The medicinal plants are of common use in traditional treatment of various diseases (Salie et al., 1996; Mc-Graw et al., 1997). Herbal medicines have been widely practiced trough out the world from ancient time. These medicines are safe and environment friendly. Nearly 80% of the world population depends upon traditional system of health care (Hutchigson et al., 1996). Parts of medicinal plants are traditionally used for the treatment of antifungal, antitumor, anthelmintic, antidiuretic, antiulcerative, diseases of heart, rheumatic pains, chest pain, dyspepsia, fever, diabetes, burning of liver and kidney diseases (Verma et al., 2011). Phytochemical is a natural bioactive compound found in plants, such as vegetables, fruits, medicinal plants, flower, leaves, roots that are rich in nutrient and fibers to protect against diseases. Phytochemicals are of two types- primary and secondary, based on their function. Primary phytochemicals comprise common sugar, amino acids, protein and chlorophyll while secondary phytochemicals consists of alkaloid, terpenoids, phenolic compounds, flavonoids; tannins, coumarin, anthroquinone etc act as good radical scavenging activities. They function by either preventing the formation of the free radicals or by inhabiting them before they can damage the cellular components (Igor et al., 2017). Vascular plants in general, harbor phyllosphere and endophytic organisms. The fungi which reside in the internal part of plant tissues are called endophytes and those residing over the leaf surfaces are recognized as phyllosphere fungi. The phyllo-sphere is a complex terrestrial habitat that is characterized by a variety of microorganisms including bacteria, filamentous fungi, yeast and algae growing on the surface of leaves (Moitinho et al., 2020). 

There are two groups of phyllosphere fungi residents and casuals. On healthy leaf surface residents can multiply, but causal cannot grow. The host remains unaffected in both cases without any adverse effect. It has been estimated, rather roughly that the total area of leaf surface on this earth is about 1 billion square kilometer and it supports about a large population of bacteria along with fungi and actionmycetes and algae. Biochemical environment of phyllosphere is mainly determined by leaf leachates, microbial activity and dusts falling on leaf surface. The composition of the leaf exudates depends on the age of the leaf and plant, therefore microbial population on phyllosphere varies with age. The leachates contain amino acids, organic acids, minerals and carbohydrates (glucose, fructose   and sucrose). [The phyllospheric microorganisms produce IAA, vitamins, different enzymes, antibiotic substances, nitrogenous substances formed through the fixation of nitrogen and other metabolites. There is an extracellular polysaccharide (EPS) deposited on the leaf surface. EPS causes formation of heterogenous aggregates of different bacterial groups and fungi. The EPS slime provides protection to the microorganisms from desiccation, reactive oxygen species (ROS) and other stress factor. The organisms found on phyllosphere are called epiphytes (Vorholt 2012; Sharif et al., 2019; Mazinani et al., 2017). 

Phyllospheric microorganisms play an important role in controlling plant disease through antagonistic activity of non-pathogenic microbe against a pathogen. Pathogens present on phyllosphere causes production of elicitors that will induce resistance in plants. The pathogen triggers production of phytoalexins in the plants to create defense. Since phyllosphere micro-organisms promotes growth of plants either by restricting pathogenic attack or by providing nutrients or both, the growth and quality of roots will increase. Ever increasing human population has failed to meet their need by plant based medicine exploiting huge amount of plant. To meet this demand researchers are involved in searching alternative means of bioactive compound. Now a days it becomes evident that endophytes produces the bioactive compounds which are used in the treatment of human diseases (Onifade, 2007). About one million endophytic species present in plants (Shekhawat et al., 2010). The endophytic fungi have a symbiotic relationship with host (Shekhawat et al., 2010) and thus it does not cause any harmful effect to the host (Saithong et al., 2010; Wei et al., 2007; Arnold et al., 2003; Selvanathan et al., 2011) found that fungal endophyte provides bioactive stress tolerance of the host plant creating defense against pathogens (Khan et al 2010). The fungal endophyte differs depending on geographical location (Fisher et al., 1994; Collado et al., 1999), age of plant and plant parts (Khan et al., 2010; Sahashi et al., 2000; Shahen et al., 2019; Clay & Schardl, 2002).

In medicinal plants fungal endophyte harbor and provide biotic stress tolerance (Zhang et al., 2006; Stro-bel, 2002; Krishnamurthy et al., 2008). In our current research, the distribution of endophytic fungi was isolated and identified from A. vasica Nees. (Acanthaceae) stems and leaves. The endophytic fungi are an important source of various secondary metabolites. It contains a bioactive compound which is useful for pharmaceutical industries (Strobel 2002; Krishnamurthy et al., 2008; Khan et al., 2010; El-hawary et al., 2020). Extensive research have been done on the bioactive compounds of fungal endophyte, such as, antitumor agents, Taxol (Stierle et al., 1993; John et al., 2018), antibacterial and antifungal agents plant growth factors, enzymes, insecticidal agents, immune-suppressive compounds and antioxidants (Strobel & Daisy, 2003; Owen & Hundley, 2004; Firoz et al., 2016; Okezie et al., 2020). Therefore the present studies have concentrated to the phylloplane and endophyte Mycoflora of a well-known medicinal plant A. vasica Nees to the family Acanthaceae. The experiments with the dominant fungal endophytes and fungal colonizers of phyllosphere isolated from A. vasica Nees were carried out along the following lines: Estimation of total phenol; Estimation of antioxidant activity; Determination of antimicrobial activity; Qualitative determination of –

a) Terpenoids; b) Cardiac glycosides; c) Saponins; d) Flavonoids; d) Anthraquinones; e) Coumarins; f) Alkalo

Isolation of Phyllosphere Organism and Endophytes.

Phylloshere Organism

Sample collection

Fresh middle aged healthy leaves of the plant Adha-toda vasica were collected carefully from our college garden and immediately placed in sterile plastic bags and brought to the laboratory. 

Determination of leaf surface area:

The surface area of the leaf was determined by placing the leaf on a graph paper (mm) followed by marking the outline of the leaf. The surface area of the leaf was calculated in cm2.

Isolation of fungi

Leaves (60cm2) were put into 250ml Erlenmeyers flasks containing 100ml of sterilized solution (prepared by mixing 98ml distilled water with 2 ml surfactant, tween 20). Three replicates were maintained. The Er-lenmeyers flasks were then put under shaking condition for 2 hours at medium speed (90 rpm.) by using rotary shaker to release the leaf surface micro-organisms into the solution. After 2 hours the three solutions obtained were mixed together by using vortex. The mixture was then diluted to 10-2 concentration. 1ml of the diluent was taken and put into the sterilized Petri plate. Followed by  pouring of sterilized PDA (Potato Dextrose Agar) media with Streptomycin sulphate (100units/ml) the Petriplates were rotated clock wise, anti-clock wise and moved to and fro for proper mixing. Three replicates were maintained. The Petriplates were then incubated at 30◦C for 7 days. 

Interim inspection was done after 3 days and 5 days for appearance of the fungal colonies. The colonization frequency of each isolate was determined following Hata & Futai, 1995. From the subculture each isolate are grown in Petri plate containing CDA by placing the inoculum in the center of the Petriplate. For each isolate two Petriplates were prepared-one for study colony characters and the other for microscopic studies. The media in the plate for microscopic study was inserted with sterilized cover slip (3Nos) at 1.5cm away surrounding the inoculum at 1.5 cm distance from the inoculum. The cover slip are taken out on 3rd, 5th and 7th day after inoculation and was placed upside down on a slide containing a drop of lacto phenol and cotton blue mixture. The cover slip was sealed with wax and observed under microscope for identification.  

Endophytes

The collected materials were washed with running tap water for 30 minutes and then washed with sterilized distilled water for 2-3 minutes, surface sterilization of the materials was done by 90% ethanol (for 1 minute), followed by 3% NaOCl (8 minutes) and finally by 90% ethanol(30 sec) and then washed with sterilized distilled water (2-3 times). After surface sterilization processes, leaf discs (0.5cm ϕ) were prepared by using sterilized cork borer. Four leaf discs were then transferred to the Petri plates containing sterilized PDA (potato dextrose agar) medium with Streptomycin (100units/ml) to isolate endophytic fungi. Same process was followed in stem (stem was cut into pieces of 1cm). The plates were then incubated at 30◦C for a period of 10days. The organisms that come out of plant materials was isolated and subcultured in slants.

Identification

The isolated phylloplane organism and fungal endophytes were identified based on characteristics and available reproductive structure following (Burnett & Hunter, 1998; Uddin et al., 2016; Whatnabe, 2010) and internet information.

Relative colony frequency of phyllosphere organism

The relative colony frequency (RCF %) of a single colony in the plate was calculated by the following formula adopted by Hata & Futai (1995, pp 384-90) after necessary modifications.

RCF % in 10^-2 diluent = The no. of individual fungal colony/Total no. of fungal colonies  × 100

Population Frequency of Fungal Isolates in Phyllosphere

Population frequency was estimated by determining number of each fungal isolate/cm2 of phyllosphere.

Colonization Frequency for Endophyte

The colonization frequency (CF %) for a single endophyte in leaf and stem tissue was calculated by the formula of Hata & Futai, 1995. 

CF% = The no. of segments colonized by endophyte species/Total no of segments ×100

Preparation of Fungal Extract

The test fungi were grown separately in 500 ml Erlenmeyer flask containing 250 ml of Czapeks dox broth (CDB). The flasks were inoculated with test fungus maintaining more or less uniform inoculum potency and incubated at 30◦C for 20 days. The flasks were subjected shaking for 4 hours on a rotary shaker.3 replicates were maintained for each set. The culture fil-trates were obtained by filtering through Whatman no.1 filter paper placed on a Buchner funnel under condition of vacuum filtration. After extraction the culture filtrates were used to perform different bio-chemical tests.                                                 

Estimation of Total Phenol

Total phenolic content was estimated in each test sample following the protocol of Bray and Thrope, (1954). A standard curve was prepared using different concentrations of catechol as standard. To prepare standard curve, 100 mg catechol solution was dissolved in 100ml of distill water. For working, 2 ml stock solution was diluted 10 times with distilled water. From this working standard, different aliquots viz- 0.2ml, 0.4ml, 0.6ml,0.8ml was taken in separate test tubes, therefore, they contained 20µg, 40µg, 60µg, 80µg of phenol respectively and their volume was adjusted to 3ml for each tube with distilled water. In a separate test tube, 3ml of culture filtrate of unknown strength was added with 0.5ml of Folin ciocalteu reagent.  After 2-3 minutes, each test tube was added with 2ml of 20% Na2CO3 solution. After thorough mixing, all the test tubes were heated for exactly 1 minute in water bath and cooled at room temperature. The absorbance was measured on a spectrophotometer at 650nm. 

Estimation of Total Antioxidant activity

The culture filtrates of each fungus grown in CDB media for 21 days were obtained by filtering through Whatman filter paper (No.1). 20 ml of culture filtrate of each fungus was evaporated and weighed. The residue was mixed with 5ml methanol to use for determining the total antioxidant activity. For this, 1ml sample was added with 2ml reagent solution (prepared by mixing Ammonium molybdate, 4mM; Sodium phosphate, 28mM and Sulphuric acid, 0.6M in a ratio 1:1:1).  3 replicates were maintained for each sample. The reaction mixture was incubated for 60 minutes at 30◦C. The absorbance was then measured on a spectrophotometer at 695 nm. The reducing capacity of the extract was expressed as the ascorbic acid (standard) equivalent.

Antagonistic Activity

Antagonistic activity of the fungal endophytes was determined by antibacterial assay against both Gram positive and Gram-negative bacteria using agar cup method. Gram positive bacterium tested was Bacillus subtilis and Gram negative bacterium tested was E. coli.                                                         

Agar cup method

The agar cups (1cm diameter) were prepared by using sterilized cork borer in nutrient agar plates pre inoculated with bacterial suspension of Gram positive and Gram negative bacteria separately. The cups were then filled with culture filtrate aseptically using 1ml pipette. For each three replicates were maintained in each set. The Petriplates were kept in an incubator for 72 hours at 30◦C and the zone of inhibition (if formed) was measured.                                       

Qualitative Analysis of Bioactive Compounds of Adhatoda vasica and Fungal Endophytes

Processing of samples

Leaves of Adhatoda vasica plant were properly washed with running tap water for 20 minutes. It was then washed with sterilized distilled water for 2-3 minutes. The rinsed leaves were dried in an oven at a tem-perature around 60°C to obtain constant dry weight. The dried leaves were pulverized by using a sterile electric blender and stored in airtight glass container, protected from sunlight until required for analysis. The mycelium (of selected dominant endophytes) harvested after 20 days of growth in CDB media, dried at 60◦C until getting constant dry weight. The subsequent steps were similar as above. Biochemical tests were done with aqueous ethanolic extract of the powdered spe-cimen by using standard protocol to identify the constituents (Harborne, 1973; Sofoware, 1993; Trease & Evans, 1989).                     

Test for Terpenoids (Salkowski test)

Extract (5 ml) was mixed with 2ml of chloroform, and concentrated sulphuric acid (3ml) was carefully added to form a layer. A reddish brown colouration of the inter face was formed to show positive results of terpenoids.

Test for Cardiac Glycosides (Keller Killani test)

Extract (5 ml) was treated with 2ml of glacial acetic acid containing one drop of ferric chloride solution and 1ml of concentrated sulphuric acid. A brown ring of the inter face was formed to show the results of cardiac glycosides.

Test for Saponins (Frothing test)

Powdered sample (2g) was boiled with 20ml of dis-tilled water in a water bath. The mixture was filtered. 10ml of the filtrate is mixed with 5ml of distilled water in a test tube and shaken vigorously for 15 minutes to develop a stable persistent froth. The froth is then mixed with 3 drops of olive oil. Formation of emulsion suggests the presence of saponins.

Test for Flavonoids

The aqueous filtrate (1ml) was mixed with 5ml dilute NaOH in a test tube. An intense yellow colour was appeared in the test tube. It becomes colourless on addition of a few drop of dilute sulphuric acid indicates the presence of flavonoids.

Test for Anthraquinones

To 1 ml of aqueous plant extract add a drop of benzene and ammonia. A pink colour appears, indicates the presence of anthraquinones.

Test for Coumarins

 To 2 ml of aqueous plant extract add 10% sodium hydroxide. A yellow colour appears indicates the presence of coumarins.

Test for Alkaloids

To 5 ml of aqueous plant extract add 10ml methanol, 1% (w/v) HCl and Wagners reagent (6 drops). A creamish or brownish red or orange precipitate appears indicates the presence of alkaloids.

Phyllosphere fungal organisms

The fungal organisms isolated from phyllosphere, are Aspergillus sp., Penicillium sp. and C. cladosporioides (Table 1). Among the isolated organism Penicilluim sp. shows highest population (1.66×103/cm2). This was followed by Aspergillus sp. (1.16×103/cm2) and C. cladosporioides (0.66×103/cm2) in descending order. The relative frequency of the isolated phyllosphere fungi was similar to population of organism. Thus Penicillium sp. represented highest frequency (47. 61%) and C. cladosporioides represented lowest frequency (19.04%) and the intermediate frequency was represented by Aspergillus sp. (33.33%). 

Fig. 1: (A) Leaves of Adhatoda vasica, (B) Cork borer, (C) Leaf disc preparation. 

Fungal endophytes

The endophytes were isolated both from the leaf discs and stem bits using PDA culture plates. Four (4) fungal endophytes were isolated from leaf discs and three (3) from stem bits (Table 2). The frequency of fungal endophytes in leaf discs and stems bits is low, which may be due to their inability to grow in PDA media. Among the 4 fungal endophytes of the leaf discs A. alternata showed higher colonization frequency (50%). This was followed by C. lunata (16.66%) and jointly by M. sterilia and A. niger in descending order. Among the fungal endophytes from stem bits Mycelia sterilia was found to be dominant as revealed from its colonization frequency (33.33%), which was followed by A. alternata (22.22%) and Penicillium sp. (11.11%) in descending order. The characteristics features noted in isolated phyllospere fungal organism and fungal endophytes were as follows:   

Fig. 2: (A) Growth of fungal endophytes from leaf discs, (B) Growth of fungal endophytes from stem bits

The author is thankful to Dr. S. K. Chatterjee, Retd. Associate Prof. and Ex-Head Post Graduate Dept. of Botany, Hooghly Mohsin College for identification of the genera.

The authors express no conflict of interest to carry for-ward this research finding to publish in this journal.