Antimicrobial potentials of endophytic fungi inhabiting three Ethno-medicinal plants of Similipal Biosphere Reserve, India
J Mohanta, K Tayung, U Mohapatra
Keywords
antimicrobial activity, ethno medicinal plants, fungal endophytes, similipal biosphere reserve, sp.
Citation
J Mohanta, K Tayung, U Mohapatra. Antimicrobial potentials of endophytic fungi inhabiting three Ethno-medicinal plants of Similipal Biosphere Reserve, India. The Internet Journal of Microbiology. 2007 Volume 5 Number 2.
Abstract
Fungal endophytes have been studied from three ethno medicinal plants of Similipal Biosphere Reserve. All the plants were found colonized with endophytic fungi. The endophytes were isolated using three different mycological media namely potato dextrose agar (PDA), malt extract agar (MEA) and water agar (WA). Maximum endophytes were isolated in PDA and minimum on WA medium. Altogether 60 fungal endophytes belonging to 14 genera were isolated from the three medicinal plants. Out of which, 31 endophytes (51.66%) were obtained as filamentous forms and 29 of them (48.33%) as yeast colonies. Species of
Introduction
The search for new antibiotics to overcome the growing human problems of drugs resistance in microorganisms and appearance of new diseases has been rapidly increasing around the world. Realizing the capability of microorganisms to produce diverse bioactive molecules and the existence of unexplored microbial diversity, research is underway to isolate and screen microbes of diverse habitat and unique environment for discovery of novel metabolites. One such unexplored and less studied microorganism is the endophytic fungi, which are defined as those microbes that colonize healthy tissues of plants, at least for a part of their life cycle, without causing apparent disease symptoms in their host (Petrini, 1991; Wilson, 1995). Different works carried out so far regarding the role of endophytes in host plants indicate that they can stimulate plant growth, increase disease resistance, improve plant's ability to withstand environmental stresses and recycle nutrient (Sturz and Nowak, 2000). Besides these, endophytes are also recognized as rich sources of bioactive metabolites of multifold importance (Tan and Zou, 2001; Strobel and Daisy, 2003).
In developing countries, the indigenous communities have been using medicinal plants in different ways for the treatment of various diseases, which in turn has resulted in scientific discoveries, with a wealth of literature on plant extracts and their biological activities. However, recently it has been reported that fungal endophytes residing within these plants could also produce metabolites similar to or with more activity than that of their respective hosts (Strobel, 2002). Therefore, it is believed that search for novel compounds should be directed towards plants that commonly serve indigenous populations for medicinal purposes and plants growing in unique environmental setting or interesting endemic locations as they are expected to harbor novel endophytes that may produce unique metabolites with diversified applications (Strobel and Daisy, 2003). With this knowledge, the present investigation was carried out to study fungal endophytes associated with three medicinal plants used as ethno medicine by the tribal communities of Similipal Biosphere Reserve, India and to evaluate these endophytes for antimicrobial activity against some human pathogens.
Materials and Methods
Location and study area
Plant materials were collected from Similipal Biosphere Reserve located at 21 o 16 / to 22 o 08 / N latitude and 86 o 4 / to 86 o 37 / E longitude. Similipal Biosphere Reserve is a rich biodiversity hotspot of eastern India representing a great aesthetic treasure as well as a grand repository of biological wealth. Samples were collected during February- March 2007 at an altitude of 80 – 869 m above Mean Sea Level (MSL). The mean temperature during the study period was 21±2 ° C. The plant species chosen for the present study were
Collection of plant parts
Four plants of each species were selected and 8 samples (rhizome and stem) from each plant were randomly cut off with an ethanol-disinfected sickle and placed separately in sterile polythene bags to avoid moisture loss. The materials were transported to laboratory within 12h and stored at 4 0 C until isolation procedures were completed.
Isolation of endophytic fungi
The collected samples were washed thoroughly with sterile distilled water and air dried before they are processed. The materials were then surface sterilized by immersing them sequentially in 70% ethanol for 3min and 0.5% NaOCl for 1min and rinsed thoroughly with sterile distilled water. The excess water was dried under laminar airflow chamber. Then, with a sterile scalpel, outer tissues were removed and the inner tissues of 0.5cm size were carefully dissected and placed on petri-plates containing different mycological media. The media were supplemented with streptomycin sulphate (100mg/L) to suppress bacterial growth. The plates were then incubated at 25±2 ° C until fungal growth appeared (Figure 1). The plant segments were observed once a day for the growth of endophytic fungi. Hypal tips growing out the plated segments were immediately transferred into PDA slant and maintained at 4 ° C. The fungal isolates were identified based on their morphological and reproductive characters using standard identification manuals (Barnett and Hunter, 1972; Subramanian, 1971). The fungal cultures that failed to sporulate were categorized as sterile mycelia. All the isolates are maintained in Potato dextrose agar slant in the department of Botany, North Orissa University.
Fungal cultivation and extraction of metabolites
The fungal endophytes were cultivated on Potato dextrose broth (Himedia) by placing agar blocks of actively growing pure culture (3mm in diameter) in 250ml Erlenmeyer flasks containing 100ml of the medium. The flasks were incubated at 25±1 ° C for 3 weeks with periodical shaking at 150 rpm. After the incubation period, the cultures were taken out and filtered through sterile cheesecloth to remove the mycelia mats. The fungal metabolites were extracted by solvent extraction procedure where ethyl acetate was used as an organic solvent. Equal volume of the filtrate and ethyl acetate was taken in a separating funnel and was shaken vigorously for 10 min. The solution was then allowed to stand, where the cell mass got separated and the solvent so obtained was collected. Ethyl acetate was evaporated and the resultant compound was dried in vacuum evaporator using MgSO4 to yield the crude extracts. The crude extracts were then dissolved in Dimethyl sulphoxide (DMSO) for antimicrobial bioassay (Figure 2).
Evaluation of antimicrobial activity
Altogether eight common human pathogens were used to evaluate the antimicrobial activity of endophytic crude extracts. The test pathogens include three Gram-positive bacteria i.e
For antimicrobial evaluation, agar cup disc diffusion method was followed (Grammer, 1976). Nutrient agar plates were inoculated with overnight culture of each bacterial suspension. Similarly for the fungal pathogens, Sabouraud's agar plates were inoculated with each fungal suspension. The plates with the inoculated organisms were evenly spread out with sterile cotton swabs. Agar cups were prepared by scooping out the media with a sterile cork borer (7mm in diameter). The cups were then filled with 100µL of the crude extract that was already dissolved in DMSO. The plates were then incubated at 35±1 ° C for 24 h and the zone of inhibition was recorded and compared with the control (i.e a cup filled with DMSO solution only).
Determination of minimum inhibitory concentration
Minimum inhibitory concentration was determined only for the crude metabolites produced by the
Results and Discussion
Three potential medicinal plants used by the tribal communities of Similipal Biosphere Reserve were selected for endophytic studies (Table 1).
Figure 1
All the plant species were found colonized with endophytic fungi. The endophytes were isolated using three different mycological media namely potato dextrose agar (PDA), malt extract agar (MEA) and water agar (WA). In many instances these media were commonly used for isolation of endophytes (Mahesh
Figure 2
Altogether 60 fungal endophytes belonging to 14 genera were isolated from the three medicinal plants (Table 3). Out of which, 31 endophytes (51.66%) were obtained as filamentous forms and 29 of them (48.33%) as yeast colonies. Colonization of endophytes was found to be variable in the three medicinal plants. In
Figure 3
Species of
Fungal endophytes have been recognized as repository of novel secondary metabolites for potential therapeutic use (Tan and Zou, 2001). Further, Daisy and Strobel (2003) necessitated that medicinal and endemic plants should use for endophytic studies as they are expected to harbor rare and interesting endophytes with novel bioactive metabolites. This has lead to the discovery of several bioactive compounds from fungal endophytes and wealth of literature on antimicrobial activity of endophytic fungi isolated from medicinal plants (Raviraja
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
The metabolite was very effective and showed highest zone of inhibition against
Figure 9
The fungus was isolated from rhizome of
Acknowledgement
The authors are grateful to Dr. A.K Biswal (Plant Taxonomist), Department of Botany, North Orissa University for collection and identification of the plants and to the field director, Similipal Tiger Reserve, Mayurbhanj, Orissa for their help and co-operation.
Correspondence to
K. Tayung (Lecturer) Department of Botany, North Orissa University, Takatpur, Baripada, Orissa, India 757003 E-mail: kumanandnou@yahoo.com Phone: +91 9938172469 (R) Fax: +91 6792255127 (O)