Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kadki 24, 80-822 Gdansk, Poland1
Author for correspondence: Ewa ojkowska. Tel: +48 58 320 22 48. Fax: +48 58 301 28 07. e-mail: lojkowsk{at}biotech.univ.gda.pl
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ABSTRACT |
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Keywords: fingerprinting, differentiation, plant-pathogenic bacteria, recombinase A
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INTRODUCTION |
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Several immunological methods have been developed for the detection and identification of Erwinia carotovora subsp. atroseptica (De Boer & McNaughton; 1987 ; Gorris et al., 1994
; Hyman et al., 1995
), Erwinia chrysanthemi (Samson et al., 1989
) and Erwinia amylovora (Vantomme et al., 1982
). Furthermore, tests based on PCR have been described only for the more economically important Erwinia species: E. carotovora subsp. atroseptica (Darrasse et al., 1994
; De Boer & Ward, 1995
; Fréchon et al., 1998
; Smid et al., 1995
), E. amylovora (Bereswill et al., 1992
, 1995
; Guilford et al., 1996
; McManus & Jones, 1995
), E. chrysanthemi (Nassar et al., 1996
) and Ligase Chain Reaction (LCR) for Erwinia stewartii (Willson et al., 1994
). Toth et al. (1999a
) described a one-step 16S rDNA PCR-based method for the detection of all soft rot Erwinia species. However, this method did not enable the identification of the species and subspecies within the genus Erwinia.
Recent intensive studies of 16S rDNA sequences suggest that this approach could be used for identification purposes and also to resolve the taxonomic relationships of different species and groups of Erwinia (Kwon et al., 1997 ; Hauben et al., 1998
; Mergaert et al., 1999
; Kim et al., 1999
). As a result, it has been proposed that the genus Erwinia should be divided into four new genera, namely Erwinia, Pectobacterium, Pantoea and Brenneria. The genus Erwinia has been restricted to six species according to Hauben et al. (1999)
, E. amylovora, Erwinia mallotivora, Erwinia persicina, Erwinia psidii, Erwinia rhapontici and Erwinia tracheiphila. The resurrected genus Pectobacterium consists of four subspecies of E. carotovora together with E. chrysanthemi, Erwinia cacticida and Erwinia cypripedii. Six species, Erwinia alni, Erwinia nigrifluens, Erwinia paradisiaca, Erwinia quercina, Erwinia rubrifaciens and Erwinia salicis, have been classified in a new genus, Brenneria. Five species previously classified in the genus Erwinia, namely Erwinia ananas, Erwinia herbicola, Erwinia milletiae, Erwinia stewartii and Erwinia uredovora, have been reclassified in the genus Pantoea. This new nomenclature has not generally been accepted by plant pathologists; nevertheless both nomenclatures are currently in use.
Regardless of the taxonomy, it is important to identify bacterial species accurately and rapidly. New approaches based on application of several molecular markers give more information about genome specificity. As well as 16S and 23S rRNA there are several other candidates: heat-shock proteins (Hsp70, GroEL, Hsp60), the ATPase ß subunit, RNA polymerases and recombinase A (RecA) can serve as molecular markers for the identification of bacterial pathogens (Ludwig & Schleifer, 1999 ). RecA is a multifunctional protein involved in homologous recombination, DNA repair and the SOS response (Eisen, 1995
). It is considered to be universally present in prokaryotic and eukaryotic cells as it shows a high degree of sequence conservation. RecA protein and recA gene sequence comparisons have been used to speculate on phylogenetic relationships among genera and species (Lloyd & Sharp, 1993
; Eisen, 1995
; Karlin et al., 1995
). The recA gene has been used for typing of acinetobacters (Nowak & Kur, 1995
) and for identification of Mycobacterium species (Blackwood et al., 2000
) and the Bulkholderia cepacia complex (Mahenthiralingam et al., 2000
). Preliminary results showing the usefulness of recA PCR-RFLP for genotyping of E. carotovora were presented by Waleron et al. (2001)
.
This paper describes a method based on the analysis of recA gene polymorphism for the identification of the different species and subspecies of the former Erwinia genus. In addition, an analysis of the differentiation between the subspecies of E. carotovora and variation within E. chrysanthemi has been done.
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METHODS |
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Primer design.
Oligonucleotide primers were designed on the basis of the sequence of the E. carotovora recA gene described by Zhao & McEntee (1990) . Regions with low variability were chosen. The sequence of each primer (5'-GGTAAAGGGTCTATCATGCG-3' and 5'-CCTTCACCATACATAATTTGGA-3') was checked for homology to other sequences that may also be amplified by them, in the GenBank and EMBL databases using the BLAST N program.
DNA amplification.
DNA amplification was performed in 50 µl reaction volumes containing 5 µl 10x reaction buffer (Fermentas), 2·5 mM MgCl2, 250 µM each of dATP, dCTP, dGTP and dTTP, 20 pmol each primer, 0·1% (v/v) Tween 20, 50100 ng DNA and 1 U recombinant Taq DNA polymerase (cloned and purified by Dr J. Osipiuk, Department of Microbiology, University of Gdansk). Amplification was performed using a UNOII Biometra thermocycler with initial denaturation (95 °C, 3 min), followed by 32 cycles of denaturation (94 °C, 1 min), annealing (47 °C, 1 min) and extension (72 °C, 2 min), with a final extension (72 °C, 5 min). The amplified products were electrophoretically separated in 6% (w/v) polyacrylamide gel at 120 V for 10 h in TBE buffer and visualized with UV light after staining in ethidium bromide (0·5 µg ml-1).
Restriction fragment length analysis.
The amplified DNA fragments were digested with four restriction endonucleases (AluI, HinfI, TasI and Tru1I), which were selected on the basis of the nucleotide sequence of the recA gene of E. carotovora using Vector NTI software. The restriction analysis was performed overnight with 2·5 U of each endonuclease using the buffer and temperature recommended by the manufacturers (Fermentas). Restriction fragments were separated in a 12 % (w/v) polyacrylamide gel at 120 V for 10 h in TBE buffer and visualized with UV light after staining in ethidium bromide (0·5 µg ml-1).
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RESULTS |
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PCR products were digested by four restriction endonucleases (AluI, HinfI, TasI and Tru1I) in four separate reactions (Fig. 1). The smallest differentiation of RFLP patterns was observed with HinfI, which gave only seven different patterns with all Erwinia strains (Fig. 1a
). The greatest differentiation was obtained after digestion of the PCR product with TasI, which gave 31 patterns (Fig. 1b
). AluI and Tru1I gave 10 and 14 RFLP patterns, respectively (Fig. 1c
, d
). The results of the recA RFLP analysis of PCR product based on the number of bands and their position revealed the presence of 57 different recA combined RFLP patterns (restriction groups) (Table 2
). The combined patterns of the restriction analysis of the recA gene were consistently different and characteristic for most of the species and subspecies tested (Table 1
).
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Strains belonging to four species (E. amylovora, E. cypripedii, E. persicina and E. stewartii) and three subspecies of E. carotovora (subsp. betavasculorum, odorifera and wasabiae) occupied single RFLP groups based on recA PCR-RFLP (Table 1). In many other cases only one strain of a species was tested and examination of additional strains may reveal different RFLP patterns. Copies of the same strain obtained from different laboratories were tested and in all cases these showed the same RFLP profile (Table 1
). Strains of E. ananas, E. carotovora subsp. atroseptica, E. herbicola and E. rhapontici occupied two different RFLP groups and those of E. cacticida three groups (Table 1
). The highest diversity of the recA gene was observed among 57 strains of E. carotovora subsp. carotovora (18 groups) and 26 strains of E. chrysanthemi (15 groups) (Tables 1
and 2
).
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DISCUSSION |
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In contrast, the 13 strains from E. carotovora subsp. atroseptica belong to only two specific RFLP groups, 1 and 2 (Table 1). The low variability in this subspecies is in agreement with serological studies that indicated the presence of only a few serogroups (De Boer & Mc-Naughton, 1987
). Similarly, only two RFLP groups were obtained after analysis of the polymorphism of the genes encoding pectate lyase (Darrasse et al., 1994
;
led
et al., 2000
). A low level of variation was also observed in PCR for enterobacterial repetitive intergenic consensus (ERIC) sequences (Toth et al., 1999b
).
Four other pectolytic Erwinia species, E. cypripedii, E. carotovora subsp. betavasculorum, E. carotovora subsp. odorifera and E. carotovora subsp. wasabiae, show only one specific combined RFLP profile each, whereas the 15 strains of E. cacticida give three different patterns (Table 1). This is in accordance with results reported by Alcorn et al. (1991)
who described differences between strains of E. cacticida based on their physiology, pathogenicity and DNA homology.
The non-pectinolytic Erwinia strains, E. amylovora and E. persicina, gave one specific restriction pattern for each species, which suggests a low degree of diversity (Table 1). Although the strains of E. amylovora used were isolated from different Rosaceae plants growing in different countries, they nevertheless belonged to the same recA PCR-RFLP group (Table 1
). The low variability among E. amylovora strains has also been observed in biochemical (Dye, 1968
; Verdonck et al., 1987
), serological (Vantomme et al., 1982
), DNA hybridization (Brenner et al., 1974
) and PFGE studies (Zhang & Geider, 1997
).
For E. rhapontici two RFLP groups were observed. Two strains were assigned to group 21 and were identical to strains of E. carotovora subsp. betavasculorum; a single strain was in the unique group 53 (Table 1). Verdonck et al. (1987)
reported that E. rhapontici strains form three groups. One of them was common for E. rhapontici and E. carotovora strains. However, in biochemical and DNA hybridization tests, the type strain of E. rhapontici was very similar to the strains of E. persicina (Hao et al., 1990
). Phylogenetic analyses of Erwinia species based on 16S rDNA sequences also confirm these observations (Kwon et al., 1997
; Hauben et al., 1998
).
Analysis of the recA gene in species which have been reclassified in the genus Pantoea (E. ananas, E. herbicola, E. milletiae, E. stewartii and E. uredovora) indicated the presence of five RFLP patterns, some common to more than one species (Table 1). Only E. stewartii strains belonged to one RFLP group. E. ananas strains belonged to two different RFLP groups, 25 and 26, but group 25 also included a single strain of E. uredovora (Table 1
). This is in agreement with the results of DNADNA hybridization (Mergaert et al., 1993
) and analysis of 16S rDNA (Kwon et al., 1997
; Kim et al., 1999
), indicating a high degree of similarity between the two species. On that basis, these two pathogens have been described as being different pathovars of the new species Pantoea ananas, pv. ananas and pv. uredovora. It is interesting to note that strains of E. ananas and E. uredovora that have been reported to be ice-nucleation-active (Watanabe & Sato, 1998
) were placed into RFLP group 25, whereas the strains without this activity were in RFLP group 26.
Six strains of E. herbicola belong to two different RFLP groups, 27 and 28, one of which, group 28, also includes single strains of E. milletiae and Pantoea sp. (Table 1). This is in agreement with data from molecular tests (Brenner et al., 1984
; Lind & Ursing, 1986
; Beji et al., 1988
) and phenotypic data (Mergaert et al., 1984
; Verdonck et al., 1987
), which suggest that E. herbicola and E. milletiae are closely related; even their names are synonymous and they have both been reclassified in the genus Pantoea (Gavini et al., 1989
).
Strains of E. nigrifluens, E. quercina, E. rubrifaciens and E. salicis, which have been reclassified into the genus Brenneria, each gave one distinct pattern (Table 1). Numerical taxonomic analysis (Verdonck et al., 1987
) and 16S rDNA sequence analysis (Hauben et al., 1998
) of these species showed that they form separate homogeneous groups and are distinct from each other.
The degree of genetic diversity in the different species indicated by the number of RFLP groups obtained per species could be tentatively associated to host specificity and host range of the bacteria, and to their geographical, spatial and temporal distributions. Species exhibiting host specificity and a narrow host range in a defined climate, such as E. amylovora and E. carotovora subsp. atroseptica, infecting rosaceous plants and potato, respectively, would tend to be genetically homogeneous. There was probably less evolutionary pressure to adapt and diversify than in the case of species with a wide host and geographical range, such as E. chrysanthemi and E. carotovora subsp. carotovora (Perombelon & Kelman, 1980 ), both with a large number of recA RFLP groups.
Nassar et al. (1996) showed in PCR-RFLP analysis of the genes encoding pectate lyases that at least some of their groupings could be associated with hosts and geographical regions. However, no correlation could be found in this study between RFLP groups and host plants or geographical origins of the E. carotovora subsp. carotovora and E. chrysanthemi strains tested. The apparent genetic homogeneity in E. amylovora and E. carotovora subsp. atroseptica could have been magnified by the fact that both bacteria were probably relatively recently distributed across the world from their centres of origin, E. amylovora from North America in the 20th century (Schroth et al., 1974
) and E. carotovora subsp. atroseptica from South America in the 16th century (Salaman, 1949
). Most of the strains of these species studied in this paper originated from countries other than the centre of origin.
In contrast to E. amylovora, E. cacticida, E. chrysanthemi, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora and E. carotovora subsp. odorifera, only a small number of strains of other species have been examined in this study. This is because only a few strains are listed in culture collections and those available tend to have been isolated usually from one host plant and from one geographical area. Therefore, the presence of only a few groups per species does not necessarily signify that the species are genetically homogeneous. Only when more strains from a wider range of sources have been studied can this point be verified.
In conclusion, the recA PCR-RFLP method can be used to rapidly identify species and subspecies of pectinolytic Erwinia. It allowed identification of all of the species of the former Erwinia genus, including members of the genus that have not been well studied and are not identified by traditional methods.
In addition, the method is a useful tool to study species diversity in relation to host specificity, host range and geographical distribution. It would also be useful to examine the distribution of bacteria connected with the worldwide exchange of plant material.
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ACKNOWLEDGEMENTS |
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Received 15 May 2001;
revised 28 September 2001;
accepted 15 October 2001.