Isolation and Characterization of Endophytic Actinomycetes from Mangrove Plant for Antimicrobial Activity

Int.J.Curr.Microbiol.App.Sci (2013) 2(11): 78-89 ISSN: 2319-7706 Volume 2 Number 11 (2013) pp. 78-89 http://www.ijcmas.com Original Research Article Isolation and characterization of Endophytic Actinomycetes from mangrove plant for antimicrobial activity P.Gayathri* and V. Muralikrishnan Department of Microbiology, Annamalai University, Annamalai Nagar, Chidambaram, Tamilnadu, India *Corresponding author: A B S T R A C T Endophytes are Microbes that colonize living, internal tissues of plants without carrying any immediate over negative effects. In this present investigation the endophytic actinomycetes were isolated from root, stem and leaf of two species of mangrove K e y w o r d s such as, Rhizopora apiculata and Avicennia marina for their antimicrobial activity. Totally 6 endophytic actinomycetes were obtained from all the samples. Population Endophytes; enumeration, phenotypic characters like substrate mycelium, aerial mycelium, Actinomycetes; aerial mass colour, reverse side pigment, melanoid pigment, spore chain Mangrove; morphology, salt tolerance on growth, growth on different pH, effect of different Antimicrobial temperature on growth, Biochemical characteristics, assimilation of different activity; carbon sources, nitrogen sources and enzymatic activity were studied. For Streptomyces antimicrobial activity 7 bacterial pathogens and 4 fungal pathogens were used. Up coelicolor. on the 6 isolate 1 endophytic actinomycetes isolate (MAR1) shows good activity against all the isolates. Based on the above characteristics the potential isolate MAR1 is identified as Streptomyces sp. and species may be Streptomyces coelicolor. Introduction Endophytes are Microbes that colonize ecological and economic significance living, internal tissues of plants without (Kathiresan, 2000). However, mangroves carrying any immediate over negative exist under condition of high salinity, effects" an inclusive and widely accepted extreme tides, strong winds, high definition of endophytes by Bacon and white temperature and muddy, anaerobic soils, (2000). Endophytes could be better These plants, and the associated microbes, protected from biotic and abiotic stresses fungi, plants and animals, constitute the than rhizosphere bacteria (Gayathri and mangrove forest community or mangal Muralikrishnan, 2013). The mangrove (Kathiresan and Bingham, 2001). forests are one among the world s most Actinomycetes are gram positive bacteria, productive ecosystems with great with a high guanine (G) plus cytosine (C) 78 Int.J.Curr.Microbiol.App.Sci (2013) 2(11): 78-89 ratio in their DNA (>55mol %), which are Sample pretreatment and endophytic phylogenetically related from the evidence actinomycetes isolation of 16S ribosomal cataloguing and DNA: rRNA pairing studies (Goodfellow and For pretreatment and isolation modified Williams, 1983). The name method of Lixiang Coa et al., (2004) were Actinomycetes was derived from Greek adopted. The root, leaf and stem samples atkis (a ray) and mykes (fungus), and were washed in running tap water to has features of both Bacteria and fungi remove soil particles and sterilized by (Das et al., 2008). In the strict taxonomic sequential immersion in 70% ethanol for 5 sense, actinomycetes are clubbed with min and a solution of sodium hypochlorite bacteria in the same class of (0.9% available chlorine) for 20 min. Schizomycetes but confined to the order Samples were washes in sterile water three Actinomycetales (Kumar et al., 2005). times to remove surface sterilization agents. Each samples was divided into small fragments and plated on starch As the frequency of novel bioactive casein agar medium (Kuster and Williams, compounds obtained from terrestrial 1964). Nystatin and Cycloheximide (50 Actinobacteria decreases with time, µg/ml of each) were added to media to Actinobacteria from diverse environments suppress the fungal growth (William and have been increasingly screened for their Davies, 1965) and incubated at room ability to produce new secondary temperature for 7 10 days. After metabolites. In the recent years, terrestrial incubation plates were observed for and marine microorganisms have known growth of endophytic actinomycetes. for antimicrobial, antiviral, antitumour, anticoagulant, antidiabetic and cardio Phenotypic characterizations active properties. Antibiotic effect of actinomycetes has been used in many The classification of actinomycetes was fields including agriculture, veterinary and originally based largely upon the pharmaceutical industry. morphological observations. So, morphology is still an important Based on the above information the characteristic for the description of taxa present study is conducted to isolate and it is not adequate in itself to bacterial endophytic actinomycetes from differentiate between many genera. In fact, different parts of mangrove plants to it was the only characteristic which was identify better antimicrobial compound. used in many early descriptions, particularly of Streptomyces species in the Materials and Methods first few editions of Bergey's Manual. These observations are best made by the Sample collection and transport variety of standard cultivation media. Several of the media suggested for the Samples were collected from Parangipettai International Streptomyces Project and by and Pichavaram. All the Samples were Pridham et al., (1957) have proven to be collected in a sterile plastic covers, useful in our hands for the characterization transferred to laboratory and processed of strains accessioned into the ARS immediately. 79 Int.J.Curr.Microbiol.App.Sci (2013) 2(11): 78-89 Actinomycetales Culture Collection pigment or a distinct brown pigment (Labeda, 1985). It includes some basic modified by other color are recorded as tests viz., Aerial mass colour, Reverse side positive (+) total absence of diffusible pigment, Melanoid pigments, Spore chain pigment, are recorded as negative (-) for morphology and Spore morphology melanoid pigment production. (Shirling and Gottileb, 1966). Spore chain morphology Aerial mass colour A characteristic of the spore bearing For the grouping and identification of hyphae and spore chains is determined by Actinomycetes sp. the chromogenicity of the direct microscopic examination of the the aerial mycelium was considered to be culture area. Adequate magnification used an important character. The colours of the to establish the presence or absence of mature sporulating aerial mycelium are spore chains and to observe the nature of white, gray, red, green, blue and violet. spore chains is 40x. By the standard When the aerial mass colour falls between protocol of cover slip culture technique, two colours series, both the colors are the plates were prepared and after the recorded. In the cases where aerial mass incubation of 7 to 10 days it was observed. colour of a strain showed intermediate During this method of spore tints, then in that place both the colour morphological study, ISP 2 medium Plates series should be noted. were prepared. After solidification, by a sharp scalpel from the central portion of Reverse side pigments the plate, medium should be scooped out making a rectangular area. Then, three The strains are divided into two groups sterile cover slips were placed on the according to their ability to produce hollow rectangular space. Slowly characteristic pigments on the reverse side Actinomycetes spores have to be of the colony, called as distinctive (+) and inoculated at the edge of the cover slips not distinctive or none (-). A colour with touching the medium. The plates were low chroma such as pale yellow, olive or incubated at 28±2°C for 5 days and yellowish brown occurs, it is included in examined periodically taking out the cover the latter group (-). slips. Melanoid Pigments Assimilation of carbon sources The grouping is made on the production of The ability of different actinomycetes melanoid pigments (i.e. greenish brown, strains in utilizing various carbon brownish black or distinct brown pigment compounds as source of energy was modified by other colours) on the medium. studied by following the method The strains are grouped as melanoid recommended in International pigment produced (+) and not produced Streptomyces Project. Stock solution of 10 (-). For the melanoid pigment observation sugars i.e., Xylose, Inositol, Sucrose, the inoculated plates were kept under Raffinose, Fructose, Rhamnose and incubator for 4 to 5 days. The strains Mannitol having concentration of 10x was which shows cultures forming a greenish prepared in autoclaved water and sterilized brown to brown to black diffusible by filtering through 0.22 m pore size 80 Int.J.Curr.Microbiol.App.Sci (2013) 2(11): 78-89 Table.1 Stress tolerance test for endophytic actinomycetes (Saurav and Kannabiran, 2009) S. Stress Concentrations or Medium Incubation Method of No tolerance test differentiation used detection 1. Different NaCl 2.5, 5.0, 7.5, 10 and Starch 30°C for 7 Growth indicates concentration 12.5 % casein agar 15 days positive 2. Different pH 4, 5, 6, 7, 8, and 9 pH Starch 30°C for 7 Growth indicates casein agar 15 days positive 3. Different 25°C, 30°C, 35°C, Starch For 7 15 Growth indicates temperature 40°C and 45°C casein agar days positive Table.2 Biochemical characteristics of endophytic actinomycetes isolates (Shriling and Gottlieb., 1966) Biochemical Medium Indicator Method of S. No Incubation Test used used detection 1. Indole Test Peptone 30°C for 3 4 Kovac s Cherry red colour broth days reagent ring indicates positive 2 Methyl red MR broth 30°C

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