Pseudonocardia endophytica sp. nov., isolated from the pharmaceutical plant Lobelia clavata

There are many endophytic micro-organisms inside healthy tissues of nearly all living plants, and endophytes are important sources of bioactive metabolites (Strobel et al., 2004; Sardi et al., 1992). Actinomycetes have been isolated from the stem and root interior of many plants species (de Araújo et al., 2000; Coombs & Franco, 2003; Okazaki et al., 1995). The genus Pseudonocardia was originally proposed by Henssen (1957) for mycolateless, nocardioform actinomycetes with a type IV cell wall (Lechevalier & Lechevalier, 1970), and its description has been emended by Warwick et al. (1994), McVeigh et al. (1994), Reichert et al. (1998) and Huang et al. (2002). In the present study, an endophytic strain YIM 56035^T was isolated from the inner tissue of Lobelia clavata. Polyphasic analysis demonstrated that the strain should be recognized as a novel species of the genus Pseudonocardia, for which the name Pseudonocardia endophytica sp. nov. is proposed.

Strain YIM 56035^T was isolated from the healthy inner tissue of L. clavata collected from Yunnan Province, south-western China. L. clavata, a traditional Chinese medicinal plant, is usually used to treat parotitis and rheumatoid arthritis. Isolation of the strain was performed as follows: plant tissues (stems, leaves and roots) were washed thoroughly in running tap water to remove all soil, and these samples were surface sterilized by the method described by Shimizu et al. (2000). The surface-sterilization process was checked by rolling the surface-sterilized plant material aseptically onto the isolation medium, tap water-yeast extract agar (TWYE; Crawford et al., 1993), and a control medium, tryptic soy agar (TSA), and then incubating the plates at 28 °C for 7 days. No contamination was found. Surface-sterilized stems, leaves and roots were cut and placed in a sterilized commercial blender and then ground into small pieces and plated onto TWYE containing nalidixic acid (10 mg ml⁻¹), nystatin and cycloheximide (each at 50 mg ml⁻¹) to repress growth of bacteria and fungi. Petri dishes were incubated at 28 °C for 4 weeks. Pure cultures were obtained by repeated streaking on plates that contained TWYE medium. Peptone-yeast extract-glycerol agar (PYGA; Martí et al., 1999) and TSA were used for cultivating the isolates. The selected isolate, YIM 56035^T, was maintained on slopes of TSA at 4 °C or room temperature. For long-term preservation, cells were stored as suspensions of mycelium fragments in 30 % (v/v) glycerol at −20 and −80 °C. Cell mass for chemical and molecular systematic studies was grown in shaken flasks (shaken at about 150 r.p.m.) of TSB (tryptic soy broth) at 28 °C for 10 days.

Extraction of genomic DNA, PCR amplification and sequencing of the 16S rRNA gene were performed as described by Li et al. (2007) and an almost-complete 16S rRNA gene sequence of strain YIM 56035^T (1452 bp) was determined. Multiple sequence alignments were performed using clustal_x software (Thompson et al., 1997) and phylogenetic analysis was done with mega 3.1 (Kumar et al., 2004). A phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, 1987). Bootstrap values were determined using the method of Felsenstein (1985) with 1000 bootstrap resamplings. The 16S rRNA gene sequences analysis indicated that strain YIM 56035^T belongs to the genus Pseudonocardia, and it was most closely related to Pseudonocardia kongjuensis LM 157^T (98.5 % similarity), Pseudonocardia autotrophica IMSNU 20050^T (97.3 %), Pseudonocardia ammonioxydans H9^T (97.3 %) and Pseudonocardia compacta IMSNU 20111^T (97.1 %) (Fig. 1⇓); sequence similarities between strain YIM 56035^T and other type strains of the genus Pseudonocardia were less than 97 %.

DNA–DNA hybridization was performed by using the fluorometric microwell method (Ezaki et al., 1989; Christensen et al., 2000; He et al., 2005). DNA–DNA relatedness values (means±sd, n=3) between strain YIM 56035^T and P. kongjuensis DSM 44525^T, P. autotrophica DSM 535^T, P. ammonioxydans H9^T and P. compacta DSM 43592^T were 62.2±4.6, 52.1±2.7, 45±2.5 and 42.6±3.2 %, respectively. These values indicated clearly that strain YIM 56035^T represents a distinct genomic species (Stackebrandt & Goebel, 1994).

Amino acids and sugars of whole-cell hydrolysates were analysed as described previously (Hasegawa et al., 1983). Menaquinones were extracted according to the method of Collins (1985) and analysed by HPLC (Groth et al., 1997). Polar lipids were extracted and identified following the method of Minnikin et al. (1979) and Collins & Jones (1980). The cellular fatty acids were determined according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The G+C content of genomic DNA was determined by the HPLC method (Mesbah et al., 1989). The chemotaxonomic data match the description of the genus Pseudonocardia (Huang et al., 2002). Strain YIM 56035^T had a type-IV cell wall composition, containing meso-diaminopimelic acid, arabinose and galactose (Lechevalier & Lechevalier, 1970), which are common to members of the genus Pseudonocardia. The menaquinones were MK-8(H₄), MK-7(H₆) and MK-9 (ratio 97.5 : 1.8 : 0.7); MK-8(H₄) is the predominant menaquinone generally found in this genus. The phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylmethylethanolamine and diphosphatidylglycerol (type PIII; Lechevalier et al., 1977; Lechevalier & Lechevalier, 1980). The major fatty acids were i-C_16 : 0 (34.03 %), C_17 : 1 cis9 (14.69 %) and i-C_15 : 0 (9.62 %) (Table 1⇓). Phospholipid type III and the presence of i-C_16 : 0 as the major fatty acid are in agreement with the description of the genus Pseudonocardia (Huang et al., 2002). The DNA G+C content of strain YIM 56035^T was 70.3 mol%, which is within the range of values for members of the genus Pseudonocardia (Huang et al., 2002).

Cultural characteristics were examined by incubating strain YIM 56035^T at 28 °C on ISP media (Shirling & Gottlieb, 1966) and the media described by Waksman (1961). Morphological characteristics of the strain were observed on ISP 2 medium (yeast extract/malt extract agar), PYGA and TSA after 7–14 days at 28 °C. Spores and mycelium were observed by light microscopy (Olympus microscope BH-2) and scanning electron microscopy (Philip XL30 ESEM-TMP). Growth at different temperatures (4, 10, 15, 20, 28, 37, 40, 45 and 55 °C), NaCl concentrations (0, 1, 3, 5, 7, 10, 15 and 20 %) and pH values (pH 5.0–10.0) was assessed on TSA, with reference type strains in parallel. Other physiological and biochemical tests were carried out as described previously (Gordon et al., 1974; Reichert et al., 1998; Liu et al., 2006).

Strain YIM 56035^T had morphological properties typical of the genus Pseudonocardia. The aerial mycelium was white and the substrate mycelium was yellowish brown on TSA and ISP2 medium. No diffusible pigment was produced. Spore chains were borne at the ends of branched aerial hyphae, and the spore surface was smooth. The substrate mycelium fragmented into rod-shaped elements on TSA (Supplementary Fig. S1, available in IJSEM Online). Growth was observed at 15–37 °C, 0–5 % NaCl (w/v) and pH 6–8. The optimal growth pH, temperature and NaCl concentration were pH 7.0, 28 °C and 1 %.

A range of phenotypic properties separated strain YIM 56035^T from the type strains of species of Pseudonocardia with validly published names (Tables 1⇑ and 2⇓). Strain YIM 56035^T can be differentiated from related Pseudonocardia species by the absence of phosphatidylglycerol and phosphatidylinositol. The isolate was distinguished from previously described species by the proportions of the major fatty acids (i-C_16 : 0, C_16 : 1 cis9, i-C_16 : 1 H and C_17 : 1 cis9) and by the presence or absence of several fatty acids, including i-C_14 : 0, i-C_18 : 0, C_18 : 1 cis9 and C_18 : 0 (Table 1⇑). Strain YIM 56035^T differed from its closest phylogenetic neighbour, P. kongjuensis DSM 44525^T, in several phenotypic properties, including acid production from some substrates, hydrolysis of casein, urease activity, H₂S production and growth at 4 °C, 7 % NaCl and pH 9.0 (Table 2⇓). The isolate is distinct from other the reference strains examined in most of the tested physiological properties (Table 2⇓).

The genotypic and phenotypic data mentioned above show that strain YIM 56035^T merits recognition as a novel species of the genus Pseudonocardia, for which the name Pseudonocardia endophytica sp. nov. is proposed.