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Paper Received: Colletotrichum, Phomopsis and Phoma Were Dominant, Their Relative Frequencies Were 23.07%, 31 December 2014 17.58% and 12.08% Respectively

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Paper Received: Colletotrichum, Phomopsis and Phoma Were Dominant, Their Relative Frequencies Were 23.07%, 31 December 2014 17.58% and 12.08% Respectively JEB Journal of Environmental Biology Website : www.jeb.co.in ISSN: 0254-8704 (Print) ISSN: 2394-0379 (Online) E-mail : [email protected] CODEN: JEBIDP Distribution and antimicrobial potential of endophytic fungi associated with ethnomedicinal plant Melastoma malabathricum L. Vineet Kumar Mishra11 , Garima Singh , Ajit Kumar Passari 1 , Mukesh Kumar Yadav 2 , Vijai Kumar Gupta31 and Bhim Pratap Singh * 1Department of Biotechnology, Mizoram University, Aizawl-796 004, India 2Korea University College of Medicine, Korea University, Seoul, 02841, South Korea 3Molecular Glyco-biotechnology Group, Department of Biochemistry, National University of Ireland Galway, Galway, H91CF50, Ireland *Corresponding Author E-mail: [email protected] Abstract Distributions of endophytic fungi associated with ethnomedicinal plant Melastoma malabathricum L. was studied and 91 isolates belonging to 18 genera were recovered. The isolates were distributed to sordariomycetes (62.63%), dothideomycetes (19.78%), eurotiomycetes (7.69%), zygomycetes (4.19%), agaricomycetes (1.09%), and mycelia sterilia (4.39%). Based on Publication Info colony morphology and examination of spores, the isolates were classified into 18 taxa, of which Paper received: Colletotrichum, Phomopsis and Phoma were dominant, their relative frequencies were 23.07%, 31 December 2014 17.58% and 12.08% respectively. The colonization rate of endophytic fungi was determined and found to be significantly higher in leaf segments (50.76%), followed by root (41.53%) and stem tissues (27.69%). All the isolates were screened for antimicrobial activity and revealed that Revised received: 26.37% endophytic fungi were active against one or more pathogens. Twenty four isolates 09 September 2015 showing significant antimicrobial activity were identified by sequencing the ITS1-5.8S-ITS2 region of rRNA gene. Results indicated that endophytic fungi associated with leaf were Accepted: functionally versatile as they showed antimicrobial activity against most of the tested pathogens. 06 October 2015 The endophytic fungi Diaporthe phaseolorum var. meridionalis (KF193982) inhibited all the tested bacterial pathogens, whereas, Penicillium chermesinum (KM405640) displayed most significant antifungal activity. This seems to be the first hand report to understand the distribution and antimicrobial ability of endophytic fungi from ethno-medicinal plant M. malabathricum. Key words Antimicrobial activity, Colonization frequency, Endophytic fungi, Melastoma malabathricum Introduction endophytic population among different host plants varies (Naik et al., 2008). However, researchers till date are able to Bioprospection of functional microbial diversity explore only limited plants for the isolation and from unusual and pristine habitat for the search of new characterization of endophytic fungal population (Hyde and secondary metabolites is an emerging area of research. Soytong, 2008; Tejesvi et al., 2011). Interestingly, some of Endophytic fungi which reside and colonize diverse atypical the endophytic fungi isolated from a particular host was niche of plant tissues are one of the important source for shown to produce same chemical compound as produced by searching of new novel bioactive agents (StrobelOnline and Daisy, their respective host (AlyCopy et al., 2010), which undoubtedly 2003; Qadri et al., 2014). Endophytes are ubiquitously showed their importance. These observations led to isolation present in broad range of host plant species and have been of Paclitaxel (TaxolTM ) from an endophytic fungi Penicillium reported from various climatic zones (Ghimire et al., 2011; Li aurantiogriseum NRRL 62431 isolated from the bark of yew et al., 2012; Verma et al., 2014). It has been shown that the Taxus brevifolia (Yang et al., 2014). Furthermore, several © Triveni Enterprises, Lucknow (India) Journal of Environmental Biology, Vol. 37, 229-237, March 2016 230 V.K. Mishra et al. studies have demonstrated the potential of endophytic fungi Tissues (leaves, stems and roots) were separated from associated with medicinal plants which synthesizes plethora plants for surface sterilization and isolation of endophytic of secondary metabolites with diverse biological activities fungi with minor modifications (Cannon and Simmons, like antimicrobial, anti-cancerous, immunosuppressive, 2002). The plant samples were washed thoroughly in running insecticidal and anti-parasitic (Daisy et al., 2002; Vieira et tap water for 10 min. The samples were surfaced sterilized al.,2012; Martinez-Luis et al., 2012). with 70% ethanol for 1 min, 3% sodium hypochlorite for 1 min and subsequently rinsed thrice with sterile distilled Melastoma malabathricum L. belongs to family water. The outer surface was removed with a sterile scalpel Melastomaceae, and it is widely distributed in south-East and samples were excised into 1.0 cm2 pieces. Tissues were Asian region. This plant is a small shrub, which has an thoroughly rinsed using sterile distilled water followed by important place in Chinese, Indian, Malay and Indonesian drying on sterile blotters to ensure complete drying. Prior to ethno-medicine. Traditionally, leaves are used as a plating, the tissues were washed again with distilled water. preventive scarring against smallpox, piles, gastric ulcer, The water obtained from the last wash was spreaded on PDA dysentery, skin scars and as a tonic; shoots are taken to treat medium to ensure the effectiveness of surface sterilization puerperal infection, high blood pressure and diabetes; while and fingerprints of the tissues were also taken on media plates roots are used to diminish rheumatism, arthritis and as control to cross check the epiphytic fungal growth. One toothache, whereas flowers are used to treat cancer (Sharma hundred and ninety five tissues were selected from each leaf, et al., 2001; Rai and Lalramnghinglova, 2010; Joffry et al., stem and root region of the plant. Tissues were placed on petri 2012). Emerging scientific findings also supports an array of dishes containing Potato Dextrose Agar (PDA), Malt Yeast medicinal and pharmacological activities of various parts of Extract Agar (MYA ) and Czapex Dox Agar (CDA) medium M. malabathricum such as antibacterial, anti-inflammatory, supplemented with streptomycin (100 mg l-1 ) to suppress wound healing, anti diarrheal, cytotoxic, antiviral, bacterial growth. Inoculated Petri plates were incubated at 28 antiparasitic, antioxidant and antipyretic (Wiart et al., 2004; 0C under 12 hr white light: 12 hr dark cycles (Bills and Zakaria et al., 2006; Susanti et al., 2007; Sunilson et al., Polishook, 1991). Petri plates were observed daily till 2-3 2009). Endophytic fungi associated with Melastoma weeks. Mycelium coming out from sterile tissues was malabathricum have also been reported to have antifungal transferred to fresh plates free from antibiotics for further activities against fungal plant pathogens and their ability to purification. Pure cultures were left to sporulate for one decolorize synthetic dyes (Kokaew et al., 2007; Ngieng, month on PDA media and then microscopic identification 2013). was carried out for vegetative and reproductive structures Keeping in view the medicinal properties of the host (Riddel, 1950). Morphology based taxonomy was used for and its unexplored nature, the present study was focused to identification of sporulating fungi (Barnett and Hunter, investigate the diversity and antimicrobial potential of 1972). endophytic fungi associated with traditional ethno-medicinal Preparation of fungal extracts : The endophytic fungi were plant M. malabathricum. cultured in Potato Dextrose Broth (PDB) medium for three weeks at 28 oC. The cultures were extracted by using Materials and Methods methanol for mycelial mat and ethyl acetate for aqueous Sample collection and isolation of endophytic fungi : The extract. Both the extracts were air dried by using rotary plant samples were collected from Phawngpui National Park, evaporator system (BUCHI, Switzerland) and mixed Mizoram (22°40'N; 93°03'E), the highest mountain peak in together to get a crude solid extract. The crude extract was Mizoram, northeast, India, rising about 2,157 meters high dissolved in methanol and filtered through Whatman filter near the Myanmar border and famous for orchids and paper to remove residues which were not dissolved in Rhododendrons. This plant is abundantly found in North methanol; then the filtrate was diluted to 10 mg ml-1 with Eastern region of India. To understand the diversity of methanol. endophytic fungi associated with M. malabthricum (n=10), plants were randomly selected. To avoid identical population Evaluation of antimicrobial activity of fungal extracts : of endophytes, distance of at least 100 m was taken into Antimicrobial activity of the extract from endophytic fungi consideration between two plants. Herbarium specimen was was tested against fungal and bacterial pathogens by agar cup prepared and submitted to the Department of Forestry, diffusion method (Tayung and Jha, 2010). The test organisms Mizoram University, India. The plant samples wereOnline placed in include two Gram Copynegative bacteria: Pseudomonas labeled polyethylene bags, transported to laboratory and aeruginosa (MTCC 2453) and Escherichia coli (MTCC placed in refrigerator at 4 0C. The samples were processed 739), one Gram positive bacteria: Staphylococcus aureus within 24 hr of collection. (MTCC 96) and three fungal pathogens: Fusarium Journal of Environmental Biology, March 2016 Antimicrobial potential of endophytic
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