Diversity of fungal endophytes in shrubby medicinal plants of Malnad region, Western Ghats, Southern India

A total of 6125 fungal endophytes were isolated from 9000 leaf segments of 15 medicinal shrubs growing in Malnad region of Western Ghats, Southern India, during winter, monsoon, and summer seasons. These fungal isolates belonged to Ascomycota (8.6 %), Coelomycetes (26.0 %), Hyphomycetes (28.0 %), Mucoromycotina (0.3 %) and sterile forms (4.9 %). Alternaria, Chaetomium, Fusarium, Colletotrichum, Cladosporium, Penicillium, Phyllosticta and Xylaria were the most frequently isolated. Significantly more isolates were obtained during the winter season than monsoon and summer seasons. a 2008 Elsevier Ltd and The British Mycological Society. All rights reserved. Introduction heavy metals in soils, propensity for vegetative reproduction Foliar fungal endophytes are a fundamental but frequently overlooked aspect of plant biology (Arnold & Lutzoni 2007). They are ubiquitous and have been found in all plant species examined to date (Stone et al. 2000). Endophyte host interactions represent a continuum from strong antagonism to obligate mutualism (Saikkonnen et al. 1998; Clay & Schardl 2002). Endophytes may also act as pathogens and saprotrophs, in some cases attacking or decaying hosts with which they do not form endophytic associations (Promputtha et al. in press). In mutualistic associations, infected plants benefit by, for example, exhibiting increased resistance to herbivore grazing through the production of various alkaloids (Owen & Hundley 2004), improved growth and competitive ability by increasing the mineral uptake potential, plant phenotypic traits, temperature and drought tolerance, leaf chemistry, tolerance of 246. (B. Shankar Naik). er Ltd and The British My (Malinowski et al. 2000; Redman et al. 2002) and defence against microbial pathogens (Arnold et al. 2003; Rubini et al. 2005). Further, fungi present in the healthy tissues of plants can promote the invasion of host plant communities with greater species diversity (Rudgers et al. 2005) and alter the nutrient cycle in individual plants and in ecosystems (Garcia & Langenheim 1990; Lodge et al. 1996). In addition, fungal endophytes have been recognized as a repository of novel compounds of immense value in agriculture, industry and medicine (Tan & Zou 2001; Strobel & Daisy 2003; Kumar et al. 2004; Kumar et al. 2005). Hence, there are major efforts to isolate and characterize endophytes from plants that have an ethnobotanical history (i.e. use by indigenous peoples) that is related to the specific uses or applications. Ongoing global efforts to discover novel compounds from endophytic fungi isolated from medicinal plants are cological Society. All rights reserved. 90 B. Shankar Naik et al. yielding good results (Liu et al. 2004; Li et al. 2005; Phongpaichit et al. 2006; Zhou et al. 2007). People in the Indian subcontinent have a long history of using medicinal plants to cure various diseases. Medicinal plants of Western Ghats of India (one of the hot spots of global biodiversity) are reported to have a diverse community of endophytic fungi (Raviraja 2005; Krishnamurthy et al. 2008). Few studies on the endophytic fungi of these plants have been conducted. The present study was undertaken to investigate the diversity of endophytic fungi and their seasonal colonization pattern in medicinal shrub species commonly used in the Malnad region, Western Ghats of Karnataka, Southern India. Materials and methods Sample collection and isolation of endophytes Apparently healthy leaf samples from 15 plant species (Table 1) were collected, brought in sterile polythene bags to the laboratory and processed within 24 h of collection. Surface sterilization of samples was done by cleaning leaves under running tap water and cutting them into 1 cm segments followed by stepwise washing with 70 % ethanol for 2 min, sodium hypochlorite solution for 5 min and 70 % ethanol for 30 s followed by two rinses in sterile distilled water, then allowed to surface dry under sterile conditions (Arnold et al. 2000). Leaf segments were placed on 9 cm Petri plates containing potato dextrose agar (PDA, Hi Media Laboratories, Mumbai, India) medium amended with streptomycin (250 mg L ) to suppress bacterial growth. The efficacy of surface sterilization was confirmed by pressing the sterilized leaf segments on to the surface of PDA medium. The absence of growth of any fungi on the medium confirmed that the surface sterilization procedure was effective (Schulz et al. 1993). Petri plates were incubated at 28 1 C with a 12 h photoperiod, and sporulation was induced by incubation in a light chamber under near UV light for 1–12 d. Fungi growing out from the leaf segments were subsequently transferred onto fresh PDA plates. Pure cultures were spread on fresh PDA slants. Endophytic fungal Table 1 – Plants from which endophytic fungal isolations were