Plant material as source of endophytic fungi

Plant material was collected randomly from fully matured Bacopa monnieri between March – April, 2011 from the farm of IIIM, Chatha, at an altitude of about 32.73°N 74.87°E in Jammu and Kashmir State, in India. This accession is collected from Miran Sahib, Jammu in 1970. The identification of species was done via leaf and flower morphology by taxonomist of the Institute (IIIM) (Dr B. M. Sharma). An identified specimen was kept in the IIIM Janaki Ammal Herbarium in India with accession no. 18554. The accession is also maintained as genetic resource in the Chatha farm of IIIM. After plant selection, disease free leaves and branches of the plant were excised with a sterile scalpel and were sealed with parafilm to preclude drying out during transport.

Isolation of endophytes

Isolation of endophytic fungi from Bacopa monnieri was carried out using the protocol described by Strobel and Daisy [4] with slight modifications. Fresh plant material (Branches and leaves) was collected. The leaves and small branches were washed under running tap water for 10 minutes and sterilized in series with 70% ethanol for 1 min, 1.0% sodium hypochlorite (NaOCl) (v/v) for 1 min and further cleaned by passing through two sets of sterile distilled water. After surface sterilization, leaves and branches were cut into small pieces, 1 cm long each, The sterile samples were placed on a plate containing water agar and potato dextrose agar (PDA) media with 250 μg/mL streptomycin to suppress bacterial contamination. The parafilm wrapped petri dishes were incubated at 25 ± 2°C till the fungal mycelia started growing on the samples. The endophytic fungi were transferred to a new potato dextrose agar slant. Endophytic fungi isolated from the leaves of Bacopa monnieri was codified as B1-B6, B8-B11, B13-B16, B18-B19, B22-B24, B9_Pink, B8_ORG, BX. Endophytes were stored at 4°C and also endophyte colonized on sterile barley seeds were air dried to be subsequently stored at −70°C for later studies. All the isolated endophytic fungi were deposited in Microbial Repository of IIIM.

Fermentation and extraction

The endophytic fungi isolates (nine) were cultured in potato dextrose liquid medium in 1000 mL Erlenmeyer flask for a period of 10 days at 25 ± 2°C at 180 rpm on an incubatory shaker (New brunswicks, USA). Ten blocks containing 10-day old fungal mycelium were used as inoculums. After ten days, crude fermentation broth was blended thoroughly in a cell disintegrator with 20% methanol. Cell homogenate was extracted four times with equal volume of DCM (HPLC grade). Solvent was stripped off in a rotary evaporator leaving behind a residue designated as organic residue (O). The retentate was filtered and supernatant was lyophilized and designated as water extract (A). The extracts were dissolved in DMSO to a final concentration of 10 mg/ml for anticancer and antimicrobial activity screening.

Cytotoxic activity

This assay is a quantitative colorimetric method for determination of cell survival and proliferation [15]. For in-vitro cytotoxic activity, colorectal carcinoma HCT-116, lung A-549, Breast MCF-7, prostate PC-3 cancer cell lines were procured from National Centre for Cell Sciences (NCCS), Pune, India. Cells were grown in RPMI-1640 medium containing 10% FCS, 100 U penicillin/100 μg per mL streptomycin in CO 2 incubator (Thermo-con Electron Corporation, USA) at 37°C with 98% humidity and 5% CO 2 gas environment. The cells were plated in a 96-well plate at a density of 2.0 × 104 in 200 μL of medium per well. Cultures were incubated with different concentrations of fungal extracts (10–100 μg per mL) for 48 h. The medium was replaced with fresh medium containing 100 μg per mL of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and plates were incubated for 3 h. The supernatant was aspirated and MTT-formazan crystals were dissolved in 200 μL DMSO and the OD of the resulting solution was measured at λ 540nm (reference wavelength, λ 620nm ) on an ELISA reader (Thermo Labs, USA). Cell growth was calculated by comparing the absorbance of treated versus untreated cells. Cells treated with equivalent concentration of DMSO were used as negative control. Clinical drugs like 5-Fu, paclitaxel, adriamycin were included as positive controls. IC 50 value was calculated by Curvfit software.

Antimicrobial activity

Antimicrobial activity was determined using the micro dilution method [16]. Lyophilized six test bacteria and a yeast were purchased from Microbial Type Culture Collection (MTCC). Cultures of Bacillus subtilis (MTCC No. 121), Pseudomonas aeruginosa (MTCC No. 424), Salmonella typhimurium (MTCC No. 98), Escherichia coli (MTCC No. 118), Klebsiella pneumonia (MTCC No. 109), Staphylococcus aureus (MTCC No. 737) were grown on Nutrient Agar media and used for measuring the antibacterial activity of isolated endophytes. Candida albicans (MTCC No. 183) was grown on Yeast extract Peptone Dextrose Agar (YEPD).

Each bacterial strain was inoculated into nutrient broth (HiMedia Biosciences) and incubated overnight at 37°C with shaking. The suspension was adjusted to 0.5 McFarland standard turbidity (equivalent to 1.5 × 108 colony forming units (CFU/ mL) [17] and finally diluted to give approximately 104 CFU/mL for all organisms. Different dilutions (10–100 μg/mL) were prepared from the stock solutions of fungal extracts (10 mg/ml) and antibiotics (1 mg/ml). 900 μL of each concentration was mixed with 100 μL of sterile nutrient broth containing 104 CFU bacteria, (obtained from a McFarland turbidity standard no. 0.5). Appropriate negative (DMSO-0.5%) and blank controls (virgin media) were used. Antibiotics like streptomycin, amphotericin B were included as positive controls. They were dissolved in sterile distilled water to a final concentration of 1 mg/mL. Tubes were incubated overnight at 37°C. The highest dilution at which 99.9% of the bacteria inoculum was killed was considered as the MBC and the lowest inhibitory dilution at which there was no visible growth was considered as MIC. The assays were replicated and the mean value of 3 experiments were recorded (n = 3) with SEM.

Identification of potential endophytes

Morphological examination of endophytes

The fungi were identified based on the morphological characteristics. Colony features were based on observation on PDA under ambient day light conditions. Endophytes were identified on the basis of microscopic characteristics such as the structure of hyphae, conidia, and conidiophores. Conidiophore structure and morphology was ascertained after obtaining them from the edge of conidiogenous pustules or fascicles during maturation of conidia, which usually occurred after 4–7 days of incubation.

Internal transcribed spacer (ITS)-based identification