Glycomyces endophyticus sp. nov., an endophytic actinomycete isolated from the root of Carex baccans Nees

The genus Glycomyces was established by Labeda et al. (1985) and its description was emended by Labeda & Kroppenstedt (2004). This genus belongs to the suborder Glycomycineae (Stackebrandt et al., 1997), family Glycomycetaceae; this family also contains the genus Stackebrandtia (Labeda & Kroppenstedt, 2005). At the time of writing, the genus Glycomyces consists of seven species with validly published names: Glycomyces algeriensis, G. arizonensis, G. harbinensis, G. lechevalierae, G. rutgersensis and G. tenuis (Labeda et al., 1985; Evtushenko et al., 1991; Labeda & Kroppenstedt, 2004) and G. sambucus (Gu et al., 2007). All of these species were isolated from soil samples, with the exception of G. sambucus (which was isolated from the inner part of a medicinal plant). During our long-term investigations on the diversity and bioactivity of endophytic actinomycetes from tropical rainforest medicinal plants, a novel strain, designated YIM 56134^T, was isolated. The aim of the present study was to characterize the taxonomic position of the novel isolate.

Strain YIM 56134^T was isolated as follows from a root sample of Carex baccans Nees (a traditional Chinese medicinal plant) collected from the tropical rainforest in Xishuangbanna, Yunnan Province, south-west China. Fresh plant samples were air-dried and washed thoroughly as described by Coombs & Franco (2003). Subsequently, the roots were subjected to a five-step surface-sterilization procedure: a 3 min wash in 5 % NaClO₃, followed by a 10 min wash in 2.5 % Na₂S₂O₃, a 3 min wash in 75 % ethanol, a 10 min wash in 10 % NaHCO₃ and a final rinse in sterile water. After air-drying, the surface-sterilized roots were then crumbled aseptically into small fragments and distributed on tap water/yeast extract agar (Crawford et al., 1993) before being incubated at 28 °C for 4 weeks.

Cultural characteristics were observed on the media of Shirling & Gottlieb (1966) and Czapek agar (Waksman, 1967) at 28 °C. Colony colour was determined with the ISCC–NBS colour charts (Kelly, 1964). Morphological characteristics of spores and mycelia were examined by using light microscopy (BH-2; Olympus) and scanning electron microscopy (JSM 5600LV; JEOL) on 14 day cultures grown on ISP 2 medium. Growth was tested over a range of temperatures and pH values, as described by Xu et al. (2005). Tolerance of various NaCl concentrations (1, 3, 5, 7 and 10 %, w/v) was tested on ISP 2 as the basal medium. Other physiological characteristics, including utilization of sole carbon sources for energy and growth, acid production from carbohydrates and decomposition of test substances, were assessed by using the media and methods of Gordon et al. (1974). Details of the physiological characteristics of strain YIM 56134^T are given in Table 1⇓ and the species description.

The procedures used to identify the cell-wall amino acids and sugars in whole-cell hydrolysates were as described by Hasegawa et al. (1983). Menaquinones were extracted using the method of Collins et al. (1977) and were analysed using HPLC (Groth et al., 1997). Polar lipids were extracted, examined by two-dimensional TLC and identified by using published procedures (Minnikin et al., 1979; Collins & Jones, 1980). Fatty acids were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification system (Sasser, 1990; Kämpfer & Kroppenstedt, 1996).

Genomic DNA extraction, PCR amplification and sequencing of the 16S rRNA gene were performed according to Li et al. (2007). A phylogenetic analysis was performed using the software package mega, version 3.1 (Kumar et al., 2004), after multiple alignment of the data using clustal_x (Thompson et al., 1997). A phylogenetic tree was constructed using the neighbour-joining (Saitou & Nei, 1987) and maximum-parsimony (Kluge & Farris, 1969) methods. Evolutionary distance matrices were generated as described by Kimura (1980). Confidence values for the branches of the phylogenetic tree were determined using bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. DNA–DNA relatedness values were determined using the fluorometric microwell method (Ezaki et al., 1989; He et al., 2005). The DNA G+C content of strain YIM 56134^T was determined to be 72 mol% by using the HPLC method (Mesbah et al., 1989).

The almost-complete sequence for the 16S rRNA gene of strain YIM 56134^T was used for phylogenetic analysis. It is evident from Fig. 1⇓ that strain YIM 56134^T consistently formed a single cluster within the genus Glycomyces: the sequence similarities between strain YIM 56134^T and G. algeriensis NRRL B-16327^T, G. lechevalierae DSM 44724^T, G. rutgersensis IFO 14488^T and G. harbinensis IFO 14487^T were 99, 99, 98.9 and 98.7 %, respectively, while the sequence similarities between strain YIM 56134^T and G. sambucus DSM 45047^T, G. tenuis IFO 15904^T and G. arizonensis NRRL B-16153^T were less than 97 %. DNA–DNA hybridization experiments were performed to establish whether the novel isolate represents a distinct species. The results demonstrated that strain YIM 56134^T showed DNA–DNA relatedness to related type strains of 53.3±4.8 % (mean±sd) to G. lechevalierae DSM 44724^T, 44.8±7.6 % to G. rutgersensis NBRC 14488^T, 50±2.3 % to G. algeriensis NRRL B-16327^T and 36±4.1 % to G. harbinensis NBRC 14487^T. These values are below the cut-off point recommended by Stackebrandt & Goebel (1994) for the circumscription of bacterial genomic species and so confirmed that strain YIM 56134^T represents a different genomic species.

Strain YIM 56134^T contained meso-diaminopimelic acid as the cell-wall diamino acid. The phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and several phosphoglycolipids of unknown composition. These chemotaxonomic traits match the characteristics of members of the genus Glycomyces. The whole-cell sugars of strain YIM 56134^T consisted mainly of galactose, glucose and xylose and a minor amount of ribose; the presence of glucose but not mannose serves to distinguish this profile from those of Glycomyces species, as reported by Labeda & Kroppenstedt (2004). The predominant menaquinone (69 %) in strain YIM 56134^T was MK-10(H₄), with MK-11(H₄) (18 %) as a minor component; the menaquinone profile therefore differed somewhat from those of recognized Glycomyces species. The whole-cell fatty acid profile of strain YIM 56134^T contained the major components (>5 %) anteiso-15 : 0 (34.4 %), iso-16 : 0 (21.7 %), anteiso-17 : 0 (16.4 %), iso-15 : 0 (9.6 %) and iso-14 : 0 (5.5 %). Detailed menaquinone and fatty acid data for the novel strain and related Glycomyces species are given in Supplementary Tables S1 and S2 (available in IJSEM Online).

On the basis of the physiological, chemotaxonomic and phylogenetic data, strain YIM 56134^T could be easily distinguished from the recognized Glycomyces species. Therefore strain YIM 56134^T represents a novel species, for which the name Glycomyces endophyticus sp. nov. is proposed.