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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 patho- var-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, respec- tively. The REP-PCR results showed the highest correlation (70%) with those obtained with multilocus sequence analy- sis (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 geneti- cally 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 identi- fication. 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.

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