RESTRICTION FRAGMENT LENGTH POLYMORPHISM-BASED IDENTIFICATION OF DICKEYA SOLANI, A NEW GENETIC CLADE OF DICKEYA spp.

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1 Journal of Plant Pathology (2013), 95 (3), Edizioni ETS Pisa, 2013 Waleron et al. 609 Short Communication RESTRICTION FRAGMENT LENGTH POLYMORPHISM-BASED IDENTIFICATION OF DICKEYA SOLANI, A NEW GENETIC CLADE OF DICKEYA spp. M. Waleron 1, R. Czajkowski 1, K. Waleron 1, * and E. Lojkowska 1 1 Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, Gdansk, Poland * Present address: Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Gdansk, Al. Gen. Hallera 107, Gdansk, Poland SUMMARY Bacteria belonging to Pectobacterium and Dickeya spp. (previously known as pectinolytic Erwinia spp.) cause soft rot diseases in a great variety of crops worldwide, particularly in potato, tomato, maize, and ornamental plants and other hosts. Infections by these pathogens may lead to severe economic losses to the crops, partially due to the lack of effective detection/identification tools and incomplete knowledge of their behavior in the environment. Since 2005, an increase in blackleg and soft rot diseases of potato has been observed in Europe due to the appearance of the new genetic clade of Dickeya spp. provisionally named Dickeya solani. This pathogen is more aggressive in most cases than bacteria of the Pectobacterium species and cause plant tissue maceration under a wide range of conditions. The present study provides a simple and reliable three-step PCR-based method for rapid differentiation of isolates from other Dickeya spp. and Pectobacterium spp. Bacterial isolates are classified as Dickeya spp. on the basis of the PCR reaction with Dickeya spp. specific primers. Isolates identified positively as Dickeya spp. are tested by PCR for the presence of reca with the primers universal for bacteria of the former genus Erwinia. The PCR product is then digested with XbaI restriction endonuclease. The differentiation of is based on the presence of a unique XbaI restriction site in the reca gene that is absent in the reca sequences of other Dickeya species. Key words: blackleg, soft rot, molecular identification, PCR, molecular diagnosis Bases on biochemical, genetic and molecular tests, the former Erwinia chrysanthemi (syn. Pectobacterium chrysanthemi) has been reclassified in 2005 into the genus Dickeya comprising six new genomic species, so-called Corresponding author: M. Waleron Fax: goniawu@biotech.ug.edu.pl genomo-species (Dickeya dianthicola, D. dadantii, D. zeae, D. chrysanthemi, D. paradisiaca and D. dieffenbachiae), within the nine biovars (Samson et al., 2005). Recently D. dieffenbachiae was reclassified as D. dadantii subsp. dieffenbachiae (Brady et al., 2012). Since 2005, strains belonging to the new genetic clade that could not be classified together with the known five Dickeya species have been isolated from blackleg-diseased plants and tubers with soft rot symptoms in different European countries, i.e. The, Poland, France, Belgium, Sweden, Spain, Finland and Germany, and in Israel (Czajkowski et al., b; Laurila et al., 2008; Slawiak et al., a; Tsror et al., ; Toth et al., 2011). Most isolates belonging to this new genetic clade characteristically express higher virulence in than the D. dianthicola isolates from potato (Czajkowski et al., 2013). This new clade has been provisionally named and perhaps constitutes a new Dickeya species although it is very closely related to D. dadantii (Van Vaerenbergh et al., 2012; Parkinson et al., ). Results from reca (Parkinson et al., ), dnax and 16S rdna sequence analyses, Rep-PCR and biochemical assays showed that all the new isolates from different European countries (from potato and hyacinth) and Israel were clonal (Slawiak et al., b; Degefu et al., 2013). can cause severe losses to potatoes due to soft rot and, in particular, the rejection and declassification of (seed) tubers, especially under certain warm growing environments. In Poland, the first strains of were isolated from potato in 2005 (Slawiak et al., a), however as early as in 2002 the presence of Dickeya strains with identical reca gene sequence was recorded in greenhouse-grown ornamental plants (unpublished information). During the last few years the number of blackleg infections caused by to seed potato crops has statistically increased (Lojkowska et al., ). The abundance of these bacteria in the field can fluctuate due to climate change, such as the extremely harsh winters. However, in Poland the number of isolation from potato has increased till 2011 (unpublished information).

2 610 The identification of Dickeya solani Journal of Plant Pathology (2013), 95 (3), Efficient control of Dickeya spp. in potato and other crops relies mainly on hygienic measures enforced to prevent infections to healthy plants and tubers (Czajkowski et al., 2012; Toth et al., 2011) for which full-proof, fast and reproducible detection and identification methods are essential. To this aim, a quantitative PCR (qpcr) based on the flic gene sequence or a TaqMan primers-probe combination predicted on the basis of genome sequence was developed (Van Vaerenbergh et al., 2012; Pritchard et al., 2012). Although these methods are now more frequently used, they still remain relatively expensive, partially because of the Real Time PCR equipment and reagents. Herewith we describe a rapid method for the identification of bacteria based on three steps: (i) bacterial isolates are first identified as Dickeya spp. by PCR with the use of specific ADE1/ADE2 primers (Nassar et al., 1996); (ii) isolates identified as Dickeya spp. are further subjected to PCR for reca with the primers from former genus Erwinia (Waleron et al., 2002); (iii) the amplified reca product is then digested with XbaI restriction endonuclease, is differentiated based on the presence of a unique XbaI restriction site in the reca gene that is absent in the reca sequences of other Dickeya species. It is worth to underline that the reca fragment amplified with primers described by Waleron et al. (2002) and Table. 1 Presence of the PCR product with Dickeya spp. specific ADE1/ADE2 primers (Nassar et al., 1996) and reca PCR-RFLP (Waleron et al., 2002) analysis with XbaI for identification of Dickeya solani (marked in bold in table) Isolate Organism Origin PCR product with ADE1/ ADE2 primers IPO2222, 2007 Ech15 Ornamental plant, Poland, 2002 IFB0099 (IPO2276) 2005 IFB0101 (IPO2278) 2005 IFB0158 IFB0159 IFB0160 IFB0203 IFB0204 IFB0205 IFB IFB0300 IPO2019 hyacinth, The S0413 T042 S432 VIC-BL W443 River water, Finland, MK8 River water, Scotland IPO2118 T D. chrysanthemi Chrysanthemum, USA + - IPO1478 D. chrysanthemi Solanum tuberosum, Spain + - IPO2117 D. chrysanthemi Parthenium, USA + - IPO2119 D. chrysanthemi Helianthus annuus, France + - IPO655 D. chrysanthemi Solanum tuberosum, Taiwan + - IPO2120 T D. dadantii Pelargonium, Comoros + - SCRI4040 D. dadantii Solanum tuberosum, Peru + - Digestion of reca PCR product with XbaI

3 Journal of Plant Pathology (2013), 95 (3), Waleron et al. 611 CFBP1240 D. dadantii Dianthus, Denmark + - IPO875 D. dadantii D. dadantii Saintpaulia, France + - IPO2125 T D. dadantii subsp. dieffenbachiae Dieffenbachia, USA + - IPO2124 D. dadantii subsp. dieffenbachiae Dieffenbachia, France + - IPO2114 T D. dianthicola Dianthus, United Kingdom + - IPO877 D. dianthicola + - IPO2116 D. dianthicola Solanum tuberosum, France + - IPO1302 D. dianthicola Solanum tuberosum, Spain D. dianthicola Dianthus, Greece + - W04K D. dianthicola river water, Finland + - IPO2127 T D. paradisiaca Musa, Colombia + - IPO2128 D. paradisiaca Zea mays, Cuba + - Ech703 * D. paradisiaca a Solanum tuberosum, Australia nd - IPO2131 T D. zeae Zea mays, USA + - SCRI4072 D. zeae Zea mays, India + - IPO647 D. zeae Solanum tuberosum, Australia + - IPO2121 (CFBP1278) D. zeae Ananas comosus, Malasia + - IPO2132 D. zeae Chrysanthemum, UK + - Ech586 * D. zeae a Philodendron, USA nd - ZJU1202 * D. zeae Oryza sativa, China nd - SCRI4061 Dickeya spp. Colocasia, Salomon Islands + - SCRI4062 Dickeya spp. Agalonema, St. Lucia + - River1 Dickeya spp. river water, Scotland + - W054 Dickeya spp. river water, Finland + - CFBP1526 T P. atrosepticum Solanum tuberosum, UK - - SCRI85 P. atrosepticum Solanum tuberosum, Peru - - SCRI1043 P. atrosepticum Solanum tuberosum, UK - - ATCC43762 T P. betavascularum Beta vulgaris, USA P. betavascularum nn, USA - - ATCC11663 T P. carotovorum subsp. carotovorum Solanum tuberosum, Denmark - - SCRI171 P. carotovorum subsp. carotovorum Solanum tuberosum, Peru - - SCRI137 P. carotovorum subsp. carotovorum Solanum tuberosum, USA - - SCRI249 P. carotovorum subsp. carotovorum Solanum tuberosum, Scotland /96 P. carotovorum subsp. carotovorum Petroselinum, Poland - - Pcc1 * P. carotovorum subsp. carotovorum Ornithogalum, Israel nd - WPP14 * P. carotovorum subsp. carotovorum Solanum tuberosum, USA nd - Pcc21 * P. carotovorum subsp. carotovorum Brassica rapa, Korea nd /00 P. carotovorum subsp. brasiliensis Solanum tuberosum, Brazil /99 P. carotovorum subsp. brasiliensis Solanum tuberosum, Brazil - + Pbr 1692 * P. carotovorum subsp. brasiliensis Solanum tuberosum, Brazil nd + CFBP1887 T P. carotovorum subsp. odoriferum Apium graveolens, France - - CFBP3259 P. carotovorum subsp. odoriferum Allium ampeloprasum, France - - CFBP3304 T P. wasabiae Armoracia rusticana, Japan - - LA242 P. wasabiae Solanum tuberosum, USA - - WPP163 * P. wasabiae Solanum tuberosum, USA nd - SCC3193 P. wasabiae Solanum tuberosum, Finland - - ATCC49485 P. cactacidum Opuntia, USA P. cactacidum Opuntia, Australia - - T type strain * in silico RFLP analysis on sequences of the reca gene retrieved from genomes available in NCBI database ( a Dickeya classification scheme, following reca and flic phylogenetic analysis (Van Vaerenbergh et al., 2012; Pritchard et al., 2012). nd not determined proposed type strain

4 612 The identification of Dickeya solani Journal of Plant Pathology (2013), 95 (3), used in this study is longer (approximate 730 bp) than that analysed by Parkinson et al. (), which contains only 481 bp and lacks the restriction site for XbaI. Forty nine isolates of Dickeya spp., 3 isolates of Pectobacterium atrosepticum, 2 of P. betavascularum, 3 of P. wasabiae, 2 of P. carotovorum subsp. odoriferum, 5 of P. carotovorum subsp. carotovorum, 2 of P. carotovorum subsp. brasiliensis and 2 of P. cacticidum together with 4 genomes of Dickeya spp. (GenBank accession Nos CP001836, CP001654, CP001655, AJVN ), 3 genomes of P. carotovorum subsp. carotovorum (CP001657, CP003776, NABVY ), 1 genome of P. wasabiae (CP001790), and 1 genome of P. carotovorum subsp. brasiliensis (AB- VX ) used is this study are listed in Table 1. Prior to analysis, bacterial strains were grown on Luria Broth agar (LA) or in Luria Broth (LB) at 28 C for 24 h. Bacterial cultures in LB were additionally shaken at 200 rpm during incubation. For long-term storage, strains were kept in 40% glycerol (v/v) at -80 C. For characterization of isolates, a colony PCR procedure was used. Cells from the suspected colony were collected from an agar plate using a sterile toothpick and resuspended in 50 ml of sterile water. Bacterial suspensions were boiled in a microwave oven for 10 sec at maximum intensity and after placed on ice for 1-2 min. One μl of a cell lysate was used as PCR template. PCR detection was done according to Nassar et al. (1996) using ADE1/ADE2 primers that yielded amplicons 420 bp in size. Amplified PCR products were analyzed by electrophoresis in a 1% agarose gel in 0.5 TBE buffer stained with 0.5 μg ml -1 of ethidium bromide. The gel was run at 75 V for ca. 40 min. at room temperature. Colony PCR procedure was also used for amplification of the reca gene from Dickeya spp. isolates. Bacterial cell lysates were prepared as described above and PCR reactions were performed as described by Waleron et al. (2002). The expected length of the PCR amplicons was 735 bp. Amplified PCR products were analyzed by electrophoresis as above. The amplified reca gene was subjected to restriction analyses with XbaI restriction endonuclease (Fermentas, Lithuania). Restriction analyses were performed with 2.5 U of XbaI for 2 h according to manufacturer s instructions. After digestion, restriction fragments were separated using a gel with 1.8% agarose in 0.5 TBE at 75 V for 2 h, and were visualized under UV light after staining with ethidium bromide (0.5 μg ml -1 ). A 100 bp Plus DNA Ladder (Fermentas, Lithuania) was used as a size marker. All tested isolates gave a 420 bp PCR product with ADE1/ADE2 primers specific for Dickeya spp. As expected, none of the other isolates belonging to Pectobacterium spp. was positive in the ADE1/ADE2 PCR. The reca gene was amplified with PCR from all tested Pectobacterium and Dickeya spp. isolates, giving a 735 bp product. From the all tested Dickeya spp. reca sequences, only PCR products amplified from isolates could be digested with XbaI restriction endonuclease to two fragments: 603 and 132 bp, repectively whereas no digestion was observed with other isolates belonging to different Dickeya species (Table 1). The analysis of 53 sequences of reca gene fragment, which overlap the restriction site for XbaI endonuclease, obtained for different Dickeya spp., and reca gene from 5 Dickeya genomes available in NCBI database ( confirmed that the specific sequence motive recognized by XbaI is not present in sequences of species other than. It should be noted here that XbaI endonuclease digested also the reca gene fragment amplified from strains of P. carotovorum subsp. brasiliensis to two fragments: 503 and 232 bp, respectively. However all P. carotovorum subsp. brasiliensis strains were negative in PCR with Dickeya spp. specific ADE1/ADE2 primers. The presence of a XbaI restriction site in reca sequences in P. carotovorum subsp. brasiliensis isolates was confirmed by in silico RFLP analysis on sequences of the reca gene of P. carotovorum subsp. brasiliensis genome retrieved from NCBI database. This study presents a simple and rapid method for identification of isolates and their differentiation from other pectinolytic bacterial species within the Dickeya and Pectobacterium genera. strains belong to a distinct genetic clade of Dickeya sp. biovar 3, which is currently more frequently associated with potato blackleg and soft rot incidences in Europe (Toth et al., 2011). The pathogen is mainly seed-borne and spreads via latently infected seed potato tubers. At present, there is no effective control management, other then cultural methods, hygiene and prevention and therefore full-proof and reliable identification methods are absolutely required and will help with the establishment of Dickeya spp.-free crops. ACKNOWLEDGEMENTS This work was supported by the National Science Centre (NCN N N ). The authors wish to thank two anonymous reviewers for their insightful comments and suggestions. 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