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Int J Syst Evol Microbiol 56 (2006), 2689-2692; DOI  10.1099/ijs.0.64214-0
© 2006 International Union of Microbiological Societies

Devosia soli sp. nov., isolated from greenhouse soil in Korea

Seung-Hee Yoo1, Hang-Yeon Weon2, Byung-Yong Kim1, Seung-Beom Hong1, Soon-Wo Kwon1, Yang-Hee Cho1, Seung-Joo Go1 and Erko Stackebrandt3

1 Korean Agricultural Culture Collection (KACC), Genetic Resources Division, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon 441-707, Korea
2 Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, Korea
3 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany

Correspondence
Soon-Wo Kwon
swkwon{at}rda.go.kr


   ABSTRACT
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A Gram-negative, obligately aerobic, rod-shaped bacterium was isolated from greenhouse soil used to cultivate lettuce. The strain, GH2-10T, was characterized on the basis of phenotypic and genotypic data. 16S rRNA gene sequence analysis revealed that the isolate belonged to the genus Devosia, with highest sequence similarity (98.5 %) to Devosia riboflavina IFO 13584T. Sequence similarities with other strains tested were below 97.0 %. Strain GH2-10T had Q-10 as the predominant ubiquinone and C18 : 1{omega}7c and C16 : 0 as the major fatty acids. The G+C content of the genomic DNA was 59.5 mol%. The results of DNA–DNA hybridization experiments (47 % relatedness between D. riboflavina DSM 7230T and strain GH2-10T) and physiological and biochemical tests suggested that strain GH2-10T represents a novel species of the genus Devosia, for which the name Devosia soli sp. nov. is proposed. The type strain is GH2-10T (=KACC 11509T=DSM 17780T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GH2-10T is DQ303125.


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Nakagawa et al. (1996)Go erected the genus Devosia with the transfer of ‘Pseudomonas riboflavina’ (Foster, 1944Go) to Devosia riboflavina. Since then, Devosia neptuniae (Rivas et al., 2003Go), ‘Candidatus Devosia euplotis' (Vannini et al., 2004Go) and Devosia limi (Vanparys et al., 2005Go) have been described within the genus.

In the present study, a soil sample was collected from a greenhouse planted with lettuce (Lactuca sativa L.) in Daejeon City, Korea. The soil sample was diluted and spread on R2A medium (Reasoner & Geldreich, 1985Go). Strain GH2-10T was isolated after incubation for 5 days at 28 °C.

The morphological, physiological and biochemical characteristics of strain GH2-10T were tested using routine cultivation on R2A medium at 28 °C. Gram staining, presence of oxidase and catalase and hydrolysis of agar, casein, DNA, gelatin and starch were determined as described by Smibert & Krieg (1994)Go. Motility testing was performed on one-tenth strength R2A broth supplemented with 0.2 % agar. Growth was assessed at 5, 10, 20, 25, 30, 37, 40 and 45 °C, at pH 4, 5, 6, 7, 8, 9 and 10 and at 0, 1, 3, 5 and 7 % NaCl. Anaerobic growth was checked by using a BBL anaerobic jar (Becton Dickinson), with incubation for up to 21 days. Carboxymethylcellulose (CM-cellulose; Sigma) (0.1 %, w/v) and tyrosine (0.5 %, w/v) were also used. Strain GH2-10T was additionally characterized by using the whole test spectra of the API 20NE, API 50CH and API ZYM (bioMérieux) systems according to the manufacturer's instructions. For tests of antimicrobial susceptibility, discs containing the following antibiotics were used: amikacin (30 µg), ampicillin (10 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), gentamicin (10 µg), kanamycin (30 µg), methicillin (5 µg), polymyxin B (300 U), rifampicin (5 µg), tetracycline (30 µg) and vancomycin (30 µg).

The strain grew rapidly (2 days) on R2A and nutrient agar (NA; Difco) and rather slowly (5 days) on trypticase soy agar (TSA; Difco) and did not grow on MacConkey agar (Difco). The pH, NaCl concentration and temperature ranges for growth were pH 4–8, 0–5 % and 10–37 °C, respectively. Strain GH2-10T was catalase-positive and oxidase-negative. The isolate hydrolysed starch, CM-cellulose and urea, but not casein, DNA, gelatin or tyrosine. Hydrolysis of urea was negative after incubation for 2 days, but positive after 7 days. According to the API 20NE test strips, strain GH2-10T did not assimilate any substrates (up to 7 days incubation). API 50CH strips did not give reproducible results even with prolonged incubation (up to 7 days). Strain GH2-10T was sensitive to all the antibiotics tested except gentamicin and polymyxin B. Details of the physiological and biochemical properties of strain GH2-10T are given in Table 1Go and in the species description below.


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  		Table 1. Differential physiological characteristics of strain GH2-10T and the type strains of recognized members of the genus Devosia

Strains: 1, strain GH2-10T; 2, D. riboflavina DSM 7230T; 3, D. neptuniae LMG 21357T; 4, D. limi LMG 22951T. All strains were positive for catalase and negative for hydrolysis of CM-cellulose. In the API 20NE and API ZYM test strips, all strains were positive for aesculin hydrolysis, alkaline and acid phosphatase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, beta-galactosidase, beta-glucosidase and N-acetyl-beta-glucosaminidase. All strains were negative for lipase (C14), cystine arylamidase, {alpha}-chymotrypsin, beta-glucuronidase, nitrate reduction, indole production, glucose fermentation, arginine dihydrolase and gelatinase. Data are from Vanparys et al. (2005)Go and this study. +, Positive; (+), weakly positive; –, negative.

 
The DNA G+C content of the isolate was determined with an HPLC method as described by Mesbah et al. (1989)Go by using a reversed-phase column (Supelcosil LC-18-S; Supelco). The cellular fatty acid profile was determined by using the standard protocol of the Microbial Identification System (MIDI; Microbial ID) after cells were grown on TSA for 48 h at 28 °C. Isoprenoid quinones were analysed by HPLC as described by Groth et al. (1996)Go. The DNA G+C content was 59.5 mol%. The predominant fatty acids of strain GH2-10T were C18 : 1{omega}7c (67.2 %), C16 : 0 (12.7 %), 11-methyl C18 : 1{omega}7c (5.8 %) and C18 : 0 3-OH (5.2 %) (Table 2Go). The major respiratory lipoquinone of strain GH2-10T was ubiquinone 10 (Q-10).


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  		Table 2. Cellular fatty acid profiles of strain GH2-10T and the type strains of recognized members of the genus Devosia

Strains: 1, strain GH2-10T; 2, D. riboflavina DSM 7230T; 3, D. neptuniae LMG 21357T; 4, D. limi LMG 22951T. Only fatty acids that represent more than 1 % of the total are included.

 
The 16S rRNA gene sequence of strain GH2-10T was amplified by PCR using conserved primers, as described by Kwon et al. (2003)Go, and direct sequencing (Hiraishi, 1992Go). Multiple alignments with sequences of isolate GH2-10T and those of members of the genus Devosia and Rhizobium leguminosarum USDA 2370T were carried out with the CLUSTAL W program (Thompson et al., 1994Go). A phylogenetic tree was reconstructed using the neighbour-joining method of Saitou & Nei (1987)Go on MEGA version 2.1 (Kumar et al., 2001Go) (Fig. 1Go). The stability of relationships was assessed by performing bootstrap analyses of the neighbour-joining data based on 1000 resamplings.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the position of strain GH2-10T. Bootstrap values are shown as percentages of 1000 replicates. GenBank accession numbers are given in parentheses. Bar, 1 substitution per 100 nucleotides.

 
The nearly complete 16S rRNA gene sequence (1417 bp) of strain GH2-10T was subjected to comparative analysis. Phylogenetically, strain GH2-10T was most closely related to D. riboflavina IFO 13584T, with a sequence similarity of 98.5 %. The sequences of other Devosia strains were shown to have similarities of <97 % to strain GH2-10T.

Levels of DNA–DNA hybridization were determined based on a membrane filter technique using a DIG High Prime DNA labelling and detection starter kit II (Roche Molecular Biochemicals) (Kwon et al., 2003Go). Relatedness between strain GH2-10T and D. riboflavina DSM 7230T was 47 %. This value was lower than the 70 % value considered to be the threshold for the delineation of genomic species (Stackebrandt & Goebel, 1994Go), and clearly indicated that strain GH2-10T represented a different species from D. riboflavina.

To test the presence of the nitrogen-fixing genes nodC and nifH, PCR was conducted according to the method of Rivas et al. (2002)Go. The primer pairs for the amplification of the nodC and nifH genes were nodCF (5'-AYGTHGTYGAYGACGGTTC-3') and nodCI (5'-CGYGACAGCCANTCKCTATTG-3') (Laguerre et al., 2001Go), and 5'-GTCTCCTATGACGTGCTCGG-3' and 5'-GCTTCCATGGTGATCGGGGT-3' (Rivas et al., 2002Go), respectively. Whereas R. leguminosarum LMG 14904T, Ensifer fredii LMG 6217T, Mesorhizobium loti LMG 6125T and Bradyrhizobium japonicum LMG 6138T formed PCR fragments of the expected size, strain GH2-10T and D. riboflavina DSM 7230T did not.

On the basis of its phenotypic characteristics, which differ from those of recognized species of the genus Devosia, 16S rRNA gene sequence analysis and levels of DNA–DNA relatedness, strain GH2-10T is considered to represent a novel species of the genus Devosia, for which we propose the name Devosia soli sp. nov.

Emended description of the genus Devosia Nakagawa et al. 1996Go
The description is as given by Rivas et al. (2003)Go, with the following amendments. Oxidase-positive or oxidase-negative. The G+C content of the DNA is 59–63 mol%.

Description of Devosia soli sp. nov.
Devosia soli (so'li. L. gen. n. soli of soil).

Cells are Gram-negative, obligately aerobic, non-spore-forming and rod-shaped (about 0.4–0.6 µm wide and 1.5–3.5 µm long). Colonies are light beige and round with clear margins after 2 days on R2A medium. Tolerates up to 5 % NaCl and grows at temperatures between 10 and 37 °C. Growth occurs at initial pH values between 4 and 8. Catalase-positive and oxidase-negative. Hydrolyses starch, CM-cellulose and urea, but not casein, DNA, gelatin or tyrosine. Contains C18 : 1{omega}7c (67.2 %), C16 : 0 (12.7 %), 11-methyl C18 : 1{omega}7c (5.8 %) and C18 : 0 3-OH (5.2 %) as major fatty acids and Q-10 as the predominant ubiquinone. The DNA G+C content is 59.5 mol%. The closest relative on the basis of 16S rRNA gene sequence similarity is Devosia riboflavina.

The type strain, GH2-10T (=KACC 11509T=DSM 17780T), was isolated from greenhouse soil planted with lettuce (Lactuca sativa L.) in Daejeon City, Korea.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant (Code #20050401034815) from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.


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Laguerre, G., Nour, S. M., Macheret, V., Sanjuan, J., Drouin, P. & Amarger, N. (2001). Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiology 147, 981–993.[Abstract/Free Full Text]

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