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1 Max-Planck-Institut für Zellbiologie, Rosenhof, 68526 Ladenburg, Germany
2 Universität Hannover, Institut für Mikrobiologie, Schneiderberg 50, 30167 Hannover, Germany
3 Friedrich-Schiller-Universität Jena, Institut für Mikrobiologie, Neugasse 25, 07743 Jena, Germany
Correspondence
Klaus Geider
K.Geider{at}bba.de
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ABSTRACT |
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Details of the fatty acid compositions of three of the novel strains and related strains and extended versions of the trees shown in Figs 1 and 2
are available as supplementary material in IJSEM Online.
Present address: Biologische Bundesanstalt, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany. 
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Here, we have selected bacteria from apple and pear trees in Australia for a white colony morphology and levan formation with sucrose. These features resemble properties of the fire blight pathogen E. amylovora (see review by Vanneste, 2000). A related Asian pear pathogen from Korea, Erwinia pyrifoliae (Kim et al., 1999, 2001), does not produce levan. The new isolates were classified by their 16S rRNA gene sequence and by partial nucleotide sequences of two housekeeping genes. Alignments of gpd gene sequences of necrogenic Erwinia and Brenneria species have been described in detail for the clarification of phylogenetic relationships (Brown et al., 2000; gpd, encoding glyceraldehyde-3-phosphate dehydrogenase, was referred to as gapDH in this earlier study). Due to multiple cellular functions of the RecA protein (Kowalczykowski et al., 1994), the recA gene is highly conserved in bacteria at the amino acid sequence level. Nevertheless, several silent changes in the nucleotide sequence can be used for species differentiation (Lloyd & Sharp, 1993). The former genus Erwinia has been dissected by microrestriction analysis of amplified recA PCR fragments (Waleron et al., 2002). The new isolates may contribute towards limiting the occurrence of fire blight in the Southern hemisphere.
Isolation of strains in Australia
In November 1999, we extracted pear flowers for levan-producing bacteria in the area of Knoxfield, Victoria, apple flowers in orchards near Hobart, Tasmania, and bark from apple trees taken in Applethorpe, Queensland, near Brisbane (Table 1), by suspending the plant tissue in a small amount of sterile water. Aliquots of the supernatants were spread on LB agar plates (with 50 µg cycloheximide ml–1 to avoid fungal growth), which were incubated for 2 days at 28 °C. From pear flowers in Victoria, 25 white colonies on agar plates with approx. 500 colonies were transferred to LB agar with 5 % sucrose and all produced levan. In similar experiments with extracts from apple flowers in Tasmania, 12 of 25 were levan-positive. From extracted bark of apple trees (cultivar ‘Fuji’) in Queensland, we obtained approx. 200 yellow and 100 white colonies per plate, some of which were levan-positive (2/25). In initial microbiological characterization, the levan-producing isolates did not stain in a Gram treatment and lysed in 3 % KOH (Gregersen, 1978; Suslow et al., 1982), both properties of Gram-negative bacteria. They were also positive in the aminopeptidase reaction (Cerny, 1976; Bascomb & Manafi, 1998) and were facultatively anaerobic, suggesting their classification in the genus Erwinia. Three isolated strains, Et1/99T (isolated in Tasmania), Et2/99 (from Knoxfield, Victoria) and Et4/99 (from the Stanthorpe area in Queensland), were studied in detail. As strains Et1/99T and Et2/99 were isolated as the dominant bacteria by suspending apple or pear flowers in water, they should exist as epiphytes that colonize plant surfaces.
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16S rRNA gene sequences
For molecular classification of the novel strains, DNA fragments were amplified with three sets of PCR primers (Table 2). DNA bearing 16S rRNA gene sequences was obtained with the primers fd2 and rP1. The 1.4 kb amplification product was cloned into vector pGEM-T (Promega) and sequenced. The nucleotide sequences of the three novel strains were aligned with a series of 16S rRNA gene sequences from other Erwinia species and species of Pectobacterium, Pantoea and Brenneria (Fig. 1 and Supplementary Fig. S1 available in IJSEM Online). They were closely related and were separated from E. amylovora, E. pyrifoliae and Erwinia billingiae. Dendrograms were constructed with CLUSTAL X for Windows version 1.81 and MEGA 3.1 (Kumar et al., 2004). Phylogenetic distances were estimated by the method of Jukes & Cantor (1969) and tree topology was inferred by the neighbour-joining method with a bootstrap value of 1000. For reconstruction of phylogeny, the neighbour-joining and maximum-parsimony methods produced similar results. The 16S rRNA gene sequences of strains Et1/99T, Et2/99 and Et4/99 possessed signature nucleotides identical to those described for the genus Erwinia (Hauben et al., 1998) [A, A, C, G, G, C, G, G, G, C, G, C, C, C and G at positions 408, 595, 599, 639, 646, 839, 847, 987, 988, 989, 1216, 1217, 1218, 1308 and 1329, according to the Escherichia coli 16S rRNA gene sequence numbering (GenBank accession no. J01695)]. A shift by one position was found for the indicative nucleotides A and C given by Hauben et al. (1998) for positions 594 and 598 in the 16S rRNA gene sequences of all Erwinia strains included in Fig. 1. Among the 16S rRNA gene sequences, those from E. amylovora, E. pyrifoliae, E. billingiae and Erwinia persicina exhibited sequence similarities to the novel strains that exceeded 97 %. Consequently, further classification steps were focused on these species.
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; Lloyd & Sharp, 1993). Consensus primers from the gpd gene (Table 2), applied by Brown et al. (2000) for classification of necrogenic Erwinia and Brenneria species, were used for PCR amplification of genomic DNA from the novel strains and strains of E. amylovora and E. pyrifoliae. The products were cloned into pGEM-T and the nucleotide sequences were analysed for their relationship to each other and other bacterial species (Fig. 2a and Supplementary Fig. S2a). The three novel strains were highly related and formed a cluster adjacent to several E. amylovora strains and to E. pyrifoliae. They were separated from those of E. billingiae, Erwinia mallotivora, Erwinia papayae, E. persicina, Erwinia psidii, Erwinia rhapontici, Erwinia toletana and Erwinia tracheiphila. The genus Brenneria and Dickeya chrysanthemi were more distantly related, with Pectobacterium carotovorum used as outgroup. From the novel strains, E. amylovora strains and related bacteria, parts of the recA gene were PCR amplified, cloned, sequenced and aligned. According to the alignment, the three novel strains formed a narrow group apart from E. pyrifoliae and E. amylovora (Fig. 2b and Supplementary Fig. S2b). Interestingly, strains within these species were not completely identical in their recA sequences. All three species were well separated from species of the genera Brenneria and Pectobacterium. Thus, the use of these two housekeeping genes confirmed the results of the previous analysis that classified the novel strains into a new species, separated from E. amylovora and E. pyrifoliae.
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. The Gram-negative, facultatively anaerobic bacteria were motile, straight rods, they metabolized glucose, lacked gas production and were oxidase-negative and catalase-positive. The isolates showed the general characteristics of strains of the genus Erwinia in the family Enterobacteriaceae (Hauben & Swings, 2005). The isolates from Australia reacted uniformly in most biochemical and physiological tests except for their reactions for galactose, inositol and xylitol. The novel strains shared some metabolic characteristics with other Erwinia species, such as acetoin production, utilization of glucose, mannitol and arabinose, the lack of arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase, no production of indole, gelatinase, urease, tryptophan deaminase or H2S and a lack of nitrate reduction. They differed in a positive reaction of 
-galactosidase, they utilized citrate, but did not metabolize sorbitol, rhamnose or amygdalin, they did not reduce nitrate and did not grow at 36 °C (Table 3). Altogether, 79 characteristics were used to construct a dendrogram showing the phenotypic distance relationships among the strains tested. Isolates Et1/99T, Et2/99 and Et4/99 clustered together in one group within 80 %. This group is phenotypically related to E. amylovora and E. pyrifoliae, but clearly different from the other groups (Fig. 3).
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7c) dominated, making up 50–65 % of the total fatty acids. The Australian isolates showed profiles that were quantitatively and qualitatively similar, without statistically significant differences. Tridecanoic acid (13 : 0) was completely absent in these isolates. The percentage of 17 : 0 cyclo was low and that of 16 : 17c was relatively high compared with other Erwinia species. The dendrogram shows that the Australian isolates are significantly separated from the other Erwinia species (Fig. 4).
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, diaminopropane was found to be the major polyamine compound of isolates Et1/99T, Et2/99 and Et4/99 (data not shown), grouping them close to E. amylovora (Zherebilo et al., 2001) by their polyamine pattern. The three strains displayed identical protein patterns when extracts of whole-cell proteins were assayed by SDS-PAGE (Fig. 5). The similarity to reference strains of the species E. amylovora and E. pyrifoliae was lower.
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). DNA–DNA relatedness provides a single species definition that can be applied equally to the genomes of all bacteria without limitation to single genes. A phylogenetic definition of a species (genomospecies) was formulated as strains with approximately 70 % or greater DNA–DNA relatedness by Wayne et al. (1987). The degree of binding in reassociation experiments with DNA from reference strains of E. amylovora, E. billingiae, E. persicina, E. pyrifoliae and E. rhapontici was well below 70 % (Table 4), indicating that the Erwinia isolates from Australia belong to a species different from the other Erwinia species.
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). Similarities above 97 % for 16S rRNA gene sequences may indicate a high relationship of strains and additional parameters may be needed for species classification (Gevers et al., 2005). Nevertheless, the 16S rRNA gene sequence trees that we constructed showed the same monophyletic groups as the trees from gpd and recA genes. Minor differences that were observed in the positions of the groups relative to each other are in agreement with previous phylogenetic analysis of these two and several other housekeeping genes (Wertz et al., 2003). Thus, we concluded that the gpd and recA genes can also be used to discriminate between E. amylovora and related species.
Description of Erwinia tasmaniensis sp. nov.
Erwinia tasmaniensis (tas.ma.ni.en'sis. N.L. fem. adj. tasmaniensis pertaining to Tasmania, where the type strain was isolated).
Cells are Gram-negative, non-spore-forming, motile, straight rods, measuring 0.5–1 by 1.5–2 µm. Facultatively anaerobic, oxidase-negative and catalase-positive. The species has all of the characteristics of the genus Erwinia. Colonies on Standard I agar (StI; Merck) are light-beige, circular, smooth, translucent, flat to slightly convex and about 3 mm in diameter after 24–48 h at 28 °C. No diffusible or fluorescent pigment is observed. Strains grow well on StI agar and nutrient agar at 28 °C, but not at 36 °C. They are able to grow in 5 % NaCl. Glucose is fermented without gas production. Arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, gelatinase and tryptophan deaminase are not produced, but -galactosidase is present. Indole and H2S are not produced. There is acetoin production. Nitrates are not reduced. The following carbon sources are utilized at 28 °C within 3 days: L-arabinose, ribose, glucose, fructose, galactose (except strain Et4/99), mannitol, N-acetylglucosamine, sucrose, trehalose, melibiose (weak) and citrate. The following carbon sources are not utilized at 28 °C within 3 days: sorbitol, rhamnose, amygdalin, glycerol, erythritol, D-arabinose, D-xylose, L-xylose, adonitol, methyl -D-xyloside, mannose, sorbose, dulcitol, inositol (except strain Et2/99), methyl 
-D-mannoside, methyl -D-glucoside, arbutin, aesculin, salicin, cellobiose, maltose, lactose, inulin, melezitose, raffinose, starch, glycogen, xylitol (except strain Et2/99), gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, 2-ketogluconate and 5-ketogluconate. The G+C contents of strains Et1/99T, Et2/99 and Et4/99 are 50.5–52.4 mol% as determined by the Tm method. The known strains were isolated from the phyllosphere, in particular apple flowers, pear flowers and bark of apple trees. The species may also occur on other plant surfaces and may also be found outside Australia.
The type strain, Et1/99T (=DSM 17950T=NCPPB 4357T), was collected in Tasmania from apple flowers. Strain Et2/99 (=DSM 17949=NCPPB 4358) was isolated from pear flowers in Victoria and strain Et4/99 was isolated from apple bark in Queensland.
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ACKNOWLEDGEMENTS |
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