Blastococcus endophyticus sp. nov., an actinobacterium isolated from Camptotheca acuminata

Abstract

A novel endophytic actinobacterium, designated strain YIM 68236 T , was isolated from healthy leaves of Camptotheca acuminata. and characterized by using a polyphasic approach. Cells of this strain occurred singly, in pairs or in tetrads. It grew at 10-45 6C, at pH 5.0-8.0 (optimum pH 7.0) and in the presence of 0-3 % (w/v) NaCl. The DNA G+C content was 71.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 68236 T belongs to the genus Blastococcus. However, it differed from its closest relatives, Blastococcus aggregatus DSM 4725 T , Blastococcus saxobsidens DSM 44509 T and Blastococcus jejuensis DSM 19597 T in many phenotypic characteristics. Moreover, the DNA-DNA relatedness values between the novel isolate and the three above-mentioned type strains were 49.0±1.6 %, 46.1±3.2 % and 39.8±1.5 %, respectively. Based on comparative analysis of physiological and chemotaxonomic data, strain YIM 68236 T represents a novel species of the genus Blastococcus, for which the name Blastococcus endophyticus sp. nov. is proposed. The type strain is YIM 68236 T (5CCTCC AA 209045 T 5DSM 45413 T 5KCTC 19998 T ).The genus Blastococcus was first proposed by Ahrens & Moll (1970) Lee, 2006), which were isolated from the Baltic Sea, limestone and a beach sand, respectively. In this study, we report a novel species isolated from Camptotheca acuminata, a traditional Chinese medicinal plant.Healthy leaves of C. acuminata were washed in running water to remove soil particles and sterilized by the established procedure (Li et al., 2008). After being surfacesterilized, the samples were sliced into pieces, followed by plating on TWYE agar (containing 0.25 g yeast extract, 0.5 g K 2 HPO 4 and 18 g agar l 21 tap water, pH 7.2) containing nalidixic acid (25 mg l 21 ), nystatin (50 mg l 21 ) and cycloheximide (50 mg l 21 ) to repress growth of Gramreaction-negative bacteria and fungi. The plates were incubated at 28 u C for 4-6 weeks until the outgrowth of endophytic actinobacterial strains was discerned. Colonies originating from leaf segments were selected and pure cultures were obtained by repeated streaking on medium YIM 38 [10 g malt extract, 4 g yeast extract, 4 g glucose, vitamin mixture (containing 0.5 mg each of thiamine-HCl, riboflavin, niacin, pyridoxine-HCl, inositol, calcium pantothenate and p-aminobenzoic acid and 0.25 mg biotin), 20 g agar; pH 7.2] . The purified strain YIM 68236 T was maintained on tryptic soy agar (TSA) slants at 4 u C and as 20 % (v/v) glycerol suspensions at 280 u C.Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were carried out as described by Li et al. (2007). Multiple alignments with sequences of the most closely related actinobacteria were carried out using the CLUSTAL X 1.8 program (Thompson et al., 1997). Phylogenetic trees were reconstructed by the

Blastococcus capsensis sp. nov., isolated from an archaeological Roman pool and emended description of the genus Blastococcus, B. aggregatus, B. saxobsidens, B. jejuensis and B. endophyticus

“…This is in accordance with the chromatographic profiles observed in this study for the references strains (Fig. 1b-e) and those published by Lee (2006) and Zhu et al (2013), although the absence of phosphatidylglycerol (present in the original descriptions of B. saxobsidens and B. jejuensis) should be noted. Such absence has already been described for other members of the family Geodermatophilaceae (Montero-Calasanz et al, 2014, 2015Hezbri et al 2015aHezbri et al , b, 2016.…”

“…1c). Phosphatidylglycerol, phosphatidylinositolmannoside and the four unidentified phospholipids listed by Zhu et al (2013) are absent. MK-9(H 4 ) is the predominant menaquinone, but MK-9 and MK-8(H 4 ) are present as minor components.…”

“…The properties are as given in the species description by Zhu et al (2013) with the following emendation. In addition to diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol listed by Zhu et al (2013) an unidentified glycolipid is present (the chromatographic mobility of which is documented in Fig.…”

“…Meier-Kolthoff et al (2013b) linked an Actinobacteria-specific 16S rRNA threshold of 99.0 %, with 1.0 % as maximum probability of error, to DNA-DNA hybridization (DDH) values above the 70 % threshold required to assign a given strain to a new species (Wayne et al, 1987 Urzì et al (2004). †Data taken from Zhu et al (2013). ‡Data taken from Lee (2006).…”

“…The genus Blastococcus is classified together with Geodermatophilus and Modestobacter in the family Geodermatophilaceae (Normand, 2006;Normand et al, 2014) of the order Geodermatophilales (Sen et al, 2014). There are four species of the genus Blastococcus with validly published names: Blastococcus aggregatus (Ahrens & Moll, 1970;Urzì et al, 2004), Blastococcus saxobsidens (Urzì et al, 2004), Blastococcus jejuensis (Lee, 2006) and Blastococcus endophyticus (Zhu et al, 2013). These were, respectively, isolated from sediment of Station Breitengrund at a depth of 20 m in the Western Baltic Sea, Marble Eleusys an archaeological site in Greece, sand sediment of a Korean beach in Jeju and and from healthy leaves of Camptotheca acuminate, a traditional Chinese medicinal plant.…”