Systematic and Applied Microbiology 37 (2014) 342–350 Contents lists available at ScienceDirect Systematic and Applied Microbiology j ournal homepage: www.elsevier.de/syapm Short communication Chryseobacterium oleae sp. nov., an efficient plant growth promoting bacterium in the rooting induction of olive tree (Olea europaea L.) cuttings and emended descriptions of the genus Chryseobacterium, C. daecheongense, C. gambrini, C. gleum, C. joostei, C. jejuense, C. luteum, C. shigense, C. taiwanense, C. ureilyticum and C. vrystaatense a,b,∗∗ a c Maria del Carmen Montero-Calasanz , Markus Göker , Manfred Rohde , a a d e Cathrin Spröer , Peter Schumann , Hans-Jürgen Busse , Michael Schmid , a a,∗ b Hans-Peter Klenk , Brian J. Tindall , Maria Camacho a Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany b IFAPA-Instituto de Investigación y Formación Agraria y Pesquera, Centro Las Torres-Tomejil, Ctra, Sevilla-Cazalla de la Sierra, Km 12.2, Alcalá del Río, 41200 Sevilla, Spain c HZI – Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany d Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität, A-1210 Wien, Austria e Research Unit Microbe-Plant Interactions, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany a r t i c l e i n f o a b s t r a c t T Article history: A novel non-motile, Gram-staining-negative, yellow-pigmented bacterium, designated CT348 , isolated Received 5 November 2013 from the ectorhizosphere of an organic olive tree in Spain and characterised as an efficient plant growth Received in revised form 21 February 2014 promoting bacterium, was investigated to determine its taxonomic status. The isolate grew best in a Accepted 22 April 2014 ◦ temperature range of 5–35 C, at pH 5.0–8.0 and with 0–1% (w/v) NaCl. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Chryseobacterium. The Keywords: DNA G + C content of the novel strain was 38.2 mol%. The strain contained a polyamine pattern with Bacteriodetes sym-homospermidine as the major compound and produced flexirubin-type pigments. MK-6 was the Flavobacteriaceae ␻ Taxonomy dominant menaquinone and the major cellular fatty acids were iso-C15:0, C17:1 9c, iso-C17:0 3-OH and PGPB iso-C15:0 2-OH. The main polar lipids were phosphatidylethanolamine and several unidentified lipids and Indole-3-acetic acid aminolipids. The 16S rRNA gene showed 92.2–97.8% sequence identity with the members of the genus Chyseobacterium. Based on the phenotypic traits and DNA–DNA hybridizations with the type strains of the most closely related species, the isolate is shown to represent a novel species, Chyseobacterium oleae, T type strain CT348 (=DSM 25575 =CCUG 63020). Emended descriptions of the genus Chryseobacterium and C. daecheongense, C. gambrini, C. gleum, C. joostei, C. jejuense, C. luteum, C. shigense, C. taiwanense, C. ureilyticum and C. vrystaatense are also proposed. © 2014 Elsevier GmbH. All rights reserved. The genus Chryseobacterium [58] whose type species is members of the genus Chryseobacterium based on the interpre- Chryseobacterium gleum [21,58] is included within the family tation of the list of all validly published names within this genus Flavobacteriaceae (emended by Bernardet et al. [4]), the main according to the “List of Prokaryotic Names with Standing in bacterial lineage in the phylum Bacteroidetes. At the time of writing Nomenclature” (http://www.bacterio.net/). Members of the genus 76 species with validly published names were considered to be Chryseobacterium can be found in a wide variety of environments, including freshwater [50], soil [32], rhizosphere [9], phyllosphere [3], sludge [43], fish [68–70], midgut of insects [26], raw dairy ∗ products [19], raw chicken [11], faeces of millipede [27], industrial Corresponding author. Tel.: +49 0531 26 16 224; fax: +49 0531 26 16 418. ∗∗ plants [20] and clinical samples [67]. Corresponding author. Tel.: +49 0531 26 16 210; fax: +49 0531 26 16 418. Based on previous studies, it is known that members of the E-mail addresses: [email protected] (M.d.C. Montero-Calasanz), [email protected] (B.J. Tindall). genus Chryseobacterium are considered to be an important bacterial http://dx.doi.org/10.1016/j.syapm.2014.04.004 0723-2020/© 2014 Elsevier GmbH. All rights reserved. M.d.C. Montero-Calasanz et al. / Systematic and Applied Microbiology 37 (2014) 342–350 343 group associated with plants [1,7,31] and currently there is strong were performed as described in Tindall et al. [55]. The utilisation evidence to show that strains of plant-associated Chryseobacterium of carbon compounds and acid production were determined using species exhibit plant-growth promoting activities [10,38]. API 20NE and API 20E strips (bioMérieux) according to the manu- ◦ During the characterisation of organisms isolated from the rhi- factures instructions, incubated at 28 C as well as using GEN III zosphere of an organically farmed olive grove (Olea europaea L. Microplates incubated in an Omnilog device (BIOLOG Inc., Hay- ◦ cv. arbequina) in Mairena del Aljarafe, Seville, Spain (37 20 18.97 ward, CA, USA). The GEN III Microplates were inoculated with a ◦ N, 6 5 8.61 W), in 2006, for application in alternative systems to cell suspension made in “gelling” inoculating fluid (IF) A at a cell T produce organically grown olive plants, strain CT348 was recov- density of 90% transmittance, yielding a running time of 4 days ◦ ered. This study based on a polyphasic approach in agreement with in Phenotype Microarray mode at 28 C. The exported data were the recommended minimal standards for describing new taxa of further analysed with the opm package for R [56,57] v.0.9.23., the family Flavobacteriaceae [4] refers the taxonomic position of using its functionality for merging subsequent measurements of a plant growth promoting bacterial (PGPB) strain representing a the same plate, statistically estimating parameters from the res- novel species within the genus Chryseobacterium isolated from the piration curves such as the maximum height, and automatically ectorhizosphere of an organically farmed olive tree. “discretizing” these values into negative and positive reactions. T Ectorhizospheric soil samples were suspended and Strain CT348 in comparison with the reference strains Chryseobac- T homogenised in sterile mineral buffer [59]. Serial dilutions terium daecheongense DSM 15235 , Chryseobacterium defluvii DSM TM T T were inoculated on Plate Count Agar (PCA; Difco ) diluted at 50% 14219 , Chryseobacterium gambrini DSM 18014 , Chryseobacterium T T supplemented with cycloheximide (100 mg/l), adjusted at pH 7.0, gleum DSM 16776 , Chryseobacterium gregarium DSM 19109 , Chry- ◦ T T at 28 C for 5 days. Strain CT348 was firstly isolated based on seobacterium indologenes DSM 16777 , Chryseobacterium joostei T T colony morphology, purified by sub-culturing on Trypticase Soy DSM 16927 , Chryseobacterium jejuense DSM 19299 , Chryseobac- TM T T Agar (TSA; Difco ) diluted at 10% and then selected by its ability terium luteum DSM 18605 , Chryseobacterium shigense DSM 17126 , T to produce indole-3-acetic acid (IAA) and siderophores, desirable Chryseobacterium taiwanense JCM 21767 , Chryseobacterium ure- T T PGPR features, and to promote rooting in model plants and olive ilyticum DSM 18017 Chryseobacterium vrystaatense DSM 25206 T cuttings [37]. The culture was suspended in 0.5% peptone and 15% and Chryseobacterium wanjuense DSM 17724 was just studied in ◦ (w/v) glycerol for storage at −80 C. the GEN III Microplates, in two independent determinations. Reac- T The colony morphology and pigmentation of strain CT348 tions that gave contradictory results between the two repetitions were observed on TSB agar after 24 and 72 h under a binocular were regarded as ambiguous. microscope according to Pelczar [42]. Exponentially growing bac- The extraction and analysis of cellular fatty acids was carried ◦ terial cultures were observed with an optical microscope (Zeiss out from biomass grown on TSA plates held at 28 C for 24 h and AxioScope A1) with a 100-fold magnification and phase-contrast harvested always from the same sector (the last quadrant streak). illumination. Micrographs of bacterial cells grown on Trypticase Analysis was conducted using the Microbial Identification System Soy Broth (TSB) for 48 h were taken with a field-emission scanning (MIDI) Sherlock Version 6.1 (results evaluated against the TSBA40 electron microscope (FE-SEM Merlin, Zeiss, Germany). Gram stain- peak naming table database) as described by Sasser [46]. The fatty ing was performed according to Halebian et al. [15] and checked by acid extracts obtained using the standard MIDI protocol were sub- the KOH test [14]. Anaerobic respiration was investigated by incu- jected to further analysis by GC–MS. GC–MS experiments were TM ◦ bation in an anaerobic pouch (Merck ) for 7 days at 28 C on 1% carried out using a Varian CP-3800 gas chromatograph, fitted with casitone, 0.3% yeast extract broth and 0.2% nitrate and 0.2% nitrite a Varian CP-8400 autosampler, coupled to a Varian 320-MS mass as terminal electron acceptors [65]. Activity of oxidase was ana- spectrometer. The gas chromatograph was fitted with a 30 m Varian lysed using filter-paper disks (Sartorius grade 388) impregnated VF-5 ms capillary column, internal diameter 0.25 mm, film thick- ◦ with 1% solution of N,N,N ,N -tetramethyl-p-phenylenediamine ness 0.25 ␮m. The run conditions were injector temperature 250 C, ◦ ◦ ◦ (Sigma–Aldrich); a positive test was indicated by the immediate transfer line 260 C, ion source 250 C, column temperature 130 C ◦ ◦ development of a blue-purple colour after applying biomass on the to 250 C at a rate of 4 C per minute, carrier gas helium at a con- filter paper. Catalase activity was tested by the observation of bub- stant flow rate of 1.3 ml/min.