Journal of Plant Pathology (2017), 99 (1), 149-160 Edizioni ETS Pisa, 2017 149 PECTOBACTERIUM CAROTOVORUM subsp. ODORIFERUM ON CABBAGE AND CHINESE CABBAGE: IDENTIFICATION, CHARACTERIZATION AND TAXONOMIC RELATEDNESS OF BACTERIAL SOFT ROT CAUSAL AGENTS* M. Oskiera, M. Kałuz˙na, B. Kowalska and U. Smolin´ska Research Institute of Horticulture, Konstytucji 3 Maja 1/3, 96-100, Skierniewice, Poland SUMMARY INTRODUCTION This study was aimed to isolate, identify and character- Cabbage (Brassica oleracea L. var. capitata L.) and Chi- ize Pectobacterium spp. causing soft rot disease of cabbage nese cabbage (Brassica rapa L. subsp. pekinensis L.) are im- and Chinese cabbage in Central Poland. Of fifty-two plant portant vegetable crops commonly cultivated in Poland. samples of cabbage and Chinese cabbage showing disease The main pathogens of Brassicaceae plants are Pectobacte- symptoms collected in Central Poland from 2007-2010, 542 rium carotovorum subsp. carotovorum (Pcc), Pseudomonas bacterial isolates were obtained. Of isolates 117 caused soft marginalis pv. marginalis, Pseudomonas syringae pv. maculi- rot on cabbage and Chinese cabbage leaves and potato cola and Xanthomonas campestris pv. campestris (Rimmer et slices and showed pectinolytic activity on crystal violet al., 2007). It is also known that Pseudomonas viridiflava can pectate medium. PCR using Y1/Y2 primers specific for occur on Brassicaceae plants, such as Chinese cabbage (Ma- Pectobacterium genus revealed that twenty-three of them ciel et al., 2010). The most common disease of Brassicaceae belonged to this genus. Phenotypic characterization in is soft rot mainly caused by highly pectinolytic bacteria combination with DNA-based typing methods (rep-PCRs) from the genus Pectobacterium (formerly Erwinia). Symp- and sequence analysis of 16S rRNA, and housekeeping toms of bacterial soft rot on cabbage include water-soaked genes gyrB, infB, rpoB, atpD, and rpoS (MultiLocus Se- lesions which later become rotted mass of macerated tis- quence Typing) done for 12 Polish representatives and ref- sue (Rimmer et al., 2007). P. carotovorum may infect the erence strains allowed for their identification and revealed plant through natural openings (in favorable conditions genetic diversity. Additional phylogeny analysis of the rpoS warm temperatures and high humidity), wounds caused by gene for Polish and worldwide well defined Pectobacte- insects or other diseases causal agent or damages caused rium spp. strains allowed to classify obtained strains into by abiotic factors such as low temperatures or mechanical Pectobacterium spp. phylogenetic clusters (PcI-PcV). The damages. Pectobacterium derives from the genus Erwinia presented studies showed that 8 of 12 isolated Pectobac- and was described as separate genus from Erwinia, Pan- terium carotovorum strains belong to the Pectobacterium toea and Brenneria on the basis of phylogenetic analyses carotovorum subsp. carotovorum (Pcc) cluster PcII and of the 16S rDNA sequences (Hauben et al., 1998). Later, other four strains belong to the subspecies Pectobacteri- Gardan et al. (2003) reclassified existed subspecies of Pec- um carotovorum subsp. odoriferum (Pco) cluster PcIII. To tobacterium carotovorum: i.e. atrosepticum, betavasculorum our knowledge, this is the first report that P. carotovorum and wasabiae to species: Pectobacterium atrosepticum, Pec- subsp. odoriferum causes soft rot of cabbage and Chinese tobacterium betavasculorum and Pectobacterium wasabiae cabbage in Central Poland. respectively. Duarte et al. (2004) described Pectobacterium carotovorum subsp. brasiliense as one of the Pectobacterium Keywords: rep-PCR, 16S rRNA, rpoS, MLST, pectino- carotovorum subspecies. Later, Nabhan et al. (2012a) and lytic bacteria. Waleron et al. (2014) described its appearance in Europe. Phylogenetic studies of the P. carotovorum pectinolytic strains performed by Nabhan et al. (2012b) reveals high heterogeneity of the species and divided it into five clus- ters (PcI to PcV) as follows: Pcc represented by two clus- teres (PcI and PcII), Pectobacterium carotovorum subsp. odoriferum (Pco) (PcIII), P. carotovorum subsp. brasiliense (PcIV) and P. atrosepticum, P. betavasculorum, P. wasabiae. Further, cluster PcV was described as Pectobacterium aroi- dearum by Nabhan et al. (2013). Corresponding author: M. Oskiera Fax: +48.46.833.31.86 * Supplementary materials can be downloaded at: E-mail: [email protected] http://dx.doi.org/10.4454/jpp.v99i1.3831.s11 150 P. carotovorum identification in Poland Journal of Plant Pathology (2017), 99 (1), 149-160 Several methods are commonly used for detection, were macerated in sterile distilled water. Serial dilutions identification and differentiation of plant pathogenic and of the mixture were plated on NA medium. The obtained pectinolytic bacteria. For many years identification of selected colonies were purified on NA medium, preserved bacteria was based on isolation of viable cells on selective in mixture of glycerol and nutrient broth (1:1) and stored culture agar media followed by serological and biochemi- in −80ºC until use. Reference strains of the Pectobacte- cal analyses, bioassays and microscopic observations in- rium carotovorum subsp. carotovorum (Pcc) (LMG 2431, cluding Gram staining (Schaad et al., 2001). Later, some LMG 2404T), Pectobacterium carotovorum subsp. odorif- improvements such as plating on CVP medium with im- erum (Pco) (LMG 17566T) and some pectinolytic strains of munomagnetic separation (IMS) step prior to plating (Van Pectobacterium atrosepticum (Eca59, Ec32), Pectobacterium Der Wolf and Perombelon, 2002), fatty acid methyl ester aroidearum (Ecc4M, Ea853), Pectobacterium sp. (Eca2M) analysis (FAME) (Dawyndt et al., 2006), immunofluores- and Dickeya dadantii (Ech0201) were included in all analy- cence staining (IF) (Van Vuurde and Van der Wolf, 2002) sis. The data on studied isolates and reference strains are and enzyme-linked immunosorbent assay (ELISA) (Lopez summarized in Table 1. et al., 2002) were developed for Pectobacterium detection. Molecular methods, such as PCR with primers specific for Selection of strains on the basis of their pectinolytic detection of Pectobacterium spp., multiplex-PCR, real-time activity on potato slices, cabbage leaves and CVP me- PCR, padlock probes and LAMP are currently widely dium. All the bacterial isolates were tested on potato tuber adapted and were reviewed by Czajkowski et al. (2015). slices, cabbage and Chinese cabbage leaves. Cabbage or Genus specific PCR primers, such as Y1/Y2 (Darrasse et Chinese cabbage leaves and potato tubers were washed al., 1994) for identification Pectobacterium spp., and species under tap water, disinfected in 70% ethanol for 1 min, specific primers, such as Eca1f/Eca2 (De Boer et al., 1995) then 5 min in 0.5% sodium hypochloride, followed by two and Y45/Y46 (Frenchon et al., 1995) for identification P. washes with sterile water. Fragments of the plants: cabbage atrosepticum are routinely used in many laboratories. As with a diameter of approx. 8 cm or 3 slices of potato 10- for many bacterial pathogens, also for soft rot causal agent, mm thick were placed on the prepared Petri dishes (9 cm fingerprinting methods through the analysis of repetitive in diameter) with wet Whatman filter paper. Each bacte- regions [rep-PCR i.e., Enterobacterial Repetitive Intergenic rial isolate grown 24 h on NA medium was placed with Consensus (ERIC), BOX and Repetitive Extragenic Pal- toothpicks on the injured tissues in 3-4 places (per plate). indromic Elements (REP)] (Weingart and Völksch, 1997) Three plates were assayed per bacterial isolate. The Petri have been successfully used for their identification and dif- dishes were then incubated at 28°C. Tissue maceration was ferentiation within the species and subspecies level (Rezaei determined by visual inspection (cabbage leaves) twice: and Taghavi, 2010). In case of Pectobacterium spp., also the after 48 h and after 5 days, while in the case of potato slices several primer pairs amplifying fragments of 16S rDNA with use of toothpicks after 24 and 48 h. Each assay was and housekeeping genes localized independently in bac- performed three times. Obtained bacterial isolates were al- terial core genome were already published and used for so tested on crystal violet pectate agar (CVP) (Perombelon sequencing (Waleron et al., 2002, 2014; Brady et al., 2008). and Burnett, 1991) with use of sodium polypectate (pectin The sequence analysis of housekeeping genes combined classic kat. CU902; Herbstreith & Fox Corporate Group, with bioinformatics tools (MultiLocus Sequence Typing) Neuenbürg/Württ, Germany), for checking their ability to allowing for identification and genotyping of the soft rot form of liquefied deep pits on the medium surface. bacteria. The objective of this study was the isolation, iden- tification and differentiation of P. carotovorum spp. strains DNA isolation. Bacterial DNA was isolated using the obtained from diseased cabbage and Chinese cabbage in method described by Aljanabi and Martinez (1997). The Poland using phenotypic tests, repetitive-sequence PCRs purity and quantity of isolated DNA were evaluated by and MLST. visualization on 0.8% agarose gel after electrophoresis with sodium-borate (NaB) buffer (Brody et al., 2004) and determined spectrophotometrically by absorbance mea- MATERIALS AND METHODS surement of A260/A280 using a BioPhotometer UV/Vis Spectrophotometer (Eppendorf, Hamburg, Germany). Plant samples and isolation of bacteria. Fifty two Finally, DNA