DIVERSITY OF ACTINOMYCETES FROM EKA KARYA BOTANICAL GARDEN BALI**, SHANTI RATNAKOMALA , PUSPITA LISDIYANTI , ,1 1 1 2* NITA R. PRAYITNO , EVI TRIANA YULIN LESTARI , RATIH D. HASTUTI , , MISA OTOGURO3 4 1 5YANTYATI WIDYASTUTI , KATSUHIKO ANDO5 4 and ENDANG SUKARA1, 1Research Center for Biotechnology Cibinong 16911, Indonesia, Indonesian Institute of Sciences, 2Research Center for Biology Cibinong 16911, Indonesia, Indonesian Institute of Sciences, 3 aculty of Mathematics and Natural Sciences, I P B ,680Department of Biology, F nstitut ertanian ogor, Bogor 16 Indonesia 4Soil Research Institute, Bogor 16002, Indonesia 5 iNITE Biolog cal Resource Center (NBRC), 2-5-8 Kasuzakamatari, Kisarazu, Chiba, Japan Received 1 July 2015/Accepted 6 May 2016 ABSTRACT A total of 2 actinomycetes were isolated 29 strains of and identified by full sequence of 16S rRNA gene analysis. Samples ed - from , Bali Island,consist of 18 soil and 20 leaf litter were collected Eka Karya Botanical Garden Indonesia. Two isolation methods, SDS- east xtract (SY) and Rehydration-Centrifugation (RC) used . i.e. Y E were in this study Based on 16S rRNA gene analysis, isolated actinomycetes may be grouped into 2 genera. B 8 ased on molecular analysis of 16S rRNA gene similarities showed that Eka Karya Botanical Garden . isolated actinomycetes of origin diverse is Analysis on from soil samples, resulted in is most 144 isolates 24 genera and more than 87 species. the Streptomyces dominant gen where or from isolated actinomycetes belong to this genus. It was followed by us 65 isolates 45% Actinoplanes (25 isolates = 17%). From leaf-litter 85 isolates 9 genera samples, the total number of may be group into ed and more than 41 species. The most dominated genus is (42 isolates = 49%) (16 Actinoplanes Catenuloplanesfollowed by isolates = 19%). Eka KaryaKeywords: 16S rRNA gene analysis, actinomycetes, biodiversity, Botanical Garden INTRODUCTION Actinomycetes are microorganisms belong to gram positive bacteria which are often saprophytic while some of them produce spores and mycelium. They play t roles n degrading importan i and decomposing organic compounds in the soil. They also produc secondary metabolites may e such as antibiotics, enzymes and other bioactive compounds for human welfare. Actinomycetes constitute a significant component of the microbial population in most soils and counted of over 1 million per gram of soil. oil is, cells S therefore this , the most prolific source of particular group. S oil represents the most intensively studied habitat of actinomycetes. Actinomycetes are thought to be most also the significant in the degradation of relatively group complex, recalcitrant polymers naturally in found plant litter and soil (Hopwood 2007; Baskaran et al. 2011). The preliminary characterization of the actinomycetes isolates was colony appearance, observing of zoospore bearing isolates, and DAP analysis. DAP analysis is a biochemical itself analysis of cell walls to observe the DAP isomers of g the cell wall of actinomycetes. Most of ram positive bacteria have lysine instead of DAP in the ir c ell wall , . T hro ugh thi s met hod actinomycetes could be into 3 groupsgrouped . According Miyadoh (2004), actinomycetes that to have LL-DAP in their cell wall generally belong to genus and ; while those Streptomyces Streptacidiphilus with -DAP type in their cell wall generally meso belong to the non- or so called rare Streptomyces actinomycetes. Actinomycetes that have both BIOTROPIA Vol. 23 No. 1, 2016: 42 - 51 DOI: 10.11598/btb.2016.2 . .5043 1 * C orresponding author: yahoo.comshanti_ratna@ ** This paper was presented at symposium on Recent Advances on Microbiological Researches and its Application (8-9 November 2011). Serpong, Indonesia. 42 LL-DAP and meso-DAP usually belong to genus Kitasatospora. Therefore, the DAP analysis could differentiate those three types of actinomycetes. Some actinomycetes share the same ability to release flagellated zoospores at a certain stage in their life cycle (Cross 1986). Current classification of motile-spored actinomycetes can identify at least six suborders containing zoosporic genera, including Micr omonosporinae, Micr ococcinae, Frankinae, Pseudonocardinae, Kineosporiineae and Streptosporanginae et al (Stackebrand . 1997). These t zoospores bearing actinomycetes have been associated with river, lake and fresh water, river sediments, desert soil (Garrity . 1996 Bredholt et al ; et al. et al 2008 Sibanda . 2010), decaying plant ; materials submerged in streams and cast up on lake shores (Kudo 1998 Tamura . 2010), et al. et al; blades of grass inhabiting streams and soils (Hasegawa 1991). It has become increasingly apparent that motile actinomycetes can produce a variety of antibiotics and other bioactive metabolites or be used for biochemical conversion of complex compounds (Hasegawa 1991 Garrity . 1996 Khamna . 2010 ; ; ;et al et al Khanna . 2011).et al According to Hayakawa (2000), et al. actinomycetes could be divided into two types based on spores produced by the non-motile and motile. Actinomycetes which bear non-motile spores that are not generally form flagella, for example, are , , , Streptomyces Nocardia Micromonospora and so on. and are Actinoplanes Catenuloplanes motile zoospores bearing actinomycetes and the zoospores can move. Several actinomycetes genera such as Actinoplanes, Amycolatopsis, Catenuloplanes, Dactylosporangium, Kineospor a, i Microbispora, Micromonospora Nonomuraeaand are often very difficult to isolate and cultivate due to their slow growth and those rare belong to the actinomycetes (Hayakawa 2008). Eka Karya Botanical Garden, Bedugul Bali in , Island is a unique plant , Indonesia ex situ conservation site for plant species of high elevated eastern tropical rain forest of Indonesia, adjoining with the tropical forest of Batukahu nature reserve. This garden is located at 1,250- 1,450 m above sea level, with area of 157.5 hectares (389 acres). Temperature is about 17- 25 C in daytime and is dropped to 10-15 C at o o night with 70-90% humidity (Mukaromah & Suparta 2007). Based on the uniq ness of the ue above location, we stud about the diversity of ied actinomycetes this location. This study wasin intended to be done as pioneer research in Eka Karya Botanical Garden. Several stud on ies diversity of actinomycetes were to be done in Indonesia, such as from Lombok Island (Lisdiyanti et al. 2012) and Cibinong Science Center (Widyastuti et al. 2013). To obtain new strains that poten ially produce new can t metabolites, it is still necessary to conduct exploration and examination of obtained samples from diverse habitats and environments. Few parts of the research had been orally presented in 2011 during Symposium on Recent Advances on Microbiological Researches and Its Application, c o n d u c t e d by I n d o n e s ia n S o c i e t y f o r Microbiology (PERMI) in Serpong. MATERIALS AND METHODS Sampling ethodsM Soil samples were obtained from Eka Karya Botanical Garden located at 1,250-1,450 m above sea level, 115 9'0-58” E and 8 15-17'0-59” N, o o with 5-10 cm depth from soil surfacesoil pH of . the soil samples was between 6.0-6.5. The samples were put in plastic bag ecaying immediately to . D leaf-litter samples were collected from soil surface were put in. The samples immediately to paper bag. All samples were air dried at room temperature for 1-2 weeks, ground using blender and filtered with 200 m mesh filter paperµ . SDS-Yeast Extract (SY) solation ethodI M SY isolation method described by was Widyastuti (201 ) A combination of 0.05% et al. 3 . SDS (Sodium Dodecyl Sulphate) as a germicide to eliminate soil bacteria, 6% yeast extract as spore activating agents and heating at 40 C for 20 o minutes increase the recovery of could also s actinomycetes from various soil samples. This method was used for isolating general actinomycetes. Rehydration and Centrifugation (RC) I Msolation ethod The RC isolation method was used for isolati . The sample is ng motile actinomycetes rehydrated by air-dried container in 10 mM phosphate buffer containing 10% soil extract, at 43 Diversity f ctinomycetes rom Eka Karya Botanical Garden Balio a f , – et al.Shanti Ratnakomala 30 C for 90 minutes, followed by centrifugation at o 1,500 x g for 20 minutes (Hayakawa . 2000 et al ; Otoguro 2001 Widyastuti . 201 )et al. et al; 3 . Humic Acid with Vitamins (HV) ediumM HV medium contained (in liter) 1 g humic was acid, 0.02 g CaCO , 0.01 g FeSO .7H O, 1.71 g 3 4 2 KCl, 0.05 g MgSO .7H O, 0.5 g NaHPO , 5 mL of 4 2 4 vitamins solution, 50 mg cycloheximide, 18 g agar, pH 7.2. The composition of vitamins solution was 0.5 mg thiamine HCl, 0.5 mg riboflavin, 0.5 mg niacin, 0.5 mg pyridoxine HCl, 0.5 mg inositol, 0.5 mg Ca-panthotenate, 0.5 mg p-aminobenzoic acid and 0.25 mg biotin in 5 mL water and sterilized by 0.22 m filtration (Hayakawa & Nonomura 1987). This vitamin solution was added after autoclave sterilization. Analysis of Diaminopimelic Acid (DAP) Preliminary biochemical test performed is the DAP determination using a method of thin layer chromatography (TLC) on cellulose to separate isomers of DAP (Hasegawa . 1983). Three et al loops of the cells was put in screw cap plastic tube, added with three drops of 6N HCl, autoclaved at temperature of 121 C, 1 atm for 15 minutes, and o then applied to cellulose chromatography plate. TLC eluent solution used was mixture of methanol: water: 6N HCl: pyridine (80: 26: 4: 10 v/v), and eluted for 12 hours. After that, the spots were sprayed with ninhydrin solution (0.3 g ninhydrin in 100 mL of butanol + 3 mL of acetic acid), and heated at 100 C for 3 minutes.o Preparation of emplate DNA and PCR T A Gmplification of 16S rRNA ene Chromosomal DNA was extracted as described by Saito & Miura (1963) from 14-day- old cell cultures grown on YG agar medium by using DNeasy Plant Maxi Kit (Qiagen). 16S rRNA gene replication reaction was performed using primer pair, 9F (forward: 5'-GAGTTTGATCCT- GGCTCAG-3' positions 9-27) and 1541R (reverse: 5'-AAGGAGGTGATCCAGCC-3' position 1541-1525) of numbering Escherichia coli system (Brosius 1978). PCR amplification et al. was performed used TaKa a ex Taq with total R volume of 50 L, consisting of 0.4 mM of each primer, 1 ng of DNA template, 2.5 mM of dNTP, 1 of Ta a a PCR buffer, and 5U of Taq K R polymerase in final volume. PCR conditions was 95 C for 3 minutes to denaturate the target DNA, o then by 30 cycles at 95 C for 3 seconds for o denaturation again, 55 C for 15 minutes for o primer annealing, and 72 C for 1 minute for o primer extension, and subsequently, 1 cycle at 72 oC for 5 minutes to complete the process of amplification. PCR reaction was conducted using a GeneAmp PCR System 9700 (Applied Biosystem). PCR products were examined by electrophoresis on agarose 2%, to assure that the target DNA had been amplified. PCR products were then purified using the GFX-96 PCR Purification Kit (Amersham Pharmacia Biotech), with reference to the protocol. 16S rRNA ene equencingG S PCR products that had been purified were cycle sequenced using the BigDye Terminator sequence with Version 3.1 Cycle Sequencing Kit. This reaction used 6 primers to amplify 1,500 bp of 16S rRNA gene, which is 9F, 515F (5'- GTGCCAAGCAGCCGCGGT-3' position 515- 531), 1099F (5'-GCAACGAGCGCAACCC-3' position 1099-1114), 536R (5'-GTATTACCGC- GGCTGCTTG-3' positions 536-519), 1115R (5'- AGGGTTGCGTCGTTG-3' position 1115- 1100), and 1541R of numbering Escherichia coli system (Brosius . 1978). In total 10 L of et al reaction sequence containing 2.0 L of Big Dye Terminator premix, 1.0 L of 5 Big Dye sequencing buffer, 0.8 L of each primer (1 pmol/L), and 0.5 L of template DNA were synthesized of the chain by using a GeneAmp PCR System 9700 (Applied Biosystem) with the following conditions pre- denaturation at 96 C for 1 minute, 45 cycles at a o temperature of 96 C for 10 seconds for o denaturation, 50 C for 5 seconds for primer o annealing, and 60 C for 90 seconds for primer o extension, and subsequent to storage at 16 C. The o product was purified using Dyeex 96 Kit (Qiagen) and sequenced using ABI Prism 3700 (Applied Biosystem) DNA sequencer. Sequence ata nalysis and lignment D A A Search 16S rDNA sequence was translated from the 16S rRNA gene by using ATGC Sequencing Analysis Software version 7.3 (ABI Prism) and corrected manually. Nucleotide sequence data of the isolates was searched the closest homology 44 BIOTROPIA Vol. 23 No. 1, 2016 with other strains in the 16S rRNA gene data base using BLAST (http://www.ncbi.nlm.nih. gov) . RESULTS AND DISCUSSION From t 38 he total number of samples consist of ed 18 soil samples and 20 leaf-litter samples 409 actinomycetes were isolated, . A total of 229 isolates based on the colony appearance, were selected and used study. in this From 229 isolates, 144 were isolated from soil samples and 85 were isolated from leaf-litter samples. From soil samples, 60 and 84 actinomycetes were isolated by SY and RC isolation method, respectively; and 85 from leaf-litter samples by using RC were isolated isolation method ( ). The DAP analysis Table 1 showed that within the actinomycetes isolated from soil source by SY isolation method, 18 isolates had LL-DAP, 31 isolates had /LL-meso meso/OH DAP, 11 isolates not but the rest d id have DAP polymers containing . Actinomycetes isolated by RC isolation method showed that 24 isolates had LL-DAP, 48 isolates had -meso DAP/LL- /OH in their cell wall, and the rest meso 12 isolates d not have DAP id containing polymers - using. From leaf litter source RC isolation method, 3 isolates had LL-DAP, 66 isolates had DAP/LL- OH on their cell meso meso/ wall, and 16 isolates not DAP containing d have id polymers. No Suborder No Family No Genus BLAST result >99 % 98 % 97 % <96 % Total 1 Corynebacterineae 1 Nocardiaceae 1 Nocardia 2 5 2 9 2 Rhodococcus 1 1 2 Frankineae 2 Cryptosporangiaceae 3 Cr yptosporangium 1 1 3 Kineosporiaceae 4 Kineosporia* 7 3 10 3 Micrococcineae 4 Intrasporangiaceae 5 Lapilicoccus 1 1 5 Promicromonosporaceae 6 Pr omicromonospora 1 1 4 Micromonosporineae 6 Micromonosporaceae 7 Actinoplanes* 8 33 22 4 67 8 Catellatospora 1 1 9 Catenuloplanes* 12 4 16 10 Dactylosporangium* 3 3 11 Krasilnikovia* 3 3 12 Micromonospora 3 4 1 8 13 Verrucosispora 1 1 5 Propionibacterineae 7 Nocardioidaceae 14 Kribbella 2 2 15 Nocardioides 1 1 2 6 Pseudonocarnineae 8 Actinosynnemataceae 16 Actinokineospora* 1 1 17 Saccharothrix 1 1 9 Pseudonocardiaceae 18 Amycolatopsis 1 2 3 19 Pseudonocardia 3 1 4 20 Saccharomonospora 1 1 7 Streptomycineae 10 Streptomycetaceae 21 Kitasatospora 5 2 7 22 Streptomyces 42 24 4 3 73 8 Streptosporangineae 11 Nocardiopsaceae 23 Nocardiopsis 4 4 12 Streptosporangiaceae 24 Acrocar pospora 1 1 25 Nonomuraea 1 2 2 5 26 Streptosporangium 1 1 13 Thermomonosporaceae 27 Actinocoraliia 1 1 28 Actinomadura 1 1 73 98 41 17 229 45 Diversity f ctinomycetes rom Eka Karya Botanical Garden Balio a f , – et al.Shanti Ratnakomala Table 1 Number of isolated and selected actinomycetes from Eka Karya Botanical Garden, Indonesia Sampling site Source No. of samples Isolation method Selected isolates DAP isomer LL M/LL-M/OH ND Eka Karya Botanical Garden Soil 18 SY 60 18 31 11 18 RC 84 24 48 12 Leaf- litter 20 RC 85 3 66 16 38 229 45 145 39 Table 2 Actinomycetes isolated from Eka Karya Botanical Garden, Bali, 2003 Indonesia, Note: * = Zoospore-bearing actinomycetes http://www.ncbi.nlm.nih. Identification of 229 isolates based on 16S rRNA gene sequencing showed that the isolates belong to 8 suborders, 13 families and 28 genera of the lass Actinomycetales (Table 2). The largest c g r oup of ac tino my ce te s f ou nd be long .to ( ) genus 73 isolates The second Streptomyces largest group genus 67 belong to (Actinoplanes isolates he third largest group ) belong to . T genus 16 isolates . About 58 Catenuloplanes ( ) isolates (25%) may be new species or new genus, because it has <98% of 16S rRNA gene similarity compared to the known strains in the database. and cultures SY isolation method incubation on HV agar plates containing nalidixic acid introduced by Hayakawa and Nonomura (1989) improve the possibilities of isolating d actinomycetes while decreasing the number of bacterial . This method proved to be an colonies effective tool for isolating act nomycete . i s RC isolation method described Hayakawa . by et al ( ) and ( found to et al2000 Otoguro . 2001) was also be an effect ve tool for the ion ofi isolat zoospore from the genera of , , Actinoplanes Actinokineospora Actinosynnema Catenuloplanes, Dactylosporangium, , Geodermatophylus Kineospor a.iand The phosphate buffer-soil extract solution significantly promoted liberation of motile zoospores from the source material, and the centrifugation eliminated Streptomyces and other non-motile actinomycetes. In general actinomycetes isolated , using SY were by many non-method dominated motile actinomycetes, while those isolated using the RC method we motile re dominated by actinomycetes. RC is an isolation method developed isolat motile zoospore for ing (Hayakawa . 2000).et al weAll 229 selected isolates re identified using m procedure olecular identification based on full sequence of 16S rRNA gene (±1,500 bp). The isolates were identified into genus and further species level by BLAST and phylogenetic tree construction. Currently, actinomycetes consisted of 24 families, 80 genera and 500 species (Liu . et al 2009). In study, identify 8 suborders, our we could 13 families and 28 genera ( )Table 3 . We predicted that there more than were 109 species. This is the first comprehensive study of actinomycetes conducted in Eka Karya Botanical Garden, Bali Island, Indonesia. Table 3 Diversity of actinomycetes in soil samples Note: * = zoospore bearing actinomycetes 46 BIOTROPIA Vol. 23 No. 1, 2016 No Suborder No Family No Genus No of species RC SY Total 1 Corynebacterineae 1 Nocardiaceae 1 Nocardia 4 1 8 9 2 Rhodococcus 1 1 1 2 Frankineae 2 Kineosporiaceae 3 Kineosporia* 1 1 1 3 Micrococcineae 3 Promicromonosporaceae 4 Promicromonospora 1 1 1 4 Micromonosporineae 4 Micromonosporaceae 5 Actinoplanes* 12 25 25 6 Catellatospora 1 1 1 7 Dactylosporangium* 1 3 3 8 Krasilnikovia* 1 3 3 9 Micromonospora 1 2 2 10 Verrucosispora 1 1 1 5 Propionibacterineae 5 Nocardioidaceae 11 Kribbella 2 2 212 Nocardioides 2 2 2 6 Pseudonocarnineae 6 Actinosynnemataceae 13 Actinokineospora 1 1 1 14 Saccharothrix 1 1 1 7 Pseudonocardiaceae 15 Amycolatopsis 2 1 2 3 16 Pseudonocardia 3 2 1 3 7 Streptomycineae 8 Streptomycetaceae 17 Kitasatospora 5 6 1 7 18 Streptomyces 36 33 32 65 8 Streptosporangineae 9 Nocardiopsaceae 19 Nocardiopsis 4 2 2 4 10 Streptosporangiaceae 20 Acrocarpospora 1 1 1 21 Nonomuraea 3 2 3 5 22 Streptosporangium 1 1 1 11 Thermomonosporaceae 23 Actinocoraliia 1 1 1 24 Actinomadura 1 1 1 87 84 60 144 Diversity of ctinomycetes on oil amplesA S S From soil samples, we obtained 144 isolates of actinomycetes that had been identified by 16S rRNA gene analysis and preserved well in liophilized form. The isolates contained 24 genera and more than 87 species. The most dominated genera in the soil samples was (65 Streptomyces isolates = 45%) and the next was (25 Actinoplanes isolates = 17%). Based on the isolation methods, 15 genera (60 isolates) were by SY isolation successfully isolated method and 16 genera (84 isolates) were isolated by RC isolation method. , Genera of Nocardia Rhodococcus Catelatospora Micromonospora Kribbella, , , , Nocardioides Streptosporangium Actinocoralia, , and Actinomadura were easily isolated using SY isolation method; while RC method was useful for isolating genera , , Actinoplanes Krasilnikovia Dactylosporangium Verrucosispora Actinokineospora, , and . Most of soil actinomycetes Saccharothrix isolated by RC isolation method belong to zoospore bearing actinomycetes. By using different isolation method, the dominant species of actinomycetes were also differe . In isolated nt this study, we proved that actinomycetes isolated using the RC method were dominated by groups of zoospore bearing actinomycetes. This result is similar to that described by et al.Hayakawa (2000) and Otoguro (2001).et al. Several ecological factors that played a role in the distribution of genera actinomycetes included humus content and pH of the soil (Nonomura & Hayakawa 1988), climate may influence the specific type of soil-inhabiting actinomycetes (Hayakawa . 2010). Soil of Eka Karya et al Botanical Garden a humus-rich soil with pH is range from 6 to 6.5. This soil type is suitable for the growth of actinomycetes. Some of actinomycetes are distributed in iverse plant species plant rhizosphere soils. D found in the garden should also support the growth of actinomycetes . Actinomycetes have been found to play an important role in rhizosphere soil (Suzuki . 2000 El-Tarabily & et al ; Sivasithamparam 2006). There is a possibility that these microorganisms can protect plant roots from plant pathogen and promote plant growth. Figure 1 Phylogenetic position based on 16S rRNA sequences of several isolates under the genera from Eka Karya Nocardia Botanical Garden. Bar, 1 substitutions per 200 nucleotides 47 Diversity f ctinomycetes rom Eka Karya Botanical Garden Balio a f , – et al.Shanti Ratnakomala For plant root protection, the modes of action of actinomycetes include antibiosis, parasitism, the production of extracellular hydrolytic enzymes and competition for iron (Getha . 2005 ;et al Errakhi . 2007). SY isolation method was et al particularly successful for isolating common actinomycetes such as spp. In natural Streptomyces habitats, streptomycetes are common and are usually a major component of the total actinomycetes population. Kim (1984) reported that within population in the soil, actinomycetes are dominated by (95 43%).Streptomyces . d indicate Identification by molecular approach that actinomycetes obtained from Eka Karya Botanical Garden have potential should value as a source find newto new species or genus. Based on the analysis of 16S rRNA gene, <97% sequence were in homology with the closest species on BLAST searching compared to the current database. N ew species and new genus among the strains studied re obviouswe . The16S rRNA gene sequence of strain ID03-0848 and ID03-0856 were aligned with those of the type species of the major and other actinomycete lineages. Nocardia The resulting phylogenetic tree is shown in Figure 1. Strain ID03-0848 and ID03-0856 formed a coherent clade within the lineage, clearly Nocardia distinguished from other described strains with highly bootstrap value. This was suspected to be new genus or new species in the lineage.Nocardia Diversity of ctinomycetes on eaf-litter A L Samples Meanwhile, from the leaf-litter as a source material, we obtained 85 isolates of actinomycetes that had been identified by 16S rRNA gene analysis and preserved well in liophilized form. The isolates contained 9 genera ( ) and Table 4 more than 41 species. The most dominated Actinoplanesgenus was (42 isolates = 49%) and Catenuloplanesthe next was (16 isolates = 19%) and (9 isolates = 10%). The same as in Kineosporia soil samples, most of the leaf-litter actinomycetes isolated by RC method belong to the zoospore bearing actinomycetes. This finding is in agreement with other reports which mentioned that actinomycetes belonging to genera Actinoplanes, Catenuloplanes Kineosporia and were frequently isolated from leaf-litter samples (Pagani 1978 Kudo . 1998 & Parenti ; ;et al Hayakawa . 2000 Ratnakomala 2011).et al et al.; They showed very similar characteristics such as possession of motility, absence or rarity of hydrophobic aerial hyphae and formation of orange colonies, similar to the color of fallen leaves (Van Hop 2011).et al. et al. et alXu (1996) and Meliani . (2012) reported that there was a positive correlation between diversity of actinomycetes with vegetation. Land of primary forest has higher diversity of actinomycetes compared with land of secondary forest and agricultural land. On dry, barren and cold land, there are less actinomycetes found (Xu 1996 Garrity . 1996). Search et al. et al; of new active compounds, especially from actinomycetes requires a large number of isolates. It would be more promising if sampling and isolation techniques more specific (Lo . are et al 2002). Therefore, it is essential to look for unique types of vegetation where the soil sample will be taken new taxonomical for finding ly important actinomycetes. It is also to find important Table 4 Diversity of actinomycetes from leaf-litter samples Note: * = zoospore bearing actinomycetes 48 BIOTROPIA Vol. 23 No. 1, 2016 No Suborder No Family No Genus No of species RC 1 Frankineae 1 Cryptosporangiaceae 1 Cryptosporangium 1 1 2 Kineosporiaceae 2 Kineosporia* 1 9 2 Micrococcineae 3 Intrasporangiaceae 3 Lapilicoccus 1 1 3 Micromonosporineae 4 Micromonosporaceae 4 Actinoplanes* 21 42 5 Catenuloplanes* 2 16 6 Micromonospora 6 6 4 Pseudonocarnineae 5 Pseudonocardiaceae 7 Pseudonocardia 1 1 8 Saccharomonospora 1 1 5 Streptomycineae 6 Streptomycetaceae 9 Streptomyces 7 8 41 85 a metaboli . cti omycete with new c propertiesn s T find a new actimomycete here is a possibility to species for the tion of newproduc antibiotics or other secondary metabolites. These microbes will specifically generate new secondary metabolites which allow them to degrade toxic compounds from these plants (Park 1999 Ho et al. ; et al . 2000). Eka Karya Botanical Garden is one place for conservation of tropical ex situ plant forests in Indonesia understood that high . It is diversity of actinomycetes will likely to be found in place.such Selection of proper method of isolation is crucial to obtain new actinomycetes species. wasIt obvious from our study that the use of RC method was useful to isolate new significantly species from leaf-litter samples, especially from genus Kineosporia . The 16S rRNA gene sequences of 10 strains (ID03-0578, ID03-0677, ID03-0678, ID03-0683, ID03-0684, ID03-0714, ID03-0716, ID03-0722, ID03-0739 and ID03-0760) were aligned with those of type species of the major Kineosporia and other actinomycete lineages. As shown in , strain ID03-0739 and ID03-Figure 2 0714 were moderately related to the type strain K. succinea AB003932. Strain ID03-0683 and ID03-0760 were related to type strain K. rhizophila AB003933. Strain ID03-0684 was closely related to type strain D86937. Strain ID03-K. aurantiaca 0677 and ID03-0678 shared the same branching position and formed a single clade with ID03- 0716 and ID03-0722. These four strains were clearly distinguished from other described strains with highly bootstrap value. This was suspected to be new genus or new species in the Kineosporia lineage. This study is significantly important to describe the diversity of actinomycete in s Indonesia. There are ample space to use isolated s actinomycete for the benefit of society. Further s research on several important taxa including proposing new species or genus is mandatory. More d ata on phenoty pe, bioche mical characterization, DNA-DNA hybridization and chemotaxonomic are required. CONCLUSIONS Selection of proper isolation method is crucial to obtain a new actinomycetes species. Using SY isolation method, this research was successfully isolated 2 new species of actinomycetes from Eka K ar ya Botanical Garden. This study is significantly important to describe the diversity of actinomycete in Indonesia. There are ample s space to use isolated actinomycete for the benefit s of society. F on important urther research some taxa including for proposing new species or genus is mandatory More. data on phenotypic, biochemical characterization, DNA hybridization and chemotaxonomic data are required to describe the other actinomycetes candidates as new species. Figure 2 Phylogenetic position based on 16S rRNA sequences of several isolates under the genera from Eka Kineosporia Karya Botanical Garden. Bar, 1 substitution per 100 nucleotides 49 Diversity f ctinomycetes rom Eka Karya Botanical Garden Balio a f , – et al.Shanti Ratnakomala 0.01 Kax 1000 1000 1000 1000 1000 665 641 938 995 644 532 438 540 567 536 1000 861 709 999 629 ID03-0739 ID03-0714 ID03-0760 ID03-0683 ID03-0684 ID03-0678 ID03-0722 ID03-0716 ID03-0677 ID03-0578 Kineosporia succinea (AB003932) Kineosporia rhizophila (AB003933) Kineosporia aurantiaca (D86937) Kineosporia aurantiaca (X87110) Kineosporia aurantiaca (AB003931) Kineosporia aurantiaca (AF095336) Kineosporia rhamnosa (AB003935) Kineosporia rhamnosa (AB003934) Kineospria radiotolerans (AF247813) Kineosporia aurantiacus (AB007420) Kineococcus mikuniensis (X92618) Cryptosporangium japonicum (D85466) Cryptosporangium sp. (AB006168) Cryptosporangium arvum (D85465) Cryptosporangium aura (AB047490) Cryptosporangium minu (AB037007) Streptomyces lavendulae (D85116) ACKNOWLEDGEMENTS This study was conducted under the Joint Research Project between Department of Biotechnology, National Institute of Technology and Evaluation, Japan and the Indonesian Institute of Sciences (LIPI) representing Indonesian Government Research Institutes. The authors thanked Eka Karya Botanical Garden, LIPI and technician in NITE and Research s Center for Biotechnology LIPI for their assistance. REFERENCES Baskaran R, Vijayakumar R, Mohan PM. 2011. Enrichment method for the isolation of bioactive actinomycetes from mangrove sediments of Andaman Islands, India. Malay J Microbiol 7(1):26-32. Bredholt H, Fjærvik E, Johnsen G Zotchev SB. 2008. , Actinomycetes from ediments in the Trondheim s Fjord, Norway: iversity and iological ctivity. d b a Mar Drugs 6(1):12 24- . Brosius J, Palmer ML, Kennedy PJ, Noller HF. 1978. Complete nucleotide sequence of a 16S ribosomal RNA gene from . Proc Natl Acad Sci Escherichia coli 75(10):4801-5. Cross T. 1986. The occurrence and role of actinoplanetes and motile actinomycetes in natural ecosystems. In: Megusar F, Gantar M, . Perspectives in (Eds) microbial ecolog y. Proceedings of the IV International Symposium on Microbial Ecology. p 265 70- . El-Tarabily KA, Sivasithamparam K. 2006. Non- streptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. Soil Biol Biochem 38: 1505-20. Errakhi R, Bouteau F, Lebrihi A, Barakate ML. 2007. Evidences of biological control capacities of Streptomyces Sclerotium rolfsi i spp. against responsible for damping disease in sugar beet ( L.). Beta vulgaris World J Microbiol Biotechnol 23:1503-9. Garrity GM, Heimbuch BK Gagliardi M. 1996. Isolation of , zoosporogenous actinomycetes from desert soils. J Ind Microbiol 17:260-7. Getha K, Vikineswary S, Wong WH, Seki T, Ward A, Goodfellow M. 2005. Evaluation of sp. Streptomyces strain g10 for suppression of wilt and Fusarium rhizosphere colonization in pot-grown banana plantlets. J Indian Microbiol Biotechnol 32:24-32. Hasegawa T, Takizawa M, Tanida S. 1983. A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29:319 22.- Hasegawa T. 1991. Studies on Motile Arthrospore-Bearing Rare Actinomycetes. Actinomycetol 5(2):64-71. Hayakawa M, Nonomura H. 1987. Efficacy of artificial h a s n aumic cid as a elective utrient in HV gar used for the solation of oil ctinomycetes. J Ferment i s a Technol 65(6):609-16. Hayakawa M, Nonomura H. 1989. A new method for the intensive isolation of Actinomycetes from soil. Actinomycetol 3(2):95-104. Hayakawa M, Otoguro M, Takeuchi T, Yamazaki T, Iimura Y. 2000. Application of a method incorporating differential centrifugation for selective isolation of motile Actinomycetes in soil and plant litter. Antonie van Leeuwenhoek 78:171-85. Hayakawa M. 2008. Studies on the isolation and distribution of rare Actinomycetes in soil. Actinomycetolo 22:12 9.- Hayakawa M, Yamamura H, Sakuraki Y, Ishida Y, Hamada M, Otoguro M, Tamura T. 2010. Diversity analysis of Actinomycetes assemblages isolated from soils in cool-temperate and subtropical areas of Japan. Actinomycetol 24:1-11. Ho CC, Tan GYA, Seow I, Ajam N, Tan EI, Goodfellow M, Ward AC, Brown R, Wong NK, Lo CW, Cheah HY, Lai NS, Suzuki KI. 2000. Isolation, characterization and biological activities of actinomycetes isolated from dipterocarp rain forest soils in Malaysia. In: Nnga BH, Tan HM, Suzuki K-I, editor. Microbiology Diversity in Asia. Singapore: World Scientific. Hopwood DA. 2007. , Streptomyces in Nature and Medicine The Antibiotic Makers. UK: Oxford University Press, Inc. Khamna S, Yokota A, Peberdy JF, Lumyong S. 2010. Indole- 3-acetic acid production by sp. isolated Streptomyces from some Thai medicinal plant rhizosphere soils. EurAsia J BioSci 4:23-32. Khanna M, Solanki R, Lal R. 2011. Selective isolation of rare Actinomycetes producing novel antimicrobial compounds. Int J Adv Biotechnol Res 2(3):357-75. Kim CJ. 1984. Isolation and screening of Actinomycetes from natural environments. Swed n: Genetic e Engineering Research Institute, KIST. Kudo T, Matsushima K, Itoh T, Sasaki J, Suzuki K. 1998. Description of four new species of the genus Kineosporia Kineosporia succinea Kineosporia : sp. nov., rhizophila Kineosporia mikuniensis sp. nov., sp. nov. and sp. nov., isolated from Kineosporia rhamnosa plant samples, and amended description of the genus . Int J Syst Bacteriol 48:1245 55.Kineosporia - Liu N, Wang H, Liu M, Gu Q, Zheng W, Huang Y. 2009. Streptomyces alni sp. nov., a daidzein-producing endophyte isolated from a root of Alnus nepalensis D.Don. Int J Syst Evol Microbiol 59:254-58. Lo CW, Lai NS, Cheah HY, Wong NKI, Ho CC. [Internet]. 2002. Actinomycetes isolated from soil samples 50 BIOTROPIA Vol. 23 No. 1, 2016 from the crocker range Sabah. Malaysia: ASEAN Review of Biodiversity and Environmental Conservation. Available from: http://www.arbec. com.my/pdf/art21julysep02.p fd . Lisdiyanti P, Tamura T, Ratnakomala S, Ridwan R, Kartina G, Lestari Y, Katsuhiko A, Widyastuti Y. 2012. Diversity of Actinomycetes from Soil Samples Collected from Lombok Island, Indonesia. Annales Bogorienses 16(1):35-40. Meliani A, Bensoltane A, Mederbel K. 2012. Microbial diversity and abundance in soil: related to plant and soil type. Am J Plant NutFert Technol Academic Journals Inc 2(1):10-8. Miyadoh S. 2004. . Antibiotic Screening from Actinomycete Isolates Bogor: Workshop on Isolation Methods and Classification of Actinomycetes. Mukaromah L, Suparta IP. 2007. Flowering biology of Paphiopedilum javanicum (Reinw. Ex Lindl) Pfitzer. In: Eka Karya Botanical Garden. Bogor (ID): Kebun Raya Bogor. p 80-4. Nonomura H, Hayakawa M. 1988. New Methods for the Selective Isolation of Soil Actinomycetes. In Biology of Actinomycetes. In: Okami Y, Beppu T, Ogawara H, editor. Tokyo (JP): Japan Scientific Societies. Otoguro M, Hayakawa M, Yamazaki T, Iimura Y. 2001. An integrated method for the enrichment and selective isolation of spp. in soil and plant Actinokineospora litter. J Appl Microbiol 91:118-30. Pagani H, Parenti F. 1978. , a new genus of Kineosporia the order Actinomycetales. Int J Syst Bacteriol 28:401-6. Park DJ, Lee SH, Kim CI, Uramoto U. 1999. Isolation of rare Actinomycetes from soil samples in specific micro-environments-natural lime cave and plant rhizosphere soil. Proceedings of International Conference on Asian Network on N Microbial Research. Thailand. p 728-37. Ratnakomala S, Ridwan R, Lisdiyanti P, Abinawanto, Utama A. 2011. Screening of Actinomycetes producing an ATPase inhibitor of RNA helicase from soil and leaf litter samples. Microbiol Indones 5(1):15-20. Saito H, Miura K. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochimica et Biophysica Acta 72:619-29. Sibanda T, Mabinya LV, Mazomba D, Akinpelu DA, Bernard K, Olaniran AO, Okoh AI. 2010. Antibiotic producing potentials of three freshwater Actinomycetes isolated from the Eastern Cape Province of South Africa. Int J Mol Sci 11:2612-23. Stackebrandt E, Rainey FA, Ward-Rainey NL. 1997. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol 47: -479 91. Suzuki S, Yamamoto K, Okuda T, Nishio M, Nakanishi N, Komatsubara S. 2000. Selective isolation and distribution of strains in Actinomadura rugatobispora soil. Actinomycetol 14:27 33.- Tamura T, Ishida Y, Otoguro M, Suzuki K. 2010. Amycolatopsis helveola Amycolatopsis sp. nov. and pigmentata sp. nov. isolated from soil. Intl J Syst Evol Microbiol 60:2629 33.- VanHop D, Sakiyama Y, Binh CTT, Otoguro M, Hang D T, Miyadoh S, Luong DT, Ando K. 2011. Taxonomic and ecological studies of Actinomycetes from Vietnam: isolation and genus-level diversity. J Antibiot 64:599 606.- Widyastuti Y, Lisdiyanti P, Ratnakomala S, Kartina G, Ridwan R, Rohmatussolihat R, Ando K. 2013. Genus diversity of Actinomycetes in Cibinong Science Center, West Java, Indonesia. Microbiology Indonesia 6(4):165. Xu LH, Li QR, Jiang CL. 1996. Diversity of soil Actinomycetes in Yunnan, China. Appl Environ Microbiol 62(1):244-8. 51 Diversity f ctinomycetes rom Eka Karya Botanical Garden Balio a f , – et al.Shanti Ratnakomala http://www.arbec.