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F Ull L Ength Original R Esearch P Aper Copyright © 2013 By IYPF All rights reserved Open Access Contents Int. J. Drug Dev. & Res . | July-September 2013 | Vol. 5 | Issue 3 | ISSN 0975-9344 | www.ijddr.in Biological activities of Streptomyces species SRDP-07 isolated from soil of Thirthahalli, Karnataka, India Prashith Kekuda T. R 1,2* Abstract: Western Ghats are one of the biodiversity hotspots in the w orld. The present study was undertaken to investigate biological activities viz., antibacterial, 1 Dileep N insecticidal, anthelmintic and antioxidant activities of a Streptomyces species SRDP-07 isolated from a soil sample collected in Thirthahalli, Karnataka (state ), 1 FullLength Syed Junaid India. Isolation of actinomycetes was carried out by plating the serially diluted soil sample on Starch casein agar. Primary screening of the actinomycete isolates for 1 antibacterial activity was done by Cross streak method. Isolate SRDP-07, showing Rakesh. K. N prom inent inhibition of test bacteria in cross streak technique, was selected for further characterization and for determining biological activities. The isolate was 2 Sunita C Mesta grown in starch casein broth for 10 days and the culture filtrate was extracted with ethyl ace tate. Antibacterial activity of ethyl acetate extract was tested against a panel of 6 bacteria by Agar well diffusion assay. Insecticidal activity of the extract Onkarappa R 2 was evaluated in terms of larvicidal effect against 2 nd instar larvae of Aedes aegypti . Anthel mintic activity of the extract was performed by determining time 1 Department of Microbiology, taken for paralysis and death of adult Indian earthworms. Antioxidant activity of Page 268 Page 268 S.R.N.M.N College of Applied the extract was determined by DPPH free radical scavenging and Ferric reducing Sciences, NES Campus, Balraj assay. A total of 9 actinomyce tes were recovered on Starch casein agar by serial Original Urs Road, Shivamogga-577201, dilution-plating technique. The isolate SRDP-07 was identified as a species of Karnataka, India Streptomyces by cultural and microscopic characteristics. Gram positive bacteria 2 Department of Post were more susceptible to extract than Gram ne gative bacteria. The extract showed dose dependent mortality of larvae of A. aegypti . Anthelmintic effect of Graduate Studies and extract was lesser and the time taken for paralysis and death of worms were Research in Microbiology, considerably higher than that of reference anthelmintic. The radical s cavenging Sahyadri Science College and reducing potential of extract was also weaker when compared with (Autonomous), Shivamogga- reference standard. The results of the present study indicate that the soils of 577203, Karnataka, India Thirthahalli are known to contain actinomycetes with potent biological activities. Research Paper Isolation of active principles present in the ethyl acetate extract of Streptomyces species SRDP-07 is under progress. Further screening programmes in soils of Corresponding Authors: Western Ghats of Karnataka can be fruitful in isolation of bioactive actinomycetes Prashith Kekuda. T. R which can be exploited for production of novel metabolites. E-mail: [email protected] Keywords : Western Ghats, Thirthahalli, Actinomycetes, Streptomyces , Biological activities endophytes) and others. They are involved in the INTRODUCTION decomposition of organic matter in soil, including Natural products produced from plants, animals lignin and other recalcitrant polymers, and can and microbes have been used for several years. degrade agricultural and urban wastes [2,3,4] . The However, the scientific knowledge of natural actinomycetes and their bioactive metabolites products has been known over the last 100 years have shown to possess antimicrobial, cytotoxic, [1] or so . Actinomycetes are high G+C containing, plant growth promotory, antiviral, antioxidant, filamentous, Gram positive bacteria that are insecticidal, antiprotozoal, anthelmintic, enzyme widespread in nature. They are distributed in a inhibitory, plant growth promoting and herbicidal variety of ecological habitats such as soil, fresh agents [5-15] . They are known to play key role in water, marine environment, plants (as Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., July-September 2013, 5 (3): 268-285 © 2013 Prashith Kekuda. T. R et al, publisher and licensee IYPF. Thi s is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited. bioremediation of dyes, petroleum products and Collection of soil sample pesticides and biosorption of heavy metals [16-20] . The soil sample was collected at Thirthahalli, The bioactive metabolites from actinomycetes Shivamogga (district), Karnataka (state), India are also active against antibiotic resistant during the month of July 2012. The soil was bacteria and plant pathogenic fungi [21-25] . collected in a sterile plastic cover from a depth of 15cm, brought to the laboratory and dried at Among actinomycetes, the genus Streptomyces is 40 oC under aseptic conditions [35] . represented in nature by the largest number of species and varieties. These organisms differ in Isolation of actinomycetes their morphology, physiology and biochemical For isolation of actinomycetes, the soil sample was characteristics. They have been distributed in subjected to serial dilution followed by plating on every possible ecological niche. Species of the Starch casein nitrate agar amended with genus Streptomyces are dominating in soil in terms antibiotic Fluconazole (to prevent fungal of number and the bioactive compounds what contamination). The plates were incubated they produce. These organisms have been aerobically at 30 oC for 10 days. Colonies of considered as potential sources of actinomycetes were selected on the basis of pharmaceutically and agriculturally important typical colony morphology. The isolates were rd bioactive agents. They have provided 2/3 of subcultured on Starch casein agar slants and Page 269 Page 269 species SRDP-07 isolated from soil of Thirthahalli, Thirthahalli, of soil from SRDP-07 isolated species naturally occurring antibiotics, such as maintained in refrigerator [35] . tetracyclines, aminoglysides, macrolides etc., discovered so far [22,26-30] . Western Ghats are one Primary screening for antibacterial potential of the of the biodiversity hotspots in the world. These are actinomycetes isolates grandly known for their diverse flora and fauna. Cross streak method was employed to screen Streptomyces Karnataka, India Karnataka, However, a very few microbiological studies have antibacterial efficacy of the actinomycete been done in this region. Few studies have been isolates. The actinomycete isolates were streaked carried out on the actinomycetes of Western at the centre of the sterile Starch casein agar Ghats of Karnataka and report antimicrobial, plates and the plates was incubated at 30 oC for 5 antioxidant, enzyme inhibitory, cytotoxic, days. After 5 days test bacteria were streaked anthelmintic, analgesic, anti-inflammatory and perpendicular to the growth of the actinomycete antipyretic activity [31-39] . The present study isolates. The extent of growth inhibition of the test describes isolation and the biological activities bacteria was observed after 24 hours of viz., antibacterial, insecticidal, anthelmintic and incubation. The absence of growth or a less dense antioxidant activity of a Streptomyces species growth of test bacteria near the actinomycete SRDP-07 from a soil sample collected at isolate was considered positive for production and Thirthahalli, Shivamogga (District), Karnataka secretion of antibacterial metabolite by the (State), India. isolates [40] . One isolate (designated as isolate SRDP-07) displaying marked inhibition of test bacteria was selected for further identification MATERIALS AND METHODS and for assessing biological activities. Covered in Scopus & Embase, Elsevier Int. J. Drug Dev. & Res., July-September 2013, 5 (3): 268-285 Prashith Kekuda T. R et al; Biological activities of Biologicalactivities of al; R et Kekuda T. Prashith © 2013 Prashith Kekuda. T. R et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited. Characteristics of the isolate SRDP-07 Fermentation and extraction of metabolite from The isolate SRDP-07 was further subjected for isolate SRDP-07 cultural, microscopic, staining and biochemical Sterile Starch casein broth containing Erlenmeyer characteristics. flasks were inoculated with the spore supsenion of well sporulated actinomycete culture and the Cultural characteristics flasks were incubated aerobically at 28 oC for 10 The isolate SRDP-07 was grown on various media days. After incubation, the broth was aseptically viz., Starch casein nitrate agar (SCA), Inorganic filtered through sterilized Whattman No. 1 filter salt-starch agar (ISSA), Actinomycetes isolation paper [35] . The culture filtrate was centrifuged and agar (AIA) and Tryptone yeast extract agar the supernatant was subjected solvent extraction FullLength (TYEA). The color of substrate and aerial mycelium in separation funnel and extracted using ethyl was noted. The plates were observed for the acetate. Equal volume (1:1) of supernatant and production of diffusible pigments. ethyl acetate were taken in a separation funnel and agitated for about 30 minutes. Solvent layer Microscopic characteristic was separated and the supernatant was again The cover slip method was followed to observe extracted
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