Characterization of Erwinia Amylovora Strains from Bulgaria by Pulsed

Characterization of Erwinia amylovora Strains from Bulgaria by Pulsed-Field Gel Electrophoresis Iliana Atanasovaa, Zoltan Urshevb, Petya Hristovaa, Nevena Bogatzevskac, and Penka Monchevaa,* a Department of General and Industrial Microbiology, Biological Faculty, Sofi a University “St. Kliment Ohridski”, 8 Dragan Tsankov Str., 1164 Sofi a, Bulgaria. Fax: +359-2-865-66-41. E-mail: [email protected]fi a.bg b LB Bulgaricum PLC, R & D Center, 12ª Malashevska Str., Sofi a, Bulgaria c Plant Protection Institute, Kostinbrod, Bulgaria * Author for correspondence and reprint requests Z. Naturforsch. 67 c, 187 – 194 (2012); received June 6, 2011/January 13, 2012 The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-fi eld gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profi le. About 82% of the strains displayed a PFGE profi le identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profi les, not reported so far, were established – one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profi les, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profi le in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum. Key words: Differentiation, Erwinia amylovora, PFGE

Introduction sence of an effective control of the disease. The data from phenotypic assays suggested that E. Fire blight, a disease that is responsible for seri- amylovora is a homogeneous species. The recent ous fruit losses of rosaceous plants, is caused by historical distribution of the pathogen may also the Gram-negative bacterium Erwinia amylovo- be a reason for this homogeneity in the genomes ra. The disease is indigenous to North America of individual strains (Zhang and Geider, 1997). but has spread to many countries in the world However, several molecular techniques such as (Van der Zwet, 1996). In Bulgaria, fi re blight polymerase chain reaction (PCR)-ribotyping was fi rst detected in 1990 on quince (Bobev, (McManus and Jones, 1995; Jeng et al., 1999), 1990). Since then, the pathogen has been pro- random amplifi ed polymorphic DNA (RAPD) gressively observed in different regions of the (Momol et al., 1995), restriction fragment length country mainly on pear, apple, and quince trees, polymorphism (RFLP) (Lecomte et al., 1997; Kim strawberry, chokeberry, cotoneaster, and pyracan- and Geider, 1999; Jock et al., 2003; Ruppitsch et tha ( Atanasova et al., 2005, 2007). The transmis- al., 2004; Barionovi et al., 2006; Atanasova et al., sion and spread of E. amylovora in the country 2009), amplifi ed fragment length polymorphism is the result of the import of contagious plants (AFLP) (Rico et al., 2004; Donat et al., 2007), and from Western Europe, as well as from the natu- pulsed-fi eld gel electrophoresis (PFGE) (Zhang ral dissemination of the bacterium and the ab- and Geider, 1997; Zhang et al., 1998; Jock et al., 2002a; Jock and Geider, 2004; Halupecki et al., Abbreviations: AFLP, amplifi ed fragment length poly- 2006; Donat et al., 2007) have proven useful in morphism; PCR, polymerase chain reaction; PFGE, pulsed-fi eld gel electrophoresis; RFLP, restriction frag- the determination of intraspecifi c diversity within ment length polymorphism; RAPD, random amplifi ed strains from different geographical origins and polymorphic DNA. hosts. Some of these approaches like RAPD and

PCR-ribotyping allowed differentiation between broth at 27 °C for about 24 h up to an optical

Maloideae and Rosoideae strains (Momol et al., density of 0.6 (A600 nm). Cells from 2 ml broth were 1995; McManus and Jones, 1995; McGhee et al., harvested by centrifugation and washed twice in 2002). SEP buffer (25 mM sodium phosphate buffer, pH Macrorestriction analysis of the bacterial ge- 8.0, with 0.3 M sucrose, 25 mM EDTA). The cells nome revealed several closely related but distin- were resuspended in 1 ml SEP buffer and mixed guishable pattern types for E. amylovora, which at 45 °C with an equal volume of 1.5% low-melt- were used to follow up the spread of the dis- ing point agarose (Sigma-Aldrich Chemie, Stein- ease through Europe, the Mediterranean region, heim, Germany) prepared in SEP buffer. The and the Balkans (Jock et al., 2002a). The PFGE agarose-cell suspension was poured into dispos- profi les obtained with XbaI- digested chromo- able Bio-Rad (Hercules, CA, USA) plug molds somal DNA of E. amylovora were used to group (10/5/1.5 mm) and allowed to solidify. Lysis of strains from different geographic regions (Zhang the agarose-embedded cells was performed in ly- –1 and Geider, 1997; Zhang et al., 1998; Jock et al., sis buffer (1 mg ml proteinase K, 1 mM CaCl2, 2002b). On the basis of this analysis, six PFGE 1% lauroylsarcosine, 0.25 M EDTA, 10 mM gly- types were established – Pt1, Pt2, Pt3, Pt4, Pt5, cine, pH 9.5) for 48 h followed by two successive and Pt6 (Jock et al., 2002a). The patterns differ in washes in 10 mM sodium phosphate buffer, pH the shift or the lack of one band. Normally, one 8.0, containing 1 mM EDTA. A quarter of a plug typical PFGE pattern is found when fi re blight is was digested overnight with either of the restric- established in a certain country. Only two strains tion enzymes XbaI, SpeI, or XhoI (20 U). After isolated from Bulgaria have been subjected to digestion, the resulting DNA fragments were re- macrorestriction analysis and were established to solved by PFGE analysis with a Bio-Rad CHEF- have Pt2 and Pt5 PFGE patterns (Zhang et al., DR II apparatus for 24 h at 14 °C with ramping 1998; Jock et al., 2002a). The genetic diversity of at 5 V cm–1 in 1% agarose gel with HEPES buffer the E. amylovora population in Bulgaria has not and a ramping time of 5 – 30 s. All strains studied been studied in detail, and very little is known were assayed, at least twice, from different DNA about the genetic variability of Bulgarian strains. extractions. The positions of the bands after di- The aim of this work was to study the genet- gestion were compared with GelCompar (Ap- ic diversity among fi fty strains of E. amylovora, plied Maths, Koptrijk, Belgium) software. isolated from different plants and locations in Bulgaria as well as in different years, by PFGE analysis. Results and Discussion To estimate the possible diversity of E. amylo- Material and Methods vora in Bulgaria, we used strains isolated from different host plants, orchards, and years (Ta- Bacterial strains ble I). The samples were derived from blight-in- Fifty strains previously identifi ed as E. amylo- fected orchards with pear, apple, and quince trees, vora (Bogatzevska, 2000; Atanasova et al., 2005, as well as from strawberry plantations, aronia, 2007; Kabadjova-Hristova et al., 2006) were used hawthorn, and ornamental plants. Restriction di- in this study. The type strain E. amylovora ATCC gests with three enzymes (XbaI, SpeI, and XhoI) 15580 and strain 2C (Serbian isolate which was and subsequent PFGE analyses of genomic DNA kindly provided to us by Prof. I. Kiryakov, Do- were applied in this study to fi fty-two E. amylo- broudja Agricultural Institute, General Toshevo, vora strains. Fifty of them were Bulgarian isolates. Bulgaria) were used as controls. The origin and year of isolation of the strains are listed in Table I. PFGE analysis of XbaI-digested chromosomal DNA PFGE analysis The restriction profi les after XbaI digestion The PFGE analysis of chromosomal DNA of (Fig. 1) were compared in order to fi nd diversity E. amylovora was performed as described by that can be used for differentiation among the Zhang and Geider (1997) with some modifi ca- strains. The molecular mass of each band was de- tions. Briefl y, the cultures were cultivated in LB termined. Four distinct patterns were distinguished I. Atanasova et al. · Characterization of Erwinia amylovora by PFGE 189

Table I. Sources, year of isolation, and PFGE patterns of E. amylovora strains. Strain Host plant Location Year XbaI SpeI designation PFGE group PFGE group Ea1, Ea2, Ea3, Ea4, Pyrus communis 42°16’44.73”N 1995 II (Pt2) II Ea5, Ea6, Ea7 22°45’36.33”E Ea39 Malus domestica “ 2000 IV (new Pt type) IV Ea51 Malus domestica 2000 II (Pt2) II Ea40 Malus domestica “ 2001 II (Pt2) II Ea42 Malus domestica “ 2002 II (Pt2) II Ea44 Malus domestica “ 2003 II (Pt2) II Ea8, Ea9, Ea10 Pyrus communis 42°18’06.07”N 1995 II (Pt2) II 22°45’24.73”E Ea49 Malus domestica “ 2000 II (Pt2) II Ea11 Malus domestica 42°16’52.47”N 1995 II (Pt2) II 22°41’17.66”E Ea16, Ea17, Ea18, Pyrus communis “ 1997 II (Pt2) II Ea19, Ea20, Ea21 Ea52 Malus domestica “ 2002 II (Pt2) II Ea54 Malus domestica “ 2003 II (Pt2) II Ea55 Malus domestica “ 2004 II (Pt2) II Ea13 Pyrus communis 42°08’37.83”N 1990 I (Pt1) I 24°44’58.42”E Ea15 Aronia melanocarpa “ 2004 I (Pt1) I Ea29 Cydonia oblonga “ 2004 I (Pt1) I Ea14 Aronia melanocarpa 42°55’46.99”N 2004 II (Pt2) II 25°52’38.92”E Ea31 Cydonia oblonga 42°18’40.81”N 2004 II (Pt2) II 23°45’56.49”E Ea22 Cydonia oblonga 42°39’17.58”N 2002 II (Pt2) II 23°16’42.14”E Ea23 Cydonia oblonga “ 2003 II (Pt2) II Ea24 Cydonia oblonga “ 2004 II (Pt2) II Ea25, Ea26, Ea27 Pyracantha coccinea 42°41’47.37”N 2003 II (Pt2) II 23°19’33.64”E Ea28 Cotoneaster integerrimus “ 2004 II (Pt2) II Ea30 Pyrus communis 42°15’44.87”N 1999 II (Pt2) II 23°06’31.51”E Ea34 Pyrus communis “ 2002 II (Pt2) II Ea32, Ea33 Pyrus communis 42°49’06.29”N 1999 II (Pt2) II 23°13’29.91”E Ea247 Fragaria moshata “ 1999 III (new Pt type) III Ea36 Crataegus sp. 42°32’01.28”N 1999 II (Pt2) II 23°21’58.12”E Ea236 Malus domestica 42°45’52.67”N 2001 II (Pt2) II 26°43’42.46”E Ea237, Ea238 Fragaria moshata “ 1999 III (new Pt type) III Ea244 Fragaria ananassa 43°08’05.42”N 2002 III (new Pt type) III 24°43’02.14”E Ea245 Fragaria ananassa 43°11’14.96”N 2003 III (new Pt type) III 25°10’22.44”E Ea246 Pyrus sp. 41°23’53.24”N 2002 II (Pt2) II 23°12’24.88”E EaATCC15580 Pyrus communis I (Pt1) I Ea2C Pyrus communis Unknown II (Pt2) II after XbaI digestion and PFGE (Table I). Single the ﬁ rst group, displaying the PFGE pattern Pt1. strains, including the type culture of E. amylovora, It is interesting to note that all strains had been isolated from three different host plants, formed isolated from the Plovdiv region (Atanasova et 190 I. Atanasova et al. · Characterization of Erwinia amylovora by PFGE 7 28 29 30 31 32 33 34 35 M m m M 35 33 34 31 32 30 28 29 7

97- 49 - 49 - 388 - 291 - 437 - 340 - 340 - 485 - 485 - 243 - 194 - 194 - 146 -

kbp M m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M m 19 20 21 22 23 24 25 26 2 25 26 24 22 23 21 M m 19 20 18 17 16 15 13 14 12 10 11 9 8 7 6 5 4 3 2 m 1 M kbp

Fig. 1. Representative PFGE patterns of Bulgarian Erwinia amylovora strains after restriction with XbaI. Lane M, pulse marker, 50 – 1000 kb (Sigma); lane m, DNA marker (γ-phage DNA, 0.1 – 200 kb, Sigma); lane 1, type culture of E. amylovora ATCC 15580; lane 2, Ea1; lane 3, Ea2; lane 4, Ea3; lane 5, Ea4; lane 6, Ea5; lane 7, Ea6; lane 8, Ea7; lane 9, Ea8; lane 10, Ea9; lane 11, Ea10; lane 12, Ea11; lane 13, Ea12; lane 14, Ea13; lane 15, Ea16; lane 16, Ea17; lane 17, Ea18; lane 18, Ea19; lane 19, Ea20; lane 20, Ea21; lane 21, Ea30; lane 22, Ea32; lane 23, Ea33; lane 24, Ea34; lane 25, Ea36; lane 26, Ea39; lane 27, Ea40; lane 28, Ea42; lane 29, Ea44; lane 30, Ea51; lane 31, Ea54; lane 32, Ea55; lane 33, Ea236; lane 34, Ea237; lane 35, Ea238. The lane with unsuccessful DNA digestion is not numbered. I. Atanasova et al. · Characterization of Erwinia amylovora by PFGE 191 al., 2009). For Bulgaria and the Balkan region in pathogenicity of the strains was confi rmed by general this profi le has not been reported so far. vacu um infi ltration of young strawberry plants In the investigations of Zhang and Geider (1997), with bacterial suspension which resulted in typi- Zhang et al. (1998), Jock et al. (2002b), and Donat cal fi re blight symptoms (Bogatzevska, 2000; et al. (2007) the PFGE type Pt1 was found for Atanasova, 2006). The occurrence of a new ge- the strains of E. amylovora isolated in England, netic pattern of strawberry isolates of E. amylo- Central Europe, Northern France, and Spain. The vora refl ects the difference between Rosoideae presence of this profi le in Bulgaria could possibly and Maloideae strains. be explained by plant imports, accidental intro- One strain (Ea39) isolated from Malus domes- duction, or introduction by natural vectors of the tica (cv. Smoothee) possessed a profi le distinctly pathogen from areas where fi re blight was caused different from all other PFGE patterns described by strains displaying type Pt1. so far. The PFGE pattern of this strain is char- The majority of the Bulgarian strains (82%) acterized by the shift of one band, characteristic as well as the Serbian strain Ea2C displayed a for the main Pt2 profi le, from 364 kbp to about PFGE profi le identical with the type Pt2, and 346 kbp, and by the appearance of a new band. formed the second group. The strains of this ma- This suggested a shift of the site of restriction re- jor group originated from different locations, host sulting in the generation of a new 177-kbp frag- plants, and orchards and were isolated in differ- ment instead of 159 kbp as it was in the other pro- ent years, which indicates genomic stability, as fi les (a fusion of fragments at 159 kbp and 18 kbp well as homogeneity of the Bulgarian population to a band at 177 kbp). Some authors explained of E. amylovora. Additionally, the strains of this similar observations by a spontaneous mutation group possessed different RFLP profi les of the affecting a single DNA fragment (Zhang and pEA29 plasmid PstI-amplifi ed fragment digested Geider, 1997; Zhang et al., 1998). with HpaII and a different number of SSR (short We did not fi nd strains with the profi le type sequence repeats) – 8, 10, 11, 12, and 13 repeats Pt5, previously reported for one Bulgarian strain (Atanasova et al., 2009). Halupecki et al. (2006) by Zhang et al. (1998). found that the type Pt2 was characteristic for The PFGE pattern identifi ed for each strain the strains of E. amylovora isolated in Croatia. was reproducible in all cases. Zhang et al. (1998) observed that the two Bulgar- The restriction with XbaI followed by PFGE ian strains included in their work possessed pro- analysis revealed relative genetic homogeneity of fi les previously found for Mediterranean strains, the Bulgarian population of E. amylovora – 82% respectively, Pt2 and Pt5. of the strains possessed the same profi le, which All fi ve isolates from Fragaria spp. (subfam- was identical to the known Pt2 profi le. Neverthe- ily Rosoideae) had a XbaI digest pattern, which less, this analysis displayed the genetic diversity in differed greatly from that of the other Bulgarian the population, since the profi le Pt1 and two new strains, as well as from all profi les reported for profi les were established. These results reveal the E. amylovora so far, and were included in the possibility that strains originating from plants of third group. This profi le is characterized by the the subfamilies Maloideae and Rosoideae can shift of one band from 400 kbp to about 414 kbp, be distinguished genetically on the basis of their the appearance of one new 348-kbp band, and PFGE profi les. The comparison of our results with the lack of two bands (146 kbp and 299 kbp). All the data published by other authors allows us to these strains showed the same RFLP profi le of suggest that the main pathogen population was the pEA29 plasmid PstI-amplifi ed fragment di- probably introduced in Bulgaria from the Eastern gested with HpaII (Atanasova et al., 2009). Zhang Mediterranean, where type Pt2 is characteristic. and Geider (1997) described a PFGE profi le of a strawberry E. amylovora strain identical to the Maloideae strains and different from the profi le PFGE analysis of SpeI-digested established by us, but emphasized that the strain chromosomal DNA was isolated from plantations near apple orchards. Genomic DNA from the same strains was ana- The E. amylovora strains from Fragaria spp. used lysed by PFGE after digestion with SpeI (Fig. 2). in our work had been isolated from a strawberry The comparison of the positions of the resulting plantation totally destroyed by the pathogen. The bands by GelCompar also distributed the strains 192 I. Atanasova et al. · Characterization of Erwinia amylovora by PFGE 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 M m m M 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19

49 - 49 243 - - 243 437 - 437 - 291 - 291 - 194 - 485 - - 485 388 - 388 - 146 -

97 - - 97 340 - 340 -

kbp M m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M m M 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 m M kbp I. Atanasova et al. · Characterization of Erwinia amylovora by PFGE 193

Fig. 2. Representative PFGE patterns of Bulgarian SpeI also allowed the differentiation between Erwinia amylovora strains after restriction with SpeI. Rosoideae and Maloideae strains. Lane M, pulse marker, 50 – 1000 kb (Sigma); lane m, DNA marker (γ-phage DNA, 0.1 – 200 kb, Sigma); lane The third restriction enzyme used in this study, 1, type culture of E. amylovora ATCC 15580; lane 2, XhoI, generated identical macrorestriction pro- Ea1; lane 3, Ea2; lane 4, Ea3; lane 5, Ea4; lane 6, Ea5; fi les for all studied isolates, including the type lane 7, Ea6; lane 8, Ea7; lane 9, Ea8; lane 10, Ea9; lane strain of E. amylovora (results not shown). 11, Ea10; lane 12, Ea11; lane 13, Ea12; lane 14, Ea13; In conclusion, this work revealed the relative lane 15, Ea16; lane 16, Ea17; lane 17, Ea18; lane 18, Ea19; lane 19, Ea20; lane 20, Ea21; lane 21, Ea30; lane genetic homogeneity of the Bulgarian population 22, Ea32; lane 23, Ea33; lane 24, Ea36; lane 25, Ea39; of E. amylovora, since about 82% of the strains lane 26, Ea40; lane 27, Ea42; lane 28, Ea44; lane 29, isolated from host plants from the subfamily Ma- Ea51; lane 30, Ea54; lane 31, Ea55; lane 32, Ea236; lane loideae showed identical PFGE patterns. For a 33, Ea237; lane 34, Ea238. The lane with unsuccessful DNA digestion is not numbered. few Maloideae strains two additional profi les were established – type Pt1 (after XbaI digestion) and, so far, one unknown pattern for E. amylovora (Malus domestica-derived isolate). All Fragaria into four groups according to their PFGE profi les spp. isolates clustered into a separate group dem- (Table I). The fi rst group included three strains onstrating the genetic difference between the as well as the type culture of E. amylovora. The strains isolated from Maloideae and Rosoideae second group, which was the major one, included plants on the base of their XbaI and SpeI restric- 82% of the strains. In this group a multiple band of tion profi les. Our study extends the data on the about 350 kbp was observed. Strain Ea39, as well genetic diversity of E. amylovora and, based on as the fi ve Rosoideae strains separated from all the results obtained, a multiple source of inocu- other isolates into a third and a fourth group, re- lum of the Bulgarian population of E. amylovora spectively. The strain structure of the SpeI groups can be assumed. was identical to that formed with XbaI. The SpeI PFGE patterns of the strains were characterized by a smaller number of fragments in comparison Acknowledgements to those with XbaI and overlapping bands cor- This study was supported by the National Sci- responding to about 350 kbp. The restriction with entifi c Foundation Project CC1403/2004.

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