IJSEM Papers in Press. Published December 24, 2010 as doi:10.1099/ijs.0.026336-0

1 oleae sp. nov., isolated from olive knots caused by Pseudomonas savastanoi pv. 2 savastanoi 3 4 Chiaraluce Moretti*1, Taha Hosni*1, Katrien Vandemeulebroecke2, Carrie Brady3, Paul De Vos2, 5 Roberto Buonaurio1 and Ilse Cleenwerck2 6 7 * C. Moretti and T. Hosni contributed equally to this work 8 1 Dipartimento di Scienze Agrarie e Ambientali, University of Perugia, Perugia, Italy 9 2 BCCM/LMG Collection, Ghent University, Ghent, Belgium 10 3 LM-UGent, Laboratory of Microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium 11 12 13 Running title: Erwinia oleae sp. nov. from olive knots 14 15 16 17 Correspondence: Chiaraluce Moretti, [email protected] 18 19 20 21 The GenBank accession number for the 16S rRNA gene sequence of DAPP-PG 531T (= LMG 22 25322T = DSM 23398T) is GU810925. The accession numbers for the atpD, gyrB, infB and rpoB 23 gene sequences of DAPP-PG 531T, DAPP-PG 672 (= LMG 25321) and CECT 5264 (= LMG 24 25328) are GU991653-GU991656, HM439616-HM439619, HM439612-HM439615 respectively. 25 26 27 Summary 28 Three endophytic bacterial isolates were obtained in Italy from olive knots caused by Pseudomonas 29 savastanoi pv. savastanoi. Phenotypic tests in combination with 16S rRNA gene sequence analysis 30 indicated a phylogenetic position of these isolates in the genus Erwinia or Pantoea, and revealed 31 two other strains with highly similar 16S rRNA gene sequences (> 99 %), CECT 5262 and CECT 32 5264, obtained in Spain from olive knots. Rep-PCR DNA fingerprinting of the five strains from 33 olive knots with BOX, ERIC and REP primers revealed three groups of profiles that were highly 34 similar to each other. Multilocus sequence analysis (MLSA) based on concatenated partial atpD, 35 gyrB, infB and rpoB gene sequences, indicated that the strains constitute a single novel species in 36 the genus Erwinia. The strains showed general phenotypic characteristic of Erwinia, and whole 37 genome DNA-DNA hybridization data confirmed they represent a single novel Erwinia species. 38 The strains showed a DNA G+C base composition ranging from 54.7 to 54.9 mol%. They could be 39 discriminated from the phylogenetically related Erwinia species by their ability to utilise potassium 40 gluconate, L-rhamnose and D-arabitol, but not glycerol, inositol and D-sorbitol. The name Erwinia 41 oleae (type strain DAPP-PG 531T = LMG 25322T = DSM 23398T) is proposed for this new taxon. 42 43 Knot formation on olive trees (Olea europaea L.) is a serious disease found in many olive 44 producing areas. It is caused by Pseudomonas savastanoi pv. savastanoi and characterized by 45 outgrowth on trunks and branches, and less frequently leaves and fruits (Sisto et al., 2004). Olive 46 knots are ideal niches for bacterial growth, not only of the causal agent of the disease, but also of a 47 number of endophytic such as Erwinia toletana (Rojas et al., 2004), Pantoea 48 agglomerans (Marchi et al., 2006; Quesada et al., 2007) and other bacteria from the genera 49 Burkholderia, Hafnia, Pseudomonas and Stenotrophomonas (Ouzari et al., 2008). In the last years, 50 several studies have focussed on the effect of these endophytes in modulating olive knot disease 51 severity (Marchi et al., 2006; Hosni, 2010). As such, it has been shown that P. agglomerans, 52 frequently isolated from olive knots when inoculated in olive plants together with P. savastanoi pv. 53 savastanoi, can either depress growth of the pathogen or produce an increase in knot size (Marchi et 54 al., 2006). 55 56 In the present study, five endophytic strains from olive knots (DAPP-PG 531T, DAPP-PG 537, 57 DAPP-PG 672, CECT 5262 and CECT 5264) were investigated using a polyphasic taxonomic 58 approach. 59 60 Strains 61 In September 2003 and May 2007, young knots from branches of diseased olive trees located in 62 orchards at Scanzano in the province of Perugia (Umbria, Central Italy) and Valenzano in the 63 province of Bari (Apulia, South Italy) were collected. Small portions of their internal water-soaked 64 tissue were excised with a scalpel and crushed in a few drops of sterile distilled water. 65 Subsequently, a loopful of these suspensions was streaked onto nutrient agar (NA; Oxoid Ltd, UK) 66 and the plates incubated at 27 ± 1 °C for 2 days. Along with circular (0.5–2.9 mm in diameter), 67 white to pale yellow colonies, resembling ‘fried egg’, typical for P. savastanoi pv. savastanoi, 68 another bacterial colony type was frequently isolated. The latter type was selected for further 69 investigation. Pure cultures of this type were obtained by picking up a single colony and streaking it 70 onto NA plates amended with 5 % of sucrose. This way, strains DAPP-PG 531T and DAPP-PG 537 71 were obtained in 2003 and strain DAPP-PG 672 in 2007. Initial microbiological characterization of 72 these strains revealed that their colonies were not fluorescent when cultivated on King’s medium B -1 -1 -1 -1 -1 73 (Peptone 20 g l , anhydrous K2HPO4 1.5 g l , MgSO4 1.5 g l , glycerol 10 ml l , agar 15 g l ). It 74 also revealed that their cells were Gram-negative (as they lysed in 3 % KOH; Suslow et al., 1982), 75 oxidase negative, catalase positive and facultatively anaerobic suggesting they belong to the family 76 Enterobacteriaceae. Additional strains used in this study were obtained from various biological 77 resource centres, and cultivated following the instructions of the provider. All bacterial strains used 78 in this study are listed in Supplementary Table 1. 79 80 16S rRNA gene sequence analysis 81 Genomic DNA was extracted from strain DAPP-PG 531T according to the protocol of Niemann et 82 al. (1997). Amplification of the 16S rRNA gene was performed with the conserved primers 16F27 83 (5’ AGAGTTTGATCCTGGCTCAG 3’) and 16R1522 (5‘ AAGGAGGTGATCCAGCCGCA 3’). 84 Purification of the amplification product was done with the NucleoFast® 96 PCR Clean-up Kit 85 (Macherey-Nagel, Düren, Germany). Sequencing reactions were performed with the internal 86 primers listed by Coenye et al. (1999) using the BigDye® Terminator Cycle Sequencing kit 87 (Applied Biosystems, Foster City, CA, USA). Purification of the sequencing reaction products was 88 done using the BigDye® XTerminatorT Purification kit (Applied Biosystems, Foster City, CA, 89 USA). Sequencing was performed using an ABI Prism® 3130XL Genetic Analyzer (Applied 90 Biosystems, Foster City, CA, USA). Sequence assembly was done using the software package 91 BioNumerics (Applied Maths, Belgium). A nearly complete 16S rRNA gene sequence (1494 nt) 92 was obtained for strain DAPP-PG 531T and compared with 16S rRNA gene sequences deposited at 93 NCBI, using BLAST. This analysis indicated that the strain belonged to the genera Erwinia or 94 Pantoea, and it revealed two strains with very similar 16S rRNA gene sequences (> 99 % pairwise 95 similarity), ‘Pantoea oleae’ CECT 5262 and CECT 5264 that were obtained in Spain from olive 96 knots. Using the software package BioNumerics (Applied Maths, Belgium), the nearly complete 97 16S rRNA gene sequences of DAPP-PG 531T, CECT 5262 and CECT 5264 were compared with 98 those of reference strains of the species of Erwinia, Pantoea and related taxa collected from EMBL. 99 Pairwise similarities were calculated using an open gap penalty of 100 % and a unit gap penalty of 0 100 %. A neighbour-joining phylogenetic tree (Fig. 1) was constructed using BioNumerics, and the 101 robustness of the branches was evaluated by bootstrap analysis (Felsenstein, 1985). A maximum- 102 likelihood phylogenetic tree was constructed (Supplementary Fig. 1) as described previously (Brady 103 et al., 2008). The three strains from olive knots showed more than 99 % 16S rRNA gene sequence 104 similarity among each other, and less than 97 % to the known Erwinia and Pantoea species. As 105 strains showing less than 97% 16S rRNA gene sequence similarity are not likely to have more than 106 60 to 70 % DNA-DNA relatedness (Stackebrandt & Goebel, 1994), these similarity values strongly 107 suggested that the strains from olive knots represented at least one novel species in the family 108 Enterobacteriaceae. 109 110 Rep-PCR DNA fingerprinting 111 Genomic DNA was extracted from strains DAPP-PG 531T, DAPP-PG 537, DAPP-PG 672, CECT 112 5262, CECT 5264 and from E. toletana CFBP 6631T with the GenElute Bacterial Genomic DNA 113 Kit (Sigma Aldrich, St Louis, MO, USA). Rep-PCR fingerprinting was performed with the BOX 114 (Versalovic et al., 1994), ERIC (Hulton et al., 1991) and REP (Higgins et al., 1982; Versalovic et 115 al., 1991) primers, according to the method described by Rademaker and de Bruijn (1997). Repeats 116 were performed, and identical results were obtained. The rep-PCR profiles are shown in 117 Supplementary Fig. 2. Irrespective of the primers used, strains DAPP-PG 531T and DAPP-PG 537 118 generated the same fingerprints as well as strains CECT 5262 and CECT 5264. The Dice’s 119 coefficient between the two groups of strains was 0.88. Strain DAPP-PG 672 had a similarity index 120 of 0.88 with DAPP-PG 531T and DAPP-PG 537 and 0.94 with CECT 5262 and CECT 5264. E. 121 toletana CFBP 6631T generated different fingerprints and with low similarity (0.36) in comparison 122 with the other tested strains. Based on previous studies (Gevers et al., 2001; De Vuyst et al., 2008), 123 the rep-PCR data suggested that strains DAPP-PG 531T, DAPP-PG 537, DAPP-PG 672, CECT 124 5262 and CECT 5264 probably constituted a single species. 125 126 Multilocus sequence analysis 127 Multilocus sequence analysis (MLSA) of concatenated partial atpD, gyrB, infB and rpoB gene 128 sequences enables the differentiation of the phylogenetically related genera Erwinia, Pantoea and 129 Tatumella from each other (Brady et al., 2008, 2009a, b, c, 2010). To refine the taxonomic position 130 of the five strains from olive knots, partial sequences of the above-mentioned housekeeping genes 131 were determined for three representative strains, DAPP-PG 531T, DAPP-PG 672 and CECT 5264, 132 and 12 reference strains from known Erwinia species (see Fig. 2 and Supplementary Fig. 3; the 133 gene sequences from accession numbers HM439612 to HM439619, GU991653 to GU991656, 134 Q3953588 to Q393635 were determined in the frame of this study). Partial fragments of the atpD, 135 gyrB, infB and rpoB genes of these strains were amplified and sequenced using the protocol of 136 Brady et al. (2008), for DAPP-PG 531T, DAPP-PG 672 and CECT 5264 with the following 137 modifications: i) primer atpD 08-R (5’-CCGAGCAGCGCGGAGACTTC-3’) was used instead of 138 atpD 04-R ; ii) primer infB 05-F (5’-ACGGBATGRTBACSTTCCTKG-3’) was used instead of 139 infB 03-F. The modifications were needed because technical good sequences could not be obtained 140 with the primers atpD 04-R and infB 03-F, probably because they couldn’t bind efficiently. Primers 141 atpD 08-R and infB 05-F were designed based on sequences obtained with the primers atpD 03-F 142 and infB 04-R, respectively. Sequence assembly was performed using the software package 143 BioNumerics (Applied Maths, Belgium), and partial nucleotide atpD, gyrB, infB and rpoB gene 144 sequences were concatenated and aligned with concatenated partial atpD, gyrB, infB and rpoB gene 145 sequences of reference strains of Erwinia, Pantoea and Tatumella species taken from EMBL. The 146 software package BioNumerics (Applied Maths, Belgium) was used for this analysis, and 147 neighbour-joining and maximum-liklihood phylogenetric trees (Fig. 2 and Supplementary Fig. 3) 148 were constructed as described for the 16S rRNA gene. MLSA revealed that strains DAPP-PG 531T, 149 DAPP-PG 672 and CECT 5264 belonged to the genus Erwinia, and also suggested that they 150 probably constituted a single novel species. 151 152 Phenotypic assays 153 Strains DAPP-PG 531T, DAPP-PG 537, DAPP-PG 672, CECT 5262 and CECT 5264 were 154 subjected to API 20E and API 50CHE systems (bioMérieux), according to the manufacturer’s 155 instructions. The results are presented in the species description below. API 50CHE tests were also 156 carried out on type and reference strains of the 12 validly named Erwinia species. The strains 157 studied are presented in Supplementary Fig. 4, and the data obtained were numerically analysed to 158 reveal the phenotypic relationship between the five strains from olive knots and the validly named 159 Erwinia species. A distant matrix was calculated from similarity matrices generated using the 160 Dice’s coefficient (Dice, 1945), and subjected to the unweighted pair-group method with arithmetic 161 average (UPGMA) clustering algorithm using the NTSYSpc software (Exeter Software, New York, 162 USA) version 2.1. A cophenetic value of 0.86 was determined for this matrix, which indicated a 163 high goodness-of-fit. The dendrogram in Supplementary Fig. 4 revealed that strains DAPP-PG 164 531T, DAPP-PG 537, DAPP-PG 672, CECT 5262 and CECT 5264 formed a very homogeneous 165 cluster with an overall similarity of about 93 % well discriminated from the 12 currently recognized 166 Erwinia species that each formed a separate cluster. It also showed that the five strains from olive 167 knots had phenotypic features common to the genus Erwinia. Table 1 lists a selected number of 168 phenotypic features that permit differentiation of strains DAPP-PG 531T, DAPP-PG 537, DAPP-PG 169 672, CECT 5262 and CECT 5264 from the known Erwinia spp. including Erwinia toletana, the 170 phylogenetically most closely related Erwinia species also isolated from olive knots. Table 1 also 171 reveals that the strains can be discriminated from each Erwinia species, including E. 172 piriflorinigrans (López et al., 2010), by at least two characteristics. 173 174 DNA-DNA hybridizations 175 To confirm whether or not strains DAPP-PG 531T, DAPP-PG 537, DAPP-PG 672, CECT 5262 and 176 CECT 5264 truly constituted a single novel Erwinia species, DNA-DNA hybridizations were 177 performed. High-molecular mass DNA for DNA-DNA hybridization studies and DNA base 178 composition determination was extracted using the method of Wilson (1987), with minor 179 modifications (Cleenwerck et al., 2002). DNA quantity and quality were determined by measuring

180 the absorptions at 260, 280 and 234 nm, and only high quality DNA with A260/A280 and A234/A260 181 ratios of 1.8 – 2.0 and 0.40 - 0.60 was selected for further use. The size of the DNA was estimated 182 by agarose gel electrophoresis. DNA-DNA hybridizations were performed with the strains DAPP- 183 PG 531T, DAPP-PG 672 and CECT 5264 and the type strain of Erwinia toletana LMG 24162T 184 using a modification (Goris et al., 1998; Cleenwerck et al., 2002) of the microplate method 185 described by Ezaki et al. (1989). The hybridization temperature was 44 °C. Reciprocal reactions (i. 186 e. A x B and B x A) were performed, and their variation was generally taken within the limits of 187 this method (Goris et al., 1998). The DNA-DNA relatedness values reported are the mean of 188 minimum 6 hybridizations. The strains DAPP-PG 531T and DAPP-PG 672 and CECT 5264 189 exhibited high levels of DNA-DNA relatedness (> 80 %) amongst each other, and low levels (< 25 190 %) with E. toletana LMG 24162T (Table 2). As levels of 60 to70% DNA-DNA relatedness are 191 generally accepted as limit for species delineation (Wayne et al., 1987), the DNA-DNA 192 hybridization results confirmed that the strains from olive knots represented a single novel Erwinia 193 species. 194 The DNA G+C content of strains DAPP-PG 531T, DAPP-PG 537, DAPP-PG 672, CECT 5262 and 195 CECT 5264, was determined by HPLC according to the method of Mesbah et al. (1989), and varied 196 from 54.7 to 54.9 mol %, which is within the range reported for the genus Erwinia (Hauben et al., 197 1998; Mergaert et al., 1999; Kim et al., 1999; Gardan et al., 2004; Rojas et al., 2004; Geider et al., 198 2006). The DNA G+C content range was also less than 2 %, the generally accepted range within a 199 species. 200 201 In conclusion, based on the genotypic data (from 16S rRNA gene sequence analysis, rep-PCR DNA 202 fingerprinting, MLSA, and DNA-DNA hybridizations) and phenotypic data obtained in this study, 203 we propose to classify the five endophytic bacterial strains DAPP-PG 531T, DAPP-PG 537, DAPP- 204 PG 672, CECT 5262 and CECT 5264 from olive knots, caused by Pseudomonas savastanoi pv. 205 savastanoi, into a novel species. The name Erwinia oleae sp. nov. is proposed, with DAPP-PG 531T 206 (= LMG 25322T = DSM 23398T) as the type strain. 207 208 Description of Erwinia oleae sp. nov. 209 210 Erwinia oleae [o´le.ae, L. gen. fem. n. oleae of olive (Olea europaea), the plant from which the 211 bacterium was isolated]. 212 213 Strains have all the characteristics of the Enterobacteriaceae. Cells are Gram-negative, rods, 214 measuring 0.9 x1.5-3.0 µm; single, pairs, motile; non-spore-forming. After growing for 24-48 h on 215 nutrient agar at 27 ± 1 °C, colonies are light-beige, circular (1-1.2 mm in diameter), convex and 216 with entire margins. They do not produce fluorescent pigment on King’s medium B. Growth in 217 Yeast salt and Liquid 523 medium (Shaad et al., 2001) occurs at 36 °C, but not at 39 °C. Strains are 218 able to grow in 5 % NaCl. The strains are facultatively anaerobic and oxidase is not produced. 219 Results obtained with API20E (bioMérieux) indicate that strains have β-galactosidase activity, but 220 no arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, tryptophane 221 deaminase, phenylalanine deaminase and gelatinase (except strain DAPP-PG 672). Citrate is not 222 utilised; hydrogen sulfide, indole and acetoin (except strains CECT 5262 and CECT 5264) are not 223 produced. Nitrate is reduced to nitrite. Results obtained with API 50CHE (bioMérieux) indicate that 224 strains utilise the following substrates as sole carbon sources at 27 ± 1 °C within 2 days: L- 225 arabinose, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, L-rhamnose, D-mannitol, N- 226 acetylglucosamine, esculin, D-trehalose, D-arabitol, potassium gluconate and potassium 2- 227 ketogluconate, and arbutin and salicin (except strains DAPP-PG 531T and DAPP-PG 537). The 228 following carbon sources are not utilised at 27 ± 1 °C within 2 days: glycerol, erythritol, D- 229 arabinose, D-xylose, L-xylose, D-adonitol, methyl β-D-xylopyranoside, L-sorbose, dulcitol, 230 inositol, D-sorbitol, methyl α-D-mannopyranoside, methyl α-D-glucopyranoside, amygdalin, D- 231 cellobiose, D-maltose, D-lactose, D-melibiose, D-sucrose, inulin, D-melezitose, D-raffinose, starch, 232 glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, L-arabitol, 233 potassium 5-ketogluconate. The DNA G+C content of the five strains ranges from 54.7 to 54.9 mol 234 % as determined by the method of Mesbah et al. (1989). 235 236 The type strain, DAPP-PG 531T (= LMG 25322T = DSM 23398T) and DAPP-PG 537 (= LMG 237 25323 = DSM 23412) were isolated in Umbria (Italy) from olive knots caused by Pseudomonas 238 savastanoi pv. savastanoi. Additional strains were isolated in Apulia (Italy) (i.e. DAPP-PG 672 = 239 LMG 25321 = DSM 23411) and Spain (i.e. CECT 5262 = LMG 25327, CECT 5264 = LMG 25328) 240 also from olive knots caused by Pseudomonas savastanoi pv. savastanoi. 241 242 Aknowledgement 243 244 This research was supported by “Fondazione Cassa di Risparmio di Perugia”, Italy, project 245 “Indagini sui batteri endofiti associati a piante di olivo affette da rogna, volte all’individuazione di 246 nuove strategie di lotta alla malattia” to R.B. The BCCM/LMG Bacteria Collection is supported by 247 the Federal Public Planning Service – Science Policy, Belgium. C. 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Phenotypic characteristics differentiating strains of Erwinia oleae sp. nov. from the other Erwinia species. 381 382 Species: 1, E. amylovora LMG 2024T; 2, E. aphidicola LMG 24877T; 3, E. billingiae LMG 2613T; 4, E. mallotivora LMG 2708T; 5, E. papayae 383 CFBP 5189T; 6, E. persicina LMG 11254T; 7, E. psidii LMG 7034T; 8, E. piriflorinigrans; 9, E. pyrifoliae ICMP 14143T; 10, E. rhapontici LMG 384 2688T; 11, E. tasmaniensis LMG 25318T; 12, E. toletana CFBP 6631T; 13, E. tracheiphila LMG 2707T; 14, Erwinia oleae sp. nov. 385 Testa 1 2 3 4 5 6 7 8c 9 10 11 12 13 14 Nitrate reductionb - + + - - + - ND - + - - - + Growth at 36°C in yeast salt and liquid 523 mediumb - + - - - + - ND - - - + - + Fermentation of (API 50-CHE): L-Arabinose + + + - + + + + + + + + + + D-Mannose - + + + + + + - - + - + + + Esculin - + + - + + + - - + - + - + L-Rhamnose - + + - - + + - - + - - - + D-Arabitol - - + ------+ + + Gluconate - + - - + - - + - - - - - + 2-Keto-gluconate - + ------+ D-Sucrose + + - + + + + + + + + - + - Glycerol - + + - - + + + + + + + + - Inositol + + + - - + - + + + + + - - D-Sorbitol + - + - - + - - + - - - - - Xylitol - + ------+ + - - - 386 387 aThe strains tested for each Erwinia species are given in Supplementary Table 1; btests performed according to Schaad et al., 2001; cData from 388 López et al., 2010; ND, not determined. 389 390 391 392 393 Table 2. DNA-DNA relatedness (%) between the strains DAPP-PG 531T, DAPP-PG 672 and 394 CECT 5264 of Erwinia oleae sp. nov. and the type strain of Erwinia toletana LMG 24162T. 395 DNA-DNA relatedness (%) with strain*: Strain 1 2 3 4 1. E. oleae DAPP-PG 672 100 2. E. oleae DAPP-PG 531T 88 ± 6 100 3. E. oleae CECT 5264 88 ± 15 89 ± 16 100 4. E. toletana LMG 24162T 17 ± 4 14 ± 5 20 ± 5 100 396 *Each value is the mean of minimum 6 hybridizations ± SD. 397 398 0.01

Erwinia tracheiphila LMG 2906T (Y13250) 20 Erwinia psidii LMG 7034T (Z96085) 81 Erwinia papayae CFBP 11606T (AY131237) 99 Erwinia mallotivora DSM 4565T (AJ233414) Erwinia toletana LMG 24162T (FN547375) 27 Erwinia oleae DAPP-PG 531T (GU810925) 100 Erwinia oleae CECT 5264 (AF130967) 98 33 Erwinia oleae CECT 5262 (AF130969) Pantoea calida 1400/07T (GQ367478) 14 100 Pantoea gaviniae A18/07T (GQ367483) Pantoea cypripedii ICMP 1591T (EF122434) 100 5 Pantoea eucrina (MLSA group I) LMG 2781T (EU216736) 59 Pantoea dispersa LMG 2603T (DQ504305) Erwinia pyrifoliae ICMP 14143T (EF122435) 97 13 Erwinia amylovora LMG 2024T (Z96088) 86 Erwinia piriflorinigrans CECT 7348T (GQ405202) 80 75 Erwinia tasmaniensis Et1/99T (AM055716) Erwinia billingiae LMG 2613T (Y13249) 82 Erwinia rhapontici DQM 4484T (AJ233417) 65 Erwinia persicina LMG 11254T (Z96086) 40 Erwinia aphidicola LMG 24877T (FN547376) T 13 Pantoea septica (MLSA group H) LMG 5345 (EU216734) Pantoea stewartii subsp. indologenes LMG 2632T (Y13251) 40 100 Pantoea stewartii subsp. stewartii LMG 2715T (Z96080) Pantoea agglomerans NCTC 9381T (AJ251466) 64 41 Pantoea vagans (MLSA group A) LMG 24199T (EF688012) 99 Pantoea eucalypti (MLSA group B) LMG 24197T (EF688009) 17 Pantoea anthophila (MLSA group C) LMG 2558T (EF688010) 35 Pantoea brenneri (MLSA group E) LMG 5343T (EU216735) Pantoea deleyi (MLSA group D) LMG 24200T (EF688011) 26 22 Pantoea ananatis ATCC 33244T (U80196) 62 Pantoea allii LMG 24248T (AY530795) 22 26 Pantoea conspicua (MLSA group F) LMG 24534T (EU216737) Tatumella citrea LMG 22049T (EF688008) Tatumella ptyseos LMG 7888T (EU344770) 93 97 T 53 Tatumella terrea LMG 22051 (EF688007) T 44 Tatumella punctata LMG 22050 (EF688006) Tatumella morbirosei (MLSA group J) LMG 23360T (EU344769) Kluyvera georgiana ATCC 51603T (AF047186) 79 Kluyvera ascorbata ATCC 33433T (AF008579) 75 Kluyvera intermedia ATCC 33110T (AF310217) 54 69 Kluyvera cryocrescens ATCC 33435T (AF310218) 23 Enterobacter nimipressuralis LMG 10245T (Z96077) 66 Citrobacter gillenii CDC 4693-86T (AF025367) 21 Citrobacter braakii CDC 80-58T (AF025368) 86 T 32 Citrobacter freundii DSM 30039 (AJ233408) 67 Citrobacter youngae ATCC 29935T (M59291) Enterobacter hormaechei CIP 103441T (AJ508302) 18 Enterobacter cancerogenus LMG 2693T (Z96078) 89 Enterobacter asburiae JCM 6051T (AB004744) 22 Enterobacter cloacae subsp. cloacae ATCC 13047T (AJ251469) 56 Enterobacter cowanii CIP 107300T (AJ508303) 57 Citrobacter sedlakii CDC 4696-086T (AF025364) 100 Citrobacter farmeri CDC 2991-81T (AF025371) 399 Brenneria alni ICMP 12481T (AJ223468) 400 401 Fig. 1. Neighbour-joining tree based on nearly complete 16S rRNA gene sequences showing the 402 phylogenetic relationship between Erwinia oleae sp. nov. and related taxa within the 403 Enterobacteriaceae family. Brenneria alni ICMP 12481T was used as outgroup. The scale bar 404 indicates 1 % nucleotide substitutions. Numbers at branching points are bootstrap percentage values 405 based on 1000 replications. 406 407 Fig. 2. Neighbour-joining tree based on concatenated partial atpD, gyrB, infB and rpoB gene 408 sequences showing the phylogenetic relationship between Erwinia oleae sp. nov. and related taxa of 409 Erwinia, Pantoea and Tatumella. Cronobacter sakazakii ATCC BAA-894 was included as 410 outgroup. The scale bar indicates 10 % nucleotide substitutions. Numbers at branching points are 411 bootstrap percentage values based on 1000 replications.