International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 3(4), 13-17, April (2014) Int. Res. J. Environment Sci. International Science Congress Association 13 Diversity of Actinobacteria in Mangrove Ecosystem of Muthupet, IndiaPriyaE.*, Thenmozhi R., NagasathyaA., ThajuddinN. and Muralitharan G.Department of Microbiology, JJ College of Arts and Science (A), Namunasamudram, Sivapuram, Pudukkottai- 622422, Tamilnadu, INDIA Department of Zoology, Govt Arts College for women, Pudukkottai- 622001, Tamilnadu, INDIA Department of Microbiology, Bharathidasan University, Tiruchirapalli-24, Tamilnadu, INDIA Available online at: www.isca.in, www.isca.me Received 12th February 2014, revised 8th March 2014, accepted 21th April 2014 AbstractTotally 57 actinobacteria isolates were obtained from four sediment samples collected from four different seasons (pre monsoon, post monsoon, summer and monsoon) of Palk Strait region situated along the, South East coast of India. Among them, 6 dominant isolates in all seasons were found to be morphologically identified on the basis of color of front mycelium and reverse side color formation and sporophore morphology. The primary identification of this isolates were based on biochemical and physiological characteristics of the isolated strains. Furthermore, the analysis of nucleotide sequence of the 16SrRNA sequencing. Six isolates were assigned to the actinobacteria Streptomyces niveoruber, S.heliomycini, S.flavomacrosporus, Lechevalieria aerocolonigenes, L.flava andDactylosporancium vinaceum. Keywords: Diversity,actinobacteria, mangrove ecosystem,16S rRNA sequencing. Introduction The mangrove forest and associated with water bodies are together called mangrove wetland. The mangrove forest is rich in biological diversity and it is a globally significant habitat for wildlife. Muthupet mangrove ecosystem is located at the South east Coast of India. Mangroves of muthupet (10° `-20° N, 79° 35`E), Tamil Nadu. Mangrove has a salty ecosystem and it is known to be large sources of organic matter due to various microbial enzymatic and metabolic activities. Actinomycetes are all Gram-positive, filamentous and are facultatively anaerobic. All species grow best under anaerobic conditions. Actinomycetes play an important role among the mangroves bacterial communities, because of its diversity and ability to produce novel chemical compounds of high commercial value. Actinomycetes participate in many important biochemical processes in the soil Actinobacteria are a well known source of various secondary metabolites such as lignocellulose, hemicellulose immunomodulators, anti-infective and anticancer agents 5,6. The present work highlights on the diversity study of potential actinobacteria in mangrove ecosystem. Material and Methods Collection of sample: Sediment soil sample collected randomly from Muthupet mangroves (Lat.10°20’N & Long.79°35’ E) South East Coast, Tamil Nadu, at different seasons (pre monsoon, monsoon, post monsoon and summer) (figure 1). Media and culture conditions: The starch casein agar medium used for the isolation and cultivation of actinobacteria. The sterilized 50ml of sea water and 50ml distilled water. After autoclaving, the medium was supplemented with nylidixic acid and amphotercin B (Himedia, Mumbai) 10 µg/ml respectively as antibacterial and antifungal agents to inhibit the bacterial and fungal contamination. The diluted sediment samples (0.1 ml) were spread over the medium with a sterilized bent (L) rod and plate spinner. The inoculated plates were incubated at 30 C for 3 to 7 days. After incubation, colonies were recorded. The pure colonies were transferred on agar slants and preserved for further analysis. Identification of isolated cultures: The growths of the 6 isolates were inoculating in to various culture media such as ISP1, ISP 2, ISP3, ISP 4, ISP 5, ISP6 and ISP7 incubated at 28C for 14 days. After growth, the slide cultures were examined under light microscope. Color of spore mass visually estimated by using the colour chart. The isolates to determine the production of acids by utilizing the different sources of carbohydrates like adonitol, arabinose, glucose, galactose, glycerol, fructose, lactose, mannitol and xylose were tested by inoculating the isolates in ISP1 broth supplemented respective sugars and incubated for 7 day at 30°C. Sodium chloride concentrations (5 and 9%) were poured to the ISP2 medium. The plates were incubated at 28 C for 7 days. After incubation growth was recorded. Physiological characterization of isolates carried out by performing the growth at different temperatures range from 15,20,28,37 and 45°C, pH range from 5 to 12 and the growth under anaerobic condition. 16S rRNA Sequencing of the Isolates: Genomic DNA was separated from isolate cultures following Pospiech and Neumann, 1995. The 16S rRNA was amplified using universal primers FD1 (5’-AGAGTTTGAT- CCTGGCTCAG-3’) and RP2 (5-ACGGCTACCTTGTT- ACGACTT-3’)10. The PCR products were purified and sequenced by Sri Bio Tech Company Limited (Hyderbad). International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(4), 13-17, April (2014) Int. Res. J. Environment Sci. International Science Congress Association 14 Figure-1 Map showing the study area of Muthupet mangrove Phylogenetic analysis of isolates: 16S rRNA sequences of 6 strains were aligned with 16S rRNA sequences of other actinomycetes retrieved from the EMBL/GenBank database. Multiple alignments were performed manually using Clustal x11. Neighbor-joining phylogenetic tree and molecular evolutionary analyses were conducted using MEGA version 412. Nucleotide sequence accession numbers: The 16S rRNA sequences of 6 isolates have been deposited in the GenBank library under accession numbers KF414963–KF414968. Results and DiscussionIn study area situated at muthupet mangroves ecosystem, South East coast of India. During the study period the seasonal distribution of actinobacteria counts were found maximum during pre monsoon season in (18.0 x 10-6CFU /g) when compared to other season and minimum during post monsoon season in (10.0 x 10-6CFU /g). Moreover, the maximum number of actinobacteria were recorded in pre monsoon season is due to the rich amount of organic substrate, light, temperature and other variable of the limiting factors determine the diversity of actinobacteria. The occurrence of microbial populatiom, plant species, nutrient status and salinity, and other environmental variables13,14. This might be attributed to nutrient accumulation, precipitation of inorganic compounds and settlement of organic matter in the mangrove sediments15. In Similar report by Sivakumar, 2001, the, counts of actinomycetes diversity were found maximum during monsoon season in mangrove forest13. International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(4), 13-17, April (2014) Int. Res. J. Environment Sci. International Science Congress Association 15 Totally 57 isolates of different color colonies of actinobacteria were isolated from the mangroves soil. Many actinomycetes reported by the present study are white colour morphology. In a study by Vanajakumar et al., 1991, reported that, out of 192 colonies belong to 68 isolates white colour series actinobacteria were the dominant forms than the gray and pink colour series16. The results obtain by the present studies correlated by the finding of Sivakumar and suganthi, 2011, the 107 actinomycetes isolated from different mangrove17. Among the isolates, six were found to be dominant in all seasons. These organisms grew well on the other growth media. The actinobacteria isolates like GACMPT4, GACMPT9, GACMPT57, GACMPT8, GACMPT44 and GACMPT 34 showed good growth was observed on different media (table1). Good growth were observed various carbon sources, pH 7 and 9, temperature 28ºC and 37ºC and salinity 5 and 9 % (table 2). After PCR amplification from strains GACMPT4, GACMPT9, GACMPT57, GACMPT8, GACMPT44 and GACMPT34, the 16S rRNA gene partical sequenced. All six Strains were deposited in Genbank, USA and the strains were assigned the accession numbers Streptomyces niveoruber (KF414963), S.heliomycini (KF414964), S.flavomacrosporus (KF414965),Lechevalieria aerocolonies (KF414966), L.flava (KF414967) and Dactylosporangiun vinaceum (KF414968). Table - 1 Cultural characteristics of GACMPT4 (Streptomyces niveoruber) on different media Media Front pigmentation Reverse side pigmentation Yeast extract agar ( ISP1) Grey Yellow Yeast extract malt extract agar (ISP2) Whitish grey Orange Oat meal agar (ISP3) Whitish grey White Inorganic salt agar ( ISP4) Whitish grey Violet Glycerol aspergine agar( ISP5) Whitish grey Pale yellow Peptone yeast extract iron agar (ISP6) Whitish grey Orange Trysine agar (ISP7) Whitish grey Pale yellow Cultural characteristics of GACMPT9 (Streptomyces heliomycini) on different media Medium Front pigmentation Reverse side pigmentation Yeast extract agar (ISP1) White Orange Yeast extract malt extract agar( ISP2) Dull white Orange Oat meal agar (ISP3) White Pale yellow Inorganic salt agar (ISP4) White Pale yellow Glycerol aspergine agar (ISP5) Yellowish white Pale yellow Peptone yeast extract iron agar( ISP6) White Pale yellow Trysine agar (ISP7) Yellowish white Pinkish white Cultural characteristics of GACMPT57 ( Streptomyces flavomacrosporus) on different media Media Front pigmentation Reverse side pigmentation Yeast extract agar( ISP1) White Pale yellow Yeast extract malt extract agar (ISP2) White Pale yellow Oat meal agar ( ISP3) White Pale yellow Inorganic salt agar( ISP4) White Pale yellow Glycerol aspergine agar( ISP5) White Pale yellow Peptone yeast extract iron agar (ISP6) White Yellowish white Trysine agar (ISP7) White Pinkish white Cultural characteristics of GACMPT8 ( Lechevalieria aerocolonies) on different media Media Front pigmentation Reverse side pigmentation Yeast extract agar(ISP1) Grey Pale yellow Yeast extract malt extract agar (ISP2) Whitish grey Orange Oat meal agar (ISP3) Dull White Pale yellow Inorganic salt agar (ISP4) Whitish grey Orange Glycerol aspergine agar (ISP5) Grey yellowish White Pale yellow Peptone yeast extract iron agar (ISP6) Whitish grey Pale yellow Tyrosine agar (ISP7) Whitish grey Pale yellow International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(4), 13-17, April (2014) Int. Res. J. Environment Sci. International Science Congress Association 16 Cultural characteristics of GACMPT44 ( Lechevalieria flava) on different media Media Front pigmentation Reverse side pigmentation Yeast extract agar (ISP1) White Pale yellow Yeast extract malt extract agar( ISP2) White Pale yellow Oat meal agar( ISP3) White Pale yellow Inorganic salt agar( ISP4) White Dark yellow Glycerol aspergine agar (ISP5) White Pale yellow Peptone yeast extract iron agar( ISP6) White Pale yellow Tyrosine agar (ISP7) White Dark yellow Cultural characteristics of GACMPT34 ( Dactylosporangium vinaceum) on different media Media Front pigmentation Reverse side pigmentation Yeast extract agar( ISP1) Whitish grey Pale yellow Yeast extract malt extract agar( ISP2) Grayish pink Reddish brown Oat meal agar (ISP3) Whitish pink Orange Inorganic salt agar (ISP4) Whitish grey White Glycerol aspergine agar( ISP5) White Pale yellow Peptone yeast extract iron agar( ISP6) Whitish pink Whitish pink Tyrosine agar (ISP7) White Pale yellow Table - 2 Physiological and biochemical characteristics of isolates Tests GACMPT 4 GACMPT9 GACMPT57 GACMPT 8 GACMPT 44 GACMPT 34 Carbon sources 1% W/V Adonitol + + + + + + Arabinose + + + + + + Glucose + + + + + + Galactose + + + + + + Glycerol + + + + + + Lactose + + + + + + Mannitol + + + + + + Fructose + + + + + + Xylose + + + + + + Effect of pH 5 _ _ _ _ _ _ 7 + + + + + + 9 + + + + + + 12 _ _ _ _ _ _ Effect of T  15C _ _ _ _ _ _ 20C _ _ _ _ _ _ 28C + + + + + + 37C + + + + + + 45C _ _ _ _ _ _ Effect of NaCl concentration W/V 5% + + + + + + 9% + + + + + + International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 3(4), 13-17, April (2014) Int. Res. J. Environment Sci. International Science Congress Association 17 ConclusionThe present study concludes, that the diversity of actinobacteria were maximum found in the pre monsoon season when compared to the other season due to the favorable nutrient, temperature, pH and salinity were present in this season. It contributes to the knowledge status of microbial diversity in mangrove ecosystem. Actinobacteria is most widely exploited microorganisms in terms of their capabilities in production of antibiotics and other compounds of biotechnology importance uses. References 1.Kathiresan K., Orissa coast: a global paradise for mangroves, Proc. Symposium on wetland and mangrove biodiversity in Orissa coast, 7-12 (2008) 2.Kathiresan K and Bingham. B.L., Biology of mangroves and mangrove ecosystems, Advances in Marine Biology., 40, 81-251 (2001) 3.Hopwood D.A., Therapeutic treasures from the deep,. 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