International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1077–1082 DOI 10.1099/ijs.0.057885-0 Sphaerisporangium rufum sp. nov., an endophytic actinomycete from roots of Oryza sativa L Ratchanee Mingma,1,2 Kannika Duangmal,1,2 Savitr Trakulnaleamsai,1,2 Arinthip Thamchaipenet,2,3 Atsuko Matsumoto4 and Yoko Takahashi4 Correspondence 1Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand Kannika Duangmal 2Center for Advanced Studies in Tropical Natural Resources, NRU-KU, Kasetsart University, [email protected] Chatuchak, Bangkok, Thailand 3Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, Thailand 4Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan An endophytic actinomycete, strain R10-82T, isolated from surface-sterilized roots of rice (Oryza sativa L.) was studied using a polyphasic approach. Strain R10-82T produced branching substrate mycelia and developed spherical spore vesicles on aerial hyphae containing non-motile spores. The major cellular fatty acids were iso-C16 : 0, iso-C14 : 0 and 10-methyl C17 : 0. The predominant menaquinones were MK-9, MK-9(H2), MK-9(H4) and MK-9(H6). Rhamnose, ribose, madurose, mannose and glucose were detected in whole-cell hydrolysates. The diagnostic phospholipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol mannosides, hydroxylphosphatidylethanolamine and ninhydrin-positive phosphoglycolipids. These morphological and chemotaxonomic data were similar to those of the genus Sphaerisporangium. Analysis of the 16S rRNA gene sequence revealed that strain R10-82T was related most closely to Sphaerisporangium cinnabarinum JCM 3291T (98.3 % similarity). The DNA G+C content of strain R10-82T was 74 mol%. DNA–DNA relatedness data in combination with differences in the biochemical and physiological properties suggested that strain R10-82T should be classified as representing a novel species of the genus Sphaerisporangium, for which the name Sphaerisporangium rufum is proposed. The type strain is R10-82T (5BCC 51287T5NBRC 109079T). An emended description of the genus Sphaerisporangium is also provided. The genus Sphaerisporangium was first proposed by Ara (Suriyachadkun et al., 2011), Sphaerisporangium siamense & Kudo (2007) for aerobic, non-acid-fast actinomycetes (Duangmal et al., 2011), Sphaerisporangium cinnabarinum, which form branched, non-fragmenting substrate and aerial Sphaerisporangium viridialbum, Sphaerisporangium rubeum hyphae. Spherical spore vesicles are produced on aerial and Sphaerisporangium melleum, the last named being the hyphae. The cell wall contains meso-diaminopimelic acid, type species of the genus (Ara & Kudo, 2007). All members and galactose, glucose, madurose, mannose and ribose of this genus have been isolated from soil samples. In are detected in whole-cell hydrolysates. The diagnostic this polyphasic taxonomic study, strain R10-82T, isolated polar lipids are phosphatidylethanolamine and ninhydrin- from the surface-sterilized roots of Oryza sativa L., is positive phosphoglycolipids and the predominant mena- described as representing a novel species of the genus quinones are MK-9, MK-9(H2), MK-9(H4) and MK-9(H6); Sphaerisporangium. mycolic acids are absent. This genus belongs to the order During the study of endophytic actinomycetes from rice ‘Sphaerisporangium’, family Streptosporangiaceae, together T with the genera Streptosporangium, Acrocarpospora, Her- plants (Oryza sativa L.), strain R10-82 was isolated from bidospora, Microbispora, Microtetraspora, Nonomuraea, surface sterilized roots collected from rice fields in Sup- Planobispora, Planomonospora, Planotetraspora and Ther- hanburi province, Thailand, in November 2010. The sample mopolyspora (Whitman et al., 2012). At the time of writing, was surface sterilized by sequential immersion in 0.1 % (v/v) the genus Sphaerisporangium comprised eight recognized Tween 20 for 5 min, 70 % (v/v) ethanol for 5 min and species: Sphaerisporangium album, Sphaerisporangium fla- sodium hypochlorite solution (1 %, w/v, available chlorine) viroseum (Cao et al., 2009), Sphaerisporangium krabiense for 10 min. Surface-treated samples were washed three times in sterile distilled water and then crushed in 0.85 % (w/v) The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene NaCl solution. The tissue suspensions were spread on starch sequence of strain R10-82T is AB842299. casein agar (Ku¨ster & Williams, 1964) supplemented with 057885 G 2014 IUMS Printed in Great Britain 1077 R. Mingma and others ketoconazole (100 mgml21), nystatin (50 mgml21) and two-dimensional TLC according to the methods proposed nalidixic acid (25 mgml21), and incubated at 28 uC for by Minnikin et al. (1977). Menaquinones were extracted 1 month. The pure culture of strain R10-82T was main- from freeze-dried biomass (100–200 mg) using the pro- tained as glycerol suspensions (20 %, v/v) of spores and cedure of Collins et al. (1977) and subsequently analysed by mycelia fragments at 220 uC. Sphaerisporangium cinna- LC/MS (JMS-T100LP; JEOL) with PEGASIL ODS column barinum JCM 3291T, Sphaerisporangium album DSM (2ø650 mm) using methanol/2-propanol (7 : 3). Mycolic 45172T, Sphaerisporangium siamense SR14.14T and Sph- acid methyl esters were examined by TLC according to the aerisporangium krabiense A-T 0308T were used for compar- method of Tomiyasu (1982). The N-acyl types of muramic ison of physiological, morphological and chemotaxonomic acid were determined by using the method of Uchida & properties and for DNA–DNA hybridization tests. Cultural Aida (1977). characteristics were determined after 3 weeks at 27 uCon Genomic DNA extraction was performed as described by various agar media according to methods recommended Kieser et al. (2000). The 16S rRNA gene was PCR amplified by the International Streptomyces Project (ISP; Shirling & from genomic DNA by using primers STR1F (59-TCAC- Gottlieb, 1966) as well as examination of growth on yeast GGAGAGTTTGATCCTG-39) and STR1530R (59-AAG- extract–starch agar (JCM medium no. 42), oatmeal nitrate GAGATCCAGCCGCA-39) (Kataoka et al., 1997) under agar (JCM medium no. 52), Czapek’s agar and Waksman the following conditions: initial denaturation for 5 min agar (Gottlieb, 1967). Colour determination was assessed at 94 uC; 30 cycles of denaturation for 1 min at 94 uC, by comparison with the Colour Harmony Manual (Jacobson annealing for 1 min at 55 uC and extension for 1 min at et al., 1958). Formation of mycelia and sporangium was 72 uC; and a final extension step for 10 min at 72 uC. PCR determined by light microscopy and scanning electron products were purified using a Gel/PCR DNA Fragment microscopy (JSM-5600 LV; JEOL) grown on soil-extract Extraction kit (Geneaid) then sent to the 1st Base Labo- agar [50 % soil extract solution (v/v) and 1.8 % agar (w/v)] ratory (Malaysia) for the DNA sequencing analysis. The at 28 uC for 1–2 months. Soil extract solution was prepared assembled sequence was compared with other sequences as described by Duangmal et al. (2008). of type strains in the EzTaxon-e server (http://eztaxon-e. Phenotypic properties were examined using standard pro- ezbiocloud.net/) (Kim et al., 2012). Evolutionary trees were cedures (Shirling & Gottlieb, 1966). The effects of various inferred using the maximum-parsimony (Fitch, 1971), temperatures (5–50 uC), pH (4.0–10.0 at intervals of 1.0 maximum-likelihood (Felsenstein, 1993) and neighbour- pH unit) and NaCl concentrations (0, 1, 2, 3, 4 and 5 %, joining (Saitou & Nei, 1987) tree-making algorithms w/v) on growth were determined on ISP medium 2 drawn from the MEGA 5 (Tamura et al., 2011) packages; (Shirling & Gottlieb, 1966) after 2 weeks. Utilization of an evolutionary distance matrix for the neighbour-joining carbohydrates as sole carbon sources at a final concen- algorithms was generated using the Jukes & Cantor (1969) tration of 1 % (w/v) was investigated on ISP medium model. The DNA G+C content was determined by HPLC 9 (Shirling & Gottlieb, 1966). The degradation of (w/v) according to the method of Tamaoka & Komagata (1984). casein (5 %), cellulose (1.0 %), chitin (0.5 %), guanine Levels of DNA–DNA relatedness were assayed using pho- (0.4 %), hypoxanthine (0.4 %), starch (1.5 %), tyrosine tobiotin and fluorometric microplates according to the (0.4 %) and xanthine (0.4 %) as well as urea hydrolysis method of Ezaki et al. (1989). were determined using standard procedures (Gordon & Strain R10-82T was Gram-stain-positive, non-acid-fast and Mihm, 1957; Gordon et al., 1974). Enzyme activities were formed branching substrate mycelia. The organism showed determined using API ZYM test kits (bioMe´rieux; 19 good growth on ISP medium 2, ISP medium 3, yeast enzyme activities tested) according to the manufacturer’s extract–starch agar and oatmeal nitrate agar. Moderate instructions. growth occurred on ISP medium 4, ISP medium 5 and Biomass for chemotaxonomic and molecular systematic Waksman agar and poor growth on Czapek’s agar and soil- studies was prepared in shake flasks of ISP medium 2 broth extract agar. Substrate mycelium ranged from red to pale at 27 uC for 14 days. Cultured cells were harvested by orange in colour and was produced on all media except centrifugation, and the pellet was washed twice with sterile on ISP 4 agar where it produced a brown substrate myce- distilled water before freeze drying. Analysis of diamino- lium. Pale red soluble pigment was produced only on ISP pimelic acid was performed according to Becker et al. medium 2. Melanoid pigment was not produced. Growth (1965) and Hasegawa et al. (1983). The whole-cell sugar was observed between 24 and 36 uC, with an optimum at compositions were determined by the method of Uchida 30 uC and pH 6–8. The strain could tolerate up to 3 % & Aida (1984). Fatty acid methyl esters were determined NaCl (w/v). Strain R10-82T produced spherical spore by GLC according to the instructions of the Sherlock vesicles (generally 3–8 mm in diameter; Fig. 1). Vesicles Microbial Identification System (Microbial ID; MIDI contained coiled chains of non-motile spores which were Version 6.1) (Sasser, 1990) and identified with the oval or spherical (0.4–0.660.6–1.0 mm). The phenotypic RTSBA6 database.