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1 Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121, USA
2 Sección de Microbiología, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo, Madrid, Spain
3 UMR de Pathologie Végétale INRA-INH-Université, BP 600 57, 42 rue Georges Morel, 49071 Beaucouzé cedex, France
Correspondence
Michael McClelland
mmcclelland{at}skcc.org
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A majority-rule consensus tree and a summary of discriminatory characters are available as supplementary material in IJSEM Online.
Present address: CNB-CSIC, Autonoma University of Madrid, Madrid, Spain. 
Present address: UR008 Pathogénie des Trypanosomatidés, IRD, 911 Avenue Agropolis, BP64501 34394 Montpellier cedex 5, France.
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, 1972) concluded that strains isolated from clinical sources and strains belonging to the Herbicola group of Dye (1969) are the same species and referred to them as the ‘Enterobacter agglomerans–Erwinia herbicola’ complex. Lelliott & Dickey (1984) defined Erwinia as associated with plants as pathogens, saprophytes or constituents of the epiphytic flora. They considered Erwinia herbicola as yellow and non-pigmented Erwinia-like organisms that exist either on plant surfaces or as secondary organisms in lesions caused by many plant pathogens, as described by Billing & Baker (1963). Gavini et al. (1989) described the new genus Pantoea and the species Pantoea agglomerans, which includes the type strains of Enterobacter agglomerans, Erwinia herbicola and Erwinia milletiae. Later, Erwinia ananatis (synonym Erwinia uredovora) and Erwinia stewartii were transferred to the genus Pantoea (Mergaert et al., 1993). Based on 16S rRNA gene phylogenetic analyses, plant-associated bacteria were reclassified into four genera: Erwinia, Pectobacterium, Brenneria and Pantoea (Hauben et al., 1998). Mergaert et al. (1999) reclassified non-pigmented Erwinia herbicola epiphytic strains isolated from trees as Erwinia billingiae, which clades within the first cluster of Hauben et al. (1998).
Samples from diseased olive trees were collected from the Navahermosa and Chozas areas in the Toledo region of central Spain. Strains from knots were isolated according to the method of García de los Ríos (1999) and were grown on King's medium B (KB) and nutrient agar for 48 h at 25 °C. Two colony types were easily distinguishable on both agar media. Pure cultures were established by single colony isolation onto fresh KB agar. The first type was identified as Pseudomonas savastanoi. The second type, which corresponded to large (3–5 mm), mucilaginous, pigmented and non-pigmented colonies, was identified as a member of Erwinia sensu lato by Gram stain, catalase, oxidase and phenylalanine deaminase activities, Kligler iron agar fermentation, methyl red reaction and motility. No Xanthomonas strains were identified. In order to determine more precisely the taxonomic status of these novel isolates, a polyphasic taxonomic study was initiated.
For sequence determination, total bacterial genomic DNA was isolated from the nine novel isolates by a CTAB miniprep procedure (Murray & Thompson, 1989). Bacteria were suspended in an extraction buffer containing 2 µl RNase A (1 mg ml–1) and incubated at 37 °C for 1 h. The samples were then microcentrifuged at 15 000 r.p.m. for 5 min. The supernatant was collected and the DNA was precipitated, resuspended in TE and then quantified spectrophotometrically and adjusted to a concentration of approximately 100 ng µl–1. An internal portion of the 16S rRNA gene sequence was obtained for each isolate using primer P16S27F, which anneals at position 27 of the Escherichia coli 16S rRNA gene (5'-ATTGAACGCTGGCGGCAGGCCTAA-3'), and primer P16S1455R (5'-CCTTGTTACGACTTCACCCCAGTC-3'), derived in this study from alignments of Erwinia and other enterobacteria species available in databases.
PCR was performed in a final volume of 25 µl containing 0·5 µM of each primer, 200 µM dNTPs, 1·5 mM MgCl2, 1·25 U Taq polymerase, 1x Taq polymerase reaction buffer and 25 ng chromosomal DNA. An initial denaturation step was performed in a thermocycler (Perkin Elmer, model 9600) at 95 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s and extension at 72 °C for 2 min, with a final extension at 72 °C for 10 min. Reactions were stored at 4 °C. A negative control contained sterile water instead of DNA template.
DNA was purified using a QIaexII gel extraction kit (Qiagen) and then ligated into a plasmid vector (TA cloning kit; Invitrogen) and transformed into cells of Escherichia coli INValpha-F'. The transformants were selected according to the blue-white screening procedure (Sambrook et al., 1989). Transformants were screened for the presence of an insert by PCR amplification using M13 phage primers (M13fwd, 5'-GGAAACAGCTATGACCAT-3'; M13rev, 5'-CGTTGTAAAACGACGGCCAG-3'). Following confirmation of inserts, plasmid DNA was extracted using Qiaquick plasmid miniprep (Qiagen). At least 1 µg DNA was used for sequencing and at least two clones of each strain were sequenced in both directions using a Li-Cor Sequencer. Sequencing reactions were prepared using the Sequenase kit with 7-deaza-dGTP (Amersham Pharmacia) according to the manufacturer's instructions. The fluorescent-labelled sequencing primers correspond to M13fwd and M13rev (see above). The sequences from each strain were aligned using the SEQUENCHER 3.0 software for Macintosh (Gene Codes Corporation) in order to trim away the vector sequence and to establish a consensus sequence for the insert.
Prior to phylogenetic analysis, representative sequences of several members of the Enterobacteriaceae were extracted from NCBI via BLAST searches (Altschul et al., 1990). Phylogenetic assays were conducted to identify the overall position of these strains in trees containing several sequences (data not shown). After this preliminary analysis, all species of Erwinia with validly published names (with the exception of Erwinia aphidicola, for which a 16S rRNA gene sequence is not available) and the closest phylogenetic neighbours from other genera were selected to perform more detailed phylogenies. All the sequences are derived from type strains with two exceptions (see Fig. 1 and Supplementary Fig. A, available in IJSEM Online). Sequence similarity comparisons were then conducted. Similarity values over 97 % were found for the following species: Erwinia billingiae (97·2 %), Pantoea ananatis (97·3 %), Pantoea agglomerans (98 %), Erwinia persicina (98 %), Erwinia rhapontici (98·1 %), Erwinia amylovora (97·4 %) and Erwinia pyrifoliae (97·1 %). As values of similarity higher than 97 % are generally not used to discriminate at the species level, DNA–DNA hybridization experiments were conducted for these strains. For other Erwinia species, the values were lower: Erwinia tracheiphila (95·6 %), Erwinia mallotivora (96·6 %) and Erwinia psidii (96·5 %). Nonetheless, we included in our analysis all species of Erwinia with validly published names, independently of the similarity value.
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) and further subjected to parsimony analysis using MEGA2.1 (http://www.megasoftware.net) as well as Bayesian probabilistic analysis using MrBayes (Huelsenbeck & Ronquist, 2001). The phylogenetic trees derived from these analyses included 1463 nucleotides and are shown in Fig. 1
(Bayesian tree) and in Supplementary Fig. A (parsimony tree). In the Bayesian analysis, 100 000 generations were run in four independent Markov chains. When convergence was reached, a total of 9000 trees were explored; Fig. 1 shows a consensus tree. The novel isolates form a very homogeneous group supported by a 99 % clade confidence value. The reliability of the parsimony tree (Supplementary Fig. A) was tested by a bootstrap analysis with 1000 replicates. Both trees showed the same topology with high confidence values.
Nucleotide signatures in the 16S rRNA genes are considered useful characters for assessing relatedness of bacteria. Hauben et al. (1998) assessed the 42 ‘signature’ nucleotide positions in the 16S rRNA gene that can be used to classify different genera of the Enterobacteriaceae. Fifteen nucleotide positions comprise the genus Erwinia signature. These positions are indicated in the species description. All the novel isolates are identical in all 15 of the characteristic nucleotide signatures of Erwinia species.
Twenty-two conventional biochemical and physiological tests and assimilation of 99 carbon sources using Biotype 100 strips (bioMérieux) were performed, as described previously (Sutra et al., 2001). A total of 121 tests were included in a numerical taxonomy analysis for 56 strains including the newly isolated strains and 43 reference or type strains representing the phylogenetic neighbours based on 16S rRNA gene sequence analysis. The distance matrix was calculated using the Jaccard coefficient (Sneath & Sokal, 1973). Cluster analysis was performed by using the unweighted pair group method with arithmetic averages (Sneath & Sokal, 1973). Discriminatory tests were selected using the diagnostic ability coefficient deduced from the numerical analysis (Descamps & Véron, 1981). The dendrogram of phenotypic distances among the 56 strains is shown in Fig. 2. At the distance level of 0·3277, 10 phena and 12 unclustered type strains were delineated. The phenotypic characteristics that differentiate the 10 phena and the unclustered type strains were deduced from the numerical taxonomy analysis (see Supplementary Table A in IJSEM Online). Phenon 1 gathered all nine endophytic strains shown in the phylogenetic tree (Fig. 1) at a distance of 0·06. Except Erwinia amylovora and Erwinia pyrifoliae, the closest species identified by 16S rRNA gene sequence comparisons (Fig. 1), i.e. Erwinia billingiae, Erwinia rhapontici and Erwinia persicina, as well as Pantoea agglomerans, Pantoea ananatis and Pectobacterium cypripedii, were found to be related to the endophytic strains on the basis of their phenotypic characteristics (Fig. 2). Erwinia amylovora and Erwinia pyrifoliae were phenotypically very distant and can be distinguished from the endophytic strains by their ability to assimilate sucrose and to produce reducing compounds from sucrose and their inability to grow at 36 °C.
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For DNA–DNA hybridization studies, DNA extractions were performed with the Qiagen Plasmid Mega kit according to an adapted protocol for genomic DNA extraction. For each strain, bacteria were grown overnight at 25 °C on four trypticase soy agar plates. Cultures were scraped off in TES buffer (50 mM EDTA, 50 mM Tris/HCl, 100 mM NaCl, pH 8·0) and centrifuged at 5000 g for 30 min at 4 °C. Bacterial pellets were washed twice in TES buffer and resuspended in 26 ml B1 buffer (50 mM Tris/HCl, 50 mM EDTA, 0·5 % Tween 20, 0·5 % Triton X-100; pH 8·0). Cell suspensions were freeze–thawed from –20 to 37 °C, diluted fourfold in B1 buffer and distributed into four tubes. Each suspension was thoroughly mixed with 1 ml of a lysozyme solution (100 mg ml–1), 1·25 ml of a 5 mg Pronase ml–1 solution (Sigma) and 900 µl of a 25 % SDS solution and incubated overnight at 37 °C. Fourteen microlitres of a 100 mg RNase ml–1 solution [98 Kunitz units (mg protein)–1] were added to each tube, which were then incubated for 1 h at 60 °C. After addition of 9·5 ml B2 buffer (3 M guanidine hydrochloride, 20 % Tween 20) to the clear lysate, the tubes were incubated for 30 min at 50 °C. The following steps of the DNA extraction are described in the Qiagen genomic DNA handbook, but volumes are adapted. After the flow of the sample through the equilibrated tip, the tip was washed twice with 100 ml Qiagen QC buffer. Genomic DNA was eluted with 35 ml Qiagen QF buffer and precipitated with 0·7 vols room-temperature 2-propanol. The DNA was then dissolved in 1 ml TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 7·5); the concentration of the extracted DNA was determined by measuring the A260 and the purity was checked by determination of the value A260/A280. Native DNA of strain CFBP 6631T was labelled in vitro with tritium-labelled nucleotides (Amersham) by random-priming (Feinberg & Vogelstein, 1983) using a Megaprime kit (Amersham). The S1 nuclease-trichloroacetic acid method was used for DNA–DNA hybridizations (Crosa et al., 1973; Grimont et al., 1980). The reassociation was performed at 60 °C in 0·42 M NaCl.
DNA–DNA hybridizations experiments with the labelled DNA of strain CFBP 6631T showed very high levels of reassociation (93–100 % hybridization) within the endophytic strains. In contrast, low levels of reassociation (9–22 % hybridization) were observed with DNAs from type strains of the closest phylogenetic neighbours: Erwinia rhapontici, Erwinia persicina, Erwinia billingiae, Erwinia pyrifoliae and Erwinia amylovora (Fig. 1). From the DNA–DNA hybridization results, we can conclude that the nine strains of phenon 1 belong to the same genomospecies (Table 1).
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, was 52±0·5 mol%. This value is in the range of the G+C contents of other Erwinia species (Hauben et al., 1998). To summarize, phenotypic tests, 16S rRNA gene sequence analysis, nucleotide signature identification and DNA–DNA hybridization procedures were performed on nine endophytic bacterial strains associated with olive trees, all of which were members of the Enterobacteriaceae with at least a superficial resemblance to the genus Erwinia. Phylogenetic analyses of 16S rRNA gene sequences, using two different methods, and nucleotide signature identification confirmed their taxonomic position in the genus Erwinia. DNA–DNA hybridizations experiments supported their status as a novel species. Numerical analysis of 121 phenotypic tests allowed determination of discriminatory characters.
Description of Erwinia toletana sp. nov.
Erwinia toletana (to.le.ta'na. L. fem. adj. toletana from Toletum, the Roman name for Toledo, the location from which the organisms were isolated).
Strains show the general characteristics of the Enterobacteriaceae and the specific characteristics of the genus Erwinia, as described by Hauben et al. (1998). Cultures are Gram-negative, oxidase-negative, catalase-positive, motile and ferment glucose without gas formation. The optimal growth temperature is 28 °C. Growth occurs at 36 °C but not at 39 °C. Colonies grown on nutrient agar are circular, slightly convex with entire margins, translucent and non-pigmented. In KB medium, colonies are circular, convex, highly mucoid, translucent and non-pigmented. Gelatin is not liquefied; indole, acetoin and hydrogen sulfide are not produced. The methyl red reaction is positive and nitrate is not reduced to nitrite. Strains exhibit 
-galactosidase, 
-galactosidase and -glucosidase activity, but no urease, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, phenylalanine deaminase, N-acetyl--glucosaminidase, -maltosidase, lipase or L-aspartic acid arylamidase activities. Acid is produced when growth medium contains mannitol, glucose, trehalose, meso-inositol, melibiose, amygdalin, L-arabinose, galacturonate, maltose, D-arabitol or cellobiose. Strains grow on the following substrates as sole carbon sources: D-glucose, L-arabinose, D-ribose, mannitol, salicin, melibiose, citrate, acetate, propionate, 2-ketogluconate, N-acetylglucosamine, malonate, L-alanine, L-proline and L-serine. Strains possess signature nucleotides identical to the signatures described by Hauben et al. (1998) for the genus Erwinia: A408, A594, C598, G639, G646, C839, G847, G987, G988, C989, G1216, C1217, C1218, C1308 and G1329, using the Escherichia coli 16S rRNA gene sequence numbering (Brosius et al., 1981). The DNA G+C content of strains CFBP 6631T and CFBP 6630 is 52±0·5 mol%.
The type strain is strain A37T (=CFBP 6631T=ATCC 700880T=CECT 5263T). Several other isolates from this species have been deposited in the CFBP (see Fig. 1) and CECT. Strains have been isolated from olive knots in association with Pseudomonas savastanoi pv. savastanoi as secondary invaders on diseased plants.
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ACKNOWLEDGEMENTS |
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