61 INTRODUCTION MATERIALSAND METHODS Isolation and Identification endophytic fungi: Aponogeton natans Aponogetonaceae et al et al et al et al et al et al et al Ascomycetes Hyphomycetes Coelomocytes Basidiomycetes et al et al Aponogeton natans Aponogeton natans et al et al et al is an aquatic and medicinally very important plant belonging to the family . This plant parts as well as their extracts were used to treat anti diabetic, anaemia and haemothermia. It grows in seasonal, permanently still or free flowing waters, rice fields and marshy places. It is an important ornamental plant for aquariums and its tubers are fit for human consumption (Jamith Basha, 2016). Endophytic fungi reside in different parts of the plants like leaf, stem, bark, flower, etc. (Suryanarayanan, 2017). They are highly localized and are transmitted horizontally. As of now, endophytes have been isolated from all groups of plants ranging from sea grasses (Alva., 2002), lichens (Li ., 2007), palms (Taylor ., 1999; Frohlich ., 2000), herbs, shrubs andlarge trees, etc. (Gonthier ., 2006; Oses ., 2008; Rajagopal, 1999). Endophytic fungi from tropical aquatic and wetland habitats were limited when compared to those growing on terrestrial moist or dry habitats, native hydrophytes and fresh water marshes (Kumar ., 2014; You ., 2016) Most endophytes isolated belongs to and their anamorphs, , and (Rungjindamai ., 2008). Endophytic fungi residing in plants serve as important sources of new and novel metabolites which are bioactive (Vellingiri Manon Mani ., 2015). Endophytes in general and endophytic fungi in particular from hydrophytes are poorly investigated even though they are important plant populations in a community. Hence, the aim of the present investigation was isolation and identification of endophytes from leaf and testing the selected endophytic fungal extracts for antibacterial activity. Ten to fifteen slender twigs of were collected from pond near Chennai city, Tamilnadu from April to June. Undamaged and healthy 5 to 10 leaves were collected from each twig. The leaves were collected in zip cover (sterile polythene bags) and processed within 24 hours of collection in the laboratory. The leaf segments of 0.5x0.5 cm were cut from the plant samples using sterile scalpel (Cabral ., 1993). These were then surface sterilized in 70% ethanol for 5 sec., immersed in 4% sodium hypochlorite (NaOCl) for 10 sec. and rinsed in autoclaved double distilled water (Dobranic ., 1995). The leaf segments were placed on Potato Dextrose Agar (PDA) medium amended with chloramphenicol (100 mg/L). The inoculated Petri plates were incubated at 27±1 C in light chamber for 2- 4 weeks for the growth of endophytic fungi. The light regime was kept at 12 hours followed by 12 hours darkness (Bills and Polishook, 1992). The endophytic fungal hyphae, which grew out from the leaf segments, were transferred to fresh PDA slants and maintained for further study. The fast growing fungi which inhibited the slow growing species, the were removed using sterile needle (Bills, 1996; Bills and Polishook, 1992). The endophytic fungi were identified morphologically using standard keys like conidia structure, conidial attachment and the fruit body structure (Onions, ., 1981). The non sporulating sterile forms were given code numbers based on their colony color, texture, and mycelial structure (Rajagopal, 1999). The colonization frequencyof each endophyte was calculated as number of leaf segments that 2 0 KAVAKA53: 61-66(2019) W. Jamith Basha , Kalyanaraman Rajagopal , B. Meenashree , R. Arulmathi , A.K. Kathiresan , G. Gayathri , G. Kathiravan and S.S. Meenambiga * Diversity and antibacterial activity of endophytic fungi associated with a hydrophyte Aponogeton natans 1 2 3 4 5 5 2 6 1 2 3 4 5 6 Department of Microbiology, Northern Border University, Arar, Kingdom of Saudi Arabia-91431. Department of Botany, Ramakrishna Mission Vivekananda College (Autonomous), Chennai- 600004, India. Asthagiri Herbal Research Foundation, Chennai- 600096 Department of Biotechnology, School of Life Sciences, Vels Institute of Science Technology and Advanced Studies (VISTAS), Chennai- 600117, India. Department of Microbiology, School of Life Sciences, Vels Institute of Science Technology and Advanced Studies (VISTAS), Chennai- 600117, India *Department of Bio-Engineering, School of Engineering, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Chennai, India. *CorrespondingAuthor Email: meenambiga.se@velsuniv.ac.in (Submitted onAugust 10, 2019;Accepted on November 12, 2019) ABSTRACT An endophytic fungus is an integral part the plant micro biome and colonizes the plant both systemically and non-systemically. In the present investigation endophytic fungi were isolated from leaf of a medicinally important hydrophyte, . Three hundred and fifty one isolates belonging to 15 different species were isolated. , sp., , and sp. showed higher colonization frequency. Alkaloids and Flavonoids were produced by all endophytic fungi in their crude extract and terpenoid was produced by 14 endophytic fungi. The antibacterial activity of ethyl acetate and diethylether extracts of four dominant endophytic fungi, , , sp. and Sterile form I were tested. Bioactive compounds produced in ethyl acetate extract was more effective than diethylether compounds in inhibiting pathogens. Thus, this study provides an insight on the diversity of endophytic fungi and their varied anti-bacterial properties. KEYWORDS: Aponogeton natans Alternaria alternata Cytospora Aspergillus fumigatus Chaetomium incomptum Phomopsis viz. Alternaria alternata Chaetomium incomptum Phomopsis Hydrophyte, leaf, alkaloid, flavonoid, gram positive bacteria, gram negative bacteria Adoi:10.36460/Kavaka/53/2019/61-66