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Int J Syst Evol Microbiol 54 (2004), 1345-1348; DOI  10.1099/ijs.0.02953-0
© 2004 International Union of Microbiological Societies

Kribbella solani sp. nov. and Kribbella jejuensis sp. nov., isolated from potato tuber and soil in Jeju, Korea

Jaekyeong Song1,3, Byung-Yong Kim1, Seung-Beom Hong1, Hyun-Suk Cho1, Kyounghee Sohn2, Jongsik Chun2 and Joo-Won Suh3

1 KACC, Genetic Resources Division, National Institute of Agricultural Biotechnology, Suwon, 441-707, Korea
2 School of Biological Sciences, Seoul National University, Seoul, 151-742, Korea
3 Department of Biological Science, Myong Ji University, Yongin 449-728, Korea

Correspondence
Joo-Won Suh
jwsuh{at}mju.ac.kr


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Two actinomycete strains, DSA1T and HD9T, were isolated from a potato tuber and soil from a potato-cultivating field in Jeju, Korea, respectively. A comprehensive 16S rDNA analysis revealed that the isolates belong to the genus Kribbella and share 97·7–98·6 % sequence similarity to Kribbella species. The strains also contained typical chemotaxonomic markers of the genus Kribbella: LL-diaminopimelic acid, alanine, glycine and glutamic acid in the cell wall peptidoglycan; mannose, glucose, galactose and ribose as whole cell sugars; diphosphatidylglycerol, phosphatidylcholine and phosphatidylinositol as characteristic phospholipids; and the major menaquinone MK-9 (H4). DNA–DNA hybridization experiments showed that the isolates represented two distinct genomic species. A number of phenotypic properties can be used to differentiate the two isolates from Kribbella species. On the basis of polyphasic evidence, two novel species are proposed: Kribbella solani sp. nov. for strain DSA1T (=KACC 20196T=JCM 12205T) and Kribbella jejuensis sp. nov. for strain HD9T (=KACC 20266T=JCM 12204T).

Abbreviations: LL-DAP, LL-diaminopimelic acid

Published online ahead of print on 30 April 2004 as DOI 10.1099/ijs.0.02953-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of strains KACC 20196T, KACC 20248T, KACC 20249T, KACC 20250T and KACC 20266T are AY253862–AY253866, respectively.

An extended neighbour-joining tree and a maximum-parsimony tree for the isolates and related actinomycete taxa are available as supplementary data in IJSEM Online.


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The genus Kribbella established by Park et al. (1999)Go contains LL-diaminopimelic acid (LL-DAP) in its cell wall peptidoglycan and lies within the family Nocardioidaceae (Nesterenko et al., 1985Go, 1990Go). Two species, Kribbella flavida and Kribbella sandramycini, included in this genus were originally assigned to the genus Nocardioides (‘Nocardioides fulvus’ IFO 14399 and Nocardioides sp. ATCC 39419, respectively) based on 16S rDNA sequence analysis. Recently, Hongia koreensis (Lee et al., 2000Go) was transferred to this genus as Kribbella koreensis on the basis of its molecular systematics (Sohn et al., 2003Go). Here, we present polyphasic characterization of two novel Kribbella strains isolated during a taxonomic survey of the potato scab caused by Streptomyces spp. from Jeju, Korea (Song et al., 2004Go).

Strain DSA1T was isolated from a potato tuber with scab lesions. For isolation, the tuber was washed twice with distilled water and scraped lesions were suspended in distilled water using a vortex mixer. The suspension was plated on GYM agar (DSMZ medium no. 65; http://www.dsmz.de/media/media.htm) using the dilution method and cultured at 30 °C for 5 days. Strain HD9T was isolated from soil of a potato-cultivating field using the same growth conditions and the dilution plating method.

Growth and morphological properties of the isolates were recorded from GYM agar, ISP media (Shirling & Gottlieb, 1966Go) and trypticase soy agar (TSA; BBL 11768). Melanin formation was examined on ISP media 6 and 7. For scanning electron microscopy, cells grown at 30 °C for 5 days on GYM agar were prepared by cutting agar blocks fixed with 1 % osmium tetroxide and observed under a scanning electron microscope (Hitachi S-2460N). Both strains grew on GYM agar, TSA, nutrient agar and all ISP media at 28 °C. The two strains did not produce melanin pigment. Their colonies were sand-pasty and had lichenous shapes with irregular edges on GYM agar. Aerial mycelium of the isolates was white and consisted of hyphae that fragmented into rod-shaped elements.

Physiological characteristics were examined using standard procedures (Lee et al., 2000Go; Williams et al., 1983Go) at 30 °C for up to 5 days. In addition, catalase activity, nitrate reduction, urease activity, hydrogen sulfide production and hydrolysis of arbutin, casein, aesculin, gelatin and starch were tested as described by MacFaddin (2000)Go. Antimicrobial activity was examined against Bacillus subtilis, Pseudomonas aeruginosa, Micrococcus luteus, Streptomyces scabiei, Candida albicans, Aspergillus niger, Trichoderma harzianum, Fusarium acuminatum and Colletotrichum gloeosporioides. Physiological characteristics are given in the species descriptions and in Table 1Go.


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  		Table 1. Differential physiological characteristics of isolates DSA1T and HD9T and type strains of Kribbella species

Strains: 1, DSA1T; 2, HD9T; 3, K. flavida KACC 20248T (data from Park et al., 1999Go unless indicated); 4, K. sandramycini KACC 20249T (Park et al., 1999Go); 5, K. koreensis KACC 20250T (Lee et al., 2000Go). +, Positive; –, negative; W, weakly positive; ND, not done.

 
Genomic DNA extraction, PCR amplification of 16S rDNA and cloning were done as described previously (Song et al., 2004Go). 16S rDNA sequences were analysed using an Applied Biosystems DNA sequencer (ABI3100) and aligned using CLUSTAL W software (Thompson et al., 1994Go). Nucleotide similarity values were calculated from the alignment. An evolutionary distance matrix was constructed using the algorithm of Jukes & Cantor (1969)Go and phylogenetic trees for the datasets were inferred from the neighbour-joining method of Saitou & Nei (1987)Go using MEGA version 2.1 (Kumar et al., 2001Go). The stability of relationships was evaluated by performing bootstrap analysis of the neighbour-joining data based on 1000 resamplings.

Phylogenetic analysis based on 16S rDNA sequences indicated that our isolates belong to the genus Kribbella with 100 % bootstrap support (Fig. 1Go). Strain DSA1T showed the highest similarity to K. sandramycini KACC 20249T (98·6 %), followed by K. flavida KACC 20248T (98·5 %), strain HD9T (98·2 %) and K. koreensis KACC 20250T (98·1 %). Strain HD9T showed the closest similarity to strain DSA1T (98·2 %), followed by K. sandramycini KACC 20249T (98 %), K. flavida KACC 20248T (97·9 %) and K. koreensis KACC 20250T (97·7 %). An extended neighbour-joining tree and a maximum-parsimony tree using PAUP version 4.0b10 (Swofford, 2000Go) are available as supplementary data in IJSEM Online.



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  		Fig. 1. Phylogenetic tree showing the relationship of isolates DSA1T and HD9T and related actinomycete taxa. Numbers at branching points indicate percentages of occurrence based on neighbour-joining analyses of 1000 resampled dataset bootstrapped trees. Bar, one nucleotide substitution per 100 nucleotides.

 
DNA–DNA relatedness between the isolates and type strains of the Kribbella species were determined using the slot-blot hybridization method of Chun et al. (1998)Go. Our isolates showed relatively low DNA–DNA relatedness of 42·5 %. Isolate DSA1T showed a low level of DNA–DNA relatedness with K. sandramycini (0 %), K. koreensis (16·4 %) and K. flavida (50·4 %). Similarly, isolate HD9T showed a low level of DNA–DNA relatedness with K. sandramycini (0 %), K. koreensis (20·9 %) and K. flavida (47·8 %).

Biomass for most of the chemotaxonomic studies was prepared following growth of the isolates and standard strains in shake flasks of GYM for 5 days at 30 °C; after a check for purity, biomass was harvested by centrifugation, washed twice in distilled water and freeze-dried. Integrated lipid and wall analysis were carried out by previously described methods: wall amino acids (O'Donnell et al., 1985Go), diaminopimelic acids (Staneck & Roberts, 1974Go), sugars (Saddler et al., 1991Go), menaquinone (O'Donnell et al., 1985Go) and polar lipids (Minnikin et al., 1984Go). Whole-cell fatty acid profile was determined on TSA (BBL 11768) using the MIDI (Microbial Identification) system. Both strains had LL-DAP, alanine, glycine and glutamic acid in the cell wall peptidoglycan, contained mannose, glucose, galactose and ribose as whole cell sugars and MK-9 (H4) as the major menaquinone, as for other Kribbella species. Polar lipid profiles of the two strains revealed diphosphatidylglycerol, phosphatidylcholine and phosphatidylinositol. Whole-cell fatty acid profiles of our isolates and other Kribbella species are given in Table 2Go. The G+C content of the DNA was determined using the thermal denaturation method of Marmur & Doty (1962)Go: values were 69·2 and 68±1 mol%, for strains DSA1T and HD9T, respectively.


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  		Table 2. Cellular fatty acid profiles of isolates DSA1T and HD9T and type strains of Kribbella species on TSA medium

Strains: 1, DSA1T; 2, HD9T; 3, K. flavida KACC 20248T; 4, K. sandramycini KACC 20249T; 5, K. koreensis KACC 20250T. Values of less than 1 % are not shown. TBSA, Tuberculostearic acid.

 
From genotypic and phenotypic characterization, it is evident that our isolates not only belong to the genus Kribbella but also represent novel species. Along with molecular data, a number of phenotypic characters can be used to differentiate the isolates from recognized Kribbella species and from each other (Tables 1 and 2GoGo). We therefore propose two novel species, Kribbella solani sp. nov. for strain DSA1T and Kribbella jejuensis sp. nov. for strain HD9T.

Description of Kribbella solani sp. nov.
Kribbella solani (so.la'ni. N.L. gen. n. solani of Solanum, the genus of the potato, Solanum tuberosum, from which the type strain was isolated).

Aerial mycelium is white and consists of hyphae that fragment into rod-shaped elements. Vegetative mycelium is creamy and shows hyphae that are broadly branched. Colonies are sand-pasty and have lichenous shapes with irregular edges. No diffusible pigment is formed. Non-motile. Nitrate is not reduced to nitrite. Catalase-positive. Urease-negative. Utilizes glucose, sucrose, D-xylose, D-arabinose, D-melezitose, D-mannose, L-rhamnose, {alpha}-lactose, trehalose, melibiose, cellobiose, raffinose and salicin as carbon sources. Utilizes L-histidine, DL-arginine, DL-{alpha}-amino-n-butyric acid, trans-4-hydroxy-L-proline and potassium nitrate as nitrogen sources. Grows at 28–30 °C but not at 15 or 37 °C. Grows at pH 7 but not at pH 4 or 10. DNA, Tween 80, aesculin, arbutin and tributyrin are hydrolysed. Xanthine, hypoxanthine, xylan, elastin, adenine, guanine, tyrosine, allantoin, casein, chitin and starch are not hydrolysed. Tolerant to 0·01 % lysozyme, 2 % NaCl, 0·1 % phenyl ethanol and 0·001 % potassium tellurite but not to 0·01 % sodium azide, 0·01 % thallous acetate and 0·1 % phenol. Resistant to 4 µg tetracycline, 10 µg gentamicin, 80 µg neomycin, 4 µg rifamficin, 20 µg streptomycin, 4 µg vancomycin and 4 µg tobramycin ml–1, but susceptible to 10 µg treoleandomycin ml–1. Weakly resistant to 10 U penicillin G ml–1. Antimicrobial activity is not observed. Cell wall peptidoglycan contains LL-DAP, alanine, glycine and glutamic acid. Mannose, glucose, galactose and ribose are present as whole cell sugars. The predominant fatty acids are 12-methyltridecanoic acid, 12-methyltetradecanoic acid and 14-methylpentadecanoic acid. The genomic DNA G+C content is about 69 mol%.

The type strain, DSA1T (=KACC 20196T=JCM 12205T), was isolated from a potato tuber with scab lesions from Jeju, Korea. The species description is based on the type strain only.

Description of Kribbella jejuensis sp. nov.
Kribbella jejuensis (je.ju.en'sis. N.L. fem. adj. jejuensis referring to Jeju, Korea).

Aerial mycelium is white and consists of hyphae that fragment into rod-shaped elements. Vegetative mycelium is creamy and shows hyphae that are broadly branched. Colonies are sand-pasty and have lichenous shapes with irregular edges. No diffusible pigment is formed. Non-motile. Nitrate is not reduced to nitrite. Catalase- and urease-positive. Utilizes glucose, sucrose, D-xylose, D-arabinose, D-melezitose, galactose, L-rhamnose, {alpha}-lactose, trehalose, melibiose, cellobiose, raffinose and inulin as carbon sources. Utilizes L-histidine, DL-arginine, DL-homoserine, DL-{alpha}-amino-n-butyric acid, trans-4-hydroxy-L-proline and potassium nitrate as nitrogen sources. Grows at 28–37 °C but not at 15 or 45 °C. Grows at pH 7 but not at pH 4 or 10. Gelatin, aesculin, arbutin and tributyrin are hydrolysed. Tween 80, xanthine, hypoxanthine, xylan, elastin, adenine, guanine, tyrosine, allantoin, casein, chitin and starch are not hydrolysed. Tolerant to 0·01 % lysozyme, 1 % NaCl, 0·1 % phenyl ethanol and 0·001 % potassium tellurite but not to 0·01 % sodium azide, 0·01 % thallous acetate or 0·1 % phenol. Resistant to 1 µg tetracycline, 0·4 µg gentamicin, 4 µg rifamficin, 1 µg streptomycin, 4 µg vancomycin and 1 µg tobramycin ml–1, but susceptible to 4 µg neomycin, 20 µg streptomycin and 10 µg treoleandomycin ml–1. Weakly resistant to 4 µg tobramycin, 4 µg streptomycin and 10 U penicillin G ml–1. Weak antimicrobial activity is observed towards Streptomyces scabiei. Cell wall peptidoglycan contains LL-DAP, alanine, glycine and glutamic acid. Mannose, glucose, galactose and ribose are present as cell wall sugars. The predominant fatty acids are 13-methyltetradecanoic acid, 12-methyltetradecanoic acid and 14-methylpentadecanoic acid. The genomic DNA G+C content is about 68 mol%.

The type strain, HD9T (=KACC 20266T=JCM 12204T), was isolated from soil from Jeju, Korea. The species description is based on the type strain only.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr J. P. Euzéby for his valuable help in assigning the specific names to novel isolates.


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