Sphaerisporangium Rufum Sp. Nov., an Endophytic Actinomycete from Roots of Oryza Sativa L

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. The analysis was performed at the characteristics of strain R10-82T compared with related Faculty of Science, King Mongkut’s Institute of Tech- species of the genus Sphaerisporangium are shown in Table nology Ladkrabang (KMITL), Thailand. Phospholipids 1. Differences between the strains analysed were as follows. were extracted from freeze-dried cells and detected by Strain R10-82T was negative for cellobiose and raffinose

1078 International Journal of Systematic and Evolutionary Microbiology 64 Sphaerisporangium rufum sp. nov.

utilization. Sphaerisporangium cinnabarinum JCM 3291T utilized D(2)sorbitol and myo-inositol whereas strain T T R10-82 did not. Strain R10-82 was able to use L(+) arabinose, D(2)fructose, D(+)galactose, glycerol, b-lactose, D(2)mannitol, D(+)mannose, melibiose, D(–)rhamnose, sucrose, D(+)trehalose and xylose as sole source of carbon for energy and growth. Casein, hypoxanthine and starch were hydrolysed. H2S production and nitrate reduction were positive. In contrast, H2S production and nitrate reduction were negative for Sphaerisporangium cinnabarinum JCM 3291T. Whole-cell hydrolysates of strain R10-82T contained meso- diaminopimelic acid, rhamnose, ribose, madurose, mannose and glucose (wall chemotype III sensu Lechevalier & Lechevalier, 1970). The predominant menaquinones were MK-9 (17.9 %), MK-9(H2) (32.6 %), MK-9(H4) (46.6 %) and MK-9(H6) (2.9 %); mycolic acids were not detected. Fig. 1. Scanning electron micrograph of spherical spore vesicles Polar lipid analysis showed that the organism contained from 45-day-old cultures of strain R10-82T grown on soil-extract phosphatidylethanolamine, diphosphatidylglycerol, phos- agar at 28 6C. Bar, 5 mm. phatidylinositol mannosides, hydroxyphosphatidylethanolamine and ninhydrin-positive phosphoglycolipids as

Table 1. Differential characteristics of strain R10-82T and the type strains of related species of the genus Sphaerisporangium

Strains: 1, R10-82T;2,Sphaerisporangium cinnabarinum JCM 3291T;3,Sphaerisporangium album DSM 45172T;4,Sphaerisporangium siamense T T SR14.14 ;5,Sphaerisporangium krabiense A-T 0308 . All data are from this study unless otherwise indicated. +, Positive; W, weakly positive; 2, negative.

Characteristic 1 2 3 4 5

Substrate mycelium colour on: ISP medium 2 Red Dull yellow orange Light brownish grey Pink Pink ISP medium 3 Light yellowish red Light yellow orange Pale orange Pale yellow orange Pink Yeast extract–starch agar Yellowish red Light yellow orange Pink Pale orange Pale orange Oatmeal nitrate agar Pink Dull orange Pale orange Pink Pale orange Utilization of: L(+)Arabinose +++22 Cellobiose 2 ++++ D(2)Fructose +++22 D(+)Galactose ++++2 myo-Inositol 2 ++22 D(2)Mannitol + W + 22 Melibiose +++22 D(2)Rhamnose +++22 Raffinose 2 W +++ D(2)Sorbitol 2 + 222 Sucrose +++22 Xylose +++22 Degradation of: Casein ++2 W + Hypoxanthine ++2 ++ Tyrosine 222++ Nitrate reduction + 2 + 22

H2S production + 22++ Source Rice roots Sandy soil* Forest soil* Rhizosphere soil* Mangrove forest soil*

*Data from Ara & Kudo (2007), Cao et al. (2009), Duangmal et al. (2011) and Suriyachadkun et al. (2011). http://ijs.sgmjournals.org 1079 R. Mingma and others major phospholipids (phospholipid pattern type IV sensu 29.5, 21.3 and 21.5 %, respectively. These values were lower Lechevalier et al., 1977). The major fatty acids were than the 70 % cut-off point recommended for assignment of iso-C16 : 0, iso-C14 : 0 and 10-methyl C17 : 0 (fatty acid type 3c bacterial strains to the same genomic species (Wayne et al., sensu Kroppenstedt, 1985). The cellular fatty acid profiles 1987) and clearly indicated that strain R10-82T represents a of strain R10-82T and the type strains of closely related novel species. On the basis of phenotypic and genotypic species are shown in Table 2. The acyl type of the pepti- characteristics, it is proposed that strain R10-82T should be doglycan was N-acetyl. The DNA base composition of classified as a representative of a novel species in the genus strain R10-82T was 74 mol% G+C. Sphaerisporangium, for which the name Sphaerisporangium The almost-complete 16S rRNA gene sequence (1484 nt) rufum sp. nov. of strain R10-82T was determined. The rooted phylogenetic tree (Fig. 2) based on the neighbour-joining method Emended description of the genus indicated that strain R10-82T formed a distinct branch with Sphaerisporangium Cao et al. 2009 the type strains of three species of the genus Sphaerispo- T In addition to the description given by Cao et al. (2009), rangium: Sphaerisporangium album DSM 45172 , Spha- the DNA G+C contents are 67–74 mol%. erisporangium siamense SR14.14T and Sphaerisporangium krabiense A-T 0308T. Strain R10-82T shared highest 16S rRNA gene sequence similarity with Sphaerisporangium Description of Sphaerisporangium rufum sp. nov. cinnabarinum JCM 3291T (98.3 %, 25 nt differences at 1445 T Sphaerosporangium rufum (ruf9um. N.L. neut. adj. rufum sites), Sphaerisporangium album DSM 45172 (98.1 %, 25/ reddish). 1333) and Sphaerisporangium siamense SR14.14T (98.0 %, 28/1354). Mean levels of DNA–DNA relatedness between Cells are aerobic, Gram-positive, non-acid-fast actinomy- strain R10-82T and the type strains of Sphaerisporangium cetes that form branching hyphae. Catalase- and oxidase- cinnabarinum, Sphaerisporangium album, Sphaerisporan- positive. The organism grows well on ISP medium 2, ISP gium siamense and Sphaerisporangium krabiense were 27.9, medium 3, yeast extract–starch agar and oatmeal nitrate

Table 2. Cellular fatty acid compositions of strain R10-82T and the type strains of related Sphaerisporangium species

Strains: 1, R10-82T;2,Sphaerisporangium cinnabarinum JCM 3291T;3,Sphaerisporangium album DSM 45172T;4,Sphaerisporangium siamense SR14.14T (data from Duangmal et al., 2011); 5, Sphaerisporangium krabiense A-T 0308T. All data are from the present study unless otherwise indicated. Values are percentages of total fatty acids. 2, Not detected.

Fatty acid 1 2 3 4 5

Saturated

C13 : 0 1.0 ,0.5 ,0.5 2 ,0.5 C14 : 0 0.6 0.6 2.2 2 0.6 C15 : 0 222 2.0 3.3 C16 : 0 2.6 2.9 13.0 3.6 2.9 C17 : 0 7.1 7.2 10.5 2.3 1.8 C18 : 0 ,0.5 1.4 2.1 2.5 1.9 Unsaturated

C15 : 1 1.0 0.6 ,0.5 2 0.8 C16 : 1 2 ,0.5 22,0.5 C17 : 1 6.2 8.0 3.0 2 1.0 C18 : 1 ,0.5 1.0 0.7 22 Branched

iso-C14 : 0 17.1 1.8 2.0 1.0 3.9 iso-C15 : 0 7.0 2.7 5.5 1.8 1.3 anteiso-C15 : 0 3.6 0.9 1.0 0.6 2.2 iso-C16 : 0 34.0 46.0 32.9 47.9 61.2 iso-C16 : 1 22 0.8 4.0 6.0 iso-C17 : 0 1.0 1.3 1.4 1.0 ,0.5 anteiso-C17 : 0 1.7 1.5 1.2 2.3 2.2 iso-C18 : 0 0.7 2.0 0.7 1.7 1.9 10-Methylated

C16 : 0 222 8.2 2 C17 : 0 12.3 16.9 12.4 14.7 2 C18 : 0 ,0.5 2 1.2 3.2 0.8

1080 International Journal of Systematic and Evolutionary Microbiology 64 Sphaerisporangium rufum sp. nov.

0.005 97/95/95 Sphaerisporangium krabiense A-T 0308T (HM037365) * * Sphaerisporangium siamense SR14.14T (HM043727) * Sphaerisporangium album DSM 45172T (EU499344) Sphaerisporangium rufum R10-82T (AB842299) Sphaerisporangium cinnabarinum JCM 3291T (X89939) Sphaerisporangium rubeum 3D-72(35)T (AB208718) 84/73/76 80/63/63 Sphaerisporangium viridialbum ATCC 33328T (X89953) * * Sphaerisporangium melleum 3-28(8)T (AB208714) Sphaerisporangium flaviroseum YIM 48771T (EU499338) 99/99/100 Microtetraspora glauca ATCC 23057T (X97891) * Microtetraspora malaysiensis H47-7T (AB062383) * 100/99/99 Planotetraspora mira NA 9211028T (D85496) 85/65/74 * Planotetraspora silvatica TT 00-51T (AB112082) * 80/69/73 ATCC 29331T (AB188150) 79/–/66 Acrocarpospora corrugata * T * Acrocarpospora macrocephala R-55 (AB025318) 82/53/83 77/81/75 Herbidospora cretacea K-319T (D85485) * Herbidospora daliensis 0385M-1T (AY749433) Actinomadura madurae ATCC 19425T (X97889)

Fig. 2. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, for strain R10-82T and species of related genera. GenBank sequence accession numbers are given in parentheses. Asterisks indicate the clades that were conserved when the neighbour-joining (NJ), minimum-parsimony (MP) and maximum-likelihood (ML) methods were used in reconstructing phylogenetic trees. Bootstrap values .50 %, based on 1000 replications, are indicated at nodes (NJ/MP/ML). Bar, 0.005 substitutions per nucleotide position. agar. Spherical sporangia are produced on aerial mycelium mannosides, hydroxyphosphatidylethanolamine and ninhy- on soil-extract agar and are usually 3–8 mm in diameter. drin-positive phosphoglycolipids. Spores are oval or spherical (0.4–0.660.6–1.0 mm) and The type strain is R10-82T (5BCC 51287T5NBRC non-motile. Melanin pigments are not produced. Growth T 109079 ), which was isolated from the surface-sterilized occurs between 24 and 36 uC, with an optimum at 30 uC. + roots of rice plants (Oryza sativa L.), collected from Grows at pH 6–8 but not with 4 % NaCl. Utilizes L( ) Suphanburi province, Thailand. The DNA G+C content D 2 D + b arabinose, ( )fructose, ( )galactose, glycerol, -lactose, of the type strain is 74 mol%. D(2)mannitol, D(+)mannose, melibiose, D(2)rhamnose, sucrose, D(+)trehalose and xylose as sole carbon sources but not adonitol, cellobiose, maltose, myo-inositol, raffinose, Acknowledgements D(2)ribose, D(2)sorbitol, sorbose or xylitol. Activities We thank Professor George M. Garrity for his kind advice on naming of alkaline phosphatase, esterase (C4), esterase lipase (C8), the species. This work was supported by the Higher Education leucine arylamidase, valine arylamidase, acid phosphatase, Research Promotion and National Research University Project of naphthol-AS-BI-phosphohydrolase, b-galactosidase and Thailand, Office of the Higher Education Commission and Kitasato a-glucosidase are positive. Activities of lipase (C14), cystine Institute for Life Sciences, Kitasato University, Japan. We are also a a b grateful to Dr Chanwit Suriyachadkun for providing the reference arylamidase, trypsin, -chymotrypsin, -galactosidase, - T glucuronidase, N-acetyl-b-glucosaminidase, a-mannosidase type strain, Sphaerosporangium krabiense A-T 0308 . and a-fucosidase are negative. Hydrolyses casein, hypoxanthine and starch but not adenine, cellulose, chitin, References guanine, tyrosine or xanthine. H2S is produced. Nitrate reduction is positive. The predominant menaquinones are Ara, I. & Kudo, T. (2007). Sphaerosporangium gen. nov., a new mem- MK-9, MK-9(H2), MK-9(H4) and MK-9(H6). Mycolic acids ber of the family Streptosporangiaceae, with descriptions of three new are not detected. The predominant cellular fatty acids are species as Sphaerosporangium melleum sp. nov., Sphaerosporangium iso-C , iso-C and 10-methyl C . The N-acyl type rubeum sp. nov. and Sphaerosporangium cinnabarinum sp. nov., and 16 : 0 14 : 0 17 : 0 transfer of Streptosporangium viridialbum Nonomura and Ohara 1960 of muramic acid is acetyl. The diagnostic amino acid of to Sphaerosporangium viridialbum comb. nov. Actinomycetologica 21, the peptidoglycan is meso-diaminopimelic acid. Whole-cell 11–21. hydrolysates contain rhamnose, ribose, madurose, mannose Becker, B., Lechevalier, M. P. & Lechevalier, H. A. (1965). Chemical and glucose. Phospholipids consist of phosphatidyletha- composition of cell-wall preparations from strains of various form- nolamine, diphosphatidylglycerol, phosphatidylinositol genera of aerobic actinomycetes. Appl Microbiol 13, 236–243. http://ijs.sgmjournals.org 1081 R. Mingma and others

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