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1 School of Biological Sciences, Seoul National University, Kwanak-gu, Seoul 151-742, Republic of Korea
2 Division of Biotechnology, The Catholic University of Korea, Puchon 420-743, Republic of Korea
3 Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, Republic of Korea
4 Department of Crop Science, College of Agriculture and Life Science, Chungnam National University, Taejon 305-764, Republic of Korea
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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ABSTRACT |
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7c and/or iso-C15 : 0 2-OH (summed feature 3, 36·4±0·4 %), C16 : 0 (27·5±0·7 %) and C18 : 17c (19·4±0·2 %). The DNA G+C content was 61·4 mol%. On the basis of the polyphasic results revealed in this study, the name Pseudomonas panacis sp. nov. is proposed for strain CG20106T. The type strain is CG20106T (=IMSNU 14100T=CIP 108524T=KCTC 12330T).
Transmission electron micrographs of Pseudomonas panacis sp. nov. CG20106T and Pseudomonas azotoformans KCCM 35487T are available as a supplementary figure in IJSEM Online.
These authors contributed equally to this work. 
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MAIN TEXT |
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, the genus Pseudomonas has been studied with increasing interest because of the importance of its members in medical, food and environmental microbiology as well as phytopathology. Members of the genus Pseudomonas are widely distributed in agricultural soils and have a variety of functions relating to the decomposition of organic matter, the promotion of plant growth and pathogenic effects (Palleroni, 1993). During the study of the induction of bacterial rusty roots, a bacterial strain, designated CG20106T, was isolated from Korean ginseng (Panax ginseng C. A. Meyer) and examined using a polyphasic taxonomic approach. On the basis of the polyphasic evidence, strain CG20106T represents a novel species of the genus Pseudomonas.
A surface-tissue sample of rusty root lesions of Korean ginseng (1 g) was collected under aseptic conditions and then suspended in 1 ml sterile water with mixing for 30 min. The suspension (0·5 ml) was spread on nutrient agar (NA; Difco) and incubated for 1 day at 30 °C. Strain CG20106T was isolated and routinely cultured on NA at 30 °C. A poorly characterized reference strain, Pseudomonas azotoformans KCCM 35487T (Anzai et al., 1997), was also cultured on NA at 30 °C and analysed in this study.
Bacterial DNA preparation and PCR amplification and sequencing of the 16S rRNA gene sequence were carried out as described previously (Chun & Goodfellow, 1995). The resulting sequence of strain CG20106T was aligned manually against sequences obtained from GenBank. Phylogenetic trees were inferred from regions available for all sequences (positions 6–1451; Escherichia coli numbering system) using the Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1993), maximum-parsimony (Fitch, 1972) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices were generated according to Jukes & Cantor (1969). The resulting tree topologies were evaluated in bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. The alignment and phylogenetic analyses were carried out using the jPHYDIT program (available at http://chunlab.snu.ac.kr/jphydit) and PAUP 4.0 (Swofford, 1998), as described previously (Chun et al., 2000).
Preliminary sequence comparisons with 16S rRNA sequences held in GenBank indicated that our isolate was closely related to the genus Pseudomonas. The newly determined sequence was then aligned manually against representatives of pseudomonads, using information on bacterial 16S rRNA secondary structures. Strain CG20106T showed the highest 16S rRNA gene sequence similarity to Pseudomonas migulae CIP 105470T (99·3 %), followed by Pseudomonas veronii CIP 104668T (99·0 %), Pseudomonas cedrina CFML 96-198T (98·6 %) and Pseudomonas azotoformans IAM 1603T (98·6 %). This relationship was also recovered in phylogenetic trees. Our ginseng isolate was clearly affiliated with the Pseudomonas fluorescens group and formed a distinct phyletic line within the clade which contained the type strains of Pseudomonas migulae, Pseudomonas cedrina, Pseudomonas azotoformans, Pseudomonas gessardii, Pseudomonas synxantha and Pseudomonas mucidolens (Fig. 1). The branching position of strain CG20106T within this clade was relatively stable, according to the multiple tree-making algorithms used in this study, in spite of the low bootstrap value (55 %). Strain CG20106T formed the deepest branch of the clade in the maximum-parsimony tree, but was recovered as a sister group of the deepest branch, Pseudomonas migulae, in the neighbour-joining, Fitch–Margoliash and maximum-likelihood trees.
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) using the DIG High Prime DNA Labelling and Detection starter kit II (Roche). DNA–DNA relatedness levels among members of the subgroup of Pseudomonas cedrina CFML 96-198T were reported to be 26–44 % (Dabboussi et al., 1999; Verhille et al., 1999). DNA–DNA relatedness levels between strain CG20106T and Pseudomonas migulae CIP 105470T and between strain CG20106T and P. cedrina CFML 96-198T were 36 and 66 %, respectively, when their DNAs were used individually as labelled DNA probes for reciprocal hybridization experiments in duplicate. These DNA relatedness values, together with 16S rRNA gene sequence-based analysis, indicate that strain CG20106T represents a novel genomic species that is distinct from Pseudomonas migulae and Pseudomonas cedrina (Wayne et al., 1987).
Cells of strain CG20106T and Pseudomonas azotoformans KCCM 35487T grown on NA at 30 °C for 1 day were used for physiological and biochemical tests. Motility and flagella shape were examined by using phase-contrast microscopy (Axioskop 40; Zeiss) and transmission electron microscopy (JEM1010; JEOL), respectively. Growth temperature (5–50 °C with 5 °C intervals), NaCl tolerance (0, 1, 2, 3, 5, 10 %, w/v) and growth in an anaerobic chamber (CO2/H2/N2, 10 : 10 : 80; Sheldon Manufacturing) were checked using NA for up to 1 week. The production of water-soluble fluorescent pigments on King's B agar was determined as described previously (Smibert & Krieg, 1994). For the determination of acetone production, test strains were grown in MRVP broth according to Smibert & Krieg (1994). The Voges–Proskauer test was performed after 48 h incubation. Other physiological and biochemical test were performed using API 20E and API 20NE (bioMérieux) and Biolog GN2 (Biolog). Enzymic activities were tested using the API ZYM kit (bioMérieux) according to the manufacturer's instructions. Our ginseng isolate and Pseudomonas azotoformans KCCM 35487T had one or more flagella (see the supplementary figure available in IJSEM Online) and possessed many characteristics in common. Unless mentioned otherwise, the phenotypic characteristics of Pseudomonas azotoformans KCCM 35487T were the same as those of strain CG20106T. The results of the biochemical and physiological tests are given in the species description and in Table 1. As shown in Table 1, our isolate can be readily differentiated from other phylogenetically related pseudomonads by several phenotypic properties.
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7c and/or iso-C15 : 0 2-OH (36·4±0·4 %) (summed feature 3) and C18 : 17c (19·4±0·2 %). In addition, C12 : 0 (3·2±0·1 %), C10 : 0 3-OH (3·3±0·4 %), C12 : 0 2-OH (3·8±0·0 %), C12 : 0 3-OH (3·8±0·3 %) and C17 : 0 cyclo (1·5±0·2 %) were detected. The major fatty acids of Pseudomonas azotoformans KCCM 35487T were C16 : 0 (25·6±0·6 %), C16 : 17c and/or iso-C15 : 0 2-OH (33·9±1·5 %) (summed feature 3) and C18 : 17c (22·7±0·2 %). In addition, C12 : 0 (3·9±0·1 %), C18 : 0 (2·5±0·6 %), C10 : 0 3-OH (3·2±0·1 %), C12 : 0 2-OH (2·8±0·1 %) and C12 : 0 3-OH (3·5±0·1 %) were present. The G+C content of the DNA was determined by using the thermal denaturation method of Marmur & Doty (1962): the values for strains CG20106T and Pseudomonas azotoformans KCCM 35487T were 61·4 and 61·0 mol%, respectively.
On the basis of the DNA–DNA relatedness and the formation of a distinctive phyletic line within the genus Pseudomonas in all trees inferred in this study, it is evident that strain CG20106T can be assigned as a novel species of the genus Pseudomonas. In addition, a number of physiological and chemotaxonomic characteristics clearly distinguished our isolate from other phylogenetically related species (Table 1
). Therefore, strain CG20106T should be classified in a novel species within the genus Pseudomonas, for which the name Pseudomonas panacis sp. nov. is proposed.
Description of Pseudomonas panacis sp. nov.
Pseudomonas panacis (pa'na.cis. L. gen. n. panacis of panax, a fabulous plant supposed to heal all diseases, and the botanical genus name of ginseng).
Gram-negative, aerobic, rod-shaped and motile with one or more flagella. Catalase- and oxidase-positive. Colonies on NA are flat, translucent, butyraceous, beige-coloured with entire margins and usually 2–3 mm in diameter within 2 days at 30 °C. Spores are not formed. Grows between 4 and 35 °C, but not at 37 °C. Tolerates NaCl up to 5 % on NA. Very poor growth detected under anaerobic conditions created by an anaerobic chamber (CO2/H2/N2, 10 : 10 : 80). Produces fluorescent pigment on King's B medium. Reduces nitrate to nitrite and decomposes gelatin. Produces aesculin and arginine dihydrolases, but not urease, H2S, indole or acetone. Produces acid from D-glucose, D-sucrose, D-melibiose and D-arabinose, but not from D-mannitol, D-sorbitol, inositol or amygdalin. Produces alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, cystine arylamidase, trypsin, valine arylamidase and acid phosphatase, but not lipase (C14), 
-galactosidase, 
-galactosidase, -glucuronidase, -glucosidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase, -fucosidase, lysine decarboxylase, ornithine decarboxylase or tryptophan deaminase. Naphthol-AS-BI-phosphohydrolase and -chymotrypsin are weakly produced. Utilizes the following substrates as sole carbon and energy sources: glucose, arabinose, mannose, mannitol, N-acetylglucosamine, gluconate, caprate, malate, malonate, citrate, D-fructose, -D-glucose, D-trehalose, methylpyruvate, cis-aconitic acid, citric acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, -hydroxybutyric acid, p-hydroxyphenylacetic acid, -ketoglutaric acid, DL-lactic acid, propionic acid, D-saccharic acid, succinic acid, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, L-ornithine, L-proline, L-pyroglutamic acid, L-serine, DL-carnitine, 
-aminobutyric acid, urocanic acid, inosine, uridine and glycerol. Does not utilize the following substrates: maltose, phenylacetate, -cyclodextrin, dextrin, glycogen, N-acetyl-D-galactosamine, cellobiose, L-fucose, gentiobiose, -D-lactose, lactulose, maltose, D-melibiose, methyl -D-glucoside, D-raffinose, L-rhamnose, turanose, formic acid, -ketobutyric acid, -ketovaleric acid, sebacic acid, glucuronamide, glycyl-L-aspartic acid, hydroxy L-proline, L-phenylalanine, D-serine, thymidine, phenylethylamine, putrescine, 2,3-butanediol, glucose 1-phosphate and glucose 6-phosphate. D-Psicose, D-alanine, D-glucosaminic acid, monomethyl succinate, acetic acid, -hydroxybutyric acid, -hydroxybutyric acid, bromosuccinic acid, succinamic acid, alaninamide, glycyl-L-glutamic acid, L-leucine, L-threonine and 2-aminoethanol are weakly utilized. Other phenotypic characteristics are given in Table 1. The major fatty acids are C16 : 0 (27·5±0·7 %), C16 : 17c and/or iso-C15 : 0 2-OH (36·4±0·4 %) (summed feature 3) and C18 : 17c (19·4±0·2 %). The DNA G+C content of the type strain is 61·4 mol%.
The type strain, CG20106T (=IMSNU 14100T=CIP 108524T=KCTC 12330T), was isolated from the surface tissue of a rusty root lesion of Korean ginseng.
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ACKNOWLEDGEMENTS |
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