Max-Planck-Institut für Zellbiologie, Rosenhof, 68526 Ladenburg, Germany1
Author for correspondence: Klaus Geider. Tel: +49 6203 106 117. Fax: +49 6203 106 122. e-mail: kgeider{at}zellbio.mpg.de
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ABSTRACT |
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Keywords: Asian pear blight, Erwinia pyrifoliae, nucleotide sequence alignment, PCR signals, PFGE
Abbreviations: EPS, exopolysaccharide; ITS, intergenic transcribed spacer; SSR, short sequence DNA repeat
The EMBL accession numbers for the sequences reported in this paper are AJ311829 (Ej546), AJ311830 (Ej617), AJ311831 (Ej557), AJ311832 (Ej562), AJ311833 (Ej556), AJ311834 (Ej546a), AJ311835 (Ep1/96), AJ311836 (Ea1/79) and AJ311837 (IL6).
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INTRODUCTION |
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Recently, a bacterial pathogen, Erwinia pyrifoliae, was characterized as a novel Erwinia species that also causes necrotic symptoms on Asian pears (Kim et al., 1999 ; Rhim et al., 1999
). The Asian pear pathogen was first detected in orchards near Chuncheon, South Korea, and has not been reported to occur in other countries. In several respects, this pathogen resembled Erwinia amylovora, the causative agent of fire blight of apple (Malus domestica L.) and European pear (Pyrus communis). The nucleotide sequence of the 16S rDNA from Erwinia pyrifoliae was almost identical with the corresponding sequence of Erwinia amylovora, and Erwinia pyrifoliae also produced ooze on slices of immature European and Asian pears. Biotype 100 and BIOLOG profiles, DNADNA hybridization kinetics, as well as nucleotide sequence analysis of the intergenic transcribed spacer region between the 16S and 23S rDNA distinguished Erwinia pyrifoliae from Erwinia amylovora. Based on these features it was classified as a new species within the genus Erwinia (Kim et al., 1999
). Additionally, Erwinia pyrifoliae does not contain a plasmid identical to pEA29 of Erwinia amylovora (Rhim et al., 1999
) and its PFGE pattern after an XbaI digest of genomic DNA is distinct from Erwinia amylovora (S. Jock & K. Geider, unpublished). Other bacteria connected to necrotic symptoms of Asian pears include Enterobacter pyrinus in Korea (Chung et al., 1993
) and Erwinia mallotivora in Japan (Goto, 1992
).
The claim of the existence of fire blight in Japan caused by Erwinia amylovora was reinvestigated in this study. Isolates from Japanese orchards described by Beer et al. (1996) and Kim et al. (1996)
were characterized for molecular properties and were found to differ from Erwinia amylovora, but to be related to Erwinia pyrifoliae, the novel species causing Asian pear blight (Kim et al., 1999
; Rhim et al., 1999
).
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METHODS |
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PCR assays for identification of Erwinia strains from Japan.
Primers for detection and identification of Erwinia amylovora as well as Erwinia pyrifoliae are listed in Table 2 and their target positions are outlined in Fig. 1
. The oligonucleotides were synthesized with a Beckman Oligo 1000M DNA Synthesizer. The PCR reactions were done in 50 µl with dilutions of bacterial cell cultures and 0·5 U Tth polymerase (Amersham Pharmacia) or 2 U Taq polymerase as described by Bereswill et al. (1992
, 1995
) for all primer pairs, and 30 cycles at 94 °C for 15 s, 52 °C for 15 s and 72 °C for 30 s were used for denaturation, annealing and polymerization, respectively. The expected product sizes are given in Table 2
.
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The Erwinia amylovora sequence overlaps with nt 2341064 in the nucleotide sequence deposited in the EMBL sequence database under accession no. X77921 for strain CFBP1430.
SDS-PAGE analysis.
Proteins from cell extracts were separated by PAGE using a 5% stacking gel and a 10% resolving gel (Sambrook et al., 1989 ). Whole-cell lysates were prepared for electrophoresis from 1·5 ml cultures, all grown overnight in LB medium. The cell pellets were resuspended in 100 µl SDS-PAGE sample buffer with ß-mercaptoethanol and boiled for 5 min. Ten microlitres of supernatant was applied and the protein bands were stained with Coomassie blue R-250. Fructose-6-phosphate kinase (85 kDa), bovine serum albumin (67 kDa), pyruvate kinase (66 kDa) and carbonic anhydrase (30 kDa) were used as size markers.
Plasmids and PFGE analyses.
Total plasmid DNA was isolated after alkaline lysis and purified with a Nucleobond AX column (Macherey and Nagel) according to the manufacturers instructions. Preparation of the plugs for PFGE, digestion with restriction enzymes and separation of the DNA fragments were as described by Zhang & Geider (1997) and Zhang et al. (1998)
.
Plant assays.
Virulence tests with slices of immature pears and with apple seedlings, as well as hypersensitive response assays, were done as described by Rhim et al. (1999) . Infiltration of three tobacco leaves (cultivar Samsun) was done with bacterial suspensions in water at 108 c.f.u. ml-1. For the other inoculations, bacterial cultures were grown for 1 d in nutrient broth and cell suspensions transferred to several pear slices or wounded leaves of pear seedlings with a toothpick. Negative controls were done with an Escherichia coli strain.
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RESULTS |
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Plant assays for confirmation as plant pathogens
The Japanese Erwinia strains produced a hypersensitive response on tobacco similar to Erwinia amylovora and Erwinia pyrifoliae. As with Erwinia pyrifoliae, they were not pathogenic on apple seedlings (cv. Golden Delicious), but produced ooze on slices of immature pears as do strains of Erwinia amylovora and Erwinia pyrifoliae (Rhim et al., 1999 ). In contrast, no ooze was produced after inoculation of pear slices with Erwinia mallotivora or Enterobacter pyrinus.
Differentiation by plasmid profiles and protein patterns
Plasmids from the Japanese Erwinia strains, Erwinia amylovora and Erwinia pyrifoliae were separated on 1% agarose gels (Fig. 2). At least one plasmid of the Erwinia strains from Japan was significantly larger than 29 kb, the size of plasmid pEA29, commonly found in Erwinia amylovora, and in all strains but Ejp556 at least one smaller plasmid was observed. Plasmid pEA29 (Fig. 2a
, lane 1) carries a single BamHI site (Falkenstein et al., 1988
) and was linearized after digestion (Fig. 2b
, lane 1). Digestion with BamHI cleaved most of the other large plasmids (Fig. 2a
) into several DNA fragments (Fig. 2b
). The plasmid patterns of four strains, Ejp546, Ejp546a, Ejp557 and Ejp562, were similar to each other, but Ejp556 did not carry the plasmid migrating as a supercoil at the position of a 6 kb DNA fragment. Ejp617 produced different plasmid profiles than the other strains and resembled that of Erwinia pyrifoliae Ep1/96 except that it contained only one low-molecular-mass plasmid migrating at the position of a 2 kb DNA fragment or below. These small plasmids were not cleaved by BamHI, similar to the small plasmids of Erwinia pyrifoliae (Rhim et al., 1999
). The complex plasmid pattern for the Erwinia strains from Japan supported the notion that they were not Erwinia amylovora.
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The 16S rDNA of the Japanese Erwinia strains was amplified with consensus primers (Weisburg et al., 1991 ) and the PCR product digested with HaeIII. For the Japanese Erwinia strains, Erwinia pyrifoliae and Erwinia amylovora an identical pattern of DNA fragments was found. Sequence analysis of the 16S rDNA from the three erwinias revealed 99% identity (data not shown). This region is apparently highly conserved for the Japanese Erwinia strains, Erwinia pyrifoliae and Erwinia amylovora, and cannot be used for their differentiation.
Sequence analysis of a DNA fragment in front of the chromosomal region encoding EPS synthesis
Biosynthesis of amylovoran depends on sugar transferases and transport proteins encoded in the ams region of Erwinia amylovora (Bugert & Geider, 1995 ). No major ORFs were detected for the nucleotide sequence up to 1 kb upstream of amsG. Promoter signals can be assumed for the region adjacent to the first gene, amsG. From this region (nt 2341064 in the sequence deposited in the EMBL nucleotide sequence database under accession no. X77921) 831 bp were subjected to sequence comparison for the Erwinia strains from Japan, Erwinia amylovora strains Ea1/79 (fruit trees/ornamentals) and IL6 (Rubus sp.), as well as Erwinia pyrifoliae strain Ep1/96, and a region with significant differences, presented in Fig. 6
, was identified. Alignment of the sequence revealed several features for the nine strains investigated: (i) the Erwinia strains from Japan were highly related to each other (9699% identity); (ii) Ejp546, Ejp546a, Ejp557 and Ejp562 can be distinguished from Ejp556 and Ejp617 by several mismatches; (iii) Erwinia strains from Japan are more closely related to Erwinia pyrifoliae (95%) than to Erwinia amylovora strains (fruit tree, 83%; Rubus sp., 84%); (iv) Erwinia amylovora strains from different hosts (fruit trees/ornamentals or Rubus sp.) carry an almost identical nucleotide sequence in this region. A dendrogram based on these nucleotide sequences placed the Erwinia strains from Japan close to Erwinia pyrifoliae and more distant from Erwinia amylovora (Fig. 7
).
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DISCUSSION |
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On the other hand, several properties, such as the lack of a PCR product with primer pairs of the ams region and primers RS1 and RS2 from pEA29 (Table 3), allowed differentiation of strains Ejp546, Ejp546a, Ejp557, Ejp562, Ejp556 and Ejp617 from Erwinia amylovora. With the Erwinia strains from Japan, the plasmid primers P29A and P29B gave rise to a DNA fragment around 1 kb (Beer et al., 1996
). Digestion with a restriction enzyme showed differences in the cleavage products in comparison to Erwinia amylovora (Table 3
), confirming that the signal with primers P29A and P29B was derived from a sequence not identical to the 1 kb fragment of pEA29 amplified from Erwinia amylovora. In contrast, the Japanese erwinias resembled Erwinia pyrifoliae in several molecular properties (Table 3
). All Japanese Erwinia strains gave a positive PCR signal with the cps primers from Erwinia pyrifoliae and produced identical DNA fragments after a restriction enzyme digest, confirming that the genes for EPS synthesis of the Japanese erwinias are related to the cps genes of Erwinia pyrifoliae. When the corresponding chromosomal regions preceding the gene clusters for EPS synthesis of Erwinia pyrifoliae, the Erwinia strains from Japan and Erwinia amylovora strains isolated from fruit trees/ornamentals and from Rubus sp. were aligned, the Japanese strains had a closer relationship to Erwinia pyrifoliae than to Erwinia amylovora. The similarity to Erwinia pyrifoliae was confirmed by PAGE of total cellular proteins and by the plasmid profiles. Plasmid pEA29 was found in all Erwinia amylovora strains investigated by DNA hybridization and by PCR assays with plasmid-derived primers (Falkenstein et al., 1988
; Bereswill et al., 1992
). In contrast to Erwinia amylovora, Erwinia pyrifoliae strains (Rhim et al., 1999
) and the Erwinia strains from Japan displayed a complex plasmid pattern. The Rubus sp. strains share a simple plasmid pattern with the Erwinia amylovora strains from fruit trees and ornamentals, and the pEA29 derivatives are largely identical for strains from different hosts (McGhee & Jones, 2000
). Cross-hybridization was observed with labelled DNA from plasmid pEA29 and DNA from Erwinia amylovora fruit tree and Rubus sp. strains, Erwinia pyrifoliae and the Erwinia strains from Japan (S. Jock & K. Geider, unpublished), limiting the use of total plasmid DNA to distinguish these sets of strains.
The PFGE patterns of Erwinia strains from Japan appeared unrelated to Erwinia amylovora, whereas they were highly conserved for all European strains assayed (Zhang & Geider, 1997 ; Zhang et al., 1998
). A relationship to Erwinia amylovora strains isolated from Rubus sp. could also be excluded by additional molecular data obtained. Rubus sp. strains differ from fruit tree strains in their PFGE pattern (S. Jock & K. Geider, unpublished), but they share an almost identical nucleotide sequence in a region upstream of the EPS-synthesis-encoding gene clusters and do not only produce PCR signals with pEA29 targeted primers, but also with ams primers used to identify strains from fruit trees and ornamentals. Similar to Erwinia pyrifoliae found in Korea, which have variable PFGE patterns, the Japanese erwinias appear to be also more variable than Erwinia amylovora. BIOLOG profiles and DNA hybridizations with the hrp region of Erwinia amylovora placed the Japanese strains close to the fire blight pathogen (Beer et al., 1996
). Other criteria, such as molecular features, including the nucleotide sequences of a DNA fragment from a chromosomal region preceding the gene cluster for EPS synthesis, seem to be quite powerful for differentiation of the Japanese Erwinia strains from Erwinia amylovora, although they are not classical taxonomic criteria. After the molecular differentiation of the Erwinia strains from Japan, molecular criteria proposed for the detection and identification of fire blight such as specific PCR primers (Bereswill et al., 1992
, 1995
; Kim & Geider, 1999
) are still valid for diagnosis of the disease and were recently successfully applied to describe a temporary infection of plants with Erwinia amylovora in the Melbourne Botanic Gardens (Jock et al., 2000
). Consequently, previous reports about fire blight in Japan on the island of Hokkaido (Beer et al., 1996
; Kim et al., 1996
) have to be reconsidered in the light of the new findings that the pear-pathogenic bacteria from Japan are more closely related to the Korean pear pathogen Erwinia pyrifoliae than to the fire blight pathogen Erwinia amylovora.
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ACKNOWLEDGEMENTS |
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Received 22 January 2001;
revised 27 June 2001;
accepted 2 July 2001.