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Summer Research Fellowship Programme of India's Science Academies

Isolation and screening of biopotential endophytic fungi from Cassia siamea tree

Divyadharsini L

Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamilnadu Agricultural University, Coimbatore 641003

Dr. Ravindra Nath Kharwar

Professor, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005

Abstract

Fungi are the important components in every ecosystem, intimately associated with crucial processes like the decomposition, recycling and transportation of nutrients in different environment. Endophytes refer to all organisms inhabiting plant organs without causing apparent harm to the host. It has been estimated that there may be over a million different fungal species on this Earth, of which only a small fraction has been identified. Endophytic fungi (EPF) may be the important contributor to fungal diversity. Diverse endophytic fungi reside in plants, representing a rich resource of bioactive natural products with potential for exploitation in pharmaceutical and agricultural field. However, it is thought that most of the endophytic fungal diversity remains to be discovered. Medicinal plants are recently being recognized as resources of endophytes with interesting bioactive compounds. Out of the 10,000 important medicinal plants, very less plants had been studied for their endophytic micro flora. So, rapid research should be required to study them because disappearance of plant species will also disappear the entire suite of associated potential endophytes. Cassia siamea possesses several medicinal properties with agricultural relevance. This medicinal plant has been used successfully over thousands of years to treat anxiety disorders, mild panic attacks, stress- and sleep disorders.

Keywords: endophytic fungi, Cassia siamea, isolation and screening, secondary metabolites, antimicrobial assay, antioxidant assay

Abbreviations

Abbreviations
 PDBPotato Dextrose Broth 
 SDASabouraud Agar 
 NANutrient Agar 
 hCMVhuman Cytomegalovirus 
 PDT Podophyllotoxin
 CPV Camptothecin
 BOD Biochemical Oxygen Demand
 SDS Sodium dodecyl sulfate
EDTA Ethylenediaminetetraacetic acid 
 CTABCetyl trimethyl ammonium bromide 
 ROSReactive Oxygen Species 
 DPPH 2,2-Diphenyl-1-picrylhydrazyl

INTRODUCTION

The word endophyte was introduced by de Bary in 1866 (endon Gr, within; phyton, plant) and was initially applied to any organism found within a plant. Over the time, the word endophyte was used to refer all organisms inhabiting plant organs that at some time in their life can colonize internal plant tissues without causing apparent harm to the host (Seigel et al., 1984; Carroll, 1986; Petrini, 1991).

Fungi described as "endophytic" characteristically exhibit a prolonged, inconspicuous period in which growth and colonization cease temporarily, resuming after a physical, or maturational, change in the host. This episodic growth is a defining feature of endophytes, whether they ultimately are considered commensal saprobes, latent pathogens, or protective mutualists. Although such a definition may seem too broad, most fungal biologists agree that the species composition of the internal mycobiota is distinct for various hosts, organs, and tissues although some species of endophytic infections also may be found in the epiphytic or rhizosphere mycobiota. In spite of the term 'endophyte' being employed for all organisms that inhabit plants without causing visible disease symptoms (Schulz and Boyle, 2005).

Ecological Roles of Endophytic Fungi

Endophytic fungi have diverse and varied ecological roles (Saikkonenet al., 1998). Endophytes can influence community biodiversity, and microbial interactions have been shown to be important determinants of plant biodiversity (Clay, 2001; Ernst et al., 2003). Dominant endophytes produce toxic alkaloids in grasses and other herbaceous plants that deter or poison herbivores (Wilkinson et al., 2000). In case of woody plants, endophytes function in specific defence roles.

Significance of Endophytic Fungi

Microbes and production of microbial allelochemicals promotes plant growth and consequently, beneficial effects on the host plant. Secondary metabolites produced by endophytes provide a variety of fitness enhancements such as increased resistance to herbivores, pathogenic fungi, bacteria, viruses, insects, nematodes (Redman et al. 2002). Endophytic fungi like Muscodor albus volatiles inhibited and killed a wide range of storage pathogen belonging to species of Botrytis, Colletotrichum, Geotrichum, Monilinia, Penicillium and Rhizopus. Since Muscodor albus is a sterile mycelium and does not require direct contact with the crops to be treated, it could be an effective biological fumigant for controlling postharvest diseases (Strobel et al., 2001).

Endophytes usually produce the enzyme necessary for the colonization of plant tissues. It was experimentally proved that most endophytes are able to utilize, xylan and pectin, show lipolytic activity and produce non-specific peroxidases and laccases (Seiberet al., 1991; Leuchtmannet al., 1992), chitinase (Li et al., 2004) and lucanase (Seiberet al., 2002) etc. In a recent study, thermostable amylotic enzymes are investigated to improve industrial processes of starch degradation. Glucoamylase produced by Streptosporangium sp. An endophytic actinomycete isolated from leaves of maize exhibited thermostable properties (Stamford et al., 2002).

Transmission of Endophytes

Endophytes enter the plants through two ways - vertical transmission and horizontal transmission. Endophytes transmit asexually through seeds by penetrating the host seeds through its hyphae in vertical transmission and during horizontal transmission, endophytes are transmitted sexually through spores.

Endophytic fungus colonies various parts of the plant. Many of them sporulate in culture indicating the potential to release spores in air. Indeed, sporulation is seen after senescence of plant tissues. In few cases as dispersal have been documented in wild however, various mechanisms remain unexplored. Nutrients are cycled between host and fungus. The endophytic fungus gains predictable environment in which nutrients are readily available.

ENDOPHYTIC FUNGI AS A SOURCE OF BIOACTIVE NATURAL PRODUCTS

The diverse endophytes residing in the plant are rich source of bioactive natural products. Large numbers of secondary metabolites are extracted from the endophytic fungus like alkaloids, flavonoids, steroids, terpenoids, and other compounds like peptides, polyketones, quinols, phenols and some chlorinated compounds also extracted. The biology of the predators is greatly affected by the endophytic fungal metabolites. There is a growing evidence that bioactive substance produced by microbial endophytes may not be involved in the host endophytic relationship, but may also ultimately have applicability in medicine, agriculture and industry (Strobel, 2000). In fact, a recent comprehensive study has indicated that 51% of biologically active substances isolated from endophytic fungi were previously unknown (Stierleet al., 1999; Strobel, 2002; Weber et al., 2004; Shen et al., 2006).

Secondary Metabolites from Endophytes as Alkaloids: Amines and Amides

Biosynthesis of ergot alkaloids such as ergo valine is better understood with the ergot fungus Claviceps purpura (Lane et al, 2000). Amines and amides are common secondary metabolites of Acremonium endophyte (Schardl and Philips, 1997).

Secondary Metabolites from Endophytes as Indole Derivatives

Indole alkaloids such as channoclavine, agroclavine and elymoclavine characterised from a culture of Neotyphodium endophyte. Some endophytes can produce plant hormone with an indole framework.

Secondary Metabolites from Endophytes as Antibiotics

The increasing occurrence of multidisciplinary pathogenic strain has limited the effect of traditional antimicrobial treatment. Hence, there is an urgent need for new therapeutic agents with infectious disease control (Stribel and Daisy, 2003; Larsen et al., 2005). Chaetoglobosin A and rhizoctonic acid, from endophytic Chaetomium globosum, in Maytenushookeri, and Rhizoctonia sp., in Cynodondactylon, respectively were reported to be active against the gastric ulcer bacterium Helicobacter pylori.

Secondary Metabolites from Endophytes as Antiviral Agents

Another fascinating use of products from endophytic fungi is the inhibition of viruses. From Cytonema sp. two novel human cytomegalovirus (hCMV) protease inhibitors cytonic acid A and B, have been isolated.

Secondary Metabolites from Endophytes as Anticancer Agents

The discovery of the paclitaxel (taxol) producing endophytic fungus Taxomyces andreanae from Taxus brevifolia (Strobel et al., 1993; Stierle and Strobel, 1995) evoked the interest in endophytes as potential new sources for therapeutic agents. Taxol is the world's first billion-dollar anticancer drug and is used to treat a number of other human tissue proliferating diseases as well (Strobel, 2002).

Secondary Metabolites from Endophytes as Antimycotic Agents

Cryptocandin A isolated from Cryptosporiopsis quercina, endophytic of Tripterigeum wilfordii(a medicinal plant) is currently used against a number of fungi causing skin and nail diseases (Strobel, 2002).

Secondary Metabolites from Endophytes as Antioxidants

Pestacin and isopestacin obtained from culture fluid of Pestalotiopsis microspora from Combretaceaous plant, Terminalia morobensis have antimicrobial as well as antioxidant activity. The antioxidant activity of pestacin is more potent than that of trolox, Vitamin E derivatives (Harper et al, 2003).

Secondary Metabolites from Endophytes as Insecticides

Several endophytes are known to have anti- insect properties. Naphthalene produced from Muscodor vitigenus isolated from liana (Paullina paullinoides) shows promising preliminary results as an insect deterrent and has exhibited potent insect repellency against the wheat stem sawfly (Cephuscinctus) (Daisy et al, 2002;2003). Since synthetic insecticides cause many ecological damage, endophytic research continues for the discovery of powerful, selective and safe alternatives.

ENDOPHYTIC FUNGI PRODUCING BIOACTIVE COMPOUNDS ORIGINALLY FROM THEIR HOST PLANTS

Endophytic Fungi for Production Paclitaxel and Itsanalogues

Paclitaxel (taxol) – Highly functionalized tetracyclic diterpenoid bioactive compound found originally from the bark of Taxus brevifolia (Wani et al 1971) has been proved with an efficient action against prostate, ovarian, breast and lung cancers. The major supply of Paclitaxel has been from the wild Taxus plants. It is found in extremely low amount in various parts such as needles, barks and roots of Taxus species. In the order of satisfy the growing demand of market and make it more widely available, the alternative resource and potential strategy should be developed.

Endophytic Fungi for Producing Podophyllotoxin

Podophyllotoxin (PDT) is known as aryltetralin lignin with potent anticancer, antiviral, antioxidant, antibacterial, immuno stimulation and anti-rheumatic properties. Mainly occurs in genera Diphylleia, Dysosma, Sabina (Juniperus) and Sinopodophyllum(Podophyllum). PDT has been used as a precursor for chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophose phosphate. At present major supply of podophyllotoxin is from the natural Sinopodophyllum plants.(Yang et al 2003; Lu et al 2006; Cao et al 2007; Kouret al 2008; Zeng et al2003; Guo et al 2004; Eybergeret al2006; Puriet al2006.).

Endophytic Fungi for Producing Camptothecine and its Analogues

Camptothecin (CPT)- Apentacyclic quinolone alkaloid was firstly isolated from the wood of Camptotheca acuminate (Nyssacesae) (Wall et al 1996). CPT and its analogue 10 -hydroxycamtothecin have been regard as two of the most effective antineoplastic agents. The primary action mechanism of CPT is by virtue of inhabiting the intranuclear enzyme topoisomerase-1, which is required in DNA replication and transcription during molecular events (Haianget al 1985). Hycamtin (topotecan) and camtostar (irinotecan) two of the famous CPT semi-synthetic drugs, have already been in clinical use against ovarian small lung and refractory ovarian cancers popularity all over the world (Sirikantaramas et al 2007). At the present the major supply of this bioactive compound CPT is still from the wild trees Camptotheca acuminata and Nothapodytes nimmoniana (lcacinaceae). As the growing demand of this compound, it has resulted in extensive cropping of the trees in China and India. It is necessary to further find high yielding candidates and alternative sources to produce this bioactive compound and its analogues (Amna et al 2006; Shweta et al 2010).

Endophytic Fungi Producing Vinblastine and its Analogues

Vinblastine and Vincristine – The terpenoid indole alkaloids derived from the coupling of vindoline and Catharanthine monomers, are two of the well-known anticancer agents (Perez et al 2002; Wang et al 2010). The primary action mechanism of vincristine is via interference with microtubule formation and mitotic spindle dynamics disruption of intracellular transport and decreased tumour blood flow, with the latter probably as a consequence of anti-angiogenesis. Guo et al in 1998 first report an endophytic fungus Alternaria sp. Isolated from the phloem of Catharanthus roseus that had the ability to produce vinblastine. Later Zhang et al 2000 successfully discovered an endophytic Fusarium oxysporumfrom the phloem of C.roseus that could produce vincristine.

Endophytic Fungi for Producing other Bioactive Compounds Originally from their Host Plants

Other pronounced bioactive compounds originated from the host plants could also be biosynthesized by their endophytic fungi mainly include huperzine A, α-irone, β-irone, diosgenin, hypericin and toosendanin. Li et al first reported an endophytic fungus Acremonium (2F09P03B) obtained from Huperzia serrate that could produce huperzine A that was a lycopodium alkaloid. They further optimized its fermentation conditions (Li et al 2007). Zhou et al in 2004 reported an endophytic fungus Penicillium chrysogenum obtained from Lycopodium serratum could also produce huperzine A as much as 4.761 mg/L in liquid culture.

Endophytic Fungi as Biocatalysts

Suryanarayanan et al in 2012 reviewed various enzymes produced by different endophytic fungi. Endophytic fungi was found to produce different extracellular enzymes Amylases, Cellulases, Laccases, Lipases, Pectinases, Pectate lyases, Proteases and Tyrosinases (Suryanarayananet al., 2012).

Why are endophytes so alluring: their cosmopolitan nature, pharmaceutical potential (Strobel and Long 1998), have capacity of mimicking host compound (Strobel 2002) and evidence that may act as mutualists with their host plant under certain conditions (Carrol 1988; Clay1991) are the fascinating reason for their study. There are many criteria for selection of plants for the isolation of endophytes; plants which hold ethnobotanical value and have been exploited by humans as traditional medicines is important one. Therefore, the present study has been proposed on investigating the presence of endophytic fungi inside bark tissues of Cassia siamea and screening of their biopotential activity.

Cassia siamea

    Cassia siamea

    Siamese Cassia is a small to medium sized tree, up to 15-20 m tall, with a short bole and low branching high crown. Leaves pinnate, alternate, rachis 25-30 cm long, with a marked furrow, 8-13 pairs of leaflets of different size. Leaflets oblong, rounded at the base and at the apex, slightly retuse. Upper side dark green and shining, underside dull-green, shortly haired. Flowers yellow, up to 3.5 cm long, in dense racemes at the end of the shoots, and in their axils.

    Agricultural relevance

    The tree is grown to provide shade along roads and in cocoa, coffee and tea plantations. It is also planted as a dense windbreak and shelterbelt. It is pruned into hedgerows and used as a live fence around food crops. When used as a hedgerow, it effectively increases topsoil infiltration, reducing runoff and combating soil erosion. The leaves are used as green manure, and a well-grown tree can yield 500 kg/year of fresh leaves. C.siamea forms ecto-mycorrhizae and provides very useful mulch, especially in alley-cropping systems. In India, it is used as a host for sandalwood (Santalum spp.), a parasitic tree producing the well-known aromatic wood.

    Medicinal properties

    Effective against anxiety, insomnia, stress, beriberi, diabetes, dysentery, gastrointestinal and urinary problems. Has analgesic, antianxiety, antibacterial, antihypertensive, antimalarial, diuretic properties.

    Objectives of the present study:

    1. Isolation of endophytic fungi from Cassia siamea bark tissues.

    2. Purification, mainantance and storage of cultures.

    3. Microscopic and molecular identification of isolated endophytic fungi.

    4. Extraction of crude secondary metabolites from isolated endophytic fungi.

    5. Qualitative and quantitative assay for extracellular enzyme production of isolated cultures.

    6. Screening of isolated cultures for antimicrobial and antioxidant assay.

    MATERIALS AND METHODS

    Collection of Plant Material

    Mature healthy, asymptomatic plant materials (bark), were collected from Cassia siamea from the botanical garden, BHU campus located in Varanasi, Uttar Pradesh, India (25o16`3`N 82o 59` 19E) Varanasi, Eastern zone of U.P. The bark samples were brought to the laboratory in sterile polythene bags and processed within a few hours after sampling. Fresh plant materials were used for isolation work to reduce the chance of contamination.

    Plant Surface Sterilization for Isolation of Endophytic Fungi

    It is necessary to sterilize the tissue before dissection to remove the surface microorganisms. The surface treatment was done adopting the methodology by Petriniet al. (1992). The bark sample were washed thoroughly in running tap water for 10 minutes to remove the debris and soil particles adhered to it and finally washed with double distilled water to minimize the microbial load from the sample surface. The samples were washed in 70% ethanol for 1 minute followed by 4% sodium hypochlorite (NaOCl) for 5 minutes and then washed with 70% ethanol for 30 seconds to remove the epiphytic microbes. Finally, the samples were washed with autoclaved distilled water for 3 times and blotted on autoclaved blotting paper. After surface treatment the bark tissues was then carefully dissected into small pieces approximately 0.5 x 0.5 mm. So that maximum possible endophytes may be obtained.

    Potato Dextrose Agar (PDA) was used as Solid Culture Plate Media
    Media components (PDA) Composition(g/500ml)
    Potato peeled 100g
    Dextrose 10g
    Agar 10g
    Distilled water 500ml
    Note
    pH-5.6

    Potato Dextrose Broth (PDB) was used as Liquid Culture Plate Media
    Media components (PDA) Composition (g/500ml)
    Potato peeled 100g
    Dextrose 10g
    Distilled water 500ml
    Note
    pH-5.6

    Isolation of Fungal Endophytes

    The imprints of surface sterilized bark pieces were taken on the PDA to check the efficacy of surface sterilization. All dissected sample tissues were then placed on separate Petri plates containing Potato Dextrose Agar (PDA) medium supplemented with Streptomycin (200mg/l). A control PDA plate without any tissue segments was also run parallel to the experiment to examine contamination. All the Petri plates were sealed with sterile parafilm to protect them from contamination during repeated handling, while examining for emerging endophytes. The plates were incubated for 21 days at 26±2ºC in BOD cum humidity incubator. Tissues were observed for fungal growth at 2 days interval for 20 days. Fungi that grew from the tissue fragments were sub-cultured on PDA Petri plates for identification and enumeration.

      Bark samples of Cassia siamea plotted on PDA plates along with imprint plates

       

        Bark samples placed on PDA plate

          Bark samples imprinted on PDA plate

          The isolates obtained from bark tissues were purified by transferring each isolate to a fresh plate.

          Preservation of Endophytic Fungi in Slants

          Slant preparation: 10ml tubes were taken to which PDA media was poured and sterilized and after sterilization the tubes were kept in tilted position. After the media solidified a loop full of endophytic fungal isolates were taken in the inoculation needle under aseptic condition and were transferred to PDA slants prepared in test tubes. They were allowed to grow for 3-4 days in BOD incubator at 27-28ºC. After considerable growth, the slants were stored at 4ºC. Two copies of each isolate were preserved.

          Microscopic and Morphological Identification

          Enzymes of microbial origin have high biotechnological importance in the processing of foods, manufacturing of detergents, textiles, pharmaceutical products, medical therapy, and in molecular biology. Among a large number of non-pathogenic microorganisms capable of producing useful enzymes, filamentous fungi are particularly interesting due to their easy cultivation and high production of extra- cellular enzymes of large industrial potency. Endophytic fungi occupy a relatively unexplored area in microorganism isolation, and thus represent a new source for obtaining enzymes of microbial origin have high biotechnological importance in the processing of foods, manufacturing of detergents, textiles, pharmaceutical products, medical therapy, and in molecular biology. Among a large number of non-pathogenic microorganisms capable of producing useful enzymes, filamentous fungi are particularly interesting due to their easy cultivation and high production of extra- cellular enzymes of large industrial potency. Endophytic fungi occupy a relatively unexplored area in microorganism isolation, and thus represent a new source for obtaining Enzymes of microbial origin have high biotechnological importance in the processing of foods, manufacturing of detergents, textiles, pharmaceutical products, medical therapy, and in molecular biology. Among a large number of non-pathogenic microorganisms capable of producing useful enzymes, filamentous fungi are particularly interesting due to their easy cultivation and high production of extracellular enzymes of large industrial potency. Endophytic fungi occupy a relatively unexplored area in microorganism isolation, and thus represent a new source for obtaining enzymes with different potentialities. Fungi endophytic on medicinal plants or plants that grow in unique and extreme habitats are likely to possess novel enzyme systems that may help in the understanding of their host tissue colonization ability, in view of the competition provided by saprophytes and plant pathogenic fungi.

          The endophytic fungal isolates were screened for cellulose, amylase and protease activity. Production of extracellular enzymes by the fungal endophytes was assessed by digestion of suspended or dissolved substrate in agar plates after inoculation with mycelia plugs and incubation for 3-9 days at 25±2ºC. The zone of enzyme activity surrounding the fungal colony was observed and recorded.

          Screening for cellulase activity

          Media composition of cellulase activity for 1 litre
          S.No Ingredients Quantity
          1. Yeast extract 0.1gm
          2. Peptone 0.5gm
          3. Agar 20gm
          4. Carboxy Methyl Cellulose 5gm

          After 3-9 days of incubation, the media plates are flooded with 0.2% aqueous Congo Red dye and further destained with 1M NaCl for 15 minutes. The clear zone surrounding the colony indicating the cellulase activity.

          Screening for amylase activity

          Media composition of amylase activity for 1 litre
          S.No Ingredients Quantity
          1. Yeast extract 0.1gm
          2. Peptone 0.5gm
          3. Glucose 1gm
          4. Agar 20gm
          5. Soluble starch 20gm(2%)

          After 3-9 days of incubation, the media plates are flooded with 1% iodine solution in 2% potassium iodide. A clear zone around the colony indicates a positive test for amylase activity.

          Screening for protease activity

          Media composition of protease activity for 1 litre
          S.No Ingredients Quantity
          1. Yeast extract 0.1gm
          2. Peptone 0.5gm
          3. Glucose 1gm
          4. Agar 20gm
          5. Distilled water 900ml

          The pH of media is adjusted to 6 and is amended with 0.4% gelatine solution (autoclaved separately).

          After 3-9 days of incubation, the media plates are flooded with saturated ammonium sulphate. A clear zone around the colony indicates a positive test for protease activity.

          Molecular Characterization

          DNA isolation

          For the isolation of DNA, Sodium Dodecyl Sulphate (SDS) method was applied to extract the genomic DNA of endophytic fungi. 500µl of lysis buffer (tris-Cl -50mM, EDTA-100Mm, NaCl-150mM) was taken in Eppendorf tube and o.2gm fresh fungus culture was added in tube and crushed gently. 0.1 vol of 10% SDS was added and incubated at 37ºC for 2 hours in water bath. After incubation, 75µl of 5M NaCl and 75µl of CTAB/NaCl solution were added into the sample and mixed thoroughly and incubated at 65ºC in water bath for 30 minutes. Equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) were added and mixed thoroughly. It was centrifuged at 10000 rpm for 10 minutes. The aqueous supernatant was taken in fresh Eppendorf tube and precipitated with 0.6 volume of ice-coldisopropanol and 0.1 volume of sodium acetate. Samples were kept in -20ºC for 4 hours/4ºC for overnight. The next day, tubes were centrifuge at 10000 rpm for 10 minutes and the pellet was washed with 70% ethanol and dried it completely. The dried pellet was dissolved in 50µl of milliQ water and stored at -20ºC. DNA was analysed in 0.8% agarose gel electrophoresis.

          Gel electrophoresis of isolated DNA

          For confirmation and qualification of the isolated genomic DNA, it was run on a gel electrophoresis unit in 0.8% agarose gel, which was then stained with ethidium bromide (0.5µl/ml), and the DNA bands were visualized under UV Transilluminator.

          Disc of agar containing mycelial growth of the fungal endophytes was inoculated in 100ml of PDB and was incubated at 27-28ºC in a BOD incubator for 21 days. The broth culture was then harvested for secondary metabolites extraction using ethyl acetate as solvent. The fungal mycelium was filtered from the broth by a four-fold layer of muslin cloth. The broth was taken in a new flask and equal volume of pure ethyl acetate was added to it. The mixture was poured into a separating funnel and was mixed well. The solvent layer was collected separately while the broth was extracted twice again with ethyl acetate. This is termed as triple extraction and it ensures maximum extraction of secondary metabolites from cultured broth. The ethyl acetate extract was evaporated using a rotary vacuum evaporator to concentrate the extracted metabolites. The concentrated metabolites were transferred in pre weighed glass vials, dried completely and final weight was taken to know the amount of secondary metabolites obtained from the culture broth.

            Extraction of Secondary Metabolites from the Fungal Endophytes

            Antibacterial assay

            Screening of the antibacterial activity was checked by disc diffusion method.

            Test organisms: Proteus vulgaris, Morganella morganii, Enterococcus faecalis, Shigella boydii, Pseudomonas aeruginosa and Escherichia coli.

            The agar plate diffusion assay method was used to evaluate the antibacterial activity against the test micro organisms. For this assay Nutrient Agar (NA) media was prepared. 10µl of metabolites are loaded onto 5mm sterile filter paper discs and were placed on the NA plates swabbed with the different organisms to be tested. A disc loaded with methanol used as control. The plates were then incubated for 18-24 hours. Zone of inhibition were measured with a measuring scale and compared with the methanol control.

            Media composition of Nutrient Agar for 1 litre
            S.No Ingredients Quantity
            1. Peptone 5gm
            2. Yeast extract 3gm
            3. Sodium chloride 5gm
            4. Agar 20gm

            Antifungal assay

            Screening of the antifungal activity was checked by disc diffusion method.

            Test organisms: Candida albicans, Microsporumgypseum and Trichophyton mentagrophytes.

            For this assay Sabouraud Dextrose Agar (SDA) media was prepared. 500µg of metabolites are loaded onto 5mm sterile filter paper discs and were placed on the PDA plates swabbed with the different organisms to be tested. A disc loaded with methanol used as control. The plates were then incubated for 18-24 hours. Zone of inhibition were measured with a measuring scale and compared with the methanol control.

            Media composition of SabouraudDextrose Agar for 1 litre
            S.No Ingredients Quantity
            1. Glucose 40 gm
            2. Peptone 10 gm
            3. Agar 20 gm

            Antioxidant assay

            The secondary metabolites of endophytic fungi were examined by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) free radical-scavenging assay to confirm the antioxidant activity. DPPH is a free radical, stable at room temperature, which produces a violet solution in methanol. It is reduced in the presence of an antioxidant molecule, giving rise to uncoloured methanol solutions. The use of DPPH provides an easy and rapid way to evaluate antioxidants.

            For antioxidant activity 10µl of metabolite dissolved in 2990µl of methanol was taken in 5ml tubes and maintained with 1000µl of DPPH reagent. 10µl of ascorbic acid dissolved in 2990µl of methanol was taken in 5ml tubes and maintained with 1000µl of DPPH reagent was used as positive control. For negative control 3ml methanol with 1 ml DPPH was used. 4ml methanol was used as blank. The tubes were then incubated in dark for 30 minutes and change in colour of DPPH was observed. The absorbance of the solution was taken at 517nm. The % inhibition of free radicals was calculated by the following formula:

            %inhibition=(Absorbance of control – Absorbance of sample) x 100 /Absorbance of control

            RESULT AND DISCUSSION

            Isolation, Purification and Preservation of Endophytic Fungi

            The present study was performed in lab of mycopathology and microbial technology, CAS botany, BHU. All the steps were done under aseptic conditions. The experiment was started with isolation of endophytic fungi from Cassia siamea bark tissues. Thirty endophytes belonging to ten morphotypes were isolated from the bark stems of the plant and were named as: JCSB1I, JCSB1M, JCSB1, JCSB3O, JCSB4O, JCSB5O, JCSB7R, JCSB9R, JCSB15R and JCSB18R. Among which JCSB7R has maximum Colonization Frequency (CF%) of 18%, followed by JCSB5O with 4% CF%, JCSB1I, JCSB1M with 2% and remaining fungus showed 1% CF% (table 8).

              Table 8 Different isolates obtained from Cassia siamea with their % colonization frequency

                Isolation, purification and preservation of fungal isolates

                Morphological and Microscopic Identification

                The isolated endophytic fungi were observed for the spores and other macroscopic and microscopic structures under the microscope and with the help of standard taxonomic manuals.

                      Fig 7 Morphological microscopic observation of fungal endophytes

                      Molecular Characterization of Endophytic Fungi

                      Isolation of genomic DNA

                      The development of molecular techniques for the identification of endophytic fungi has been opening a new perspective for taxonomic characterization and relationship between complex groups of organisms. Endophytic fungal isolates were subjected to molecular characterization based on ITS sequencing. DNA isolation was performed by CTAB-NaCl method and it was confirmed by agarose gel electrophoresis.

                        Total DNA of Cassia siamea endophytic fungal isolates


                        1- Ladder, 2- JCSB1M (a), 3- JCSB1M (b), 4- JCSB1O (a), 5- JCSB1O (b), 6- JCSB2O (a), 7- JCSB2O (b), 8- JCSB15R (a), 9- JCSB15R (b), 10- JCSB18R (a), 11- JCSB18R (b), 12- JCSB5O (a), 13- JCSB5O (b), 14- JCSB9R (a), 15- JCSB9R (b), 16- JCSB1I (a), 17- JCSB1I (b), 18- Ladder

                        Qualitative analysis of enzyme production

                        Fungal enzymes are gaining importance in agriculture, industry and human health, as they are often more stable (at high temperature and extreme pH) than the enzymes derived from plants and animals. Fungal enzymes are used in manufacturing food, beverages, confectioneries, textile and leather and help simplifying the processing of raw materials. In order to establish the functional role of endophytes it would be useful to establish their patterns of substrate utilization and which enzymes they produce (Carroll and Petrini, 1983). If they are weak parasites or latent pathogens, they may produce proteinase and pectinase (Brett, 1990b; Reddy et al, 1997), while if they are mutualistic, eventually being saprobes, they are likely to produce cellulase, and xylanase (Pointing SB 1999). The selected isolates were screened for cellulase, amylase and protease activity. The diameter of the zone produced by different isolates may be correlated with their ability to hydrolyse different substrates present in the medium by different enzymes like Cellulase for cellulose, Amylase for starch and Protease for gelatin. Among eight isolates, 62.5% isolates have given positive result for amylase and protease and 75% isolates for cellulase. For amylase, protease, cellulase the activity range was 0.06cm- 1.3cm, 1- 1.3cm and 0.3- 1.2cm respectively (table 10). Hence the extracellular enzyme producing fungi may be utilized for commercial production of fungi.

                          Table 9 Different enzyme activity of endophytic fungi from Cassia siamea

                                Pictures showing clear zone of enzyme activity by isolates in the media plate

                                Antimicrobial assay

                                Antibacterial activity

                                Secondary metabolites of different morphotypes of isolated fungi were tested against human pathogenic bacteria namely Proteus vulgaris, Morganella morganii, Enterococcus faecalis, Shigella boydii, Pseudomonas aeruginosa and Escherichia coli.JCSB5 Outer shows antibacterial activity against almost all the bacteria except Shigella boydii, a gram-negative bacterium causing dysentery in humans through fecal-oral contamination. None of the endophytic fungi shows antibacterial activity against Shigella boydii. JCSB1 Inner ** shows bacteriostatic and antibacterial activity against Proteus vulgaris, Morganella morganii and Pseudomonas aeruginosa, Escherichia coli and have maximum (19mm) zone of inhibition towards E.coli. JCSB1 Inner shows bacteriostatic and antibacterial activity against Proteus vulgaris and Pseudomonas aeruginosa, Escherichia coli and have maximum (13mm) zone of inhibition towards Proteus vulgaris. JCSB1 Middle, JCSB2 Middle, JCSB1 Outer shows bacteriostatic activity against Proteus vulgaris and antibacterial activity against Pseudomonas aeruginosa. JCSB2 Outer shows bacteriostatic activity against Proteus vulgaris and antibacterial activity against Pseudomonas aeruginosa and E.coli. JCSB3 Outer and JCSB7 Rind shows antibacterial activity against Pseudomonas aeruginosa and Morganella morganii and the maximum (17mm) zone of inhibition was shown by JCSB7 Rind against Morganella morganii. JCSB15 Rind and JCSB18 Rind shows antibacterial activity against Pseudomonas aeruginosa, Morganella morganii and Proteus vulgaris. The result was shown in the table below:

                                  Table 10 Table for Antibacterial activity by different Isolates

                                      Antibacterial assay

                                      Antifungal activity

                                      Secondary metabolites of six endophytic fungal isolates were screened for antifungal activity (JCSB1 Inner**, JCSB1 Inner, JCSB2 Middle, JCSB1 Outer, JCSB15 Rind and JCSB18 Rind) against human pathogenic fungi like Candida albicans, Microsporum gypseum and Trichophyton mentagrophytes. The range of inhibition zone is 9mm. Metabolites of JCSB1 Inner showed a zone of inhibition against Candida
                                      albicans
                                      and JCSB1 Inner** showed a zone of inhibition against Microsporum gypseum. None of the fungal isolates exhibits antifungal property against Trichophyton mentagrophytes. The results are shown in Fig 11 and table 11.

                                        Table 11 Table for Antifungal activity by different Isolates

                                          Antifungal assay

                                          Antioxidant activity

                                          There are many diseases due to the effect of reactive oxygen species (ROS). In the prevention and treatment of disease linked to ROS, one of the strategies involves use of antioxidants as supplement. Natural antioxidants are commonly found in medicinal plants, vegetables, and fruits. Studies revealed that many endophytic actinomycetes can be the better antioxidant than the synthetic antioxidants. There are many more chances of getting very good active antioxidant compounds from endophytic actinomycetes based upon their occurrence in the plants having antioxidant property. The free radical scavenging activity of fungal extract was carried out by using DPPH (2, 2-Diphenyl-1-picrylhydrazyl). Eight endophytic fungi and standard (Ascorbic acid) were assayed for antioxidant activity at 517nm in UV spectrophotometer. Among eight isolates, JCSB15 Rind showed maximum activity (70.27%) followed by JCSB5 Outer showing 67.49% activity. JCSB2 Middle shown moderate activity (54.75%) while JCSB1 Inner** shown 50.95%. JCSB1 Inner, JCSB1 Outer, JCSB3 Outer, JCSB18 Rind shown below 50% activity.

                                            Table 12 Antioxidant activity by different isolates

                                            CONCLUSION AND FUTURE PERSPECTIVES

                                            Endophytic fungi are like treasure for novel natural compounds with interesting biological activities, a high level of biodiversity and may also produce several compounds of pharmaceutical significance, which is currently attracting worldwide scientific investigations toward isolation and exploration of their biotechnological promise. They represent a relatively unexplored ecological source, and their secondary
                                            metabolism is particularly active because of their metabolic interactions with their hosts. Endophytic fungi have received more attention as they can sometimes produce bioactive compounds analogous to their hosts. Isolation of endophytic fungi from medicinal and other plants to produce biologically active agents for biological utilization on a large commercial scale is possible because they can easily culture in laboratory instead of harvesting plants and affecting the environmental biodiversity. Due to their great importance were showed in plants, scientists have exploited endophytic fungi very much attention for detection of bioactive compounds in the form of antimicrobial activity, anticancer activity etc. Considering the above, I isolated endophytic fungi from Cassia siamea bark. Isolated endophytic fungi were found to exhibit good antimicrobial activity against various human pathogenic bacteria. Screening of cellulase and protease activity was also remarkable. Although antioxidant study was not good, as only 50% isolates gave above 50% free radical scavenging activity. Instead of this the finding of this study provides a strong platform for the isolation and purification of antibacterial compounds and enzymes from endophytic origin. Also there are further need to study antimicrobial activity and mechanisms for phytopathogens, quantification of enzyme and isolation of endophytic bacteria and endophytic actinomycetes from Cassia siamea bark tissues.

                                            ACKNOWLEDGEMENTS

                                            I feel highly obliged and honoured in expressing my indebtedness to my respected supervisor Prof. R. N. Kharwar, CAS in Botany, Institute of Science, BHU, for his valuable guidance and encouragement, inspiration, warm, sympathy and valuable criticism that lead to successful, completion of this dissertation.

                                            I am thankful to IASc Academy, who selected and gave me this golden opportunity by providing the Summer Fellowship 2019. It’s a great pleasure to express my gratitude and thankfulness to Miss. Monika Yadav for her constant support and technical help during the dissertation.

                                            I also thank to other lab members, Mrs. Arti Singh, Mr. Jayhind Nishad, Mr. Veer Singh Gautam, Miss. Puja Kumari, Mr. Rajnish Bharati and Miss. Priyanka Prajapati for their valuable and continuous support.

                                            I dedicate my overall work to omnipotent God, my parents, and to my teachers who provided me everlasting support and financial assistance according to read.

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