Diversity of Actinomycetes from Eka Karya Botanical Garden, Bali

BIOTROPIA Vol. 23 No. 1, 2016: 42 - 51 DOI: 10.11598/btb.2016.23.1.504 DIVE RSITY OF ACTINOMYCE TE S FROM E KA KARYA BOTANICAL GARDE N , BALI** 1* 1 1 2 SHANTI RATNAKOMALA , PUSPITA LISDIYANTI , NITA R. PRAYITNO , E VI TRIANA , 3 4 1 5 YULIN LE STARI , RATIH D. HASTUTI , YANTYATI WIDYASTUTI , MISA OTOGURO , 5 1,4 KATSUHIKO ANDO and E NDANG SUKARA 1 Research Center for Biotechnology, Indonesian Institute of Sciences, Cibinong 16911, Indonesia 2 Research Center for Biology, Indonesian Institute of Sciences, Cibinong 16911, Indonesia 3 Department of Biology, F aculty of Mathematics and Natural Sciences, Institut Pertanian Bogor, Bogor 16680 , Indonesia 4 Soil Research Institute, Bogor 16002, Indonesia 5 NITE Biological Resource Center (NBRC), 2-5-8 Kasuzak amatari, Kisarazu, Chiba, Japan Received 1 July 2015/Accepted 6 May 2016 ABSTRACT A total of 229 strains of actinomycetes were isolated and identified by full sequence of 16S rRNA gene analysis. Samples consisted of 18 soil and 20 leaf-litter were collected from E ka K arya Botanical Garden, Bali Island, Indonesia. Two isolation methods, i.e. SDS-Y east E xtract (SY) and Rehydration-Centrifugation (RC) were used in this study. Based on 16S rRNA gene analysis, isolated actinomycetes may be grouped into 28 genera. B ased on molecular analysis of 16S rRNA gene similarities showed that isolated actinomycetes of Eka Karya Botanical Garden origin is diverse. Analysis on 144 isolates from soil samples, resulted in 24 genera and more than 87 species. Streptomyces is the most dominant genus where 65 isolates or 45% from isolated actinomycetes belong to this genus. It was followed by Actinoplanes (25 isolates = 17%). From leaf-litter samples, the total number of 85 isolates may be grouped into 9 genera and more than 41 species. The most dominated genus is A ctinoplanes (42 isolates = 49%) followed by Catenuloplanes (16 isolates = 19%). Keywords: 16S rRNA gene analysis, actinomycetes, biodiversity, Eka K arya 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 important roles in degrading and decomposing organic compounds in the soil. They may also produce secondary metabolites 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 cells per gram of soil. Soil is, therefore, the most prolific source of this particular group. Soil represents the most intensively studied habitat of actinomycetes. * Corresponding author: shanti_ratna@ yahoo.com ** This paper was presented at symposium on Recent Advances on Microbiological Researches and its Application (8-9 November 2011). Serpong, Indonesia. 42 Actinomycetes are also thought to be the most significant group in the degradation of relatively complex, recalcitrant polymers found naturally in 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 itself is a biochemical analysis of cell walls to observe the DAP isomers of the cell wall of actinomycetes. Most of gram positive bacteria have lysine instead of DAP in their c ell wall . T hro ugh this method, actinomycetes could be grouped into 3 groups. According to Miyadoh (2004), actinomycetes that have LL-DAP in their cell wall generally belong to genus Streptomyces and Streptacidiphilus; while those with meso-DAP type in their cell wall generally belong to the non-Streptomyces or so called rare actinomycetes. Actinomycetes that have both Diversity of actinomycetes from E ka Karya Botanical Garden, Bali – Shanti Ratnakomala et al. 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 Micromonosporinae, Micrococcinae, Frank inae, Pseudonocardinae, Kineosporiineae and Streptosporanginae (Stackebrandt et al. 1997). These zoospores bearing actinomycetes have been associated with river, lake and fresh water, river sediments, desert soil (Garrity et al. 1996; Bredholt et al. 2008; Sibanda et al. 2010), decaying plant materials submerged in streams and cast up on lake shores (Kudo et al. 1998; Tamura et al. 2010), 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 et al. 1996; Khamna et al. 2010; Khanna et al. 2011). According to Hayakawa et al. (2000), 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. A ctinoplanes and Catenuloplanes are motile zoospores bearing actinomycetes and the zoospores can move. Several actinomycetes genera such as A ctinoplanes, A mycolatopsis, Catenuloplanes, Dactylosporangium, Kineosporia, Microbispora, Micromonospora and Nonomuraea are often very difficult to isolate and cultivate due to their slow growth and those belong to the rare actinomycetes (Hayakawa 2008). E ka K arya Botanical Garden, in Bedugul, Bali Island, Indonesia is a unique ex situ plant 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 1,2501,450 m above sea level, with area of 157.5 hectares (389 acres). Temperature is about 17o o 25 C in daytime and is dropped to 10-15 C at night with 70-90% humidity (Mukaromah & Suparta 2007). Based on the uniqueness of the above location, we studied about the diversity of actinomycetes in this location. This study was intended to be done as pioneer research in E ka K arya Botanical Garden. Several studies on 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 can potentially produce new metabolites, it is still necessary to conduct exploration and examination of samples obtained 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 nduc ted by I ndo nesian So c iety f o r Microbiology (PE RMI) in Serpong. MATE RIALS AND ME TH ODS Sampling Methods Soil samples were obtained from E ka K arya Botanical Garden located at 1,250-1,450 m above o o sea level, 115 9'0-58” E and 8 15-17'0-59” N, with 5-10 cm soil depth from soil surface. pH of the soil samples was between 6.0-6.5. The samples were immediately put into plastic bag. D ecaying leaf-litter samples were collected from soil surface. The samples were immediately put into paper bag. All samples were air dried at room temperature for 1-2 weeks, ground using blender and filtered with 200 µm mesh filter paper. SDS-Yeast E xtract (SY) I solation Method SY isolation method was described by Widyastuti et al. (2013). A combination of 0.05% SDS (Sodium Dodecyl Sulphate) as a germicide to eliminate soil bacteria, 6% yeast extract as spore o activating agents and heating at 40 C for 20 minutes could also increases the recovery of actinomycetes from various soil samples. This method was used for isolating general actinomycetes. Rehydration and Centrifugation (RC) I solation Method The RC isolation method was used for isolating motile actinomycetes. The sample is rehydrated by air-dried container in 10 mM phosphate buffer containing 10% soil extract, at 43 BIOTROPIA Vol. 23 No. 1, 2016 o 30 C for 90 minutes, followed by centrifugation at 1,500 x g for 20 minutes (Hayakawa et al. 2000; Otoguro et al. 2001; Widyastuti et al. 2013). Humic Acid with Vitamins (HV) Medium HV medium was contained (in liter) 1 g humic acid, 0.02 g CaCO 3, 0.01 g FeSO 4.7H2O, 1.71 g KCl, 0.05 g MgSO 4.7H2O, 0.5 g NaHPO 4, 5 mL of 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 et al. 1983). Three loops of the cells was put in screw cap plastic tube, added with three drops of 6N HCl, autoclaved at o temperature of 121 C, 1 atm for 15 minutes, and 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 o acid), and heated at 100 C for 3 minutes. Preparation of T emplate DNA and PCR Amplification of 16S rRNA Gene Chromosomal D NA was extracted as described by Saito & Miura (1963) from 14-dayold 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'-GAGTTTGATCCTG GCTCAG-3' positions 9-27) and 1541R (reverse: 5'-AAG G AG G T G AT CCAG CC-3' position 1541-1525) of E scherichia coli numbering system (Brosius et al. 1978). PCR amplification was performed used TaK aR a ex Taq with total volume of 50 L, consisting of 0.4 mM of each primer, 1 ng of DNA template, 2.5 mM of dNTP, 1 of TaK aRa PCR buffer, and 5U of Taq 44 polymerase in final volume. PCR conditions was o 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 primer extension, and subsequently, 1 cycle at 72 o C for 5 minutes to complete the process of amplification. PCR reaction was conducted using a G eneAmp 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 Gene Sequencing 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 515531), 1099F (5'-GCAACGAGCGCAACCC-3' position 1099-1114), 536R (5'-GTATTACCGCGGCTGCTTG-3' positions 536-519), 1115R (5'AGG G TTGCG TCG TTG -3' position 11151100), and 1541R of E scherichia coli numbering system (Brosius et al. 1978). In total 10 L of 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 preo 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 product was purified using Dyeex 96 Kit (Qiagen) and sequenced using ABI Prism 3700 (Applied Biosystem) DNA sequencer. Sequence Data Analysis and Alignment 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 Diversity of actinomycetes from E ka Karya Botanical Garden, Bali – Shanti Ratnakomala et al. leaf-litter samples were isolated by using RC isolation method (Table 1). The DAP analysis showed that within the actinomycetes isolated from soil source by SY isolation method, 18 isolates had LL-DAP, 31 isolates had meso/LLmeso/OH DAP, but the rest 11 isolates did not have DAP containing polymers. Actinomycetes isolated by RC isolation method showed that 24 isolates had LL-DAP, 48 isolates had mesoDAP/LL-meso/OH in their cell wall, and the rest 12 isolates did not have DAP containing polymers. From leaf-litter source using RC isolation method, 3 isolates had LL-DAP, 66 isolates had meso DAP/LL-meso/ OH on their cell wall, and 16 isolates did not have DAP containing polymers. with other strains in the 16S rRNA gene data base using BLAST (http://www.ncbi.nlm.nih. gov). RE SULTS AND DISCUSSION From the total number of 38 samples consisted of 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 in this study. 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 Table 1 Number of isolated and selected actinomycetes from Eka K arya Botanical Garden, Indonesia Sampling site Eka Karya Botanical Garden Source Soil Leaflitter No. of samples 18 18 Isolation method SY RC 20 RC 60 84 LL 18 24 DAP isomer M/LL-M/OH 31 48 ND 11 12 85 3 66 16 229 45 145 39 Selected isolates 38 Table 2 Actinomycetes isolated from Eka K arya Botanical Garden, Bali, Indonesia, 2003 BLAST result No Suborder 1 Corynebacterineae 2 Frankineae 3 Micrococcineae 4 5 6 7 8 Micromonosporineae Propionibacterineae No Family 1 Nocardiaceae 2 3 4 5 Cryptosporangiaceae K ineosporiaceae Intrasporangiaceae Promicromonosporaceae 6 7 Micromonosporaceae Nocardioidaceae 8 Actinosynnemataceae 9 Pseudonocardiaceae Pseudonocarnineae Streptomycineae Streptosporangineae 10 Streptomycetaceae 11 Nocardiopsaceae 12 Streptosporangiaceae 13 T hermomonosporaceae No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 G enus N ocardia Rhodococcus Cryptosporangium Kineosporia* L apilicoccus Promicromonospora A ctinoplanes* Catellatospora Catenuloplanes* Dactylosporangium* Krasilnik ovia* Micromonospora Verrucosispora Kribbella N ocardioides A ctinok ineospora* Saccharothrix A mycolatopsis Pseudonocardia Saccharomonospora Kitasatospora Streptomyces N ocardiopsis A crocarpospora N onomuraea Streptosporangium A ctinocoraliia A ctinomadura > 99 % 2 98 % 5 97 % 1 7 1 8 33 1 12 3 3 1 22 2 1 1 1 5 42 4 4 4 3 4 1 3 < 96 % 2 1 1 1 1 1 2 3 1 2 24 4 2 2 1 98 41 3 1 1 1 1 73 17 Total 9 1 1 10 1 1 67 1 16 3 3 8 1 2 2 1 1 3 4 1 7 73 4 1 5 1 1 1 229 Note: * = Zoospore-bearing actinomycetes 45 BIOTROPIA Vol. 23 No. 1, 2016 Table 3 Diversity of actinomycetes in soil samples No Suborder No Family Rhodococcus 1 Kineosporiaceae Promicromonosporaceae 3 4 Kineosporia* Promicromonospora 1 1 1 1 A ctinoplanes* Catellatospora Dactylosporangium* Krasilnik ovia* Micromonospora Verrucosispora 12 1 1 1 1 1 25 Micromonosporaceae 5 6 7 8 9 10 Nocardiaceae 2 3 Frankineae Micrococcineae 2 3 5 6 7 8 Propionibacterineae Streptosporangineae 1 SY Total 8 9 1 1 1 1 1 3 3 2 1 Nocardioidaceae 11 12 Kribbella Nocardioides 2 2 6 Actinosynnemataceae 13 14 A ctinok ineospora Saccharothrix 1 1 1 1 7 Pseudonocardiaceae 15 16 A mycolatopsis Pseudonocardia 2 3 1 2 2 1 3 3 8 Streptomycetaceae 17 18 Kitasatospora Streptomyces 5 36 6 33 1 32 7 65 9 Nocardiopsaceae 19 Nocardiopsis 4 2 2 4 10 Streptosporangiaceae 20 21 22 A crocarpospora Nonomuraea Streptosporangium 1 3 1 1 2 3 1 1 5 1 11 Thermomonosporaceae 23 24 A ctinocoraliia A ctinomadura 1 1 1 1 1 1 60 144 87 2 2 25 1 3 3 2 1 5 Pseudonocarnineae Streptomycineae RC Nocardia 1 4 No of species 4 2 Corynebacterineae Micromonosporineae Genus 1 1 4 No 84 2 2 1 1 Note: * = zoospore bearing actinomycetes 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 class Actinomycetales (Table 2). The largest group of ac tino mycetes f ound belong to genus Streptomyces (73 isolates). The second largest group belong to genus A ctinoplanes (67 isolates). T he third largest group belong to genus Catenuloplanes (16 isolates). About 58 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. SY isolation method and cultures incubation on HV agar plates containing nalidixic acid introduced by Hayakawa and Nonomura (1989) improve d the possibilities of isolating actinomycetes while decreasing the number of bacterial colonies. This method proved to be an effective tool for isolating actinomycetes. RC isolation method described by Hayakawa et al. (2000) and Otoguro et al. (2001) was also found to be an effective tool for the isolation of zoospore from the genera of A ctinoplanes, A ctinok ineospora, A ctinosynnema, Catenuloplanes, Dactylosporangium, 46 Geodermatophylus and Kineosporia. 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 method were dominated by many nonmotile actinomycetes, while those isolated using the RC method were dominated by motile actinomycetes. RC is an isolation method developed for isolating motile zoospore (Hayakawa et al. 2000). All 229 selected isolates were identified using molecular identification procedure based on full sequence of 16S rRNA gene (± 1,500 bp). The isolates were further identified into genus and species level by BLAST and phylogenetic tree construction. Currently, actinomycetes consisted of 24 families, 80 genera and 500 species (Liu et al. 2009). In our study, we could identify 8 suborders, 13 families and 28 genera (Table 3). We predicted that there were more than 109 species. This is the first comprehensive study of actinomycetes conducted in E ka K arya Botanical Garden, Bali Island, Indonesia. Diversity of actinomycetes from E ka Karya Botanical Garden, Bali – Shanti Ratnakomala et al. Diversity of Actinomycetes on Soil Samples 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 Streptomyces (65 isolates = 45%) and the next was A ctinoplanes (25 isolates = 17%). Based on the isolation methods, 15 genera (60 isolates) were successfully isolated by SY isolation method and 16 genera (84 isolates) were isolated by RC isolation method. Genera of Nocardia, Rhodococcus, Catelatospora, Micromonospora, Kribbella, Nocardioides, Streptosporangium, A ctinocoralia and A ctinomadura were easily isolated using SY isolation method; while RC method was useful for isolating genera A ctinoplanes, Krasilnik ovia , Dactylosporangium, V errucosispora, A ctinok ineospora and Saccharothrix . Most of soil actinomycetes isolated by RC isolation method belong to zoospore bearing actinomycetes. By using different isolation method, the dominant species of actinomycetes isolated were also different. In 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 Hayakawa et al. (2000) and Otoguro et al. (2001). 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 et al. 2010). Soil of E ka K arya Botanical Garden is a humus-rich soil with pH range from 6 to 6.5. This soil type is suitable for the growth of actinomycetes. Some of actinomycetes are distributed in plant rhizosphere soils. D iverse plant species found in the garden should also support the growth of actinomycetes. Actinomycetes have been found to play an important role in rhizosphere soil (Suzuki et al. 2000; E l-Tarabily & 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 Nocardia genera from Eka K arya Botanical Garden. Bar, 1 substitutions per 200 nucleotides 47 BIOTROPIA Vol. 23 No. 1, 2016 For plant root protection, the modes of action of actinomycetes include antibiosis, parasitism, the production of extracellular hydrolytic enzymes and competition for iron (Getha et al. 2005; E rrakhi et al. 2007). SY isolation method was particularly successful for isolating common actinomycetes such as Streptomyces spp. In natural 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 Streptomyces (95.43%). Identification by molecular approach indicated that actinomycetes obtained from E ka K arya Botanical Garden should have potential value as a source to find new species or new 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. New species and new genus among the strains studied were obvious. The16S rRNA gene sequence of strain ID03-0848 and ID03-0856 were aligned with those of the type species of the major Nocardia and other actinomycete lineages. The resulting phylogenetic tree is shown in Figure 1. Strain ID03-0848 and ID03-0856 formed a coherent clade within the Nocardia lineage, clearly distinguished from other described strains with highly bootstrap value. This was suspected to be new genus or new species in the Nocardia lineage. Diversity of Actinomycetes on L eaf-litter 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 (Table 4) and more than 41 species. The most dominated genus was A ctinoplanes (42 isolates = 49%) and the next was Catenuloplanes (16 isolates = 19%) and Kineosporia (9 isolates = 10%). The same as in 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 A ctinoplanes, Catenuloplanes and Kineosporia were frequently isolated from leaf-litter samples (Pagani & Parenti 1978; Kudo et al. 1998; Hayakawa et al. 2000; Ratnakomala et al. 2011). 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 et al. 2011). Xu et al. (1996) and Meliani et al. (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 et al. 1996; Garrity et al. 1996). Search of new active compounds, especially from actinomycetes requires a large number of isolates. It would be more promising if sampling and isolation techniques are more specific (Lo et al. 2002). Therefore, it is essential to look for unique types of vegetation where the soil sample will be taken for finding new taxonomically important actinomycetes. It is also important to find Table 4 Diversity of actinomycetes from leaf-litter samples No Suborder 1 Frankineae 2 No Family Micrococcineae 1 2 3 Cryptosporangiaceae Kineosporiaceae Intrasporangiaceae 3 Micromonosporineae 4 Micromonosporaceae 4 Pseudonocarnineae 5 Pseudonocardiaceae 5 Streptomycineae 6 Streptomycetaceae Note: * = zoospore bearing actinomycetes 48 No 1 2 3 4 5 6 7 8 9 Genus Cryptosporangium Kineosporia* L apilicoccus A ctinoplanes* Catenuloplanes* Micromonospora Pseudonocardia Saccharomonospora Streptomyces No of species RC 1 1 1 21 2 6 1 1 7 41 1 9 1 42 16 6 1 1 8 85 Diversity of actinomycetes from E ka Karya Botanical Garden, Bali – Shanti Ratnakomala et al. 0.01 Kax 1000 1000 1000 665 1000 1000 ID03-0739 ID03-0714 540 Kineosporia succin ea (AB003932) ID03-0760 861 ID03-0683 Kineosporia rhizophila (AB003933) ID03-0684 567 Kineosporia aurantiaca (D86937) 709 Kineosporia aurantiaca (AB003931) 999 629 Kineosporia aurantiaca (X87110) Kineosporia aurantiaca (AF095336) 641 644 ID03-0722 ID03-0716 938 ID03-0678 995 ID03-0677 ID03-0578 Kineosporia rhamnosa (AB003935) 1000 Kineosporia rhamnosa (AB003934) Kineospria radiotolerans (AF247813) Kineosporia aurantiacus (AB007420) Kineococcus mikuniensis (X92618) Cryptosporangium arvum (D85465) 532 Cryptosporangium japonicum (D85466) Cryptosporangium sp. (AB006168) Cryptosporangium aura (AB047490) 438 Cryptosporangium minu (AB037007) Streptomyces lavendulae (D85116) 536 Figure 2 Phylogenetic position based on 16S rRNA sequences of several isolates under the Kineosporia genera from E ka K arya Botanical Garden. Bar, 1 substitution per 100 nucleotides actinomycetes with new metabolic properties. T here is a possibility to find a new actimomycete species for the production of new antibiotics or other secondary metabolites. These microbes will specifically generate new secondary metabolites which allow them to degrade toxic compounds from these plants (Park et al. 1999; Ho et al. 2000). E ka K arya Botanical Garden is one place for ex situ plant conservation of tropical forests in Indonesia. It is understood that high diversity of actinomycetes will likely to be found in such place. Selection of proper method of isolation is crucial to obtain new actinomycetes species. It was obvious from our study that the use of RC method was significantly useful to isolate new 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 Figure 2, strain ID03-0739 and ID030714 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 K. aurantiaca D86937. Strain ID030677 and ID03-0678 shared the same branching position and formed a single clade with ID030716 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 actinomycetes in Indonesia. There are ample spaces to use isolated actinomycetes for the benefit of society. Further research on several important taxa including proposing new species or genus is mandatory. More data on phenotype, biochemical 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 E ka K arya B otanical G arden. T his study is significantly important to describe the diversity of actinomycetes in Indonesia. There are ample space to use isolated actinomycetes for the benefit of society. F urther research on some important 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. 49 BIOTROPIA Vol. 23 No. 1, 2016 ACKNOWLE DGE ME NTS Hasegawa T. 1991. Studies on Motile Arthrospore-Bearing Rare Actinomycetes. Actinomycetol 5(2):64-71. This study was conducted under the Joint Research Project between D epartment of Biotechnology, National Institute of Technology and E valuation, Japan and the Indonesian Institute of Sciences (LIPI ) representing Indonesian Government Research Institutes. The authors thanked Eka K arya Botanical Garden, LIPI and technicians in NITE and Research Center for B iotechnology LI PI for their assistance. Hayakawa M, Nonomura H. 1987. Efficacy of artificial humic acid as a selective nutrient in HV agar used for the isolation of soil actinomycetes. J Ferment Technol 65(6):609-16. RE FE RE NCE S Hayakawa M. 2008. Studies on the isolation and distribution of rare Actinomycetes in soil. Actinomycetolo 22:12-9. 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 sediments in the Trondheim Fjord, Norway: diversity and biological activity. 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 E scherichia coli. Proc Natl Acad Sci 75(10):4801-5. Cross T. 1986. The occurrence and role of actinoplanetes and motile actinomycetes in natural ecosystems. In: Megusar F, Gantar M, (E ds). Perspectives in microbial ecology. Proceedings of the IV International Symposium on Microbial E cology. p 265-70. E l-Tarabily K A, Sivasithamparam K . 2006. Nonstreptomycete 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 spp. against Sclerotium rolfsii responsible for damping disease in sugar beet (Beta vulgaris L.). 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 Streptomyces sp. strain g10 for suppression of Fusarium wilt and 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. 50 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, 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 Mak ers. UK: Oxford University Press, Inc. Khamna S, Yokota A, Peberdy JF, Lumyong S. 2010. Indole3-acetic acid production by Streptomyces sp. isolated 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. Sweden: Genetic 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 sp. nov., Kineosporia rhizophila sp. nov., Kineosporia mik uniensis sp. nov. and Kineosporia rhamnosa sp. nov., isolated from plant samples, and amended description of the genus Kineosporia. Int J Syst Bacteriol 48:1245-55. 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 A lnus 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 Diversity of actinomycetes from E ka Karya Botanical Garden, Bali – Shanti Ratnakomala et al. from the crocker range Sabah. Malaysia: ASEAN Review of Biodiversity and E nvironmental Conservation. Available from: http://www.arbec. com.my/pdf/art21julysep02.pdf . Lisdiyanti P, Tamura T, Ratnakomala S, Ridwan R, K artina 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. A ntibiotic Screening from A ctinomycete 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. NewMethods for the Selective Isolation of Soil A ctinomycetes. 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 A ctinok ineospora spp. in soil and plant litter. J Appl Microbiol 91:118-30. Pagani H, Parenti F. 1978. Kineosporia, a new genus of 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 Actinomadura rugatobispora strains in soil. Actinomycetol 14:27-33. Tamura T, Ishida Y, Otoguro M, Suzuki K. 2010. Amycolatopsis helveola sp. nov. and A mycolatopsis 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, K artina 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