Liu et al.
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Bacillus wuyishanensis sp. nov., isolated from rhizosphere soil of a
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medical plant, Prunella vulgaris, in Wuyi mountain of China
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Bo Liu1,·Guo-Hong Liu1,·Cetin Sengonca2, Peter Schumann3, Jian-Yang Tang1, Mei-Chun Chen1, Jian-Mei
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Che1
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Agricultural Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003,
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China.
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Institute of Crop Sciences and Resource Conservation, INRES University of Bonn, Meckenheimer Allee
166A D-53115 Bonn, Germany.
3Leibniz
Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B,
38124 Braunschweig, Germany.
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Correspondence:
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Prof. Dr. Bo Liu
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E-mail: fzliubo@163.com
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Tel: + 86 591 87884601
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Fax: +86 591 87884262
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Subject category: New Taxa-Firmicutes and Related Organisms
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Running title: Bacillus wuyishanensis sp. nov.
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The GenBank accession number for the 16S rRNA gene sequence of strain FJAT-17212T was KF040589.
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Bacillus wuyishanensis sp. nov.
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A Gram-positive-staining, rod-shaped, endospore-forming, aerobic bacterium (FJAT-17212T) was isolated
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from the rhizosphere soil of a medical plant, Prunella vulgaris, in Wuyi mountain of China. Strain
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FJAT-17212T grew at 10–50 °C (optimum 30 °C), pH 5–11 (optimum pH 7) and 0–6% (w/v) NaCl
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(optimum 2%). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain
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FJAT-17212T was a member of the genus Bacillus and was most closely related to Bacillus
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galactosidilyticus DSM 15595T (97.3 %). DNA–DNA relatedness between strain FJAT-17212T and B.
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galactosidilyticus DSM 15595T was low (35.2% ± 2.3). Diagnostic diamino acid of the peptidoglycan of
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strain FJAT-17212T was meso-diaminopimelic acid and the predominant isoprenoid quinone was MK-7
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(80.8%). The major cellular fatty acids were iso-C15:0 (35.7%), anteiso-C15:0 (29.8%), iso-C14:0 (9.9%) and
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iso-C16:0 (9.9%) and the G+C DNA content was 39.8 mol%. Phenotypic, chemotaxonomic and genotypic
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properties clearly indicated that strain FJAT-17212T represents a novel species within the genus Bacillus,
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for which the name Bacillus wuyishanensis sp. nov. is proposed. The type strain is FJAT-17212T ( = DSM
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27848 T = CGMCC1.1 2709T).
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Bacillus species contain rod-shaped, aerobic or facultative anaerobic, Gram-positive,
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endospore-forming bacteria, which exhibited a wide range of physiological abilities that
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enable them to live in every natural environment, such as soil, hot springs, water, marine
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sediments or airborne dust (Berkeley, 2002). Bacillus species in the rhizosphere and
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rhizoplane of plants have attracted a great deal of attention (Singh et al., 2014) and increasing
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numbers of new species have been isolated, such as Bacillus rhizosphaerae sp. nov., an novel
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diazotrophic bacterium isolated from sugarcane rhizosphere soil (Madhaiyan et al., 2011),
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Bacillus methylotrophicus sp. nov., a methanol-utilizing, plant-growth-promoting bacterium
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isolated from rice rhizosphere soil (Madhaiyan et al., 2010) and Bacillus koreensis sp. nov., a
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spore-forming bacterium, isolated from the rhizosphere of willow roots in Korea (Lim et al.,
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2006). It was of great significance that Bacillus species could have a role to further define the
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diversity, ecology, and biocontrol activities in the promotion of plant growth and the
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suppression of soil-borne pathogens (Kaki et al., 2013; Lee et al., 2014). In this study, a novel
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isolate designated strain FJAT-17212T was isolated from the rhizosphere of Chinese medical
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plant roots of Prunella vulgaris in Wuyi mountain of Fujian Province, China, describing its
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phylogenetic and phenotypic characteristics for classification.
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Strain FJAT-17212T was isolated from rhizosphere soil samples of Prunella vulgaris in Wuyi
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mountain of Fujian Province, China. For isolation, the sample was suspended in sterilized
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water, serially diluted, spread on nutrient agar (NA) and incubated at 30 °C for 48 h (Atlas
Liu et al.
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1993). Pure cultures were obtained by several successive single colony isolations. The strain
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was stored both on NA slants at 4 °C and as suspensions in Luria–Bertani broth (LB) with 20%
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(v/v) glycerol at –80 °C. The reference strain Bacillus galactosidilyticus DSM 15595T was
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obtained from the culture collections indicated and used as controls in the phenotypic tests.
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For phenotypic tests, the isolate FJAT-17212T and B. galactosidilyticus DSM 15595T were
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performed as described by Logan et al., (2009). Cell morphology was observed by light
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microscopy (Leica DMI3000B, Germany). The Gram staining and the KOH lysis test were
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carried out according to the methods described by Smibert & Krieg (1994) and Gregersen
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(1978). Endospores were examined according to Schaeffer–Fulton staining method (Murray
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et al. 1994). Motility was examined on motility agar (Chen et al., 2007). Catalase activity was
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determined by investigating bubble production with 3% (v/v) H2O2, and oxidase activity was
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determined using 1% (v/v) tetramethyl p-phenylenediamine (Chen et al., 2007). Cell growth
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under anaerobic conditions was determined in a CO2 incubator on anaerobically prepared
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maintenance medium. Physiological characteristics, such as Voges–Proskauer test, hydrolysis
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of gelatin, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, tryptophan
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deaminase, and urease, citrate utilization, ONPG, H2S and indole production were performed
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using API 20E strips (BioMérieux). Nitrate reduction, hydrolysis of casein, starch, Tween 20,
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40, and 80 were determined as described by Cowan and Steel (1965), Smibert & Krieg (1994).
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The tests of carbon source utilization and acid-production were performed using the API
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50CHB system (BioMérieux). Growth at different temperatures (5–50 °C, in increments of
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5 °C), pH (5.0–10.0, in increments of 1 pH units) and NaCl concentrations (0–10% (w/v), in
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increments of 1% NaCl) was tested in NB medium, respectively.
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For phylogenetic analysis, the 16S rRNA gene sequence was amplified by PCR with the
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universal primers 27F (5'-AGAGTTTG ATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTT
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GTTACGACTT-3'). Amplification was carried out with a DNA thermal cycler (Gene Amp
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PCR System 2700; Applied Biosystems) according to the following program: 95 °C for 10
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min, 30 cycles of 94 °C for 0.5 min, 50 °C for 1 min and 72 °C for 2 min and final extension
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at 72 °C for 10 min. PCR products were purified and sequenced by Shanghai Biosune
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(Shanghai, PR China) with an Applied Biosystems automatic sequencer (ABI 3730). Pairwise
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sequence similarities were calculated using a global alignment algorithm implemented in the
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EzTaxon -e database (http://eztaxon-e.ezbiocloud.net/, Kim et al., 2012). After multiple
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sequence alignment using CLUSTAL_X (Thompson et al., 1997), phylogenetic analysis was
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Bacillus wuyishanensis sp. nov.
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performed using MEGA version 4 (Tamura et al., 2007). Distances were calculated using
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distance options according to the Jukes-Cantor model (Jukes & Cantor, 1969) and clustering
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was performed with the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis
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by means of 1000 resamplings was used to evaluate tree topologies (Felsenstein, 1985).
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For DNA–DNA hybridization, it was performed to determine the relatedness of isolate
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FJAT-17212T to the reference strain using the optical renaturation method (De Ley et al.,
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1970; Huß et al., 1983) with three replications. Hybridization of extracted DNA was carried
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out using a DIG High Prime DNA Labeling and Detection Starter kit (Roche), according to
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the manufacturer’s instructions, with a hybridization temperature of 40 °C (calculated with
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correction for the presence of 50 % formamide).
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For calculation of DNA G+C content, it was analysed by HPLC (Young Lin??), as described
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by Shin et al. (1996). Genomic DNA was isolated according to Hopwood et al. (1985) and the
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DNA G+C content was determined using the thermal denaturation method described by
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Marmur & Doty (1962) using Escherichia coli K-12 DNA as calibration standard. ??
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For measurement of chemotaxonomic characteristics, polar lipids were extracted and analysed
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by two-dimensional TLC according to Minnikin et al. (1984). The respiratory quinone system
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was extracted and determined by HPLC according to established methods (Nakagawa &
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Yamasato, 1993). The stereoisomer of diaminopimelic acid in the peptidoglycan of isolate
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FJAT-17212T was determined as described by Staneck & Roberts (1974).
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For measurement of chemotaxonomic characteristics, constituents of peptidoglycan were
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determined using the method of Hasegawa et al. (1983). Isoprenoid quinones were analyzed
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by reversed-phase HPLC (Groth et al., 1996).
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For the analysis of cellular fatty acids, strains were harvested after cultivation on TSA at
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30 °C for 48 h and the cellular fatty acids in the cell walls were extracted and analysed
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according to the standard protocol of the Microbial Identification System (MIDI; Microbial
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ID) (Sasser, 1990). Extracts were analysed by GC (Agilent 7890) as described by Miller
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(1982).
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Strain FJAT-17212T formed cream-yellow, smooth, flat, opaque, circular colonies and 2–9
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mm in diameter on NA plates. The strain was Gram-positive, moderately halophilic and
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facultatively alkaliphilic. Ellipsoidal endospores were formed and cells were motile
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(Supplementary Fig. S1). The strain grew at salt concentrations in the range 0–6% (w/v) NaCl,
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with optimum growth occurring at 2% (w/v) NaCl. Growth was observed at 10–50 oC
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(optimum 30
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catalase-positive and did not reduce nitrate to nitrite. The phenotypic properties that
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differentiate strain FJAT-17212T from its closest phylogenetic neighbours are given in Table
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1.
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C) and pH 5.0–11 (pH 7.0). The strain was oxidase-negative and
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An almost-complete 16S rRNA gene sequence (1440 bp) of the isolate was determined. The
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neighbour-joining phylogenetic tree (Fig. 1) revealed that the isolate FJAT-17212T belongs to
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the genus Bacillus. The type strain in the genus Bacillus with the greatest pairwise similarity
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to the isolate was B. galactosidilyticus DSM 15595T (97.3% 16S rRNA gene sequence
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similarity). The value is low enough (<98%) to justify defining the isolate as a representative
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of a novel species (Devereux et al., 1990; Fry et al., 1991; Ko et al., 2006 ). These results are
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also supported by phylogenetic tree reconstructed using maximum-parsimony algorithms
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(Supplementary Fig. S2) and maximum-likelihood and maximum-parsimony method
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(Supplementary Fig. S3). The isolate showed 35.2% ± 2.3 DNA–DNA relatedness to B.
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galactosidilyticus DSM 15595T, which is lower than the cut-off point (70%) for the
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delineation of novel species. These results support the view that strain FJAT-17212T
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represents a novel species in the genus Bacillus (Stackebrandt & Goebel, 1994; Wayne,
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1988).
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The DNA G+C content was calculated to be 39.8 mol%, which is within the range for the
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genus Bacillus (35.6%–44.8%; Yoon et al., 2001; Heyrman et al., 2004; Ten et al., 2007;
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Zhang et al., 2010; Seiler et al., 2012). These characteristics confirm that strain FJAT-17212T
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belongs to the genus Bacillus (Albert et al., 2007).
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Strain FJAT-17212T contained MK-7 (80.8%) as the major menaquinone, with MK-6 (1.8%)
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and MK-8 (14.9%) present as minor constituents. Analysis of the cell-wall peptidoglycan
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Bacillus wuyishanensis sp. nov.
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showed that the strain contained meso-diaminopimelic acid as the diagnostic diamino acid, as
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it is typical of the vaste majority of members of the genus Bacillus (Priest et al., 1988).
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The cellular fatty acid profile of the isolate comprised iso-C15:0 (35.7%), anteiso-C15:0 (29.8%),
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iso-C14:0 (9.9%), iso-C16:0 (9.9 %) and anteiso-C17:0 (4.7%) as the major fatty acids (>4 %).
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The fatty acid profile of the isolate is clearly qualitatively and quantitatively different from
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this of the closest related type strain B. galactosidilyticus DSM 15595T (Table 1) with the
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fatty acids iso-C15:0 (31.9%), C16:0 (21.5%), anteiso-C15:0 (17.5%), iso-C16:0 (5.8%), iso-C14:0
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(4.2%) and C14:0 (4.0%) as major components.. Acid Summed Feature 1 and anteiso-C14:0
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found in the isolate were not detected in strain B. galactosidilyticus DSM 15595T. These iso-
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and anteiso-branched fatty acids of the 14– and 17-carbon series are typical of those observed
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in profiles of the type strains of the genus Bacillus (Kämpfer, 1994; Albert et al., 2005).
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Based on physiological, biochemical and phylogenetic properties, we consider the strain
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FJAT-17212T represents a novel species of the genus Bacillus, for which we propose the name
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Bacillus wuyishanensis sp. nov.
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Description of Bacillus wuyishanensis sp. nov.
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Bacillus wuyishanensis [wu.yi.shan.en'sis. N.L. masc. adj. wuyishanensis, belonging to Wuyi
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mountain of Fujian Province in China, where a rhizosphere soil sample in a medical plant,
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Prunella vulgaris,was collected for isolation of the organism]
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Cells are Gram-positive, aerobic, moderately halophilic, facultatively alkaliphilic, straight,
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motile rods (0.4-0.5×3-5.0µm), with rounded ends and one flagella at the end and occurred
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singly or in pairs. Oval endospores are located subterminally and give rise to swollen
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sporangia. Colonies on NA are cream-yellow, flat, opaque, smooth, circular margins and 2–9
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mm in diameter. Growths at salinities of 0–6 % (w/v) NaCl (optimum 0–2 %), pH 5.0–10
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(optimum pH 7.0) and 10–50 °C (optimum 30 °C). catalase-positive and oxidase-negative.
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Nitrate is not reduced to nitrite. H2S and indole are not produced. Gelatin and arginine are not
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hydrolyzed. Reactions for ONPG, urease, Voges–Proskauer, citrate utilization, lysine
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decarboxylase, ornithine decarboxylase and tryptophan deaminase are negative. Acids are
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produced from ribose, D-xylose, galactose, glucose, mannose, N-acetyl-D-glucosamine,
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amygdalin, esculin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose,
Liu et al.
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raffinose, but not from methyl D-xyloside, rhamnose, methyl a-D-glucoside, inulin,
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melezitose, starch, gentiobiose, glycerol, erythrol, D-arabinose, L-xylose, adonitol, sorbose,
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dulcitol, inositol, mannitol, sorbitol, glycogen, xylitol, D-Lyxose, D-tagatose, D-fucose,
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D-arabitol, L-arabitol, gluconate, 2-keto-D-gluconate, 5-keto-D-gluconate. Acids are weakly
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produced from L-arabinose, fructose, L-fucose, arbutin and D- turanose. The cell-wall
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peptidoglycan contains meso-diaminopimelic acid and the major isoprenoid quinone is MK-7.
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The major fatty acids are iso-C15:0 (35.7%), anteiso-C15:0 (29.8%), iso-C14:0 (9.9%) and
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iso-C16:0 (9.9%). The DNA G+C content of the type strain is 39.8 mol%.
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The type strain, FJAT-17212T ( = DSM 27848 T = CGMCC1.1 2709T) was isolated from the
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rhizosphere of Prunella vulgaris roots in Wuyi mountain of Fujian Province in China. The
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GenBank accession number for the 16S rRNA gene sequence of strain FJAT-17212T is
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KF040589.
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Bacillus wuyishanensis sp. nov.
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Acknowledgement:
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We thank Professor J. P. Euzéby for his suggestion on the spelling of the specific epithet. We thank
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also the Agricultural Bioresources Institute, Fujian Academy of Agricultural Sciences, PR China, and the
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international cooperation project of Chinese Ministry of Science and Technology (2012DFA31120), Natural
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Science Foundation of China (NSFC) (31370059), 948 project of Chinese Ministry of Agriculture
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(2011-G25), 973 program earlier research project (2011CB111607), Chinese Special Fund for
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Agro-scientific Research in the Public Interest（201303094）and project of agriculture science and
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technology achievement transformation (2010GB2C400220) for the supporting, respectively.
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Liu et al.
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Srivastava, A. K., Singhal, P. K., Arora, D. K. (2014). Multifarious plant growth promoting
314
characteristics of chickpea rhizosphere associated Bacilli help to suppress soil-borne pathogens. Plant
315
Growth Regulation 73: 91-101.
316
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S
317
rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44,
318
846-849.
319
320
Tamura, K., Dudley J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics
Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-1599.
321
Ten, L. N., Baek, S. H., Im, W. T., Larina, L. L., Lee, J. S., Oh, H. M. & Lee, S. T. (2007). Bacillus
322
pocheonensis sp. nov., a moderately halotolerant, aerobic bacterium isolated from soil of a ginseng
323
field. Int J Syst Evol Microbiol 57, 2532–2537.
324
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The
325
CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality
326
analysis tools. Nucleic Acids Res 25, 4876-4882.
327
Wayne, L. G. (1988). International Committee on Systematic Bacteriology: announcement of the report of
328
the ad hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Zentralbl Bakteriol
329
Mikrobiol Hyg [ A ] 268, 433–434.
330
Yoon, J. H., Kang, S. S., Lee, K. C., Kho, Y. H., Choi, S. H., Kang, K. H. & Park, Y. H. (2001). Bacillus
331
jeotgali sp. nov., isolated from jeotgal, Korean traditional fermented seafood. Int J Syst Evol Microbiol
332
51, 1087–1092.
333
Zhang, J., Wang, J. W., Fang, C. Y., Song, F., Xin, Y. H., Qu, L., & Ding, K. l (2010). Bacillus
334
oceanisediminis sp. nov., isolated from marine sediment. Int J Syst Evol Microbiol 60, 2924–2929.
335
11
Bacillus wuyishanensis sp. nov.
336
Table 1. Characteristics used to distinguish strain FJAT-17212T from its closest phylogenetic
337
neighbours
338
All data were obtained from this study. All strains are endospore-forming, Gram-positive rods and positive for
339
catalase, All strains are negative for indole and H2S production, Voges–Proskauer, Citrate utilization, casein,
340
Tween 20, 40 and Tween 80, starch, gelatin, arginine double enzyme hydrolysis, lysine decarboxylase, ornithine
341
decarboxylase and tryptophan deaminase. All strains are negative for acid production from glycerol, erythrol,
342
D-arabinose, L-xylose, adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol, Methyl a-D-mannoside, glycogen,
343
xylitol,
344
5-keto-D-gluconate. All strains are weakly positive for acid production from L-arabinose, fructose, arbutin and
345
D-turanose. +, positive; -, negative.
D-Lyxose,
D-tagatose,
Characteristics
D-fucose,
D-arabitol,
FJAT-17212T
Temperature (°C)
L-arabitol,
gluconate,
2-keto-D-gluconate,
Bacillus galactosidilyticus DSM 15595T
-
10
+
-
20
+
-
30
+
+
40
+
+
50
+
-
0
+
+
2
+
+
4
+
+
6
+
+
8
-
-
10
-
-
5
-
-
6
+
+
7
+
+
8
+
+
9
+
+
10
+
+
ONPG
-
+
Urease
-
+
Nitrite reduction
-
+
Ribose
+
w
D-xylose
+
-
Methyl D-xyloside
-
w
Galactose
+
w
NaCl
pH
Liu et al.
FJAT-17212T
Bacillus galactosidilyticus DSM 15595T
Glucose
+
w
Mannose
+
w
Rhamnose
-
w
Methyl a-D-glucoside
-
w
N-acetyl-D-glucosamine
+
w
Amygdalin
+
w
Esculin
+
w
Salicin
+
w
Cellobiose
+
w
Maltose
+
w
Lactose
+
w
Melibiose
+
w
Sucrose
+
w
Trehalose
+
w
Inulin
-
w
Melezitose
-
w
Raffinose
+
w
Starch
-
w
Gentiobiose
-
w
L-fucose
w
-
Characteristics
346
347
13
Bacillus wuyishanensis sp. nov.
348
Table 2. Fatty acid compositions of strain FJAT-17212T and related species of the genus Bacillus
349
All data were taken from this study.
Fatty acid (%)
FJAT-17212T
Bacillus galactosidilyticus DSM 15595T
iso-C15:0
35.7
31.9
anteiso-C15:0
29.8
17.5
iso-C16:0
9.9
5.8
iso-C14:0
9.9
4.2
C16:1ω7c alcohol
1.7
0.5
C16:1ω11c
1.5
2.1
C16:0
1.5
21.5
iso-C17:1 ω10c
0.4
0.4
iso-C17:0
0.8
4
anteiso-C17: 0
4.7
3.8
C14:0
1.3
4.0
C10:0
0.3
0.3
C12:0
0.2
0.2
iso-C13:0
0.6
0.3
anteiso-C13:0
0.2
0.1
anteiso-C14:0
0.3
-
C18:1ω9c
0.2
0.4
C18:0
0.1
1.1
1
0.1
-
3
0.1
0.3
4
0.3
0.2
8
0.1
0.1
Summed feature*
350
*Summed features represent two or three fatty acids that cannot be separated by the Microbial Identification
351
System. Summed feature 1 consisted of C13:0 3OH and/or iso- C15:1 H; Summed feature 3 consisted of C16:1ω7c
352
and/or C16:1ω6c; Summed feature 4 consisted of iso-C17:1 I and/or anteiso-C17:1 B; Summed feature 8 consisted of
353
C18:1ω6c and/or C18:1ω7c. -, not detected.
354
Liu et al.
355
Bacillus galactosidilyticus LMG 17892T (AJ535638)
100
Bacillus ruris LMG 22866T (AJ535639)
58
Bacillus wuyishanensis FJAT-17212T (KF040589)
63
Bacillus panacisoli CJ32T (JQ806742)
55
Bacillus graminis YC6957T (GU322908)
93
Bacillus farraginis R-6540T (AY443036)
95
Bacillus fortis R-6514T( AY443038)
100
Bacillus thermophiles SgZ-9T (JX274437)
68
Bacillus lentus IAM 12466T (D16272)
95
Bacillus purgationiresistens DS22T (FR666703)
Bacillus horneckiae DSM 23495T (FR749913)
Bacillus oceanisedimins H2T (GQ292772)
Bacillus firmus NCIMB9366T (X60616)
91
Bacillus circulans ATCC 4513T (AY724690)
81
Bacillus benzoevorans DSM 5391T (D78311)
Bacillus foraminis CV53T (AJ717382)
99
Bacillus horneckiae DSM 23495T (FR733689)
Falsibacillus pallidus CW 7T (EU364818)
Bacillus subtilis DSM 10T (AJ276351)
Ornithinibacillus contaminans CCUG 53201T (FN597064)
80
356
0.005
357
358
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences, indicating the
359
position of strain FJAT-17212T among related members of the genus Bacillus. Bootstrap values
360
based on 1000 resampled datasets are shown at branch nodes. Bar, 0.005 substitutions per
361
nucleotide position.
362
15
Bacillus wuyishanensis sp. nov.
363
364
Supplementary Fig. S1 Scanning electron micrograph showing strain FJAT-17212T grown on
365
NA medium for 2 days at 30 oC, Bar 1 μm, Ellipsoidal endospores are formed subterminally,
366
swollen sporangia.
367
368
Liu et al.
369
Bacillus foraminis CV53T (AJ717382)
77
Bacillus horneckiae DSM 23495T (FR733689)
45
Bacillus subtilis DSM 10T (AJ276351)
T
Bacillus purgationiresistens DS22 (FR666703)
Bacillus horneckiae DSM 23495T (FR749913)
98
Falsibacillus pallidus CW 7T (EU364818)
Bacillus benzoevorans DSM 5391T (D78311)
54
Ornithinibacillus contaminans CCUG 53201T (FN597064)
55
Bacillus circulans ATCC 4513T (AY724690)
54
Bacillus oceanisedimins H2T (GQ292772)
Bacillus firmus NCIMB9366T (X60616)
Bacillus lentus IAM 12466T (D16272)
Bacillus farraginis R-6540T (AY443036)
60
Bacillus thermophiles SgZ-9T (JX274437)
100
73
Bacillus fortis R-6514T( AY443038)
Bacillus graminis YC6957T (GU322908)
53
Bacillus panacisoli CJ32T (JQ806742)
58
Bacillus wuyishanensis FJAT-17212T (KF040589)
40
Bacillus galactosidilyticus LMG 17892T(AJ535638)
50
Bacillus ruris LMG 22866T (AJ535639)
100
370
5
371
372
Supplementary Fig. S2.
Maximum Parsimony phylogenetic tree based on 16S rRNA gene
373
sequences, showing the position of strain FJAT-17212T. Bootstrap values are shown as
374
percentages of 1000 replicates. Bar, 5 substitutions per nucleotide position.
375
17
Bacillus wuyishanensis sp. nov.
376
Bacillus foraminis CV53T (AJ717382)
68
Bacillus horneckiae DSM 23495T (FR733689)
Bacillus subtilis DSM 10T (AJ276351)
80
Bacillus purgationiresistens DS22T (FR666703)
Bacillus horneckiae DSM 23495T (FR749913
99
Bacillus oceanisedimins H2T (GQ292772)
66
Bacillus firmus NCIMB9366T (X60616)
79
Falsibacillus pallidus CW 7T (EU364818)
Bacillus benzoevorans DSM 5391T (D78311)
52
Ornithinibacillus contaminans CCUG 53201T (FN597064)
54
52
Bacillus circulans ATCC 4513T (AY724690)
Bacillus lentus IAM 12466T (D16272)
Bacillus farraginis R-6540T (AY443036)
58
100
Bacillus thermophiles SgZ-9T (JX274437)
Bacillus fortis R-6514T( AY443038)
Bacillus graminis YC6957T (GU322908)
Bacillus panacisoli CJ32T (JQ806742)
59
Bacillus wuyishanensis FJAT-17212T (KF040589)
Bacillus galactosidilyticus LMG 17892T(AJ535638)
99
Bacillus ruris LMG 22866T (AJ535639)
0.005
377
378
Supplementary Fig. S3. Minimum-likelihood phylogenetic tree based on the 16S rRNA gene
379
sequence of strain FJAT-17212T and closely related species within the genus Bacillus. The
380
significance of each branch is indicated by a bootstrap value calculated for 1000 subsets. Bar,
381
0.005 substitutions per site.
382
Liu et al.
383
Appendix 1. The G+C content of strain FJAT-17212T was determined using the thermal
384
denaturation method described by Marmur & Doty (1962) using Escherichia coli K-12 DNA as
385
calibration standard
386
387
388
389
19
Bacillus wuyishanensis sp. nov.
390
391
Liu et al.
392
393
394
395
396
397
21
Bacillus wuyishanensis sp. nov.
398
Appendix 2. The test report of chemical composition of cell wall of strain FJAT-17212T used the
399
400
401
TLC method
402
403
404
405
Liu et al.
406
Appendix 3 The nomenclature of strain FJAT-17212T by Pro. J. P. Euzeby
407
23