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Erwinia Pyrifoliae Sp. Nov., a Novel Pathogen That Affects Asian Pear Trees (Pyrus Pyrifolia Nakai)

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Erwinia Pyrifoliae Sp. Nov., a Novel Pathogen That Affects Asian Pear Trees (Pyrus Pyrifolia Nakai) International Journal of Systematic Bacteriology (1 999), 49, 899-906 Printed in Great Britain Erwinia pyrifoliae sp. nov., a novel pathogen that affects Asian pear trees (Pyrus pyrifolia Nakai) Won-Sik Kim,’ Louis Gardan,’ Seong-Lyul Rhim3and Klaus Geiderl Author for correspondence : Klaus Geider. Tel : + 49 6203 106 117. Fax : + 49 6203 106 122. e-mail : kgeider azellbio.rnpg.de Max-Planck-lnstitut fur A novel pathogen from Asian pears (Pyruspyrifolia Nakai) was analysed by Zel Ibiologie, Rosenhof, sequencing the 16s rDNA and the adjacent intergenic region, and the data D-68526 Ladenburg, Germany were compared to related Enterobacteriaceae. The 165 rDNA of the Asian pear pathogen was almost identical with the sequence of Erwinia amylovora, in INRA, Pathologie Vegetale et Phytobacteriologie, contrast to the 165-235 rRNA intergenic transcribed spacer region of both 42, rue Georges Morel, species. A dendrogram was deduced from determined sequences of the spacer F-49071 Beaucouze, regions including those of several related species such as Erwinia amylovora, Cedex, France Enterobacter pyrinus, Pantoea stewartii subsp. stewartii and Escherichia coli. Department of Genetical Dendrograms derived from 121 biochemical characteristics including Biotype Engineering, College of Natural Science, Hallym 100 data placed the Asian pear pathogen close to Erwinia amylovora and more University, 1 Okcheon- distantly to other members of the species Erwinia and to the species Pantoea Dong, Chuncheon-Si, and Enterobacter. Another DNA relatedness study was performed by DNA Kangwon-Do, 200-702, South Korea hybridizations and estimation of AT,,, values. The Asian pear strains constituted a tight DNA hybridization group (89-100%) and were barely related to strains of Erwinia amylovora (40-50%) with a AT,,, in the range of 5-2-6.8. The G+C content of DNA from the novel pathogen is 52 mol%. Therefore, it is proposed that strains isolated from Asian pears constitute a new species and the name Erwinia pyrifoliae is suggested; the type strain is strain E~16/96~(= CFBP 4172T = DSM 121633. Keywords : Erwinia pyrifoliae, taxonomy, DNA-DNA hybridization, rDNA sequencing INTRODUCTION pathogen Enterobacter pyrinus (Chung et al., 1993) especially by its lack of nitrate reduction and for A recently observed necrotic disease of Asian pear growth at high temperature, as well as acid production fruit trees (Pyrus pyrifolia cv. ‘Shingo’ and in the presence of aesculin or cellobiose (Rhim et al., ‘ Mansamgil ’) near Chuncheon in Korea has resulted 1999). in isolation and partial characterization of a bacterial The purpose of this work was the differentiation of the pathogen (Rhim et al., 1999). Symptoms on pear trees novel pathogen from Erwinia amylovora, Pantoea and in the Korean orchards remotely resembled those of Enterobacter by using DNA sequence analysis of the fire blight, but microbiological assays and the absence 16s rDNA and the adjacent intergenic transcribed of the PCR signals expected for the presence of Erwinia spacer region, by numerical analysis of phenotypical amylovora distinguished the novel Erwinia sp. from the characteristics, DNA-DNA hybridization and the fire blight pathogen. The new pathogen was also G + C content. Our findings resulted in the description different from the recently described Korean pear of a new species 1 Erwinia pyrifoliae. METHODS Abbreviations: ITS, intergenic transcribed spacer region; RCS, reducing compounds from sucrose. Strains. The strains investigated in the experiments and used The EMBL accession number for the 165 rRNA sequence of Erwinia for differentiation are listed in Tables 1 and 2. They were pyrifoliae strain Ep1196 is AJ009930. used under authorities mentioned in Young et al. (1996). We 00919 0 1999 IUMS 899 W.-S. Kim and others Table 1. Strains used for molecular characterization of Erwinia pyrifoliae Strain No. in collection" Plant, origin, year of isolation Reference/source Erwinia amylovora Eal/79 Cotoneaster sp., Germany, 1979 (obtained from Falkenstein et al., 1988 W. Zeller) Enterobacter pyrinus 90.2' KCTC 2520T Pyrus pyrifoliu, South Korea (from Korean strain Chung et al., 1993 collection) Erwin ia pyr ifoliae Ep8/95 DSM 12393 Pyrus pyrifolia, South Korea, 1995 S.-L. Rhim, unpublished Epl/96 CFBP 4171, Pyrus pyrifoliu, South Korea, 1996 S.-L. Rhim, unpublished DSM 12162 Ep 16/96' CFBP 4172T, Pyrus pyrifolia, South Korea, 1996 S.-L. Rhim, unpublished DSM 12163T Ep28/96 CFBP 4173 Pyrus pyrifolia, South Korea, 1996 S.-L. Rhim, unpublished Ep3 1/96 CFBP 4 174 Pyrus pyrifolia, South Korea, 1996 S.-L. Rhim, unpublished Ep4/97 DSM 12394 Pyrus pyrifolia, South Korea, 1997 S.-L. Rhim & K. Geider, unpublished Ep102/98 Pyrus pyrifolia, South Korea, 1998 S.-L. Rhim, unpublished * CFBP, Collection FranCaise des Bactiries Phytopathogenes ; DSM, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen ; KCTC, Korean Collection of Type Cultures. assayed four strains isolated from Pyrus pyrifolia, the type raffinose, D( -)-tartrate and utilization of malonate were strain of Erwinia arnylovora, and 20 type strains of phyto- tested on the basal medium of Ayers et al. (1919) pathogenic and saprophytic Erwinia, Pantoea and Entero- supplemented with 0.1 O/O (w/v) of carbohydrates and or- bacter sp. for constructing a dendrogram. ganic acid sodium salts. The other tests were classical Nucleotide sequencing. The 16s rRNA genes and the techniques (Smibert & Krieg, 1981 ; Lelliott & Stead, 1987; 16s-23s rRNA intergenic transcribed spacer region (ITS) Schroth & Hildebrand, 1988) and included: Simmons were cloned with consensus primers into vector pGEM-T citrate, gelatin hydrolysis, lecithinase, casein hydrolysis, (Promega) and pBluescript KS, respectively. The primers for indole production, arginine dihydrolase (Mraller), reducing PCR amplification were for 16s rDNA fd2 ( 5' AGA GTT compounds from sucrose (RCS), growth on NaCl (5% TGA TCA TGG CTC AG) and rP1 (5' ACG GTT ACC w/v), growth at 36 "C/39 "C in liquid King's B, liquefaction TTG TTA CGA CTT) (Weisburg et al., 1991) and for the of calcium polygalacturonate in Sutton medium and ITS PIGL (5' GCG CGC GTCGAC ACC TGC GGT TGG utilization of inulin. AAC ACC) and PIGR (5' GCG CGC TCTAGA CAC CGT Numerical taxonomy. A total of 121 characteristics were GTA CGC TTA GTC). They were synthesized with included in the numerical taxonomy analysis. The matrix of Beckman Oligo lOOOM DNA Synthesizer. PCR cloning of distances was calculated by using the Jaccard coefficient ITS was done with pfu DNA polymerase and primers with (Sneath & Sokal, 1973) and cluster analysis was done with SalI and XbaI restriction sites (underlined) and three GC the unweighted pair group method with averages (UPGMA) oligonucleotide pairs. PCR was performed in 50 ml con- (Sneath & Sokal, 1973). ITS sequence were aligned with the taining 30 pmol each primer, 3 U cloned pfu DNA poly- program ALIGN version 2.0 (Scientific & Educational merase (Stratagene), 10 ng isolated DNA or 10000 cells, and Software) using a mismatch penalty of 2, an open gap 20mM dATP, dCTP, dGTP and dTTP (Boehringer penalty of 4 and an extend gap penalty of 1. Phylogenetic Mannheim) in the 10 x buffer provided by Stratagene. interferences were derived from multiple alignments of the Amplification was carried out in an Eppendorf mastercycler ITS region using programs CLUSTREE and CLUSTAL w with 5330 in 30 cycles. Denaturation was done at 94 "C (in the the neighbour-joining method of Saitou & Nei (1987). first cycle for 45 s and in subsequent cycle for 30 s) and 45 s at 52 "C for annealing followed by 2min at 72 "C for DNA-DNA hybridization and thermal stability. Extraction polymerization. For cloning, the PCR product was eluted and purification of DNA were performed with previously from a 1.0% ultrapure agarose gel, digested with SaZI and described methods (Brenner et al., 1982). Native DNAs were XbaI and ligated into the vector pBluescript KS. The labelled in vitro by using the Megaprime DNA-labelling insertions were repeatedly sequenced with an automatic system (Amersham International). DNA-DNA hy- sequencer (ALF ; Pharmacia). bridization experiments were done with labelled DNA from Erwinia pyr$oliae E~16/96~(CFBP 4172T) at a Biochemical and physiological tests. Assimilation of 99 temperature of 65 "C for reassociation. The S1 nuclease- carbohydrates, organic acids and amino acids was studied TCA method was used for hybridization (Brenner et ul., using Biotype 100 strips (bioMirieux) which were incubated 1982; Crosa et al., 1973). at 28 "C; the growth was recorded after 4 and 6 d incubation. Twenty-two additional tests were also performed. Acid The temperature at which 50 % of reassociated DNA became production from lactose, methyl a-glucoside, melibiose, hydrolysable by enzyme S, (T,) was determined by the D( +)-arabitol, sorbitol, D( -)-arabinose, mannitol, method of Crosa et al. (1973). The AT, is the difference 900 International Journal of Systematic Bacteriology 49 Erwinia pyrifoliae sp. nov. from Pyrus pyrifolia Nakai between the Tm of the homoduplex and the Tm of the Enterobacter pyrinus and Pantoea stewartii subsp. heteroduplex. stewartii (data not shown). To further analyse the 16s DNA base composition. The G+C content of Erwinia rDNA sequences of Erwinia pyrfoliae, Enterobacter pyrifoliae CFBP 4172T was determined by a thermal pyrinus and Pantoea stewartii subsp. stewartii on the denaturation temperature method (Marmur & Doty, 1962) nucleotide level, the amplified fragments were cloned and was calculated with the equation of
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