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Int J Syst Evol Microbiol 56 (2006), 433-438; DOI  10.1099/ijs.0.63825-0
© 2006 International Union of Microbiological Societies

Chryseobacterium soldanellicola sp. nov. and Chryseobacterium taeanense sp. nov., isolated from roots of sand-dune plants

Myung Soo Park1, Se Ra Jung1, Kang Hyun Lee1, Myung-Sook Lee1, Jin Ok Do1, Seung Bum Kim2 and Kyung Sook Bae1

1 Korea Research Institute of Bioscience and Biotechnology, 52 Oun-dong, Yusong, Daejon 305-333, Republic of Korea
2 Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, 220 Gung-dong, Yusong, Daejon 305-764, Republic of Korea

Correspondence
Seung Bum Kim
sbk01{at}cnu.ac.kr


    ABSTRACT
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Two Gram-negative, yellow-pigmented bacteria designated PSD1-4T and PHA3-4T, isolated from two sand-dune plant species inhabiting coastal areas in Tae-an, Korea, were subjected to taxonomic investigation. 16S rRNA gene sequence analysis indicated that both isolates should be placed in the genus Chryseobacterium of the family Flavobacteriaceae. The phenotypic properties of the strains were also consistent with their classification into this genus. The levels of 16S rRNA gene sequence similarity between strain PSD1-4T and other Chryseobacterium species were 95·2–97·2 %; those between PHA3-4T and others were 93·7–97·8 %. The DNA–DNA relatedness data indicated that strains PSD1-4T and PHA3-4T were clearly different from the nearest species, Chryseobacterium indoltheticum and Chryseobacterium taichungense. The major fatty acids were 13-methyltetradecanoic acid (iso-C15 : 0), 3-hydroxy-15-methylhexadecanoic acid (iso-C17 : 0 3-OH) and omega-9-cis-15-methylhexadecenoic acid (iso-C17 : 1{omega}9c) for both strains. On the basis of polyphasic taxonomic analysis results, it is evident that each of these strains represents a novel species of Chryseobacterium, for which the names Chryseobacterium soldanellicola sp. nov. (type strain PSD1-4T=KCTC 12382T=NBRC 100864T) and Chryseobacterium taeanense sp. nov. (type strain PHA3-4T=KCTC 12381T=NBRC 100863T) are proposed.


Published online ahead of print on 28 October 2005 as DOI 10.1099/ijs.0.63825-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains PSD1-4T and PHA3-4T are AY883415 and AY883416, respectively.


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The genus Chryseobacterium currently includes the initially described species Chryseobacterium balustinum, C. gleum, C. indologenes, C. indoltheticum and C. scophthalmum (Vandamme et al., 1994Go) and several more recently described species, Chryseobacterium defluvii (Kämpfer et al., 2003Go), C. joostei (Hugo et al., 2003Go), C. daecheongense (Kim et al., 2005Go), C. formosense (Young et al., 2005Go), C. taichungense (Shen et al., 2005Go), C. shigense (Shimomura et al., 2005Go) and C. vrystaatense (de Beer et al., 2005Go). Two former Chryseobacterium species, Chryseobacterium meningosepticum (Vandamme et al., 1994Go) and Chryseobacterium miricola (Li et al., 2003Go), have been reclassified in a new genus Elizabethkingia (Kim et al., 2005Go). Strains belonging to this genus have been found in a wide variety of environments such as soil, sewage, freshwater, marine sediment and clinical samples.

During a study of bacterial diversity associated with sand-dune plants using a culture-dependent approach, a number of bacterial strains were isolated from root samples of two sand-dune plant species (Park et al., 2005Go). Two strains designated PSD1-4T and PHA3-4T, forming yellow colonies on R2A agar (Difco) at 30 °C, were isolated from the roots of Calystegia soldanella (beach morning glory) and Elymus mollis (wild rye), respectively, and subjected to further taxonomic investigation. Analysis of the 16S rRNA gene sequences showed that both of the isolates could be placed within the phylogenetic clade encompassed by the genus Chryseobacterium of the family Flavobacteriaceae. The strains were subcultured on tryptic soy agar (TSA; Difco) at 30 °C for 48 h and subsequently investigated for fatty acid methyl ester profiles, 16S rRNA gene sequences and phenotypic characteristics. The strains were also maintained as glycerol suspensions (20 %, w/v) at –80 °C.

Cellular morphology was examined using a Nikon MICROPHOT-FAX phase-contrast microscope with cells grown for 3 days at 30 °C on TSA. Gram staining was carried out using the modification of Hucker's method as described by Gerhardt et al. (1994)Go. Growth was investigated at different NaCl concentrations (0–7 %, w/v), at various temperatures (5–42 °C) and on MacConkey agar. Biochemical tests were performed using the API 20E, API 20NE, API CHB and API ZYM systems (bioMérieux). Oxidation of 95 selected carbon sources was tested using Biolog GN2 microplates according to the manufacturer's instructions. Antifungal activities were screened using four plant-pathogenic fungi, namely Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani and Pythium ultimum (cultures provided by the Korea Research Institute of Chemical Technology, Daejon, Korea).

Strains PSD1-4T and PHA3-4T were Gram-negative and formed visible colonies (diameter of about 2 mm) on nutrient agar within 24 h at 30 °C. No growth was observed at temperatures above 42 °C within 14 days. At 5 °C, small colonies were seen on the agar plates. The colonies were yellowish, translucent and shiny with entire edges, becoming mucoid after prolonged incubation.

Strains PSD1-4T and PHA3-4T shared a number of phenotypic characteristics tested in common, with the following differential characteristics: pH ranges for growth, tolerance to NaCl and degradation of Tween 80. Comparison of physiological and biochemical characteristics also enabled differentiation of each isolate from other representative species of the genus Chryseobacterium (Table 1Go). Strain PSD1-4T was shown to inhibit growth of the plant pathogen Fusarium oxysporum, whereas strain PHA3-4T exhibited no antifungal activity.


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Table 1. Distinctive phenotypic characteristics of strains PSD1-4T and PHA3-4T and species of the genus Chryseobacterium

Species: 1, C. soldanellicola (strain PSD1-4T); 2, C. taeanense (strain PHA3-4T); 3, C. taichungense (n=1); 4, C. balustinum (n=1); 5, C. daecheongense (n=1); 6, C. defluvii (n=1); 7, C. formosense (n=1); 8, C. gleum (n=2); 9, C. indologenes (n=7); 10, C. indoltheticum (n=1); 11, C. joostei (n=11); 12, C. scophthalmum (n=2). Data for reference species were taken from Kämpfer et al. (2003)Go, Li et al. (2003)Go, Hugo et al. (2003)Go, Kim et al. (2005)Go, Shen et al. (2005)Go and Young et al. (2005)Go. +, All strains positive; W, weakly positive; –, all strains negative; V, variable; D, delayed; NA, no data available. All species hydrolyse aesculin.

 
Fatty acid methyl esters were extracted and prepared by the standard protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids analysed by a gas chromatograph (Hewlett Packard 6890) were identified by the Microbial Identification software package (Sasser, 1990Go). The fatty acid contents of PSD1-4T and PHA3-4T indicated that 13-methlyltetradecanoic acid (iso-C15 : 0) was the most abundant fatty acid for both strains, which was followed by 3-hydroxy-15-methylhexadecanoic acid (iso-C17 : 0 3-OH) and omega-9-cis-15-methylhexadecenoic acid (iso-C17 : 1{omega}9c) (Table 2Go). These fatty acid profiles were similar to those of the type strains of other Chryseobacterium species. However, strains PSD1-4T and PHA3-4T differed from each other and from other Chryseobacterium species in relative abundance of shared components (Table 2Go).


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Table 2. Fatty acid content (%) of strains PSD1-4T and PHA3-4T and species of the genus Chryseobacterium

Species: 1, C. soldanellicola (strain PSD1-4T); 2, C. taeanense (strain PHA3-4T); 3, C. taichungense (n=1); 4, C. balustinum (n=1); 5, C. daecheongense (n=1); 6, C. defluvii (n=1); 7, C. formosense (n=1); 8, C. gleum (n=5); 9, C. indologenes (n=45); 10, C. indoltheticum (n=1); 11, C. joostei (n=11); 12, C. scophthalmum (n=7). Data for reference species were taken from Kämpfer et al. (2003)Go, Li et al. (2003)Go, Hugo et al. (2003)Go, Kim et al. (2005)Go, Shen et al. (2005)Go and Young et al. (2005)Go. tr, Trace (less than 1·0 % of the total); ND, not detected.

 
The 16S rRNA gene sequences of strains PSD1-4T (1426 nucleotides) and PHA3-4T (1425 nucleotides) were determined as described previously (Han et al., 2003Go). The resultant sequences were aligned manually, based on 16S rRNA secondary structure, with representative sequences of Chryseobacterium species obtained from the GenBank database. The phylogenetic tree based on the 16S rRNA gene sequences was inferred using the neighbour-joining method (Saitou & Nei, 1987Go). Evolutionary distance matrices for the neighbour-joining method were generated according to Kimura's 2-parameter distance model (Kimura, 1980Go). The resultant neighbour-joining tree topology was evaluated by bootstrap analysis (Felsenstein, 1985Go) based on 1000 resampled datasets. Alignment with representative sequences of Chryseobacterium and subsequent phylogenetic analyses were carried out using the PHYDIT program (jPHYDIT is available at http://chunlab.snu.ac.kr/jphydit/) as described previously (Han et al., 2003Go).

The final set of sequence data consisted of 1402 nucleotide positions. Strains PSD1-4T and PHA3-4T shared 96·2 % 16S rRNA gene sequence similarity. Strain PSD1-4T shared between 95·2 and 97·2 % similarity with other established Chryseobacterium species, among which C. indoltheticum ATCC 27950T was the closest neighbour. The similarity values between strain PHA3-4T and other type strains of Chryseobacterium species were in the range 93·7–97·8 %, C. taichungense CC-TWGS1-8T being the closest neighbour. The close relationship between the two organisms was reflected by the 100 % bootstrap support and the recovery of the same topology using other algorithms (Fig. 1Go). However, the phylogenetic tree and DNA–DNA relatedness data clearly indicated that each of the two strains forms a distinct phyletic lineage. The level of DNA–DNA relatedness was determined according to the procedure described by Lim et al. (2003)Go and the resultant data supported the separation between PSD1-4T and C. indoltheticum ATCC 27950T (23 %), and also between PHA3-4T and C. taichungense CC-TWGS1-8T (25 %), as the relatedness levels were well below the suggested cut-off value of 70 % for species differentiation.


Figure 1
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Fig. 1. Neighbour-joining tree based on nearly complete 16S rRNA gene sequences showing relationships among strains PSD1-4T and PHA3-4T and other species of the genus Chryseobacterium. The values above each branch indicate the percentage levels of bootstrap support (>50 %) for the branch point based on 1000 resamplings. The asterisks indicate branches that were also recovered in the maximum-likelihood and maximum-parsimony trees. Bar, 0·01 substitutions per nucleotide position.

 
On the basis of the results of the phenotypic and genotypic study, it is evident that PSD1-4T and PHA3-4T should be placed in the genus Chryseobacterium as the type strains of two novel species, for which the names Chryseobacterium soldanellicola sp. nov. and Chryseobacterium taeanense sp. nov. are proposed.

Strains of Chryseobacterium were found to form the second largest group after Pseudomonas among the culturable microbial populations retrieved from the root and rhizosphere of sand-dune plants (Park et al., 2005Go). Since little information is available on the possible roles of bacteria belonging to Chryseobacterium associated with plants (Bernardet et al., 2001Go; McSpadden Gardener & Weller, 2001Go), it is not easy to speculate on their ecological significance to the sand dune vegetation. More detailed studies on these bacteria will be necessary to elucidate their significance in that specific ecosystem.

Description of Chryseobacterium soldanellicola sp. nov.
Chryseobacterium soldanellicola (sol.da.nel'li.co.la. N.L. n. soldanella the species epithet of a plant belonging to the genus Calystegia; L. suff. verbal n. cola dweller; N.L. masc. n. soldanellicola dweller of Calystegia soldanella).

Cells are non-spore-forming rods. Gram-negative. Good growth is observed on R2A agar, TSA and nutrient agar at 25–30 °C. Grows at 5 °C, but not at 42 °C. No growth is observed on MacConkey agar. Colonies are yellowish, circular and shiny. The pH range for growth is pH 5–7 and that for optimal growth is pH 5. Growth occurs aerobically, but not anaerobically. Growth occurs in the presence of 0–4 % (w/v) NaCl within 14 days. The most abundant cellular fatty acids are iso-C15 : 0 and iso-C17 : 0 3-OH. Indole, H2S and acetoin are not produced. Nitrate and nitrite are not reduced. Positive for cytochrome oxidase but negative for arginine dihydrolase, lysine decarboxylase, citrate utilization, tryptophan deaminase and urease. Capable of hydrolysing aesculin and gelatin. Weakly assimilates glucose, arabinose, mannose and maltose. The following compounds are utilized as sole carbon sources: {alpha}-cyclodextrin, dextrin, glycogen, Tween 40, L-arabinose (weakly), D-fructose, gentiobiose, {alpha}-D-glucose, maltose, D-mannose, L-rhamnose, D-trehalose, turanose, mono-methyl succinate, acetic acid, D-galacturonic acid, {alpha}-ketobutyric acid, {alpha}-ketovaleric acid, propionic acid, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-glutamic acid, L-phenylalanine, L-proline, L-serine, L-threonine, 2,3-butanediol (weakly) and glycerol (weakly). The following compounds are not utilized: Tween 80, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, D-arabitol, cellobiose, i-erythritol, L-fucose, D-galactose, myo-inositol, {alpha}-L-lactose, lactulose, mannitol, D-melibiose, methyl beta-D-glucoside, D-psicose, D-raffinose, D-sorbitol, sucrose, xylitol, methyl pyruvate, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, {alpha}-, beta- and {gamma}-hydroxybutyric acid, p-hydroxyphenylacetic acid, itaconic acid, {alpha}-ketoglutaric acid, DL-lactic acid, malonic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, alaninamide, D-alanine, glycyl L-aspartic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-pyroglutamic acid, D-serine, DL-carnitine, {gamma}-aminobutyric acid, urocanic acid, inosine, uridine, thymidine, phenylethylamine, putrescine, 2-aminoethanol, DL-{alpha}-glycerol phosphate, glucose 1-phosphate and glucose 6-phosphate. Results from API ZYM tests are given in Table 3Go. The G+C content is 28·8 mol%.


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Table 3. API ZYM profiles of strains PSD1-4T and PHA3-4T and other species of the genus Chryseobacterium

Species: 1, C. soldanellicola (strain PSD1-4T); 2, C. taeanense (strain PHA3-4T); 3, C. taichungense; 4, C. balustinum; 5, C. daecheongense; 6,C. defluvii; 7, C. formosense; 8, C. gleum; 9, C. indologenes; 10, C. indoltheticum; 11, C. joostei; 12, C. scophthalmum. All species showed positive reactions for 2-naphthyl phosphate, 2-naphthyl caprylate, L-leucyl 2-naphthylamide, L-valyl 2-naphthylamide, 2-naphthyl phosphate and naphthol-AS-BI-phosphate and showed negative reactions for 6-Br-2-naphthyl {alpha}-D-mannopyranoside. Data for strains PSD1-4T and PHA3-4T and C. taichungense were obtained in this study; those for other reference species were taken from Hugo et al. (2003)Go, Kim et al. (2005)Go and Young et al. (2005)Go. The intensity of the developed colour was measured on scale from 0 to 5 and interpreted as negative (–) when values ranged between 0 and 1 and positive (+) for values between 2 and 5 (Mudarris et al., 1994Go).

 
The type strain is PSD1-4T (=KCTC 12382T=NBRC 100864T), which was isolated from the roots of Calystegia soldanella growing on a coastal area in Tae-an, Korea. The type strain displays antifungal activity against Fusarium oxysporum.

Description of Chryseobacterium taeanense sp. nov.
Chryseobacterium taeanense (tae.an.en'se. N.L. neut. adj. taeanense of Tae-an, a geographical region in Chungnam, Korea, where the type strain was isolated).

Cells are non-spore-forming rods. Gram-negative. Good growth is observed on R2A agar, TSA and nutrient agar at 25–30 °C. Grows at 5 °C, but not at 42 °C. No growth is observed on MacConkey agar. Colonies are yellowish, circular and shiny. The pH range for growth is pH 5–9 and that for optimal growth is pH 5. Growth occurs aerobically, but not anaerobically. Growth occurs in 0–6 % (w/v) NaCl within 14 days. The most abundant cellular fatty acids are iso-C15 : 0 and iso-C17 : 0 3-OH. Indole, H2S and acetoin are not produced. Nitrate and nitrite are not reduced. Positive for cytochrome oxidase but negative for arginine dihydrolase, lysine decarboxylase, citrate utilization, tryptophan deaminase and urease. Capable of hydrolysing aesculin and gelatin. Weakly assimilates glucose, arabinose, mannose and maltose. The following compounds are utilized as sole carbon sources: {alpha}-cyclodextrin, dextrin, glycogen, Tween 40, Tween 80, L-arabinose, cellobiose, D-fructose, L-fucose, D-galactose, gentiobiose, {alpha}-D-glucose, myo-inositol, {alpha}-D-lactose, lactulose, maltose, mannitol (weakly), D-mannose, methyl beta-D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, methyl pyruvate, monomethyl succinate, cis-aconitic acid, citric acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, {alpha}-hydroxybutyric acid, beta-hydroxybutyric acid (weakly), itaconic acid, {alpha}-ketobutyric acid, {alpha}-ketoglutaric acid, {alpha}-ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, sebacic acid, succinamic acid, glucuronamide, alaninamide, L-alanine, L-alanyl glycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, hydroxy-L-proline (weakly), L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-serine, L-threonine, DL-carnitine, inosine (weakly) and uridine (weakly). The following compounds are not utilized; N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, adonitol, D-arabitol, i-erythritol, D-melibiose, acetic acid, {gamma}-hydroxybutyric acid, p-hydroxyphenylacetic acid, succinic acid, bromosuccinic acid, D-alanine, L-histidine, D-serine, {gamma}-aminobutyric acid, urocanic acid, thymidine, phenylethylamine, putrescine, 2-aminoethanol, 2,3-butanediol, glycerol, DL-{alpha}-glycerol phosphate, glucose 1-phosphate and glucose 6-phosphate. Results from API ZYM tests are given in Table 3Go. The G+C content is 32·1 mol%.

The type strain is PHA3-4T (=KCTC 12381T=NBRC 100863T), which was isolated from the roots of Elymus mollis growing on a coastal area in Tae-an, Korea.


    ACKNOWLEDGEMENTS
 
This research was undertaken with the support from the Eco-Technopia 21 Project sponsored by the Ministry of the Environment, Republic of Korea.


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S. C. Park, M. S. Kim, K. S. Baik, E. M. Kim, M. S. Rhee, and C. N. Seong
Chryseobacterium aquifrigidense sp. nov., isolated from a water-cooling system
Int J Syst Evol Microbiol, March 1, 2008; 58(3): 607 - 611.
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Int. J. Syst. Evol. Microbiol.Home page
P. Herzog, I. Winkler, D. Wolking, P. Kampfer, and A. Lipski
Chryseobacterium ureilyticum sp. nov., Chryseobacterium gambrini sp. nov., Chryseobacterium pallidum sp. nov. and Chryseobacterium molle sp. nov., isolated from beer-bottling plants
Int J Syst Evol Microbiol, January 1, 2008; 58(1): 26 - 33.
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Int. J. Syst. Evol. Microbiol.Home page
S. Campbell, R. M. Harada, and Q. X. Li
Chryseobacterium arothri sp. nov., isolated from the kidneys of a pufferfish
Int J Syst Evol Microbiol, January 1, 2008; 58(1): 290 - 293.
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Int. J. Syst. Evol. Microbiol.Home page
M. Vaneechoutte, P. Kampfer, T. De Baere, V. Avesani, M. Janssens, and G. Wauters
Chryseobacterium hominis sp. nov., to accommodate clinical isolates biochemically similar to CDC groups II-h and II-c
Int J Syst Evol Microbiol, November 1, 2007; 57(11): 2623 - 2628.
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Int. J. Syst. Evol. Microbiol.Home page
E. Hantsis-Zacharov and M. Halpern
Chryseobacterium haifense sp. nov., a psychrotolerant bacterium isolated from raw milk
Int J Syst Evol Microbiol, October 1, 2007; 57(10): 2344 - 2348.
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Int. J. Syst. Evol. Microbiol.Home page
U. Behrendt, A. Ulrich, C. Sproer, and P. Schumann
Chryseobacterium luteum sp. nov., associated with the phyllosphere of grasses
Int J Syst Evol Microbiol, August 1, 2007; 57(8): 1881 - 1885.
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Int. J. Syst. Evol. Microbiol.Home page
J.-H. Yoon, S.-J. Kang, and T.-K. Oh
Chryseobacterium daeguense sp. nov., isolated from wastewater of a textile dye works
Int J Syst Evol Microbiol, June 1, 2007; 57(6): 1355 - 1359.
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Int. J. Syst. Evol. Microbiol.Home page
V. Gallego, M. T. Garcia, and A. Ventosa
Chryseobacterium hispanicum sp. nov., isolated from the drinking water distribution system of Sevilla, Spain.
Int J Syst Evol Microbiol, July 1, 2006; 56(Pt 7): 1589 - 1592.
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