International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2223–2228 DOI 10.1099/ijs.0.057737-0

Mucilaginibacter pineti sp. nov., isolated from Pinus pinaster wood from a mixed grove of pines trees

Gabriel Paiva,1 Pedro Abreu,1 Diogo Neves Proenc¸a,1 Susana Santos,1 Maria Fernanda Nobre2 and Paula V. Morais1,3

Correspondence 1IMAR-CMA, University of Coimbra, 3004-517 Coimbra, Portugal Paula V. Morais 2CNC-Center for Neuroscience and Cell Biology, University of Coimbra, [email protected] 3004-517 Coimbra, Portugal 3Department of Life Sciences, FCTUC, University of Coimbra, 3004-517 Coimbra, Portugal

Bacterial strain M47C3BT was isolated from the endophytic microbial community of a Pinus pinaster tree branch from a mixed grove of pines. Phylogenetic analysis of 16S rRNA gene sequences showed that this organism represented one distinct branch within the family , most closely related to the genus . Strain M47C3BT formed a distinct lineage, closely related to Mucilaginibacter dorajii KACC 14556T, with which it shared 97.2 % 16S rRNA gene sequence similarity. The other members of the genus Mucilaginibacter included in the same clade were Mucilaginibacter lappiensis ATCC BAA-1855T sharing 97.0 % similarity and Mucilaginibacter composti TR6-03T that had a lower similarity (95.7 %). The novel strain was Gram-staining-negative, formed rod-shaped cells, grew optimally at 26 6C and at pH 7, and was able to grow with up to 0.3 % (w/v) NaCl. The respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids of the strain were summed feature 3

(C16 : 1v7c/iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 3-OH, representing 73.5 % of the total fatty acids. The major components of the polar lipid profile of strain M47C3BT consisted of phosphatidylethanolamine, three unidentified aminophospholipids, one unidentified aminolipid and three unidentified polar lipids. The G+C content of the DNA was 40.6 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics we propose the name Mucilaginibacter pineti sp. nov. for the novel represented by strain M47C3BT (5CIP 110632T5LMG 28160T).

The genus Mucilaginibacter of the family Sphingobac- myungsuensis (Joung & Joh, 2011), M. angelicae (Kim teriaceae, phylum , was first described by et al., 2012a), M. litoreus (Yoon et al., 2012), M. lutimaris Pankratov et al. (2007) to group Gram-staining-negative, (Kim et al., 2012b), M. polysacchareus (Han et al., 2012), M. non-spore-forming and non-motile rods producing large soli (Jiang et al., 2012) and M. jinjuensis (Khan et al., 2013). amounts of extracellular polymeric substances. The genus Members of the genus Mucilaginibacter have been isolated was proposed to include two novel species, Mucilaginibacter from very different environments including peat bogs, soils, paludis and Mucilaginibacter gracilis. At the time of writing, dried rice straw, wetland freshwater (An et al., 2009; Baik the genus Mucilaginibacter comprises 22 species with validly et al., 2010; Madhaiyan et al., 2010; Pankratov et al., 2007; published names: M. paludis and M. gracilis (Pankratov Urai et al., 2008) and also cold environments (Ma¨nnisto¨ et al., 2007), M. kameinonensis (Urai et al., 2008), M. daejeo- et al., 2010). nensis (An et al., 2009), M. ximonensis (Luo et al., 2009), M. oryzae (Jeon et al., 2009), M. rigui (Baik et al., 2010), Pinus pinaster trees from a mixed grove of pines in Malhada, M. gossypii and M. gossypiicola (Madhaiyan et al., 2010), Oliveira do Hospital, Portugal, were sampled and the bark M. frigoritolerans, M. lappiensis and M. mallensis (Ma¨nnisto¨ and sapwood of each cross-section were removed under sterile conditions for the study of the diversity of the et al., 2010), M. boryungensis (Kang et al., 2011), M. composti T (Cui et al., 2011), M. dorajii (Kim et al., 2010), M. endophytic community. Strain M47C3B , forming mucoid, light-pink colonies, was isolated from dilutions of wood The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene chips plated on R2A agar (Difco) incubated at 25 uC, for sequence of strain M47C3BT is KF483876. three days. The strain was maintained in the same medium Two supplementary figures are available with the online version of this supplemented with 15 % (v/v) glycerol at 280 uC after paper. subculture and purification.

057737 G 2014 IUMS Printed in Great Britain 2223 G. Paiva and others

Cell morphology and motility were examined by phase- elastin, starch, DNA, chitin, and Tweens 20, 40, 60 and 80 contrast microscopy (Leitz Diaplan, UK) after growth on at a concentration of 1.0 % (w/v or v/v) in R2A agar, after R2A agar at 26 uC for 48 h. Growth on several bacterio- incubation at 26 uC for up to 5 days, was determined as logical media was tested using R2A agar, tryptic soy agar described by Tindall et al. (2007). Other physiological (TSA; Difco), nutrient agar (NA; Difco) and R2 liquid properties and enzyme activities were determined using the (Difco) incubated at 26 uC for 5 days. The temperature API ZYM and API 20NE test strips (bioMe´rieux) at 26 uC range (4, 15, 20, 22, 26, 30, 37, 40, 42, 45 uC) and optimum according to the manufacturer’s instructions. Single- temperature for growth were examined on R2A agar and in carbon-source assimilation was determined using API 50 R2 liquid incubated for up to 5 days. Salt tolerance was CH test strips (bioMe´rieux) after incubation at 26 uC for tested in R2A agar and R2 liquid supplemented with NaCl up to 7 days, as described by Morais et al. (2004). API 50 at the final concentrations of 0 to 3 % (w/v), in 0.5 % CH strips were also used for evaluation of acid production increments (agar) and 0.1 % increments (liquid), at 26 uC, from single carbon sources after incubation at 26 uC for up incubated for up to 5 days. The pH range for growth was to 5 days, according to the manufacturer’s instructions. The examined at 26 uC in the same medium by using 50 mM ability of the strain to oxidize different carbon sources was MES (pH 3, 4, 5, 6, 7), HEPES (pH 6, 7, 8), TAPS (pH 8, assessed using Biolog GN2 MicroPlates, incubated at 26 uC. 9) and CAPSO (pH 10) over a pH range from pH 5.0 to The results were recorded daily for up to 7 days using a 10.0, with intervals of 0.5 pH units. The Gram-staining MicroPlate reader (Sunrise Xread Plus version V 4.30; reaction and the presence of cytochrome oxidase and Tecan). The test for flexirubin-like pigments was performed catalase were determined after 24 h of incubation on R2A by soaking cells grown on R2A agar at 26 uC for 2 days with agar as described by Smibert & Krieg (1994). The ability to 20 % (w/v) KOH (Fautz & Reichenbach, 1980). Congo red hydrolyse agar aesculin, casein, xylan, gelatin, arbutin, adsorption was tested by using R2A-Congo red agar (25 mg

Table 1. Differential characteristics of strain M47C3BT compared with the type strains of Mucilaginibacter genus

Strains: 1, M47C3BT (data from this study); 2, M. dorajii KACC 14556 T;3,M. lappiensis ATCC BAA-1856T. All strains are positive for (API ZYM) alkaline phosphatase, leucine arylamidase, Valine arylamidase, cystine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase, a-fucosidase and are negative for lipase (C14), a-chymotrypsine. All strains cannot assimilate (API 20NE and API 50 CH) D-Mannitol. Data for reference strains are from Kim et al. (2010) and Ma¨nnisto¨ et al. (2010). +, positive; w+, weakly positive; –, negative; ND, not determined.

Characteristic 1 2 3

Colony colour Light Pink Light Yellow Light Pink Catalase/oxidase +/++/++/+ Cell length (mm) 1–3 1.1–1.8 1–3 Temperature range (uC) 15–30 4–30 0–31 Max. NaCl (%, w/v) tolerance 0.3 1 1.5 pH range 5.5–8.0 5.0–8.0 4.5–8.0 Acid production from: D-Arabinose – ++ D-Galactose – ++ D-Glucose 2 ++ N-Acetylglucosamine 2 w+ w+ Amygdalin 2 w++ D-Cellobiose 2 ++ D-Maltose 2 ++ D-Lactose 2 ++ D-Trehalose 2 ++ Assimilation of: L-Arabinose – ++ N-acetylglucosamine – ++ Maltose – ++ D-Glucose – ++ D-Mannose – w+ w+ Enzyme activities: Esterase C4 – w+ w+ Esterase lipase C8 – w++ DNA G+C content (mol%) 40.64 42.6 43.5

2224 International Journal of Systematic and Evolutionary Microbiology 64 Mucilaginibacter pineti sp. nov. from pine wood

Congo red l21) incubated at 26 uC for 2 days (Freeman was positive for catalase and weakly positive for oxidase. et al., 1989). Antibiotic-sensitivity tests were performed for Strain M47C3BT could grow in R2A agar and R2 liquid strain M47C3BT by using discs (Oxoid) containing the containing up to 0.3 % (w/v) NaCl, while its closest relatives following: lincomycin (15 mg), ampicillin (10 mg), amox- grew with 1 to 1.5 % (w/v) NaCl. Colonies on R2A-Congo red icillin+clavulanic acid (30 mg), gentamicin (30 mg), poly- agar were non-pigmented and flexirubin-type pigments were myxin B (300 U), chloramphenicol (100 mg), erythromycin not formed. Strain M47C3BT was able to assimilate very few (15 mg), vancomycin (30 mg), streptomycin (50 mg), rifam- carbon sources in comparison with M. dorajii and M. picin (30 mg), tetracycline (30 mg) or kanamycin (30 mg). lappiensis (Table 1). The strain was able to hydrolyse casein and starch, and was weakly urease-positive. The major polar Cells for polar lipids and lipoquinone analysis were grown lipids were phosphatidylethanolamine, three unidentified on R2A agar at 26 uC for 48 h and then harvested and aminophospholipids, one unidentified aminolipid and three lyophilized. Polar lipids were extracted and two-dimensional unidentified polar lipids. No glycolipids were detected (Fig. TLC was performed on silica gel G plates (10610 cm, S1, available in the online Supplementary Material). 0.25 mm thickness; Merck) using chloroform/methanol/water T (65 : 25 : 4, by vol.) in the first direction and chloroform/acetic The major fatty acids of strain M47C3B were summed acid/methanol/water (80 : 15 : 12 : 4, by vol.) in the second feature 3 (C16 : 1v7c/iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 direction (da Costa et al., 2006). The polar lipids were identified using differential staining with molybdophosphoric T acid (total polar lipids), ninhydrin (aminolipids) and a- Table 2. Fatty acid composition of strain M47C3B and the naphthol/sulfuric acid (glycolipids). Phosphatidylethano- type strains of closely related members of the genus laminewasusedasastandard.Lipoquinones were extracted Mucilaginibacter from freeze-dried cells, purified by TLC and separated by Strains: 1, M47C3BT (data from this study); 2, M. dorajii KACC HPLC (da Costa et al., 2011a). Cells for fatty acids analysis 14556T (Kim et al., 2010); 3, M. lappiensis ATCC BAA-1855T were grown on R2A agar in sealed plastic plates for 48 h (Ma¨nnisto¨ et al., 2010). Values are percentages of total fatty acids. TR, (Morais et al., 2004). The fatty acid methyl esters (FAMEs) Traces (,0.5 %); –, not detected. were obtained from fresh wet biomass and were separated, identified and quantified using the standard MIS Library Fatty acid 1 2 3 Generation Software (Sherlock Microbial ID System, 2TSBA 6 database, version 6.0; MIDI) as described previously (da Costa iso-C13 : 0 3-OH – 0.9 TR et al., 2011b). The DNA G+C content of the genome was C14 : 0 0.7 1.2 1.1 C 2-OH 0.3 – – determined by HPLC as described by Mesbah et al. (1989). 14 : 0 C15 : 0 0.8 6.5 3.4 The 16S rRNA gene was amplified by PCR and sequenced C15 : 0 2-OH – 1.2 1.1 as described by Morais et al. (2004), and aligned against C15 : 0 3-OH – TR – representative reference sequences of the most closely related iso-C15 : 0 17.7 22.6 28.9 members of the genus Mucilaginibacter obtained from the iso-C15:0 3-OH 2.2 1.3 1.4 EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., C15 : 1v6c 0.2 1.2 2.9 anteiso-C 0.3 0.8 – 2012c) and then aligned by SINA (v1.2.9) using the SILVA SEED 15 : 0 as reference alignment (http://www.arb-silva.de/aligner/; C16 : 0 7.7 6.5 6.4 C 3-OH 3.3 2.3 – Pruesse et al., 2012). Evolutionary distances were calculated 16 : 0 iso-C16 : 0 – 1.2 – (Jukes & Cantor, 1969), and phylogenetic dendrograms were iso-C 3-OH – 0.6 – reconstructed using the neighbour-joining (Saitou & Nei, 16 : 0 C16 : 1v5c 6.5 4.7 2.9 1987) and maximum-likelihood (Olsen et al., 1994) algo- C17 : 0 – 0.6 – ARB rithms included in the software package (Ludwig et al., C17 : 0 3-OH – 0.6 – 2004). Tree topologies were evaluated by performing boot- iso-C17 : 0 0.5 TR 0.8 strap analysis (Felsenstein, 1985) of 1000 datasets by using the iso-C17 : 0 3-OH 10.2 6.5 7.8 ARB software package. C17 : 1v8c – 0.9 TR iso-C v9c – 1.7 2.4 Strain M47C3BT was Gram-staining-negative, non-spore- 17 : 1 C – 3.4 – forming and appeared as rod-shaped cells (0.09–0.1 mmin 18 : 0 C18 : 1v7c – 1.6 – width and 1–3 mm in length). After 48 h of incubation on Summed feature 2* 0.2 – – R2A agar, colonies were circular, smooth, glistening and light Summed feature 3* 45.5 30.9 39.9 pink. Biochemical and physiological characteristics of strain Summed feature 9* 2.2 – – M47C3BT are summarized in Table 1 and in the species T description. Strain M47C3B had an optimum temperature *Summed features combine groups of two or more fatty acids that forgrowat26uC and was not able to grow at temperatures cannot be separated by GLC with the MIDI system. Summed feature 2 below 15 uC, in contrast to its closest relatives, M. dorajii and contains C12 : 0 aldehyde, C14 : 0 3-OH and/or iso-C16 : 1. Summed T M. lappiensis (Table 1). Strain M47C3B was able to grow feature 3 contains C16 : 1v7c and/or iso-C15 : 0 2-OH. Summed feature from pH 5.5 up to pH 8.0 with optimal growth at pH 7, and 9 contains iso-C17 : 1v9c and/or C16 : 0 10 methyl. http://ijs.sgmjournals.org 2225 G. Paiva and others

3-OH, which accounted for 73.5 % of the total fatty Cells on R2A agar are small, smooth, glistening and light acids (Table 2). Menaquinone 7 (MK-7) was the major pink. Cells are Gram-staining-negative, rod-shaped (0.09– respiratory quinone. The almost complete 16S rRNA gene 0.1 mm in width and 1–3 mm in length) after 48 h of sequence of strain M47C3BT (1496 bp) was aligned with incubation on R2A agar and non-spore-forming. Grows on those of representative members of all species of the genus NA and on R2A agar but not on TSA. Growth occurs Mucilaginibacter of the family Sphingobacteriaceae, showing between 15 and 30 uC (optimal at 26 uC). Growth occurs at this organism to be a representative of a novel species pH 5.5–8.0 (optimal at pH 7). Growth occurs with up to belonging to the genus Mucilaginibacter. According to the 0.3 % (w/v) NaCl. Catalase- and oxidase-positive. Colonies phylogenetic tree reconstructed by using the neighbour- are not coloured on R2A-Congo red agar and flexirubin- joining method, the closest phylogenetic relatives were type pigments are not formed. Resistant to ampicillin M. dorajii KACC 14556T (97.2 %) and M. lappiensis ATCC (10 mg), gentamicin (30 mg), polymyxin B (300 U) and BAA-1855T (97.0 %) (Fig. 1). The tree topology was con- kanamycin (30 mg), and susceptible to rifampicin (30 mg), firmed by using a maximum-likelihood analysis (Fig. S2). streptomycin (50 mg), vancomycin (30 mg) and tetracycline The G+C content of the DNA, as determined by HPLC (30 mg). Positive for hydrolysis of aesculin, starch and (Mesbah et al., 1989), was 40.6 mol%. The phenotypic DNA (weakly positive). Negative for hydrolysis of xylan, characterization, enzymic activities, biochemical and physio- elastin, arbutin, gelatin, Tween 20, Tween 40, Tween 60 logical characteristics (Table 1), fatty acid profile (Table 2) and Tween 80. Negative for nitrate reduction, indole and phylogenetic evidence indicate that strain M47C3BT production and glucose fermentation. Positive for alkaline represents a novel species of the genus Mucilaginibacter, for phosphatase, leucine arylamidase, valine arylamidase, tryp- which we propose the name Mucilaginibacter pineti sp. nov. sin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, a-galactosidase, b-galactosidase, a-glucosidase, b-glucosi- Description of Mucilaginibacter pineti sp. nov. dase, N-acetyl-b-glucosaminidase, a-mannosidase and a- fucosidase. Weakly positive for cystine arylamidase. Negative Mucilaginibacter pineti (pi.ne9ti. L. gen. n. pineti of a pine for esterase (C4), esterase lipase (C8), lipase (C14), a- wood, pine grove). chymotrypsin and b-glucuronidase (API ZYM). Positive for

72 Mucilaginibacter gossypiicola Gh-48T (EU672805) 0.10 Mucilaginibacter gossypii Gh-67T (EU672804) Mucilaginibacter kameinonensis SCKT (AB330392) Mucilaginibacter oryzae B9T (EU109722) Mucilaginibacter angelicae GG-w14T (HM627214) Mucilaginibacter polysacchareus DRP28T (HM748604) Mucilaginibacter pineti M47C3BT (KF483876) Mucilaginibacter dorajii DR-f4T (GU139697) Mucilaginibacter lappiensis ANJLI2T (DQ234446) Mucilaginibacter composti TR6-03T (AB267719) 99 Mucilaginibacter rigui WPCB133T (EU747841) Mucilaginibacter lutimaris BR-3T (HQ455786) Mucilaginibacter soli R9-65T (JF701183) Mucilaginibacter litoreus BR-18T (JF999998) 97 Mucilaginibacter gracilis TPT18T (AM490403) TPT56T (AM490402) Mucilaginibacter frigoritolerans FT22T (FN400860) Mucilaginibacter gynuensis YC7003T (KC247157) 72 Mucilaginibacter mallensis MP1X4T (FN400859) Mucilaginibacter myungsuensis HMD1056T (GQ144415) Mucilaginibacter ximonensis XM-003T (EU729366) Mucilaginibacter boryungensis BDR-9T (HM061614) 86 Mucilaginibacter daejeonensis Jip 10T (AB267717) Mucilaginibacter jinjuensis YC7004T (JQ765855) 71 Parasegetibacter luojiensis RHYL-37T (EU877263) Segetibacter koreensis Gsoil 664T (AB267478) Sediminibacterium salmoneum NJ-44T (EF407879)

Fig. 1. Phylogenetic dendrogram based on a comparison of the 16S rRNA gene sequences of M47C3BT strain and the closest phylogenetic relatives. The tree was created using the neighbour-joining method. Numbers indicate percentages of bootstrap sampling, derived from 1000 replications; values below 70 % are not shown. Filled circles indicate node branches conserved when the tree was reconstructed using the maximum-parsimony algorithm. The isolate characterized in this study is indicated in bold type. Bar, one inferred nucleotide substitution per 10 nt.

2226 International Journal of Systematic and Evolutionary Microbiology 64 Mucilaginibacter pineti sp. nov. from pine wood aesculin and p-nitrophenyl-b-D-galactopyranoside (API 20 da Costa, M. S., Nobre, M. F. & Wait, R. (2006). Analysis of lipids from NE). Assimilates the carbon sources aesculin ferric citrate extremophilic . Methods Microbiol 35, 127–159. (API 20 NE and API50 CH), p-nitrophenyl-b-D-galactopyr- da Costa, M. S., Albuquerque, L., Nobre, M. F. & Wait, R. (2011a). The anoside (PNPG) (API 20 NE), and methyl a-D-mannopyr- extraction and identification of respiratory lipoquinones of prokar- anoside, arbutin, potassium 2-ketogluconate and potassium yotes and their use in . Methods Microbiol 38, 197–206. 5-ketogluconate (API 50 CH). The other organic substrates da Costa, M. S., Albuquerque, L., Nobre, M. F. & Wait, R. (2011b). The included in API 50 CH and API 20 NE are not utilized. identification of fatty acids in bacteria. Methods Microbiol 38, 183– 196. Acid is produced from (API 50 CH) D-ribose, D-xylose, Fautz, E. & Reichenbach, H. (1980). A simple test for flexirubin-type D-galactose, D-glucose, D-mannitol, D-sorbitol and aesculin pigments. FEMS Microbiol Lett 8, 87–91. ferric citrate. Acid is produced weakly from methyl b- Felsenstein, J. (1985). Confidence limits on phylogenies: an approach D-xylopyranoside, D-mannose, melibiose, sucrose, gentio- biose and potassium 5-ketogluconate. Tests using the Biolog using the bootstrap. Evolution 39, 783–791. GN2 system show dextrin is oxidized, a-cyclodextrin, Freeman, D. J., Falkiner, F. R. & Keane, C. T. (1989). New method for detecting slime production by coagulase negative staphylococci. J Clin glycogen, N-acetyl-D-galactosamine, N-acetyl-D-glucosa- Pathol 42, 872–874. mine, cellobiose, D-fructose, L-fucose, D-galactose, gentio- Han, S.-I., Lee, H.-J., Lee, H.-R., Kim, K.-K. & Whang, K.-S. (2012). biose, a-D-glucose, a-lactose, lactulose, maltose, D-mannose, Mucilaginibacter polysacchareus sp. nov., an exopolysaccharide- melibiose, methyl b-D-glucoside, D-psicose, raffinose, suc- producing bacterial species isolated from the rhizoplane of the herb rose, trehalose, turanose, D-gluconic acid, D-glucosaminic Angelica sinensis. Int J Syst Evol Microbiol 62, 632–637. acid, a-ketobutyric acid, L-alaninamide, L-alanine, L-alanyl Jeon, Y., Lee, S.-S., Chung, B. S., Kim, J. M., Bae, J.-W., Park, S. K. & glycine, L-glutamic acid, L-proline, L-serine and L-threonine Jeon, C. O. (2009). Mucilaginibacter oryzae sp. nov., isolated from soil are weakly oxidized, and the remaining substrates are not of a rice paddy. Int J Syst Evol Microbiol 59, 1451–1454. oxidized. The major components of the polar lipid profile of Jiang, F., Dai, J., Wang, Y., Xue, X., Xu, M., Guo, Y., Li, W., Fang, C. & T strain M47C3B consisted of phosphatidylethanolamine, Peng, F. (2012). Mucilaginibacter soli sp. nov., isolated from Arctic three unidentified aminophospholipids, one unidentified tundra soil. Int J Syst Evol Microbiol 62, 1630–1635. aminolipid and three unidentified polar lipids. No glycoli- Joung, Y. & Joh, K. (2011). Mucilaginibacter myungsuensis sp. nov., pids are detected. The major fatty acids are summed feature isolated from a mesotrophic artificial lake. Int J Syst Evol Microbiol 61, 3(C16 : 1v7c/iso-C15 : 0 2-OH), iso-C15 : 0, iso-C17 : 0 3-OH, 1506–1510. C16 : 0 and C16 : 1v5c. The major respiratory lipoquinone is Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In menaquinone 7 (MK-7). Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press. T T T The type strain, M47C3B (5CIP 110632 5LMG 28160 ) Kang, S.-J., Jung, Y.-T., Oh, K.-H., Oh, T.-K. & Yoon, J.-H. (2011). was isolated from the wood of a Pinus pinaster in Malhada, Mucilaginibacter boryungensis sp. nov., isolated from soil. Int J Syst Coimbra, Portugal. The DNA G+C content of the type Evol Microbiol 61, 1549–1553. strain is 40.6 %. Khan, H., Chung, E. J., Kang, D. Y., Jeon, C. O. & Chung, Y. R. (2013). Mucilaginibacter jinjuensis sp. nov., with xylan-degrading activity. Int J Syst Evol Microbiol 63, 1267–1272. Acknowledgements Kim, B.-C., Lee, K. H., Kim, M. N., Lee, J. & Shin, K.-S. (2010). We are indebted to Professor J. P. Euze´by (E´ cole National Ve´te´rinaire, Mucilaginibacter dorajii sp. nov., isolated from the rhizosphere of Toulouse, France) for the etymology of the new organisms’ name. Platycodon grandiflorum. FEMS Microbiol Lett 309, 130–135. This research was financially supported by Program of National Kim, B.-C., Poo, H., Lee, K. H., Kim, M. N., Kwon, O.-Y. & Shin, Action for Pine Wood Nematode control: ‘The disease of Pine Wood K.-S. (2012a). Mucilaginibacter angelicae sp. nov., isolated from the Nematode’, by Instituto de Financiamento da Agricultura e Pescas rhizosphere of Angelica polymorpha Maxim. Int J Syst Evol Microbiol (IFAP) and Autoridade Florestal Nacional (AFN) and by the 62, 55–60. Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT), Portugal, under the Kim, J.-H., Kang, S.-J., Jung, Y.-T., Oh, T.-K. & Yoon, J.-H. (2012b). PTDC/AGR-CFL/115373/2009 project. G. P. was supported by FCT, Mucilaginibacter lutimaris sp. nov., isolated from a tidal flat sediment. Portugal, fellowship DRH36/11/448. D. N. P. was supported by FCT, Int J Syst Evol Microbiol 62, 515–519. Portugal, graduate fellowship SFRH/BD/61311/2009. Kim, O.-S., Cho, Y.-J., Lee, K., Yoon, S.-H., Kim, M., Na, H., Park, S.-C., Jeon, Y. S., Lee, J.-H. & other authors (2012c). Introducing EzTaxon- References e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. 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