Keywords

1 Introduction

Microorganisms are an important component of the environment, they affect their surroundings in various ways and forms, one of them are endophytes. The term endophyte was first introduced by de Bary in 1866 for all those microbes that reside inside the living healthy tissues. Many workers define endophytes in various ways, but the definition given by Bacon and White (2000) was perhaps most acceptable as ‘microbes that colonize living, internal tissues of plants without causing any immediate and overt negative symptoms’. This is a topographical term and includes bacteria, fungi, actinomycetes and algae, which spend their whole or a period of life cycle either in symplast or apoplast region of healthy plant tissues without producing any disease or clinical symptoms. On the basis of their nature, endophytes may be categorized in three groups: (1) pathogens of another host that are nonpathogenic in their endophytic relationship, (2) nonpathogenic microbes, (3) pathogens that have been rendered nonpathogenic but are still capable of colonization by selection methods or genetic alteration (Backman and Sikora 2008). Among all the endophytic microbes, fungi are the most studied group so far. Endophytic fungi play an important role in plant/host community health by providing resistance from herbivores (Brem and Leuchtmann 2001), pathogenic fungi, bacteria, viruses, insects, nematodes (Gond et al. 2010), illness (Clay 1990), reduced seed production (Rice et al. 1990), temperature and salinity (Redman et al. 2002) and also against drought and minerals (Malinowski et al. 1997), heavy metal (Li et al. 2012). Endophytic fungi are also able to produce a considerable number of useful enzymes and this ability can make enzymes cost effective because approximately 60 % of the currently used industrial enzymes are of fungal origin (Østergaard and Olsen 2010). Interestingly, Suryanarayanan and his colleagues observed the number of foliar fungal endophytes associated with trees of forests in the Western Ghats mountain (in Southern India) produced a range of extracellular enzymes including amlyases, cellulases, chitinases, chitosanases, laccases, lipases, pectinases and proteases (Suryanarayanan et al. 2012).

An irrational and irregular use of antibiotics makes pathogen more resistant and it is a serious impediment for microbiologists providing the required demand of antibiotics. To cope with this problem, there is ultimate necessity for an alternative and novel source of effective drugs without destroying biodiversity. In such respect, endophytic fungi became an effective solution because one can isolate the compound of plant/host origin without destroying the plant population. After the discovery of taxol (billion dollar drug) from the endophytic fungus Taxomyces andreanae (Stierle et al. 1993), it proved itself as a novel source of taxol production without loss of the Taxus plant. After this discovery, the endophytic research came to light and microbes have been considered as a novel and alternative source for new biologically active compounds and/or compounds of host origin such as taxol (Stierle et al. 1993), vincristin (Tung et al. 2002), camptothecin (Shweta et al. 2010), piperin (Verma et al. 2011), azadirachtin (Kusari et al. 2012), etc. Today, credits go to endophytic microbes for producing a number of new and effective bioactive natural compounds that can be used in agriculture, medicine and industry. In addition, more than 100 anticancer compounds have been (57 % novel and 43 % known) isolated only from endophytic fungi (Kharwar et al. 2011). In this chapter, we have focused mainly on the diversity of endophytic fungi of eight medicinal (Azadirachta indica, Agele marmelos, Catharanthus roseus, Eucalyptus citriodora, Nyctanthes arbor-tristis, Adenocalymma alliaceum, Tinospora cordifolia, Cinnamomum camphora) plants of Uttar Pradesh, India, with their antimicrobial potential.

2 Transmission of Endophytic Fungi

Transmission describes the spreading of microbes within and among host population. Endophytic fungi have two transmission modes, vertical and horizontal. Vertical transmission occurs when fungi travel from host to their offspring via host tissues such as host seeds and vegetative propagules. Systemically infected endophytic fungi have vertical transmission mode that differs from horizontal transmission where fungus travels by its sexual or asexual spores.

3 Ecology and Biodiversity of Endophytic Fungi

Endophytic fungi are important, hidden, highly diverse, less exploited and highly potential component of the environment. Almost all plant species studied to date for endophytic diversity were found to act as a reservoir for potential of microbes to be used to resolve the problems of mankind. The endophytes were observed in all green biota ranging from algae (Yang et al. 2006), bryophytes (Chambers et al. 1999), pteridophytes (Schmid and Oberwinkler 1995), gymnosperms (Huang and Wang 2011) and to angiosperms (Mishra et al. 2012a), including underground root to all aerial parts of host (Kharwar et al. 2008). Endophytic fungi isolated from water-stressed deserts (Bashyal et al. 2005), cold-stressed arctic (Fisher et al. 1995), Antarctic ocean (Rosa et al. 2009; Wang et al. 2006), geothermal soils (Redman et al. 2002), highly diverse rain forests (Strobel 2002), dry deciduous and coastal forests (Suryanarayanan et al. 2003) and mangrove swamps (Maria et al. 2005). Fungal endophytes were isolated from either all or specific organs of selected hosts showing the impacts of environmental variables on their colonization frequency (CF), diversity and antimicrobial activity (Hyde and Soytong 2008; Mishra et al. 2012a; Verma et al. 2011; Verma et al. 2013). Our earth harbours almost 300,000 higher plants species, and each species represents either one or plethora of endophytic community which is well proved by the previous reports of higher plants fungal endophytes (Strobel 2002). Out of these plants that exist on the earth, only a few dozen, have been studied related to their endophytic biology, and every plant studied has an endophytic community. Including fungal endophytes, the ratio of fungal to plant species will reach up to 33:1 from 6:1 (Hawksworth and Rossman 1987).

3.1 Endomyco Diversity in Adenocalymma alliaceum Miers

A. alliaceum, is commonly known as garlic creeper or lahsun lata plant. It is a member of the family Bignoniaceae, a highly medicinal, evergreen tropical shrubby vine plant that is native to the Amazon rainforest. In the absence of garlic, its leaf can be used as a substitute for cooking purposes. Every part of the plant is well used by the indigenous people of the Amazon as folk medicine for curing various disorders. Despite having several compounds, it is considered analgesic, anti-inflammatory, depurative, purgative and widely used against arthritis, rheumatism, body aches, muscle pain, cholesterol and injuries. Its leaves are also used to cure flu, pneumonia, cough, fever and headache. Kharwar et al. (2011) reported the isolation of total 149 fungal endophytic isolates belonging to 17 fungal taxa from 270 segments of leaf, stem and petiole (90 segments of each tissue). Collectively, among the total isolates recovered, hyphomycetes were more frequent (74.47 %) followed by mycelia sterilia (10.07 %), ascomycetes (8.05 %) and coelomycetes (4.03 %) (Table 3.1, Fig. 3.1). Among all tissues studied, leaves showed greater colonization of endomycobiota (72.22 %) compared to stem (67.78 %) and petiole (25.54 %). A. alternata (6.30 %), A. niger (5.93 %), Stenella agalis (5.20 %), Fusarium oxysporum (5.18 %), C. lunata (4.18 %) and Fusarium roseum (4.07 %) were recovered as the dominant genera. However, Penicillium sp. and Rhizoctonia sp. were the least frequent with equal CF of 1.85 %. Out of 17 taxa, Penicillium sp., C. globosum and Rhizoctonia sp. were only restricted to stem tissue, and as per authors this may be because of displacement of their spores from root and substrate specificity supported by stem.

Fig. 3.1
figure 1

Per cent recovery of different classes of endophytic fungi

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Table 3.1 Endophytic fungal diversity among eight different medicinal plants
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3.2 Endomyco Diversity in Aegle marmelos

A. marmelos is an Indian plant having medicinal and religious importance as well. The plant is used in Indian system of ayurvedic medicine against variety of diseases including diarrhoea, dysentery and dyspeptic symptoms. Green leaves of the plant are used for lowering blood sugar level. The plant was also reported to possess antifungal and antibacterial properties. Gond et al. (2007, 2011) isolated total of 511 endophytic fungal isolates representing 32 endophytic fungal taxa from 550 segments of bark, leaf and root. In the study, bark was found to harbour greater number of endophytic fungi followed by leaf and root. Among total taxa recovered, the Aureobasidium sp. (11.45 % CF) was found to be the highly dominated taxon. Among different endophytic classes, hyphomycetes showed maximum colonization 57.92 % followed by 28.57 % coelomycetes, 7.82 % ascomycetes and 5.67 % mycelia sterilia (Table 3.1, Fig. 3.1).

3.3 Endomyco Diversity in Azadirachta indica

A. indica is native to India and one of the most effective and popular medicinal plant, commonly known as neem, belongs to family Meliaceae. Different parts or extracts of the plant are used as antibacterial, antiretroviral, antiarthritic, anti-inflammatory and antiulcer. Over 400 bioactive compounds from neem plant and 32 from its endophytes have been reported so far. Verma et al. (2007) isolated 495 endophytic fungal isolates from 600 segments of leaf, stem and bark, root and fruit of neem collected from Varanasi region. The total endophytic fungal isolates recovered belonged to 44 fungal species including mycelia sterilia. In whole of the study, hyphomycetes dominated with 76.56 % followed by 17.37 % coelomycetes, 3.63 % mycelia sterilia, 2.22 % ascomycetes and interestingly only a single isolate (0.02 %) of zygomycete (Table 3.1, Fig. 3.1). However, genera like Cladosporium Aspergillus, Acremonium, Pestalotiopsis, Phomopsis, Curvularia and Trichoderma were observed as dominant fungi. Among 495 isolates, 223 isolates were recovered from 200 segments of leaf, bark and stem while 272 isolates were isolated from 400 segments of root and fruit (Verma et al. 2007, 2011; Verma 2009).

3.4 Endomyco Diversity of Catharanthus roseus

C. roseus is commonly known as Madagascar periwinkle or sadabahar belonging to family Apocynaceae. A number of anticancer vinca alkaloids such as vincristine, vindesine, vinorelbine, vinblastin and vinflunine have been isolated from the plant. It has also been used as a folk remedy to cure diabetes and high blood pressure. Kharwar et al. (2008) reported the isolation of 183 fungal endophytic isolates under 19 fungal species from 300 segments of stem, leaf and root of Varanasi region. Hyphomycetes showed maximum recovery (86.88 %) followed by mycelia sterilia or unidentified groups (8.19 %), 3.27 % coelomycetes and least by ascomycetes 1.63 % (Table 3.1, Fig. 3.1). The CF was found higher in root sample followed by leaf and stem. Root tissues were heavily colonized by genera such as Alternaria, Cladosporium and Aspergillus. Leaf tissues showed a greater diversity of endophytes and Drechslera, Curvularia, Bipolaris, Alternaria and Aspergillus spp. were the dominant fungi.

3.5 Endomyco Diversity in Cinnamomum camphora

C. camphora is commonly known as camphor or kapoor plant. The plant belongs to the family Lauraceae and is native to Taiwan, southern Japan, Southeast China and Indochina. The oil of camphor is used as an anti-inflammatory, antiseptic, a cardiac, carminative, diuretic, febrifuge, an insecticide, a laxative, rubefacient, stimulant and vulnerary agent. Kharwar et al. (2012) claimed the isolation of 162 endophytic fungal isolates belonging to 26 species from more than 100 segments of leaf, stem and petiole. Among isolates recovered, hyphomycetes ranked first with 62.96 % isolation frequency (IF) followed by coelomycetes 16.66 %, mycelia sterilia 15.43 % and least from ascomycetes 4.93 % (Table 3.1, Fig. 3.1). Among all the segments studied, leaf harbour maximum (40.44 %) endomyco isolates followed by stem (29.04 %) and petiole (30.24 %). Among all the species observed, A. niger (10.49 %) was found to be most dominated followed by Phyllosticta nobilis and Trichoderma harzianum with equal IF of 6.79 % while Arthrinium sp. and C. lunata were recorded as rare isolates with IF value of 0.61 %.

3.6 Endomyco Diversity of Eucalyptus citriodora

Basically E. citriodora is a long tree and native to Australia, but it is frequently grown in the northeastern states of India. The bluish-green leaves of the plant contain fragrant volatile oil that have antiseptic, expectorant, antibacterial, anti-inflammatory, deodorant, diuretic and antispasmodic properties. Commonly used and a very important essential oil, it is known as eucalyptol, isolated from the leaves and used as an anti-cough syrup, for aromatherapy, dentistry, and to treat bronchitis, sinusitis, chronic rhinitis and asthma, etc. (Gond et al. 2010; Gond 2011). A total of 552 fungal endophytic isolates belonging to 32 fungal species from 600 segments of leaf and stem at Varanasi and Sonbhadra regions were isolated. Hyphomycetes was found to be the highly dominated group (67.02 %) followed by coelomycetes (18.29 %), mycelia sterilia or unidentified taxa (11.05 %) while ascomycetes represented the least IF (3.62 %; Table 3.1, Fig. 3.1). Cladosporum cladosporioides with an IF of 11.77 % was the most dominant taxon followed by P. glomerata at 10.50 %.

3.7 Endomyco Diversity of Nyctanthes arbor-tristis

N. arbor-tristis is a well-known medicinal plant native to the Indian subcontinent and grows abundantly in all parts of the country. It is commonly known as Harsinghar, Parijata, or night jasmine and belongs to the family Oleaceae. The flowers and leaves of N. arbor-tristis are well known for their interesting antibacterial, antifungal, antileishmanial and cytotoxic activity. Gond 2011 described the endomyco diversity of leaf and stem of N. arbor-tristis collected from Varanasi and Sonbhadra regions. From 800 segments (400 segments for each tissue) of leaf and stem, the author reported the isolation of 799 endophytic isolates. In this study, the recovery of hyphomycetes was found maximum with 72.09 % followed by coelomycetes 16.64 %, mycelia sterilia 8.13 % and least from ascomycetes 3.12 % (Table 3.1, Fig. 3.1). A total of 34 endophytic fungal species were observed from both tissues collectively. Among the total 34 species recorded, 32 were isolated from the leaves while only 19 species from the stem. C. cladosporioides (11.63 %), A. alternata (7.38 %), Phomopsis helianthi (6.25 %) were observed as dominated taxa. C. cladosporioides, C. lunata, C. dematium, Drechslera ellisii, Acremonium sp., N. oryzae, Phomopsis sp. and Rhizoctonia sp. were isolated as common species for both tissues; Aspergillus fumigates, A. niger, Helicosporium sp. Scytilidium sp. and Stachybotrys sp. were only isolated from the leaf segments while isolate NAH3 only reported from stem segments; however, these results are a fine example of tissue specificity of endophytic fungi. Gond 2011 concluded that leaves harbour a higher number and high diversity of endophytic fungi in comparison to the stem, and this may be due to the large surface area of leaves exposed to the outer environment and the presence of stomata providing passage to the entry of fungal mycelia.

3.8 Endomyco Diversity in Tinospora cordifolia Miers

T. cordifolia is a widely used medicinal plant in the Indian Ayurvedic system of medicine. It is commonly known as Guduchi, Gurch, Giloe or Amrita, having a large, glabrous, deciduous, shade-loving climbing shrub belonging to family Menispermiaceae. A number of chemical constituents such as alkaloids, diterpenoids, lactones, phenolics, glycosides, aliphatic compounds and steroids have been isolated from T. cordifolia. It is used as an anti-inflammatory, antiperiodic, antifever, antidyspepsia, antiarthritic, anti-allergic and antidiabetic agent. The plant is also used to cure scorpion stings, and its watery extract used in febrifuge which is called ‘Indian quinine’ (Chopra et al. 1982; Singh and Panda 2005). The plant contains a polyclonal B cell mitogen with antioxidant activity which can be used as an immunomodulator (Venna et al. 2002). Mishra et al. (2012a) isolated 1,151 endophytic fungal isolates representing 29 taxa from 7,200 segments of leaf, stem, petiole and root (1800 segments of each tissue) collected at three locations of Varanasi district in three different seasons (winter, summer and monsoon). The IF of hyphomycetes (74.80 %) was found greater followed by coelomycetes(14.07 %) and ascomycetes (11.12 %; Table 3.1, Fig. 3.1). Leaf tissues harbour maximum endophytes (29.38 % of the isolates), followed by stem (18.16 %), petiole (10.11 %) and root segments (6.27 %). The leaf segments harbour greater species (29) followed by stem (26), petiole (23) and root (18). CF was maximal during monsoon (23.23 %) followed by winter (15.35 %) and minimal during summer (8.85 %). Among the isolates, Penicillium spp. were dominant (12.62 % of all isolates), followed by Colletotrichum spp. (11.75 %), Cladosporium spp. (8.93 %), C. globosum (8.06 %), Curvularia spp. (7.55 %) and A. alternata (6.75 %). Trichoderma viride, Monilia sp., Acremonium sp. and Guignardia sp. were rare (0.69 %, 0.86 %, 0.52 % and 0.52 %). The paper suggested that some endophytes are season specific for example Colletotrichum linicola occurred almost exclusively in winter and F. oxysporum only in winter and summer but never during monsoon while C. lunata was found only in winter and during monsoon but never in summer. It was concluded that the effect of season and tissue type on CF and species diversity was much more pronounced than the effect of the location.

4 Biopotential of Endophytic Fungi

Microbes have played an important role in the discovery of novel and effective drugs. More than 22,000 secondary metabolites from natural sources are reported with various bioactive properties, but not more than 200 compounds could reach the market which certainly is a discouraging figure (Bérdy 2005). Due to the rising resistance ability in pathogens against existing antibiotics and ingress of newer diseases in society, there is an urgent need to discover the novel and potent antimicrobials. For this, one should go with a novel alternative source. This is the reason that endophytic fungi are getting attention from the scientific community for their ability to produce novel natural metabolites. As the literature suggests, the microbes residing in special niches may be able to produce novel and potent compounds as well. After the discovery of taxol from an endophytic fungi, T. andreanae isolated from the Pacific yew (Stierle et al. 1993), the endophytic research came to light as an alternative source and till today over 100 (57 % novel and 43 % known) anticancer compounds have been isolated and characterized from endophytic fungi (Kharwar et al. 2011a). Not only anticancer but a number of effective and potential antibacterial and antifungal compounds were also isolated from endophytic fungi against a range of Gram +ve and Gram −ve strains. Some of them are colletotric acid isolated from Colletotrichum gloesporioides, an endophytic fungus of Artimisia mongolica active against Bacillus subtilis, Staphylococcus aureus and Sarcina leutea (Zou et al. 2000); Javanacin isolated from endophytic fungus Chloridium sp. resident of A. indica showed strong antibacterial property against Bacillus sp., Escherichia coli, Pseudomonas fluorescens and Pseudomonas aeruginosa. The compound was also active against several fungal pathogens (Kharwar et al. 2009). Cryptocandin isolated from endophytic Cryptosporiopsis quercina showed avtivity against human pathogenic fungi Trichophyton rubrum (ATCC 28188), Trichophyton mentagrophytes (ATCC 28185), Candida albicans (ATCC 90028), Candida parapsilosis and Histoplasma capsulatum (Strobel et al. 1999). Excluding other diseases, malaria alone is responsible to kill about 1 million people throughout the world every year. Endophytic fungi produce several antimalarial compounds such as phomoxanthones A and B from an endophyte Phomopsis sp., which are known to display antimalarial activity against Plasmodium falciparum K1 (Isaka et al. 2001). Pestacin and isopestacin, obtained from endophytic Pestalotiopsis microspora from the interior of Terminalia morobensis, displayed an antioxidant activity (Strobel et al. 2002). Cytonic acids A and B are novel protease inhibitors, isolated from Cytonaema sp., an endophyte of Quercus sp. against human cytomegalovirus (hCMV) (Guo et al. 2000). L-783 and 281 are nonpeptidal fungal metabolites isolated from endophytic Pseudomassaria sp. The compound acts as an insulin mimetic, but without destroying the digestive tract (Zhang et al. 1999). Subglutinol A and B are immunosuppressive, noncytotoxic diterpene pyrones isolated and characterized from an endophytic fungus Fusarium subglutinans of Tripterygium wilfordii (Lee et al. 1995). Nodulisporic acid A is a potential insecticide obtained from an endophytic fungus Nodulisporium sp. of Bontia daphnoides (Ondeyka et al. 1997). 3-Hydroxypropionic acid was isolated from Phomopsis phaseoli endophytically present in Betula pendula and Betula pubescens showed selective nematicidal activity against the plant-parasitic nematode Meloidogyne incognita (Schwarz et al. 2004). In addition to endophytic fungal diversity of eight plants, this chapter also covers the biopotential of the endophytic diversity. Literatures reveal that of the total endophytic community reported to have bioactive potential, 35 % belong to medicinal plants, followed by crops at 29 %, and the rest is equally divided between plants with special niches and other plants, each at 18 % (Selim et al. 2012).

4.1 Biopotential of Endophytic Fungi of Adenocalymma alliaceum

Out of 17 endophytic taxa, only 12 taxa were tested for their antibacterial activity against five human bacterial pathogens. Among 12 endophytic taxa, nine were found to be active against at least one bacterial pathogen. A. alternata, C. globosum, C. lunata and Penicillium sp. were active against 4 of 5 tested pathogens. Salmonella enteritidis (IMS/GN3) was found to be the most susceptible pathogen (Kharwar et al. 2011b).

4.2 Biopotential of Endophytic Fungi of Aegle marmelos

4.2.1 Antibacterial Activity

Seventeen endophytic fungi isolated from A. marmelos were tested for antibacterial activity against human pathogenic bacteria. Fifteen (88.23 %) endophytic fungi showed antibacterial activity against one or more pathogenic bacteria. Out of 17 endophytic fungi, four were active against five bacteria (Shigella flexnii, Shigella boydii, S. enteritidis, Salmonella paratyphi and P. aeruginosa). Phoma herbarum had exhibited an impressive antibacterial activity against seven of eight bacteria tested. The extract of P. herbarum showed strongest activity (inhibition zone 23 mm) against S. boydii. Among the endophytes of A. marmelos, P. herbarum gave least minimum inhibitory concentration (MIC; 40 µg/ml) against S. flexnii and S. boydii. S. boydii was found to be most susceptible followed by P. aeruginosa towards the extract of endophytic fungi. Fifteen endophytic fungal extracts were active against S. boydii and 13 against P. aeruginosa (Gond 2011).

4.2.2 Antifungal Activity

Seventeen endophytic fungi of A. marmelos were also tested against eight pathogenic fungi by dual culture assay. Out of 17 endophytic fungi, 10 were found to be active against one or more fungal pathogens. P. herbarum was most active that inhibited growth of five out of eight fungal pathogens. It inhibited 54.47 % growth of C. lunata. Pestalotia macrotricha was most active against C. cladosporioides showing 47.03 % growth inhibition, while C. globosum showed 24.03, 26.90, 27.07 and 39.13 % growth inhibition against C. cladosporioides, F. oxysporum, Fusarium udum and C. lunata, respectively. Colletotrichum dematium showed activity against C. cladosporioides, F. oxysporum, F. udum and C. lunata with 29.03, 32.7, 33.73 and 43.00 % inhibition, respectively. The endophytic Phomopsis sp. showed inhibitory activity against A. alternata (40.73 %), C. cladosporioides (30.03 %) and C. lunata (38.89 %). The pathogenic C. lunata was found most susceptible whereas Microsporium gypseum was resistant against all endophytic fungi tested (Gond 2011).

4.2.3 Antimarial Activity

P. herbarum (A. marmelos) was assessed for antimalarial activity against 3D7 strain of P. falciparum. The extract of P. herbarum gave only 55 % Schizont maturation inhibition of 3D7 strain of P. falciparum at the concentration of 50 µg/ml (Gond 2011).

4.2.4 Antioxidant Activity

The free radical-scavenging activity of fungal extract was carried out by using 2,2-diphenyl-1-picrylhydrazyl (DPPH). The IC50 of P. herbarum isolated from A. marmelos was 125.63 µg/ml (Gond 2011), which could further be studied for detail and precise activity.

4.2.5 Extracellular Enzyme Production

Out of 32 endophytic fungi, only A. alternata, C. globosum, P. herbarum, C.olletotrichum dematium, T. viride of A. marmelos were tested for extracellular production of amylase, xylanase and phosphate solubilization. All five endophytic fungi were found to produce amylase, while only P. herbarum, C. dematium secreted xylanase whereas no fungi were observed for solubilizing the phosphate in solid media (Gond 2011).

4.3 Biopotential of Endophytic Fungi of A. indica

Among endophytic isolates of A. indica, six endophytic fungi (Alternaria sp., Colletotrichum sp., Chloridium sp., Nigrospora sp., Pestalotiopsis sp., Scytalidim sp.) were evaluated for their anti-dermatophyte activity. Among the six endophytic taxa, ethyl-acetate-extracted Pestalotiopsis metabolite was found more effective against dermatophytes at MIC 80 µg/ml while acetone-extracted Scytalidium sp. exhibited least activity with 400 µg/ml. Javanicin, a napthaqunone isolated from Chloridum sp., an endophytic fungus resident of neem tree root (Kharwar et al. 2009), showed antibacterial as well as antifungal activity. Among all tested pathogens, P. fluorescens and P. aerugenosa were observed more sensitive at MIC 2 µg/ml followed by Cercospora arachidicola at 5 µg/ml. At the rate of 10 µg/ml, the compound inhibited the growth of Rhizoctonia solani and Verticillium dahalae, and F. oxysporum at 20 µg/ml, whereas the suppression of Bacillus sp., E. coli and C. albicans were observed at 40 µg/ml (Kharwar et al. 2009). The isolation of azadirachtin was previously only known from A. indica but Kusari et al. (2012) described the isolation and characterization of azadirachtin A and B from Eupenicillium parvum isolated from A. indica.

4.4 Biopotential of Endophytic Fungi of C. roseus

The endophytic fungi isolated from C. roseus collected in China are known to produce vinka alkaloids. Endophytic Alternaria sp. and F. oxysporum isolated from the phloem of C. roseus were able to produce vinblastine and vincristine. These alkaloids have anticancer property (Guo et al. 1998; Zhang et al. 2000).

4.5 Biopotential of Endophytic Fungi of Cinnamomum camphora

Five out of 26 endophytic taxa were tested against 11 fungal (five human and five phytopathogens) and single bacterial pathogens. Pestalotiopsis sp. showed significant inhibition against Phytophthora cryptogea (57.7 %), Pythium aphanidermatum (54.5 %), Microsporum nanum (51.4 %), T. rubrum (49.7 %), Microsporum gypseum (48.5 %) and P. fluorescence (47.1 %), while Phomopsis sp. showed significant inhibition only to P. aphanidermatum (50.6 %) (Kharwar et al. 2012).

4.6 Biopotential of Endophytic Fungi of Eucalyptus citriodora

4.6.1 Antibacterial Activity

Thirteen (72.22 %) out of 18 endophytic fungi were found active against one or more human bacterial pathogens. C. globosum, Rhizoctonia sp., P. glomerata and T. viride were found to be active against four bacteria. Pestalotia sp. was most active against S. flexnii and S. boydii with an inhibition zone of 16.33 mm and 16.00 mm, respectively. Periconia sp. showed the activity only against S. enteritidis. S. paratyphi showed most susceptibility against Rhizoctonia sp. with 10 mm diameter of inhibition zone. The extract of seven endophytic fungal species inhibited P. aeruginosa. An unidentified species ECB2 (mycelia sterilia) gave maximum inhibition to P. aeruginosa with 12 mm diameter. Citrobacter freundii was only inhibited by extract of Pestalotia sp. P. vulgaris was inhibited by P. glomerata and T. viride. However, Morganella morganii was resistant against all the fungal extracts (Kharwar et al. 2010).

4.6.2 Antifungal Activity

Out of 18 endophytic fungi, eight were found active against at least one phytopathogenic fungus. Phomopsis sp. was the most active taxon against C. lunata followed by an unidentified fungus ECB1 and with 48.88 and 47.1 % radial growth inhibition, respectively. C. globosum also inhibited 32.87 % growth of T. rubrum. Phomopsis sp. and ECB1 inhibited growth of four pathogenic fungi out of the eight tested. Pathogenic A. alternata was inhibited only by endophytic F. oxysporum (34.57 %) while F. udum was inhibited (30.7 %) only by Phomopsis sp. (Kharwar et al. 2010).

4.6.3 Extracellular Enzyme Production

Eight endophytic fungi of E. citriodora were tested for amylase, xylanase and phosphate solubilization activity. Except C. globosum, all seven were found to produce amylase. Among them, Periconia sp. gave maximum zone of amylase production on solid agar medium. Only Colletotrichum gloeosporioides and Aspergillus terreus were observed to produce xylanase. Like A. marmelos, none of the endophytic fungus of E. citriodora had exhibited phosphate-solubilization activity (Gond et al. 2012).

4.7 Biopotential of Endophytic Fungi of Nyctanthes arbor-tristis

4.7.1 Antibacterial Activity

Sixteen endophytic fungi isolated from N. arbor-tristis were tested for antibacterial activity by disc diffusion assay against eight clinical isolates of human pathogenic bacteria (S. flexnii, S. boydii, S. enteritidis, S. paratyphi, P. aeruginosa, C. freundii, M. morganii and P. vulgaris). Among them, 12 (75 %) were found active at a rate of 5 mg/disc. C. dematium and Chaetomiun globosum exhibited antibacterial activity against five pathogens with inhibition ranged from 6.00 to 14.00 mm while Nigrospora sp. inhibited the growth of four bacterial pathogens, i.e. S. paratyphi (22.00 mm), S. flexnii (15.00 mm), S. boydii (18.00 mm) and P. aeruginosa (15.66 mm). In a study, C. freundii, M. morganii and Proteus vulgaris were found resistant against all the fungal extracts. S. boydii was found most susceptible and was inhibited by ten endophytic fungal extracts (Gond et al. 2012).

4.7.2 Antifungal Activity

Nine out of 16 endophytic fungi exhibited antifungal activity. C. dematium displayed the inhibitory activity against five phytopathogens, however its maximum activity was pronounced against C. lunata producing 55.87 % radial growth inhibition in dual culture. Acremonium sp. and N. oryzae inhibited the growth of three of eight pathogenic fungi. C. cladosporioides was found to be the most susceptibile species that was inhibited by Aspergillus fumigatus, and F. oxysporum with 39.66 %, 39.57 % while 31.60 % by Dreschlera rostrata. The growth of F. oxysporum was restricted by a single unidentified fungus MS/NAB2 up to 38.47 %.

4.7.3 Antimalarial Activity

N. oryzae isolated from N. arbor-tristis showed 100 % Schizont maturation inhibition of a malarial parasite 3D7 strain of P. falciparum at the concentration of 50 µg/ml (Gond 2011).

4.7.4 Antioxidant Activity

The free radical-scavenging activity of fungal extract of N. oryzae was carried out by using DPPH . The IC50 for N. oryzae was found at 265.53 µg/ml (Gond 2011), which was quite higher than P. herbarum isolated from A. marmelos.

4.7.5 Extracellular Enzyme Production

Nine endophytic fungi of N. arbor-tristis were tested for amylase, xylanase and phosphate-solubilization activity. Only N. oryzae, Helicosporium sp., Diatrype sp., Macrophoma sp. were found to produce amylase. Except N. oryzae, none were observed to produce xylanase. Unlike A. marmelos and E. citriodora, three endophytic fungi of N. arbor-tristis, i.e. P. glomerata, Scytilidium sp. and Diatrype sp. were able to solubilize phosphate (Gond 2011).

4.8 Biopotential of Endophytic Fungi of Tinospora cordifolia

Twenty nine endophytic taxa were tested for their antibacterial activity against eight human bacterial pathogens. More than 50 % (15 out of 29) of the endophytic taxa exhibited antimicrobial activity. Botryosphaeria rhodina (JQ031157) and C. globosum showed activity against all bacterial human pathogens tested, with the former showing higher activity than the latter. B. rhodina (JQ031157) exhibited strongest activity against C. freundii (IMS/GN5) producing an inhibition zone of 45.66 ± 0.33 mm whereas lowest against M. morganii (IMS/GN6) with an inhibition zone of 12.83 ± 0.16 mm at the rate of 5 mg/ml. C. linicola, A. alternata, C. cladosporioides, N. oryzae and Pseudofusicoccum violaceum (JQ031159) were active against a single pathogen. S. flexnii IMS/GN1 was observed to be the most susceptible bacterial pathogen, inhibited by 11 endophytic taxa followed by E. coli ATCC 25922, inhibited by six endophytic taxa, S. paratyphi and P. vulgaris inhibited by five endophytic taxa whereas S. enteritidis IMS/GN3, P. aeruginosa ATCC 27853, C. freundii IMS/GN5, M. morganii IMS/GN6 were found to be the least susceptible and were only inhibited by three endophytic taxa (Mishra et al. 2012a).

5 Future Prospective

Endophytic fungi are relatively less studied, unexploited and hidden microbes of the microbial community. All the plants studied to date for their endophytic fungi were found to harbour either at least a single or plethora of fungi. Hawksworth and Rossman (1987) estimated there may be as many as 1.5 million different fungal species, while only about 100,000 have been described, and this study raises the question, Where are the rest of the fungi? Are they in form of endophytes or somewhere else? These are some of the basic questions regarding the diversity of endophytic fungi that require more endophytic research which may help in the isolation and characterization of new fungal species and/or bioactive compounds. Since a considerable number of novel fungal genera and species have been reported from this relatively hidden (inside healthy plant tissues) source that may be a good repertoire for filling the gap between reported and estimated fungal diversity. Literatures suggest that endophytes enhance resistance in their hosts against herbivores, pathogenic fungi, bacteria, viruses, insects, nematodes illness, reduced seed production, temperature and salinity and also against drought and minerals (Mishra et al. 2012b). Today a major problem in the front of scientists is the development of resistance in pathogenic microbes (bacteria, fungi and other microbes, malarial parasite, viruses, etc.), pests and weeds that have become a serious trouble for humans, animals and agriculture. To overcome this problem, there is an urgent need for a novel and alternative source for drug discovery. Endophytic fungi can serve as a good alternative because a number of antibacterial, antidiabetic, antifungal, antimalarial, antioxidant, antiviral and other bioactive compounds exhibited promising activity isolated and characterized from this source. The endophytic fungi isolated from the above described eight medicinal plants are under the process of isolation and characterization of antibacterial, antifungal, antioxidant and antimalarial compounds. Suryanarayanan and his colleagues found endophytic fungi as a prolific source for production of extracellular amlyases, cellulases, chitinases, chitosonases, laccases, lipases, pectinases and proteases (Suryanarayanan et al. 2012).

The isolation of several fungi and their isolates (Alternaria, Phomopsis, Chaetomium, Cholletotrichum, Fusarium, etc) from these medicinal plants indicate that some endophytic fungi may be probable candidates to produce some cytotoxic compounds. Another interesting aspect of fungal endophytes is to produce antimicrobial volatile organic compounds (VOCs) reported from mitosporic xylariales fungus Muscodor albus and Muscodor vitigenus isolated from Cinamomum zeylanicum (Strobel et al. 2001). Recently, some hydrocarbon derivatives as major constituents of diesel fuel (Mycodiesel) were reported for the first time from a fungal endophyte Gliocladium roseum, NRRL 50072 (Strobel et al. 2008); however, some genuine technical questions on mycodiesel production were raised (Stadler and Schulz 2009). Nevertheless, the successful trial of running a Honda (100 cc) motorbike using eucalyptol, a better and safe alternative of gasoline received from a fungal endophyte of Eucalyptus sp. (Tomsheck et al. 2010) furthered our understanding towards other interesting aspects.

Very recently, some interesting works have been published in order to enhance the production of cryptic and known bioactive compounds through epigenetic modulations, and these works may point the way in future that can reduce the stigma of reduced yield of fungal endophytes in successive generations (Sun et al. 2012; Hassan et al. 2012). A huge diversity of endophytic fungi isolated from these plants and significant antimicrobial and biochemical activity of crude extracts provide us potential fungal endophyte pools to isolate pure and novel bioactive compounds. In future, epigenetic modulation may play a very crucial role in isolating the cryptic secondary metabolites which are not either known, or it may enhance multifold production of known compounds from fungal source which may serve the need of society.