3.

MATERIALS AND METHODS

Isolation of Endophytic Fungi from Plant Endophytic fungi were isolated from the H. rosa-sinensis plant. Different part i.e.stems, leaves and roots of these plants were sampled for the investigation of endophytic fungal communities. (A) Collection of Plant Material In the present studies fungal species were isolated from different parts of Hibiscus rosa -sinensis a medicinal plant, commonly known as Arundi Collected from R. D. V. V. campus, Jabalpur (M.P.). Healthy and mature plants were carefully chosen for sampling. Leaves, stems and roots were collected from Hibiscus rosa-sinensisc plant. The plant material was brought to the laboratory in sterile bags and processed within a few hours after sampling. Fresh plant materials were used, to reduce the chances of contamination. (B) Isoltion of Endophytic fungi Isolation of endophytic fungi was done according to the method described by (Petrini et al., 1986). Isolation of endophytic fungi was done by modification of Radu Methods (2003). (c) Surface Sterilization and Incubation
1. The plant materials were rinsed gently in running water to remove

dust and debris.

2. After proper washing stem and root samples of Hibiscus rosa

-sinensis

were cut into small fragments using sterile

surgical blades into 1 cm long pieces, whereas leaves were cut into 3-4 x 0.5-1 cm. pieces with and without midrib under aseptic conditions.
3. Each sample was surface sterilized with 70% ethanol for 1 minute

by immersion in sodium hypochlorite (NaOCl) for 30 seconds.

4. The samples were then rinsed in sterile distilled water for 30

second.
5. The pieces were blotted dry on sterile blotting paper. 6. After proper drying in each Petri plate, 5-6 segments were placed

on

potato

dextrose

(PDA)

supplemented

with

antibiotic

(Tetracycline).
7. Plates were incubated at 28C for 7 days. 8. Pure colonies of endophytic fungi appearing from the edge of the

segments were transferred to PDA slants. (D) Media Used for Isolation of Endophytic Fungi Potato dextrose agar (PDA) is the most useful selective medium for the culture of endophytes. The antibiotic (Tetracycline) was added to prevent the growth of bacterial contamination. Media Preparation Peeled potatoes, sliced finely, boil in 500 ml distilled water till it become soft, passed through cheese cloth, volume adjusted to 1000 ml.

dextrose and agar- agar pH has adjusted to 5.5 - 6.0 with 20% lactic acid. Fungi were grown on specified under specified culture condition, for identification. The fungi were identified on the basis of available literature (Domsch et al., 1980).

(E) Preservation of Endophytic Strains The fungal strains in the pure culture were preserved on potato dextrose agar (PDA) slant at 4 to 5C with proper labeling and were sub cultured from time to time. Identification of Endophytic fung The colonies appearing on Petri -plates were sub culture tube containing potato dextrose agar medium for identification, fungi were cultured in Petridis containing potato dextrose agar- agar medium without tetracycline antibiotic for 7 days. To describe colony characteristics (Domsch et al., 1980, Ellis 1971, Kenneth et al., 1965, Sutton 1980). The each fungus was identified on the basis of cultural characteristics and microscopic characters, as seen under the microscope for morphological observation slides were prepared.

(a) Slide Culture Technique From the screening, one strains endophytic fungus Hibiscus rosa-sinensi were found to be a potential strain. Thus identification of these fungi was done by slide culture technique. 1. A sterilized moist chamber was prepared with a thin wetted cotton a slide inside a sterilized Petri -plate.

pad a wet filter paper and

2. one drop of sterilized PDA (potato dextrose agar) was placed on side, inside the moist distilled water). 3. One loop of fungal conidial suspension was inoculated in it and the slide was incubated at 281C. 4. After sporulation the slides were stained with cotton blue and mount in one drop of lacto phenol and observed under microscope for identification. Table 3.1 the Composition of Lactophenol Component Phenol (pure crystals) Lactic acid Glycerol Water Cotton blue Amount 20mg 20ml 40ml 20ml 10ml chamber (after moistening the filter paper with sterile

Principal:-lacto-phenol serves as the mounting fluid the dye is cotton blue, organisms suspended in the mounting fluid are rapidly killed by the presence of phenol which acts a gross cytoplasm poison. Which precipitate cellular protein and essential enzyme systems, cotton blue is an acid dye, which stains chitin and cellulose staining of fungi by cotton blue is due to presence of chitin in their cell wall. Identification was carried out with the available literature (Subramanian 1972, Barnett and Hunter 1981, Raper and Tham 1984, and Cur rah 1985). (c) Source of Bacterial Strains Six strains of clinical isolates of bacteria were used in screening for antibacterial activities were kindly provided by Prof. S. S. Sandhu fungal biotechnology and invertebrate pathology laboratory Deptt. of biological sciences R.D.V.V. Jabalpur (M.P.) Table the list of different bacteria used Gram positive 1. 2. Bacillus subtilis Staphylococcus epidermidis Strain B01 B02

Gram negative
1. Escherichia coli

Strain B03

2. Klebsiella pneumonia 3. Pseudomonas arugenosa 4. Salmonella typhimurium

B04 B05 B06

(d)Maintenance of Bacterial Strains Bacterial cultures were maintained on slant of nutrient agar medium and incubated at 37C for 24 hrs. In incubator and then stored at 40C. Production of Antibacterial Metabolites
1. 25 ml of potato dextrose broth was prepared in 50 ml flasks and

autoclaved at 15 lb psi for half hrs.

2. The medium was inoculated with various fungi culture and

incubated at 281C in the incubator.

3. After 14 days of incubation the crude culture broth was filtered

and tested for antibacterial activity against all the test bacterial agar well diffusion methods.
4. This procedure was followed till the 13 day of incubation. 5. The zone of inhibition was measured with the help of transparent

ruler. Screening of Endophytic Fungi Total number of metabolites isolated from 6 endophytic fungi were screened for their antibacterial activity against 6 Pathogenic bacteria , which were , used i.e. Bacillus subtillis (B01) , Staphylococcus

epidermidis (B02) , Escherichia coli (B03), Klebsiella pneumonia (B04) and Pseudomonas arugenosa (B05) by agar well diffusion method (Egorov 1995) from the screening , strain ends was found to be Hibiscus rosa-sinensis, potential strains. Extration of Mycelial Free Culture Filterate (MFCF) Under aseptic conditions, the 5 ml of the fungal culture broth was filtered through a reweighed whatman filter paper no. 1 and was centrifuged at 6000 rpm for 10 min. the pellet was discarded and the supernatant was used for antibacterial bioassay. Antibacterial Bioassay Agar well diffusion method (Egorov 1995) was used.
1. Nutrient agars were prepared. 2. Agar plates were seeded with 100 l of bacterial culture and lawn

was prepared by spread plate method.

3. The surface of the seeded media was allowed to dry for 30 min. in

the cooling incubator.

4. Wells of 4mm. diameter were aseptically made in the seeded media

using sterile cork borer (Azoro 2002).

5. 40 ml, 60 ml, 80 ml of the antibacterial metabolite was dropped in

the prepared wells and plates were kept at 8-10C for 2-4 hrs. For diffusion those plates were incubated at 37C in bacteriological incubator for 24 hrs.

Finally plates were observed for zones of inhibition and their diameter was measured with a transparent ruler. Chromatographic Analysis Detection of Class of Compounds Compounds were separated using thin layer chromatography (TLC). n butanol and acetic acid were used for detection of type of compounds and TLC was visualized under ultraviolet (UV) light at 254 nm and 366 nm. Thin Layer Chromatography (TLC) TLC (thin layer chromatography) was chosen as our analytical technique. TLC is a standard technique, which separates the organic compounds of lower molecular weights according to the polarity, and again it is most common technique used for separation of natural substances (Hoagland and Johnson 1999). TLC is inexpensive method, thus, allowing us to run many analyses in optimizing the experimental parameters. (A) Preparation of the Slurry and TLC Plate The glass plate on which the thin layer is prepared was thoroughly washed and dried before layer application. The material of which the thin layer was to be made (silica gel) was usually mixed with water in such a proportion that a thick suspension known as slurry results. this

slurry was applied to a plate surface as a uniform thin layer by means of a plate spreader starting at one end of the plate and moving to the under in an unbroken uniform motion the nature of the desired chromatographic separation dictates the thickness of the slurry layer used. Thus, for analytical separation of the thickness of the layer is usually about 0.25 mm, thickness about 5 mm. (B) Making of TLC Plate A silica gel TLC plate that is approximately 10 cm wide and 20 cm long was obtained. The TLC plate was marked using a pencil (pencil must be used rather pen because inks are moved by many developing solvents). First a straight line parallel to the short dimension of the plate was drawn, about 1 cm from one end of the plate. (C) Activation the TLC Plate The marked TLC plate is placed in an oven at 110C for 60 minutes to activate it activation involves driving off water molecules that bond to the polar sites on the plate. (D) Preparation of Solvent System TLC analyses were carried out on 0.25 mm silica gel plates, developed in the following solvents.
(i)

Ethyl acetate

: :

2 propanol (95:5) water (30:10:10

(ii) n-butanol: acetic acid

TLC of Fungal Metabolites Two fungal metabolites of 10 ml and 25 ml of sample was loaded on TLC plate through micropipette the TLC plates were developed using different solvents i.e. ethyl acetate : 2 propanol (95:5), n - butanol : acetic acid : water (30:10:10), and after run then plate were air dried . TLC plates were run in duplicate and one set was used as the reference chromatogram and other set was used as bioautography. The separated components were visualized under visible and ultraviolet light (at 254 and 366 nm). (E) Calculation of Rf Value The Rf value was calculated by measuring the distance from the point of application of each spot to the top of the solvent front (solvent distance) the distance from the point of application of each spot to the centre of each spot was measured (Migration).

The Rf values was calculated by following formula.