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, anthel-minthic, 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, ter-penoid, phenolic compounds, flavonoids; tannins, coumarin, anthroquinone etc act as good radical sca-venging 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 phyl-losphere 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 chara-cterized 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 mul-tiply, but causal cannot grow. The host remains un-affected 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. Bio-chemical environment of phyllosphere is mainly de-termined 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 pro-duce IAA, vitamins, different enzymes, antibiotic sub-stances, 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 phyllo-sphere 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 acti-vity 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 res-tricting 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 endo-phytes 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 toler-ance of the host plant creating defense against patho-gen (Khan et al 2010). The fungal endophyte differs depending on geo-graphical 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. (Acan-thecae) 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; De-termination 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  × 100

 Total no. of fungal colonies

Population Frequency of Fungal Isolates in Phyllo-sphere

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 ×100

Total no of segments

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

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

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 fre-quency (19.04%) and the intermediate frequency was represented by Aspergillus sp. (33.33%).

Fungal endophytes

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

Fig. 3: Aspergillus sp. (A) Upper surface of the Aspergillus colony, (B) Lower surface of the Aspergillus colony (C) Microscopic view of Aspergillus Conidia with conidiophores, (D) Microscopic view of Aspergillus Conidia.

Fig. 4:   Penicillium sp. (A) Upper surface of the Penicillium colony, (B) Lower surface of the Penicillium colony, (C) Microscopic view of Penicillium Conidiophore, (D) Microscopic view of Penicillium conidia.

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. alter-nata (22.22%) and Penicillium sp. (11.11%) in descending order. The characteristics features noted in iso-lated phyllospere fungal organism and fungal endophytes were as follows:                                      

Fig. 5: Cladosporium cladosporioides, (A) Lower surface of the C. cladosporioides colony, (B) Upper surface of the C. cladosporioides colony, (C) Microscopic view of C. cladosporioides Conidiophore with conidia.

Phyllosphere fungal organisms

Aspergillus sp. (Fig. 3)

 Fig 6: Alternaria alternate, (A) Upper surface of the A. alternata colony (After 5 days), (B) Lower surface of the A. alternata colony (After 5 days), (C) Microscopic view of A. alternata Conidia with conidiophores, (D) Microscopic view of A. alternata Conidia showing germination.

Growth restricted (2.9cm/7days), upper surface of the colony is differentiated into central dull white followed by a sporulation zone which is blue green in colour; finally there is a white ring of margin colony adpressed to the media and compact. Reverse brownish white at the center surrounded by a whitish periphery presence of no hyaline margin. Hyphae loosely arranged, no aggregation of hyphae forming tuft. Vesicle club-shaped, sterigmata in 2 whorls, conidiophore smooth surfaced long septate (length 299-356 µm, breadth 4.21-4.93µm), basal sterigmata ranges 14.97µm to 21.32µm and upper sterigmata ranges from 18.93µm to 19.03µm, vesicle length 12.42µm and breadth 9.04 µm. spores round, surface spiny (3.97-4.03µm).

Fig. 7:  Curvularia lunata, (A) Upper surface of the C. lunata colony (After 5 days), (B) Lower surface of the C. lunata colony, (C) Upper surface of the C. lunata colony (After 10 days), (D) Lower surface  of the C. lunata colony, (E) Microscopic view of C. lunata conidiophore with conidia, (F) Microscopic view of C. lunata conidia.

Penicillium sp. (Fig. 4)

Growth restricted (4cm/-7days), upper surface of the colony highly greenish yellow or lemon in colour. Reverse orange to orange-yellow in color with hyaline margin, colony compact adpressed to the media. Hyphae loosely arranged hyaline, septate.

Fig. 8:  Mycelia sterilia 1, (A) Upper surface of the M. sterilia 1 colony, (B) Lower surface of the M. sterilia 1colony, (C) Microscopic view of formation of Chlamydospores in M. sterilia 1, (D) Microscopic view of tuft of hyphae in M. sterilia 1.

Conidiophore branched bearing 2 (12.00 to 12.23µm and 11.96 to 12.33µm) to 3 (10.26, 10.91 and 10.56µm), each branch terminating in a whorl of sterigmata (9.36 – 10.33µm), each sterigma bear a chain of conidia. Long conidiophore with septa (51.70 to 52.24µm-length; 2.34 to 2.51µm - breadth). Conidia round (2.32µm) or oval (length-2.31-2.81µm; breadth-2.58µm), smooth surfaced.

Fig. 9: Aspergillus niger,  (A) Upper surface of the A. niger colony   (After 5 days), (B) Lower surface of the A. niger colony, (C) Upper surface of the A. niger colony (After 10 days), (D) Lower surface of the A. niger colony,  (E) Microscopic view  of A. niger conidiophore with conidia, (F) Microscopic view  of A. niger conidia.

Cladosporium cladosporioides (Fresenius) (Fig. 5)

Fig. 10:    Mycelia sterilia2, (A) Upper surface of the Mycelia sterilia2colony, (B) Lower surface of the Mycelia sterilia2colony, (C) Microscopic view of the Mycelia sterilia2hyphae.

Fig. 11:  Penicillium sp. (A) Upper surface of the Penicillium sp. colony, (B) & (C) Microscopic view of Penicillium sp. conidiophore with conidia.

Growth restricted (3.5cm/13days), upper surface of the mycelia mats adpressed to the media, central inoculum portion elevated, olive-brown in color, uniform out-line, velvety and tufted. The edges of the mycelia are olive-grey to white, and feathery. The colonies are diffuse and the mycelia form mats and rarely grow upwards on the surface of the colony. Reverse Dark brown, margin hyaline, compact. Hyphae branched; darkly pigmented hyphae are not constricted at the septal region. Mature conidiophores are treelike and comprise many long, branched chains of conidia. It produces brown to olive-brown coloured, solitary conidiophores that branch irregularly, forming many ramifications. The sporophores (length- 9.26 to 9.76µm; breadth- 4.31- 4.41µm) are thin-walled and cylindrical and are formed at the end of ascending hyphae. The conidia are small, lemon-shaped and smooth-walled, bi-celled (11.81-12.68µm).

Fig. 12: Total Phenol Content in the Culture Filterate of Dominant Fungal Endophytes.

Fig. 13: Total Antioxident Activity of Dominant Endophytic Culture Filterate.

Leaf endophytes

Alternaria alternate (Fr.) Keissler (Fig. 6)

Growth moderate (3.5cm/5days), upper surface of the colony brownish grey followed by narrow greyish margin, extreme margin is more or less uniform in out-line, extreme center at the point of inoculum dark brown in color presence of ill-defined concentric zones. Reverse greyish brown with concentric zon-ation. Colony ad pressed to the media very slightly floccose. Hyphae light brown in color, short septate. 

Conidiophore short, septate (length 54.76µm and breadth 4.21µm). Conidia borne single on conidio-phore. Conidia brown in color, presence of trans-verse and longitudinal septa. Number of transverse septa is 2-6 and longitudinal septum is 1-2. Conidia club sha-ped, round at the apex (length 31.141µm-31.22µm and breadth 10.99µm-13.66µm).     

Curvularia lunata (Walker) Boedijn (Fig.7)

Table 1: Determination of Relative Colony Frequency and Population Frequency of Fungal Organism in Phyllosphere.

Growth restricted (4.5cm/5days), upper surface of the colony grey colored compact margin white and narrow extreme margin hyaline with uniform outline. After 10 days, upper surface of the colony is velvety in appe-arance elevated from the media, greenish grey in color, and presence of concentric rings. Grayish white ring present in between the central and peripheral part.  Reverse light grayish brown with a dark narrow zone or ring, margin white or hyaline. The extreme center at the point of inoculum is dark brown in color or grayish brown in color with concentric ring. Hyphae loosely arranged, medium septate. Conidia straight or curved (length- 24.96µm to 26.20µm and breadth- 9.10µm to 9.74µm). Second cell from the tip is large. Conidia 3, septate, brown. Conidiophore short, short septate, smooth (length- 141.38µm to 141.68µm and breadth-3.84µm to 4.91µm). Conidia are borne at the tip of the conidiophore.

Table 2: Colonization Frequency of Fungal Endophytes.

Table 3:  Qualitative Tests for Some Biochemical Constitutes of A. vasica and Dominant Fungal Endophytes.

Table 4: Antagonistic Activity.

Mycelia sterilia 1 (Fig. 8)

Growth moderate (3.3cm/5days), upper surface of the colony cottony and compact in appearance, marginal part is more compact than the center part, white in color extreme margin thin and hyaline, margin outline is narrow or less irregular. Reverse very light, creamy white. Hyphae thick walled, short septate bearing pro-fuse chlamydospores (length- 7.25 to 7.75µm and breadth- 5.52 to 6.51µm).No other spores were found (breadth of hyphae- 2.96 to 3.55µm).

Aspergillus niger van Tieghem (Fig. 9)

Growth rapid (6.4cm/5days), upper surface of the colony adpressed to the media slightly floccose, central part black, margin broad white to hyaline, margin out-line more or less uniform. Reverse whitish at the center which was followed by a hyaline zone, broad margin white in color. Conidiophores (199.35-250µm) septate, smooth, vesicle round and sterigmata present in single whole, sterigmata7-9µm in length. Spores globose, diameter 3.90-4.59µm.

Stem endophytes

Mycelia sterilia 2 (Fig. 10)                                                         

Growth very rapid (8.4cm/2days), colony at upper side is hyaline, for a diameter of 3cm followed by cottony white elevated mycelia zone represented by 1-1.2cm zone subsequently hyaline margin with scattered white cottony hyphae. Reverse reddish white, while as the inoculum region is reddish brown. Hyphae loosely arranged thin; no chlamydospore or other spores were found; Breadth of hypha 1.76 to 1.88µm.

Penicillium sp. (Fig. 11)

Growth restricted (2.5/7days), upper surface of the colony slightly elevated from the media, olive green in color, central inoculum portion slightly elevated and dark in color, margin white, hyaline, margin irregular. Reverse light brown, margin white, hyaline, central inoculum part dark brown in color. Conidiophore (69.86µm) unbranched bearing a whorl of phyallide (10.87, 11.57, 13.21µm) at the tip i.e. monoverticilate. Spores are smooth walled and oval (length- 4.44 to 4.50µm and breadth- 3.14 to 3.32µm). Phyllosphere mycoflora was extensively studies by a number of researchers (Vacher et al., 2016; Chauhan & Navneet, 2015; Angela & Shri, 2016; Nayak & Anandhu, 2017). Occurrence of Aspergillus sp. in the phylloplane is considered as very common (John & James, 2017). Penicillium sp. in phylloplane was reported by (Chau-han & Navneet, 2015; Pandey et al., 1993; Nayak and Anandhu, 2017; Hilber & Bodmer, 2017). C. clado-sporoides as phyllosphere mycoorganism was reported by (Hussain et al., 2015). Existence of fungal endo-phytes have been studied by a number of researchers (Gond et al.,  2012; Potshangbam et al.,  2017; Ilic et al., 2017; Bhattacharyya et al., 2017). Presence of Alternaria alternate as fungal endophyte was reported by (Murthy et al., 2011).                                                     

Curvularia lunata as endophyte was reported by (Lar-ren et al., 2002) from Soya bean leaf. Mycelia sterilia was reported by Cai et al. (2004). Penicillium sp. was reported by John et al. (2017). A. niger is considered as a very common endophyte (Khan et al., 2010). It is evident from Table 3 that A. vasica contained terpenoids, cardiac glycosides, saponins, flavonoids and alkaloids as revealed from qualitative assay and showed no occurrence of anthroquinons & coumarins. All the endophytes subjected to qualitative test for the above mentioned seven biochemical constituents. It has been found that A. alternata and C. lunata were able to produce terpenoids, saponins and alkaloids. A. alternata alone have the capability to produce cardiac glycosides. M. sterilia 2 also produce saponins and alkaloids. As revealed from Fig. 12 that the total phenol content among the fungal endophytes tested shows highest in A. alternata (19.97 mg/100ml), followed by C. lunata (7.72 mg/100ml) and M. sterilia 2 (3.9 mg/ 100ml) in descending order. 

Phenol being antioxidant their production by fungal endophytes has significance in the practical field. Murthy et al. (2011) reported efficient products of phenolics by numerous fungal endophytes. Endophytes are closely related with their host plant in stress tolerance through production of phenolics (Huang et al., 2007; White & Torres 2009). Total antioxidant activity (Fig. 13) of the culture filtrate of fungal endophytes tested was evaluated by the Phospho-Molybdate method (Murthy et al., 2011). In culture filtrate, the Mo (VI) is reduced to Mo (V) and as a result of green colored phosphor-molybdenum-(V)-complex is formed, which shows maximum absorbance at 695nm. All the organisms tested showed anti-oxidant activity within a range in between 25.9g equivalent and 8.01g equivalent to Ascorbic acid. The highest level of antioxidant activity among endophytes was found in A. alternata which is followed by C. lunata (11.41g) and M. Sterilia 2 in descending order. It is noteworthy to mention that the organism A. alternata which shows maximum phenol content is capable in showing maximum anti-oxidant activity. Similar such positive correlation exists in C. lunata In M. sterilia 2 however, the phenol content was found to be low while antioxidant activity is high. This lack of correlation indicates that the antioxidant activity is possibly contributed by the other antioxidant cons-tituents. Such positive correlation was also put for-warded by (Murthy et al., 2011; Huang et al., 2007). 

The antagonistic activity of endophytes is tested against B. subtilis (Gram positive) and E. coli (Gram negative) following Agar cup method. The culture filtrate obtained from the broth culture of endophytes showed no zone of inhibition against the bacteria (both gram positive and negative

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.