB1, lacF and lepA according to Almeida et al. (2010); and four Pathogenicity tests were carried out by spraying a bacterial sus- genes rpoD, dnaK, fyuA and gyrB2 given by Young et al. pension on the surface of leaves (Popovic et al., 2013b, 2014) of

(2008). The list of primer pair sequences for these genes is the seven tested brassica plants – broccoli hybrid F1 cv. Maraton shown in Table S1. (Sakata), and domestic cultivars of cabbage (Futoski), kale The PCR amplification was performed in a total reaction (Gvozdena Glava), cauliflower (Snezna Grudva), collard greens volume of 25 lL, containing 12.5 µL of DreamTaq Green PCR (unknown), kohlrabi (Backa Bela) and winter oilseed rape Master Mix (Thermo Fisher Scientific), 1 µL of sample total (Banacanka) obtained from the Institute of Field and Vegetable DNA, 1 µL of each of the primers (10 µM) and 9.5 µLof Crops (Novi Sad, Serbia). Plants were at the 2–3 true leaves ultrapure DNase/RNase-free water. Cycling programmes for stage (3-week-old plants). fusA, gap-1, gltA, gyrB1, lacF and lepA primers were per- The seeds used in the tests were not chemically treated or dis- formed according to Kyeon et al. (2016), starting with an ini- infected, and so to avoid any potential contamination from tial denaturation step at 94 °C for 5 min; followed by 35 seedborne pathogens (mostly fungi) that could affect seed ger- cycles of denaturation at 94 °C for 30 s, annealing at 58 °C mination and development, seeds were first sterilized by soaking for 30 s and polymerization at 72 °C for 30 s; with a final in a 3% sodium hypochlorite (NaOCl) solution for 3–5 min, extension cycle at 72 °C for 7 min. The remaining four genes washed in tap water and then dried at room temperature. (rpoD, dnaK, fyuA and gyrB2) were amplified according to the Potato dextrose agar (PDA) was used to test the success of dis- method described by Young et al. (2008). The initial denatura- infection. Plates with a few seeds were incubated at 25 Æ 1 °C tion step was performed at 94 °C for 3 min; followed by 30 for 15 days in the dark to check disinfection success; untreated cycles of denaturation at 94 °C for 30 s, annealing at 54 °C seeds were used as a control treatment. After 15 days, plates for 30 s and polymerization at 72 °C for 1 min; with a final with developed seedlings were checked for their health status

Plant Pathology (2019) 68, 1448–1457 1452 T. Popovic et al.

using a stereomicroscope (Carl Zeiss Jena). After checking seed sterility, young and healthy seedlings were sown in plastic Statistical analysis boxes containing 66 wells (11 9 6), each measuring 4 9 5cm Basic statistical parameters were calculated and are presented in in size, filled with a sterile growth substrate. The experiment the tables. The Kolmogorov–Smirnov test of normality was used was conducted in a completely randomized design in three for analysis of variance, as was Levene’s test for homogeneity of replicates, with six host plants per replicate for each of the variance. The data obtained were subjected to analysis of vari- seven hosts (a total of 18 plants for each tested host). A total ance (ANOVA), and separation of mean values of plant tissue of 18 representative isolates, the same as used for molecular necrosis in planta was accomplished by Tukey’s HSD (honest tests, and reference strain NCPPB 1144, were checked for their significant difference) test. Values were considered significant at pathogenicity. Inoculation was made by spraying plants with P < 0.05 for all tests. Statistical analyses were conducted by the 8 À1 the bacterial suspension (10 CFU mL of a 48 h YDC culture general procedures of STATISTICA v. 7 (StatSoft, Inc.) and SPSS in SDW). SDW was used as a negative control treatment, and STATISTICS v. 20 (SPSS, Inc.). plants treated with reference strain Xcc NCPPB 1144 were used as a positive control. After the inoculation, plants were kept under the plastic cover for the first 24 h to maintain RH at 90– Results 100%. After 24 h and until the end of the experiment, the plastic cover was removed, and RH was reduced to c.50–60%. Identification of X. campestris pv. campestris During the whole experiment, controlled conditions in the greenhouse were kept at 30 Æ 2 °C (day), and 20 Æ 2 °C All 147 isolates showed only one type of colony mor- (night), with a 12 h photoperiod. These conditions were similar phology on YDC agar plates, which was characterized as to the usual conditions for brassica cultivation in Serbia, which pale yellow, convex and mucoid. Isolates showed Gram- are also optimal for Xcc development. Irrigation was carried negative staining, a catalase-positive reaction and an oxi- out once a day using a microsprayer. To avoid splash transfer dase-negative reaction, as well as oxidative metabolism among isolates (cross contamination), treatments were separated of glucose (aerobic). They produced H2S and indole, did with plastic barriers. not reduce nitrate, and hydrolysed starch, gelatin and Disease was visually checked for symptom development esculin. 7 days after inoculation. Inoculated plants were scored on the All tested isolates and the control reference strain 15th day after inoculation by rating five leaves per plant using the following scale: 1 = healthy leaves; 2 =< 10%; 3 => 10– NCPPB 1144 were identified as Xcc after PCR amplifica- 25%; 4 => 25–50%; 5 => 50–75%; 6 => 75–100% infected tion with XCF/XCR pathovar-specific primers, which leaf area. generated the specific band of 535 bp.

Figure 1 Concatenated neighbour-joining phylogenetic tree based on gltA, gyrB1, gap-1, lepA, lacF, fusA, rpoD, dnaK, gyrB2 and fyuA partial sequences obtained from 18 representative Serbian Xanthomonas campestris pv. campestris isolates and reference strain NCPPB 1144 used for comparison. The tree was rooted with an outgroup strain, X. campestis pv. vesicatoria (CP017190), from the NCBI database.

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pathogenic only to oilseed rape, as the host from which Genotyping of X. campestris pv. campestris they were originally sampled. In contrast, Xc20 (also Repetitive element PCR fingerprinting from winter oilseed rape) showed a broader spectrum of The UPGMA phylogenetic trees obtained from rep-PCR pathogenicity, as it caused symptoms on all of the tested and constructed on the basis of differences in generated hosts. Isolates Xc23 (winter oilseed rape), Xc48 and patterns of the 147 isolates are presented in Figure S1 Xc75 (cabbage), Xc170 (broccoli) and Xc178 (kohlrabi), (BOX-PCR), Figure S2 (ERIC-PCR) and Figure S3 (REP- as well as reference strain NCPPB 1144, produced no PCR). Six (BOX-PCR), 10 (ERIC) and nine (REP-PCR) symptoms of any kind within this time period. Isolates different profiles, ranging from 300 to 3000 bp, could be from the other brassicas caused the appearance of symp- generated after visual analysis of the amplified PCR toms on all of the tested hosts, regardless of the host products and construction of fingerprinting profile from which they were originally isolated. groups (Table 1). For combined results of BOX-, ERIC- The appearance of symptoms and their intensity on and REP-PCR, one isolate from each fingerprinting seven brassica hosts 15 days after inoculation are pre- group, and within each group one isolate from each host sented in Table 2 as mean values of the rated necrotic in the group, were taken as representative for further leaf lesions on all hosts in comparison with those of indi- study, in total 18 isolates. After software comparison of vidual isolates. All 18 representative isolates and refer- the patterns, most of the groups were found to contain ence strain NCPPB 1144 induced lesions on all tested Xcc isolates originating from winter oilseed rape, show- hosts. Even though isolates from winter oilseed rape ing their genetic polymorphism (BOX-PCR groups: I–V; (Xc6, Xc9, Xc23, Xc27, Xc30, Xc31 and Xc32) caused REP-PCR groups: I–VI; ERIC-PCR groups: I–VI). In con- characteristic symptoms on all of the tested hosts, the trast, most of the isolates from the six brassica vegetables highest virulence with statistical significance (P < 0.05) produced the same banding patterns (BOX-PCR groups: was on their original host, winter oilseed rape, compared VI; REP-PCR groups: VII-IX; ERIC-PCR groups: VII-IX; to the other hosts. Among them, isolate Xc30 caused the Table 1). There was no relationship between the geo- significantly largest necrotic lesions on oilseed rape graphical distribution of isolates and their rep-PCR (2.7 Æ 0.13). The only exception among the winter oil- group. seed rape isolates was isolate Xc20, which also showed statistically significant differences on kale (2.4 Æ 0.07) Multilocus sequence analysis (MLSA) and collard greens (2.3 Æ 0.11). Among the other tested Amplification of all the studied genes identified the 18 isolates, only Xc110 (cauliflower), Xc120 (kale) and tested isolates as Xcc, based on BLAST analysis with glob- Xc178 (kohlrabi) showed a host–isolate relationship, ally available strains from the NCBI database. Individual expressed as appearance of the most pronounced symp- phylogenetic trees of the 10 housekeeping genes (Fig. S4) toms on the original host. and a tree based on the concatenated sequences of all 10 A characteristic host–isolate specificity pattern was not genes (Fig. 1) showed heterogeneity among isolates from observed for the other isolate–host pairs. For example, winter oilseed rape on the one hand and homogeneity of an isolate obtained from broccoli (Xc160) caused the lar- B. oleracea hosts on the other. The gltA and rpoD genes gest lesions on the surface of cauliflower leaves, rated as most clearly point to these differences, distinguishing iso- 3.3 Æ 0.09. lates in two separate clusters. Apart from fusA and fyuA, The reference strain NCPPB 1144 caused symptoms where all of the isolates showed homogeneity, the rest of on all hosts, which were statistically significant on cab- the genes (gap-1, gyrB1, lacF, lepA, dnaK and gyrB2) bage, kale and collard greens, compared to other tested showed sequence changes that could be grouped into hosts, with rated disease severity of 1.6 Æ 0.08, three clusters. Two of these included winter oilseed rape 1.5 Æ 0.05 and 1.5 Æ 0.06, respectively (Table 2). None isolates, and the other included all remaining B. oleracea of the tested hosts treated with sterile distilled water isolates together with Xc9 B. napus isolate, which (which served as a negative control treatment) showed showed no differences among themselves (Fig. 1). Only any symptoms. isolate Xc9 from winter oilseed rape showed more simi- larities with isolates from other hosts than with the host Discussion from which it originated (Fig. 1). An outgroup strain [X. campestris pv. vesicatoria (CP017190)] was placed Brassicas belong to a plant family that includes a wide on a separate branch in all of the phylogenetic trees. variety of economically important vegetables and some crops with worldwide distribution, mostly significant for their richness in nutritional composition and oil produc- Pathogenicity tion. In view of their importance as agricultural crops, as The majority of brassica hosts did not have clear visual well as their susceptibility to various plant-pathogenic symptoms of infection 7 days after artificial inoculation bacteria causing diseases that can lead to severe yield with the 18 representative Xcc isolates and reference losses, it is of great importance to study the pathogen– strain NCPPB 1144, and showed only up to 10% of leaf plant interactions in more detail. Black rot, caused by surface covered with necrotic lesions. Winter oilseed rape the plant-pathogenic bacterium Xcc, is considered to be isolates Xc6, Xc9, Xc27, Xc30, Xc31 and Xc32 were one of the most important diseases of crucifers, attacking

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Table 2 The mean values of leaf infections (mean Æ SE, n = 6) on seven brassicas hosts tested against 18 Xanthomonas campestris pv. campestris isolates in planta rated 15 days after inoculation, based on a scale of 1–6.

NCPPB 1144 Xc6 (winter Xc9 (winter Xc20 (winter Xc23 (winter Xc27 (winter Xc30 (winter Xc31 (winter Xc32 (winter Xc40 Xc48 Host/isolate (cabbage) oilseed rape) oilseed rape) oilseed rape) oilseed rape) oilseed rape) oilseed rape) oilseed rape) oilseed rape) (cabbage) (cabbage)

Winter oilseed rape 1.2c Æ 0.01 2.3a Æ 0.08 2.3a Æ 0.08 2.3a Æ 0.09 1.9a Æ 0.05 2.5a Æ 0.05 2.7a Æ 0.13 2.4a Æ 0.16 2.6a Æ 0.15 1.6b Æ 0.05 1.2b Æ 0.07 Cabbage 1.6a Æ 0.08 1.8b Æ 0.17 1.6b Æ 0.06 1.7bc Æ 0.05 1.7bc Æ 0.03 1.3bc Æ 0.05 1.2bc Æ 0.05 1.5b Æ 0.09 1.3c Æ 0.04 1.9b Æ 0.10 1.8a Æ 0.08 Kale 1.5a Æ 0.05 1.2c Æ 0.04 1.4b Æ 0.08 2.4a Æ 0.07 1.3de Æ 0.02 1.3bc Æ 0.08 1.1c Æ 0.02 1.3b Æ 0.03 1.1c Æ 0.03 2.3a Æ 0.09 1.9a Æ 0.06 Cauliﬂower 1.2bc Æ 0.04 1.3c Æ 0.02 1.4b Æ 0.05 1.8b Æ 0.10 1.5cd Æ 0.04 1.1cÆ 0.04 1.1c Æ 0.06 1.2bÆ 0.04 1.1c Æ 0.04 1.9b Æ 0.06 1.1b Æ 0.04 .Popovi T. Broccoli 1.4ab Æ 0.07 1.5bc Æ 0.04 1.5b Æ 0.05 2.1ab Æ 0.13 1.7b Æ 0.03 1.5b Æ 0.06 1.2bc Æ 0.03 1.4b Æ 0.07 1.6b Æ 0.03 1.8b Æ 0.07 1.8a Æ 0.04 Collard greens 1.5a Æ 0.06 1.5bc Æ 0.02 1.4b Æ 0.04 2.3a Æ 0.11 1.4de Æ 0.05 1.4b Æ 0.04 1.4b Æ 0.04 1.3b Æ 0.04 1.3c Æ 0.03 1.8b Æ 0.03 1.6a Æ 0.04 Kohlrabi 1.4abc Æ 0.06 1.4c Æ 0.03 1.5b Æ 0.08 1.3c Æ 0.07 1.2e Æ 0.07 1.3bc Æ 0.09 1.2bc Æ 0.04 1.3b Æ 0.05 1.1c Æ 0.04 1.7b Æ 0.04 1.8a Æ 0.06 c Host/isolate Xc75 (cabbage) Xc110 (cauliﬂower) Xc120 (kale) Xc150 (collard greens) Xc160 (broccoli) Xc170 (broccoli) Xc175 (kohlrabi) Xc178 (kohlrabi) al. et

Winter oilseed rape 1.1b Æ 0.04 1.0e Æ 0.00 1.2d Æ 0.07 1.0e Æ 0.00 1.6d Æ 0.11 1.2e Æ 0.06 1.2d Æ 0.04 1.2c Æ 0.06 Cabbage 1.5a Æ 0.04 2.5bc Æ 0.05 2.4bc Æ 0.17 2.3cd Æ 0.08 2.7bc Æ 0.19 2.0a Æ 0.06 2.0c Æ 0.05 1.5abc Æ 0.09 Kale 1.5a Æ 0.05 2.7ab Æ 0.12 3.1a Æ 0.07 2.9a Æ 0.06 2.8ab Æ 0.13 1.6cd Æ 0.07 2.6ab Æ 0.17 1.2bc Æ 0.11 Cauliﬂower 1.5a Æ 0.08 3.0a Æ 0.11 2.8ab Æ 0.10 2.9a Æ 0.10 3.3a Æ 0.09 1.5cde Æ 0.07 3.0a Æ 0.10 1.3abc Æ 0.07 Broccoli 1.5a Æ 0.08 2.2cd Æ 0.09 2.1c Æ 0.10 2.6ab Æ 0.06 2.6bc Æ 0.08 1.8ab Æ 0.06 2.4bc Æ 0.09 1.5ab Æ 0.05 Collard greens 1.5a Æ 0.05 2.0d Æ 0.05 2.7ab Æ 0.11 2.1d Æ 0.06 2.2c Æ 0.10 1.4de Æ 0.03 2.5ab Æ 0.13 1.4abc Æ 0.03 Kohlrabi 1.6a Æ 0.09 2.6ab Æ 0.09 2.0c Æ 0.10 2.5bc Æ 0.08 3.1ab Æ 0.09 1.7bc Æ 0.05 2.4bc Æ 0.14 1.6a Æ 0.05

Values followed by the same letter within columns (according to the monitoring) are not signiﬁcantly different (P < 0.05), according to Tukey’s HSD test. The values in bold represent the highest scale of ln Pathology Plant necrotic lesions to particular host after 15 days of inoculation as a result of pathogenesis of an individual isolates. (2019) 68 1448–1457 , Diversity of Xanthomonas campestris 1455

many cultivated cruciferous crops (Popovic et al., 2013a, Xc27, Xc31 and Xc32 compared to all other isolates 2014). In the light of its destructive potential, 147 iso- tested. This could possibly indicate more oriented lates collected from seven brassicas (broccoli, cabbage, microevolution (evolution within species) established on cauliflower, collard greens, kale, kohlrabi and winter oil- this ‘core’ gene, arisen in response to certain selection seed rape) were subjected to examination of their genetic pressures. Young et al. (2008) explained that MLSA is a and pathogenic features in the present study. Fingerprint- precise and accurate method for differentiation of most ing techniques, with primers such as BOX, ERIC and Xanthomonas species and emphasizes that MLSA results REP, but also with some novel rep-PCR primers (Tsy- generally confirm the groupings obtained with methods gankova et al., 2004), have been previously used to like AFLP and rep-PCR. In this study, REP-PCR results reveal genetic heterogeneity between different Xan- showed the highest correlation, of about 70%, with the thomonas species and pathovars of these species (Jensen results obtained with MLSA. A recent Xcc study (Bella et al., 2010). In this study, a high level of genetic diver- et al., 2019) used MLST of seven loci (atpD, dnaK, efp, sity was found particularly in winter oilseed rape iso- fyuA, glnA, gyrB and rpoD) to test whether the long tra- lates, as compared to isolates from other hosts, especially dition of cultivation of brassica crops (broccoli, cabbage, as determined by REP- and ERIC-PCR. This indicates cauliflower, seakale, kale, kohlrabi, Savoy cabbage and that they are possibly good tools for separating groups rutabaga) in Italy has influenced the evolution of differ- of isolates of Xcc. The results confirm the genetic hetero- ent Xcc populations, and the presence of two Xcc geneity among Xcc isolates that was established by genetic groups was revealed in that study. The genetic Popovic et al. (2013a), where two major clusters of Ser- diversity among Serbian oilseed rape isolates may eventu- bian Xcc isolates from broccoli (first), kale (first) and ally be explained through their adaptation to the weather cabbage (second) isolated in 2010 were revealed using and environmental conditions of winter oilseed rape cul- GTG5 and ERIC rep-PCR primers. Subsequently, ERIC tivation (winter form), which is different from other and GTG5 proved to be useful tools for fast estimation brassicas (spring form). Unlike B. oleracea vegetable of pathovar diversity within 10 Serbian oilseed rape Xcc crops, oilseed rape is a natural hybrid, allelotetraploid strains, also isolated in 2010, separating them into two (n = 19, AACC), spontaneously derived after interspecific (98% similarity) and three (92% similarity) groups, hybridization of two diploid sets of chromosomes, one respectively (Popovic et al., 2014), thereby anticipating from B. rapa (n = 10, AA genome) and the second from the differences at the genetic level obtained in the present B. oleracea (n = 9, CC genome) (Sharma et al., 2014). study with isolates obtained 4 years later. In the study of Genetic complexity and oil production make oilseed rape Singh et al. (2011), rep-PCR (REP-, BOX- and ERIC- different from B. oleracea crops and form a completely PCR) oligonucleotide primers also showed an ability to different ecological niche for bacterial colonization. separate five different groups of Xcc isolates from differ- Another similar study (Fargier & Manceau, 2006) per- ent Brassica crops – cauliflower, cabbage, broccoli, kohl- formed with eight housekeeping genes (atpD, dnaK, efP, rabi and turnip – from the northern region of India, glnA, gyrB, rpoD, tpiA and fyuA), revealed that four of indicating the presence of genetic diversity within this X. them (dnaK, gyrB, rpoD and fyuA) are identical in their campestris pathovar. Zaccardelli et al. (2008) demon- usefulness for identifying Xanthomonas genus level, spe- strated extensive genetic polymorphism within Italian cies, pathovar and below pathovar. Ngoc et al. (2010) Xcc strains from different crucifers (broccoli, Brussels used MLSA for genotypic classification of two citrus sprout, cabbage, cauliflower, kale, kohlrabi, rutabaga pathogens, X. citri pv. citri and X. citri pv. bilvae. Use and Savoy cabbage) using the RAPD-PCR (randomly of rpoB or gyrB alone can provide better insight into amplified polymorphic DNA-polymerase chain reaction) Xanthomonas taxonomy than the use of (partial) technique, but no obvious relations were obtained sequences of 16S and 16S-23S rRNA (Parkinson et al., between them and their geographic origin or host plant. 2007; Ferreira-Tonin et al., 2012). Also, for identifica- Advanced, mostly PCR-based, techniques for accurate tion of Xcc, just a single gene such as gyrB (Parkinson identification and characterization of various plant et al., 2007) could be used as a reliable method, or a pathogenic bacteria to the species, pathovar and strain pathogenicity test in a susceptible host. level are nowadays increasingly being used (Valverde One of the most powerful aspects of MLSA is its abil- et al., 2007; Singh et al., 2011; Bella et al., 2019). Simi- ity to evaluate evolution within certain species by moni- lar to results of the rep-PCR fingerprinting analysis per- toring different types of recombination. Evaluation of formed in the present work, MLSA of 10 sequenced the recombination and its rate can help to understand genes indicated greater diversity of Xcc isolates from how bacterial strains evolve within species, how clones winter oilseed rape as compared to isolates from the adapt to new environmental and host challenges, and other tested hosts, separating them into three genetically thus how new pathogenic strains emerge (Fargier et al., different populations. Genes gltA and rpoD showed the 2011). ability to separate Xcc isolates into two different clus- Even though the pathogenicity results obtained in the ters, in contrast to fusA and fyuA, which were not dis- present study showed that all isolates were capable of criminative. Also, apart from changes in individual inducing symptoms after artificial inoculation on seven nucleotides, specific three-nucleotide changes were different brassicas, differences in their virulence were observed after lepA gene sequencing of isolates Xc6, noticed. Host–pathogen specificity was especially evident

Plant Pathology (2019) 68, 1448–1457 1456 T. Popovic et al.

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rep-PCR primers. European Journal of Plant Pathology 110, in further tests are marked with rectangles. Members of each of the 845–53. groups presented in the figure are listed in Table 1. Valverde A, Hubert T, Stolov A, Dagar A, Kopelowitz J, Burdman S, Figure S2. Dendogram obtained after UPGMA analysis of ERIC-PCR 2007. Assessment of genetic diversity of Xanthomonas campestris pv. results obtained from 147 Xanthomonas campestris isolates from seven campestris isolates from Israel by various DNA fingerprinting brassica crops, viz. Brassica oleracea varieties capitata (cabbage), botrytis techniques. Plant Pathology 56,17–25. (cauliflower), italica (broccoli), sabauda (kale), gongylodes (kohlrabi) and Versalovic J, Schneider M, de Bruijn FJ, Lupski FR, 1994. Genomic acephala (collard greens); B. napus (winter oilseed rape); and Xan- fingerprinting of bacteria using repetitive sequence-based polymerase thomonas campestris pv. campestris reference strain NCPPB 1144 after chain reaction. Methods in Molecular and Cellular Biology 5, performing ERIC-PCR. Representative isolates used in further tests are 25–40. marked with rectangles. Members of each of the groups presented in the Young JM, Park DC, Shearman HM, Fargier E, 2008. A multilocus figure are listed in Table 1. sequence analysis of the genus Xanthomonas. Systematic and Applied Figure S3. Dendogram obtained after UPGMA analysis of REP-PCR – Microbiology 31, 366 77. obtained from 147 Xanthomonas campestris isolates from seven brassica Zaccardelli M, Campanile F, Moretti C, Buonaurio R, 2008. crops, viz. Brassica oleracea varieties capitata (cabbage), botrytis (cauli- Characterization of Italian populations of Xanthomonas campestris flower), italica (broccoli), sabauda (kale), gongylodes (kohlrabi) and ace- pv. campestris using primers based on DNA repetitive sequences. phala (collard greens); B. napus (winter oilseed rape); and Xanthomonas – Journal of Plant Pathology 90, 375 81. campestris pv. campestris reference strain NCPPB 1144 after performing REP-PCR. Representative isolates used in further tests are marked with Supporting Information rectangles. Members of each of the groups presented in the figure are listed in Table 1. Additional Supporting Information may be found in the online version of Figure S4. Individual neighbour-joining phylogenetic trees based on this article at the publisher’s web-site. gltA, gyrB1, gap-1, lepA, lacF, fusA, rpoD, dnaK, gyrB2 and fyuA gene Figure S1. Dendogram obtained after UPGMA analysis of BOX-PCR sequences obtained from 18 representative Xanthomonas campestris pv. results obtained from 147 Xanthomonas campestris pv. campestris iso- campestris isolates from Serbia and reference strain NCPPB 1144 used lates from seven brassica crops, viz. Brassica oleracea varieties capitata for comparison. All trees were rooted with an outgroup strain, X. cam- (cabbage), botrytis (cauliflower), italica (broccoli), sabauda (kale), gongy- pestris pv. vesicatoria (CP017190), from the NCBI database. lodes (kohlrabi) and acephala (collard greens); B. napus (winter oilseed Table S1. Primers used in this study. rape); and Xcc reference strain NCPPB 1144. Representative isolates used Table S2. GenBank accession numbers.

Plant Pathology (2019) 68, 1448–1457