Fungal endophytes as prolific source of phytochemicals and other bioactive natural products: A review

Microbial Pathogenesis 82 (2015) 50e59

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Review

Fungal endophytes as prolific source of phytochemicals and other bioactive natural products: A review Humeera Nisa a, *, Azra N. Kamili a, Irshad A. Nawchoo b, Sana Shafi a, Nowsheen Shameem a, Suhaib A. Bandh a a b

Centre of Research for Development and Department of Environmental Sciences, University of Kashmir, Srinagar 190006, India Department of Botany, University of Kashmir, Srinagar 190006, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 December 2014 Received in revised form 9 March 2015 Accepted 8 April 2015 Available online 9 April 2015

Endophytic fungi are those that live internally in apparently healthy and asymptomatic hosts. Endophytic fungi appear to be ubiquitous; indeed, no study has yet shown the existence of a plant species without endophytes. High species diversity is another characteristic of endophytic mycobiota which is depicted by the fact that it is quite common for endophyte surveys to find assemblages consisting of more than 30 fungal species per host plant species. Medicinal plants had been used to isolate and characterize directly the bioactive metabolites. However, the discovery of fungal endophytes inside these plants with capacity to produce the same compounds shifted the focus of new drug sources from plants to fungi. Bioactive natural products from endophytic fungi, isolated from different plant species, are attracting considerable attention from natural product chemists and biologists alike which is clearly depicted by the steady increase of publications devoted to this topic during the recent years. This review will highlight the chemical potential of endophytic fungi with focus on the detection of pharmaceutically valuable plant constituents as products of fungal biosynthesis. In addition, it will cover newly discovered endophytic fungi and also new bioactive metabolites reported in recent years from fungal endophytes. It summarizes the up-to-date and comprehensive information on bioactive compounds from endophytic fungi by having done a thorough survey of literature. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Endophytic fungi Bioactive metabolites Phytochemicals Antimicrobial activity

1. Introduction Endophytes are microorganisms living in the internal tissues of the plants without causing any overt symptoms [1]. The term “Endophyte” was introduced by De Bary in 1866 [2] and was initially applied to any organism found within a plant that causes asymptomatic infections entirely within plant tissues without any symptoms of disease [3]. By definition, an endophytic fungus lives in mycelial form in biological association with living plant at least for some time. Therefore, the minimal requirement before a fungus to be termed as an endophyte should be the demonstration of its hyphae in the living tissue [4]. During the past 30 years the terms endophyte and endophytic fungi have appeared frequently in the mycological literature to describe the internal mycota of living plants. Although the origin of the terms can be traced back to the nineteenth century, their contemporary meaning is different from

* Corresponding author. E-mail address: [email protected] (H. Nisa). http://dx.doi.org/10.1016/j.micpath.2015.04.001 0882-4010/© 2015 Elsevier Ltd. All rights reserved.

the original one [5,6]. The terms often are combined with modifiers to refer to a specific host type, a taxonomic group of hosts, or the type of tissue occupied (e.g., systemic grass endophytes, bark endophytes). Contemporary applications of the terms are not always consistent nor are they accepted by all investigators [1,3,7e9]. In general, however, the terms apply to fungi capable of symptomless occupation of apparently healthy plant tissue. In the broadest sense, endophytic fungi are fungi that colonize living plant tissue without causing any immediate, overt negative effects [10]. This definition includes virtually the entire spectrum of symbiotic interactions in which fungi and plants participate: parasitism, commensalism, and mutualism. Fungi are plant-like organisms that lack chlorophyll. An endophytic fungi is a fungal microorganism, which spends the whole or part of its life cycle colonizing inter and/or intra-cellularly inside the healthy tissues of the host plants, typically causing no apparent symptoms of diseases [7]. These are fungal microorganisms which asymptomatically inhabit plant tissues and have been isolated from many species of woody plants and grasses [7,11]. Endophytic fungi

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are found in all kinds of plants, i.e. trees, grasses, algae and herbaceous plants. Endophytic fungi, a polyphyletic group of highly diverse, primarily ascomycetous fungi that are defined functionally by their occurrence within tissues of plants without causing any immediate overt effects [11,12], are found in liverworts, hornworts, mosses, lycophytes, equisetopsids, ferns, and seed plants from the arctic tundra to the tropics [11,13e18]. Endophytes are microorganisms that live in the intercellular spaces of stems, petioles, roots and leaves of plants causing no discernible manifestation of their presence and have typically gone unnoticed [19]. The symbiosis between plant and endophyte was ascertained, namely, the former protects and feeds the latter which produces ‘in return’ bioactive (plant growth regulatory, antibacterial, antifungal, antiviral, insecticidal, etc.) substances to enhance the growth and competitiveness of the host in nature [20]. Accordingly, some endophytes could be reliable sources of materials of the agricultural and/or pharmaceutical potential as exemplified by taxol [21], subglutinol A and B [22], and peptide leucinostatin A [23] (all these could be produced by both endophytes and the hosts). The colonisation of plant tissues by endophytic fungi occurs in a manner similar to those of plant pathogens and mycorrhizae [24]. Colonisation comprises a sequence of steps involving host recognition by the fungus, spore germination, penetration of the epidermis, and tissue colonisation. Once inside their host plant, endophytes usually assume a quiescent (latent) state either for the whole lifetime of the infected plant tissue or for an extended period of time, i.e. until environmental conditions are favourable for the fungus or the ontogenetic state of the host changes to the advantage of the fungus which may then turn pathogenic [25,26]. Although the first discovery of endophytes already dates back to 1904, this group of microorganisms at first did not receive much attention in the decades to follow. This changed dramatically after the detection of paclitaxel (taxol) in the endophytic fungus Taxomyces andreanae that had been isolated from Taxus brevifolia, the latter being the original source of this important anti-cancer drug [21,27]. To date, only a few plants have been extensively investigated for their endophytic biodiversity and their potential to produce bioactive secondary metabolites. Endophytic fungi generally live peacefully with their host, while these fungi under different conditions may act as facultative pathogen. One of the important roles of endophytic fungi is to initiate the biological degradation of dead or dying host-plant, which is necessary for nutrient recycling [28]. Medicinal plants are reported to harbour endophytes [28], which in turn provide protection to their host from infectious agents and also provide adaptability to survive in adverse environmental conditions. Endophytes may contribute to their host plant by producing a plethora of compounds that provide protection and survival value to the plant [29e31]. Colonization of host plants by endophytic fungi is believed to contribute to host plant adaptation to biotic and abiotic stress factors [32e38]. It is of special interest that in many cases host plant tolerance to biotic stress has been correlated with fungal natural products [9,35,39,40]. The nature and biological role of endophytic fungi with their plant host is variable. Endophytic fungi are known to have mutualistic relations to their hosts, often protecting plants against herbivory, insect attack or tissue invading pathogens [41e43]; and in some instances the endophyte may survive as a latent pathogen, causing quiescent infections for a long period and symptoms only when physiological or ecological conditions favours virulence [6,44]. Hawksworth and Rossman in 1987 [47] estimated that nearly one million species of endophytes may exist in the unexplored plants [30,45]. Ever since the discovery of the rich diversity of the endophytic fungi, their population dynamics, their role in improving plant growth, plant health [46], their distribution in the plant, the metabolites they secrete and their potency to produce novel

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compounds within the plants [39], have formed an important aspect of many present day research studies. There are approximately 300,000 plant species on earth and each individual plant is the host to one or more endophytes, and many of them may colonize certain hosts. It has been estimated that there may be as many as one million different endophytic fungal taxa, thus endophytes may be hyperdiverse [7,30]. The described populations of endophytic strains are few, which means the opportunity to find new strains and targeting natural products from endophytic microorganisms that colonize plants in different niches and ecosystems is abundant. 2. Fungal endophytes recovered as new to science Endophytes constitute a major portion of the unexplored fungal diversity. A large part of work carried out on endophytic fungi is based on diversity studies whereas; only some studies report their practical biotechnological applications. However, attempts for searching taxonomic novelty in endophytic taxa are comparatively less, partly due to unavailability of appropriate expertise in fundamental systematics. Endophytic fungi represent an important and quantified component of fungal biodiversity, and are known to have an effect on and be affected by plant community diversity and structure [154e156]. Rodrigues and Samuels [48], described a new species Idriella licualae from a tropical palm tree, Licuala ramsayi (F. Muell.) Domin occurring in rainforests of Queensland. Subsequently after 2 years [49], they described three new species of Idriella (I. euterpes, I. asaicola and I. amazonica) from Euterpe oleracea in Brazilian Amazon forest. Liu et al. [50] described Colletotrichum yunnanense as an endophytic species isolated from Buxus sp. from China. Penicillium coffeae was described as a new endophyte from Coffea arabica L., collected in Hawaii by Peterson et al. [51]. Similarly, Ceratopycnidium baccharidicola [52] from Argentina and Preussia mediterranea (Sporormiaceae) from Mediterranean region [53] were also described as new endophytes. Although most of the newly described species are largely mitosporic fungi, there are few reports on new ascomycetous species. Jacob and Bhat [54] described two new endophytic conidial fungi, Kumbhamaya indica and Gonatobotryum bimorphosporum from India. Later on Echinosphaeria pteridis and its anamorph Vermiculariopsiella pteridis have been described as endophytes of a pteridophyte by Dhargalkar and Bhat [55]. Singh et al. [56,57] studied endophytic assemblages of two medicinal plants collected in India and described two species, viz., Gnomoniella pongamiae from Pongamia pinnata and Thielavia icacinacearum from Nothapodytes nimmoniana. Mirjalili et al. [58], successfully isolated a total of 25 endophytic fungi from the inner bark of Taxus baccata grown in Iran by the aseptic technique. An isolate SBU-16 was identified as Stemphylium sedicola SBU-16, according to its morphological characteristics as well as the internal transcribed spacer nuclear rDNA gene sequence analysis. Interestingly, this was the first report of the genus Stemphylium as a taxolproducing taxon. In a preliminary report the species diversity and the frequency of colonization of endophytic fungi in the aerial parts of Chamaecyparis thyoides was presented by Bills and Polishook [59]. A total of 961 fungal isolates were cultured representing 88 species of filamentous fungi, yielding 10 ± 2 species per tree. One hundred and thirty (130) endophytic fungi were isolated from 12 Chinese traditional medicinal plants collected in Yunnan province, southwest China by Li et al. [60] and were further tested for antitumour and antifungal activities. A survey of the endophytic fungi in fronds of Livistona chinensis was carried out in Hong Kong by Guo et al. [61]. The endophyte assemblages identified using morphological characters consisted of 16 named species and 19 morphospecies, the latter group based on cultural morphology and growth rates. The phylogenetic analysis showed that these morphospecies

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were filamentous Ascomycota, belonging in the Loculoascomycetes and Pyrenomycetes. Results showed that isolate MS704 belonged to the genus Diaporthe and its anamorph Phomopsis of the Valsaceae. The isolate MS594 was inferred to be Mycosphaerella and its anamorph Cladosporium of the Mycosphaerellaceae. Also, isolates MS339, MS366, MS370, MS395, MS1033, MS1083 and MS1092 were placed in the genus Xylaria of the Xylariaceae. MS194, MS375 and MS1028 were close to the Clypeosphaeriaceae. MS191 and MS316 were closely related to the Pleosporaceae within the Dothideales. The other 5 morphospecies; MS786, MS1043, MS1065, MS1076 and MS1095, probably belonged in the Xylariales. A survey of fungal endophytes associated with xylem of presumably healthy trees was conducted by Oses et al. [17]. Wood-inhibiting fungal endophytes of Prumnopitys andina, Podocarpus saligna, Drimys winteri, and Nothofagus oblique were isolated from surface sterilised xylem core samples. Five basidiomycetes (Inonotus sp., Bjerkandera adusta and 3 unknown strains), 2 ascomycetes (Xylaria sp. and Bipolaris sp.) and 1 anamorphic strain were detected. Xylaria sp. and B. adusta were the most frequent fungal isolates. Ultra-structural observations of wood core samples by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Light Microscopy showed the presence of fungal hyphae attached to the inner cell surface inhabiting xylem elements even before the induction of wood degradation. Quite recently, endophytic fungi were isolated from the seagrass, Enhalus acoroides, in Thailand. Forty-seven endophytic fungal isolates cultured were classified into 17 phylo-genetically diverse genera, based on their morphology and molecular analysis. Most common species were Penicillium (6 isoaltes), Nigrospora (5), and Fusarium (4) and 2 with unknown taxonomic affinity. The crude extracts including culture media and cells of all the isolates were also tested for their antimicrobial activities [151]. 3. Biodiversity of endophytic fungi isolated from different medicinal plant species Fungi are one of the most diverse life forms on this planet and predicting the number of fungal species is considered important among mycologists [62]. Hawksworth [150], predicted that there are 1.5 million species of fungi; of these, about 74,000 are currently known [63]. Recent studies from tropical forests [64e66] suggest that fungal diversity is greater in the tropics than in the temperate regions, and many tropical mycologists view 1.5 million as a conservative figure [63]. Some researchers however, feel that the figure of 1.5 million is too high [67e69]. Endophytes of tropical plants are among the groups of fungi that have been studied to arrive at the predicted figure of 1.5 million. Based on their studies on nine neotropical trees, Arnold et al. [70] concluded that fungal endophytes are hyperdiverse in the tropics and that the figure of 1.5 million may markedly underestimate fungal diversity. More recently, studies on a forest in Guyana [71] and four forests in Mudumalai Wildlife Sanctuary, southern India [72] revealed that certain tropical forests are not hyperdiverse with reference to fungal endophytes. Of the myriad of ecosystems on earth, those having the greatest biodiversity seem to be the ones also having endophytes with the greatest number and the most bio-diverse microorganisms. Tropical and temperate rainforests are the most biologically diverse terrestrial ecosystems on earth. The most threatened of these spots cover only 1.44% of the land's surface, yet they harbour more than 60% of the world's terrestrial biodiversity [73]. As such, one would expect that areas of high plant endemicity also possess specific endophytes that may have evolved with the endemic plant species [30]. Khan et al. [74] investigated Calotropis procera (Ait.) R. Br. for endophytic mycoflora as a possible source of bioactive secondary

metabolites. Four hundred seventy three segments from 9 plants of C. procera were analysed for the presence of endophytic fungi. A total of 8 fungal species viz., Aspergillus flavus, Aspergillus niger, Aspergillus sp., Penicillium sublateritium, Phoma chrysanthemicola, Phoma hedericola, Phoma sp., and Candida albicans were isolated. Among the endophytic flora, Phoma was the most prominent genus. Interestingly, no endophyte was isolated from 118 leaves samples and overall colonization frequency from surface sterilized stem was 8.86%. Endophytic fungi of inner bark and leaf tissue of Kigelia pinnata (Lam) Benth., also called sausage tree, from South India, was studied by Maheswari and Rajagopal, [75]. Four hundred bark and leaf segments were analysed and a total of 732 isolates representing 28 taxa, including 3 morphotypes were isolated. The genera Glomerella and Gibberella were first reported in Taxus in a study by Xiong et al. [76]. Eighty-one endophytic fungi were isolated from Taxus media which were grouped into 8 genera based on their morphological and molecular identification. It was found that Guignardia and Colletotrichum were the dominant genera, whereas the remaining genera were infrequent groups. Three representative species of the distinct genera gave positive results by molecular marker screening and were capable of producing taxol which were validated by HPLC-MS. An ecological investigation of foliar endophytic fungal communities on a Musa acuminate (Banana) species complex was undertaken in Hong Kong and Queensland, Australia by Brown et al. [77]. The study yielded twenty-four taxa. Colletotrichum gloeosporioides, Pestalotiopsis palmarum and Nigrospora oryzae were the dominant endophytes isolated from banana in Hong Kong. Isolates of the family Xylariaceae and a Phoma species were most frequently isolated from indigenous banana in the wet tropics of north Queensland. Sarocladium species are frequently associated with grasses as mutualistic endophytes. A species of Sarocladium (anamorphic Hypocreales) was isolated as endophytic fungus from the coastal grass Spinifex littoreus (Poaceae) by Yeh and Kirschner, [78]. According to characterization by LSU and ITS rDNA sequences and culture morphology and micromorphology, the species differed from the species hitherto described in Sarocladium. A key to the known species of Sarocladium was given. Sarocladium spinificis was proposed as a new species. The mycota and decomposing potential of endophytic fungi associated with Acer truncatum, a common tree in northern China, were investigated by Sun et al. [79]. A total of 58 endophytic taxa were recovered using two isolation methods and these were identified based on morphology and ITS sequence data. The results indicated that the composition and diversity of endophytic fungi obtained differed using two isolation methods. This study suggested that endophytic fungi play an important role in recycling of nutrients in natural ecosystems. Khan et al. [80] isolated five endophytic fungi from the roots of Capsicum annuum, Cucumis sativus and Glycine max. The culture filtrates of these endophytes were screened on dwarf mutant rice (Waito-C) and normal rice (Dongjin-byeo). Endophyte CAC-1A significantly inhibited the growth of Waito-C and Dongjin-byeo. This endophyte was identified as Paraconiothyrium sp. by sequencing the ITS rDNA region and phylogenetic analysis. The ethyl acetate fraction of Paraconiothyrium sp. suppressed the germination of Lactuca sativa and Echinochloa crus-galli seeds. This fraction of the endophyte was also subjected to bioassay-guided isolation and a phytotoxic compound ascotoxin was obtained which was characterized through NMR and GC/MS techniques. Ascotoxin revealed 100% inhibitory effects on seed germination of Echinochloa crus-galli. The compound ascotoxin was isolated for the first time from Paraconiothyrium sp. Khan et al. [81] conducted a study on the identification of communities of endophytic fungi in medicinal plant Withania somnifera. A total of 643 segments (202 leaf, 391 stem and 50 root samples) from 20 different plants of W. somnifera were screened for their endophytic

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mycoflora. Thirty-three fungal strains of 24 species were isolated of which four belonged to the class Ascomycetes and 20 to class Deuteromycetes. The highest species richness as well as frequency of colonization was in stem; with the exception of A. niger, Aspergillus terreus and Alternaria alternata, all the other fungi were found to be organ-specific and the most dominant endophyte in this study was found to be A. alternata. Overall colonization frequency was measured as 14.15%. Avicennia marina a dominant mangrove species in Karankadu, Tamil Nadu in India was investigated for endophytic mycoflora by Bharathidasan and Panneerselvam, [82]. Four hundred seventy three segments from 7 plants of A. marina were processed for the presence of endophytic fungi. A total of 10 fungal species viz. A. flavus, A. niger, Aspergillus sp., P. sublateritium, Phomo chrysanthemicola, P. hedericola, Phoma sp. and C. albicans were isolated. Among the endophytic flora, Phoma was the most prominent genus. Interestingly no endophyte was isolated from 110 leaf samples and overall colonization frequency from surface sterilized stem was 8.85%. Diverse fungal species live inside plant tissues, some of which presumably occur in a mutualistic association. Some fungal endophytes are widespread and can be found in many different plant species, whereas others are highly specific to single hosts. In a study, Huang and his co-workers [16] investigated the taxonomic identities and phylogenetic relationships of fungal endophytes isolated from three plant species; Artemisia capillaris, Azadirachta indica, and A. lactiflora, using a combination of morphological and molecular approaches. Morphological differences among the fungal isolates indicated that diverse distinct morphotypes might be present within the hosts. Their results suggested that Alternaria, Colletotrichum, Phomopsis, and Xylaria species were the dominant fungal endophytes in the Artemisia hosts, and some of these endophytes exhibited host and tissue specificity. It is this aspect that could be further explored to understand the relationships between plant hosts and their fungal endophytes. It is well known that diverse endophytes colonize internal tissue of plants. Recently, endophytic fungi residing in medicinal plants have gained unequivocal attention, thus requiring their systematic identification and characterization. In a study by Ruma et al. [83], 106 endophytic fungi from Artocarpus hirsutus Lam. and Vateria indica Linn. two endemic medicinal plants of Western Ghats, India were documented using traditional morphological methods. The frequency dominant genera were Coniothyrium sp (96.5%), Trichoderma sp. (84.5%), Mortierella sp. (36.75%), Phyllosticta sp. (19%) and Acremonium sp. (21.5%). The other endophytes recovered belonged to class Ascomycetes and Hyphomycetes i.e., Aspergillus spp. Colletotrichum spp., Fusarium spp. and Penicillium spp. higher number of isolates was recovered from the bark of the plants than from twigs. Fifty-two isolates of endophytic fungi were collected from the bark of Cephalotaxus mannii (plum-yew) trees located in the north of Thailand and the south of China by Saithong and his co-workers, [84]. All isolates were identified based on colony morphology and examination of spores and fruiting bodies using stereo and light microscopes. Thirty-five isolates (67.3%) belonging to 13 genera were recorded, viz. Cladosporium sp., Acremonium sp., Trichoderma sp., Monilia sp., Fusarium sp., Spicaria sp., Humicola sp., Rhizoctonia sp., Cephalosporium sp., Botrytis sp., Penicillium sp., Chalaropsis sp. and Geotrichum sp., while 17 strains (32.7%) were unidentified. The dominant genera found both in northern Thailand and southern China were Acremonium sp., Monilia sp. and Fusarium sp. The Cladosporium sp. and Trichoderma sp. were found only in southern China, whereas Spicaria sp., Humicula sp., Rhizoctonia sp., Botrytis sp., Penicillium sp., Geotrichum sp., Chalaropsis sp. and Cephalosporium sp. were found only in northern Thailand. Thus, it was inferred from this study that there exists a significant difference in the genera of endophytic fungi from C. mannii trees of different

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sources. 418 endophyte morphospecies (estimated 347 genetically distinct taxa) were isolated from 83 healthy leaves of Heisteria concinna and Ouratea lucens in a lowland tropical forest of central Panama, and proposed that tropical endophytes themselves could be hyperdiverse with host preference and spatial heterogeneity [66]. Various species of endophytic fungi as Cladosporium cladosporoides, Phoma spp., Phomopsis spp., Xylaria spp. had been reported in four types of tropical forests: dry thorn forest, dry deciduous forest, moist deciduous forest and semi-evergreen forest [72]. Thus, of the myriad of ecosystems on earth, those having the greatest biodiversity also have endophytes with the greatest number and the most biodiverse microorganisms. Almost all vascular plant species examined to date are found to anchorage endophytic bacteria and/or fungi [66,85]. Moreover, the colonization of endophytes in marine algae [86,87], mosses and ferns [88,89] have also been detected. Based on this fact, endophytes are important components of microbial biodiversity [90]. 4. Endophytic fungi as sources of plant secondary metabolites Fungi are key resources for exploiting bioactive metabolites [64,91]. Among fungi, endophytes are important to screen biologically active metabolites [92]. Endophytic fungi inhabit within their host plant without causing any disease symptoms [92]. In endophyte-host symbioses, secondary metabolites produced by endophytes contribute positively to their host [93]. Secondary metabolites, defined as low molecular weight compounds not required for growth in pure culture, are produced as an adaptation for specific functions in nature. They play vital role in vivo, e.g., numerous metabolic interactions between fungi and their plant hosts, such as signalling, defence and regulation of the symbiosis [92]. Diverse classes of chemical substances like steroids, xanthones, phenols, isocoumarines, perylene derivatives, quinones, furandiones, terpenoids, depsipeptides and cytochalasines have been isolated from endophytic fungi [92,94]. Such substances are synthesized through polyketide pathway from mevalonate-derived C5 units and (or) using the non-ribosomal protein synthesis. A literature survey reveals that the number of novel chemical structures produced by endophytes (51%) is significantly higher than the soil fungus (38%), suggesting that these frequently overlooked endophytes are the novel source of bioactive secondary metabolites [92,94]. Endophytes are synergistic to their host. At times they are known to prevent the host from successfully attacking fungi and pests by producing special substances such as secondary metabolites and in return demanding nutrition [30]. The array of metabolites and other chemicals synthesized by the endophytes endow the plants with more resistance to nematodes, insects and livestock. Plants inhabited with specific endophytes are often able to grow faster due to the production of phytohormones and become so competitive that they dominate in a particular environment. Endophytes are the chemical synthesizers inside plants [95]. The secondary metabolites produced by endophytes associated with medicinal plants can be exploited for curing many diseases [96]. Strobel [28], isolated about 6500 endophytic fungi and concentrated over novel endophytic microbes. As discussed earlier, a large number of bioactive metabolites of endophytic fungi have been extracted and characterized over 12 years, and those isolated belong to diverse structural groups i.e. alkaloids, steroids, terpenoids, peptides, polyketones, flavonoids, quinols, phenols, xanthones, chinones, isocumarines, benzopyranones, tetralones, cytochalasines, perylene derivatives, furandiones, depsipeptides and enniatines. Some of them represent novel structural groups, e.g., the palmarumycins and a new benzopyroanone [39]. These secondary metabolites of endophytes are synthesized via various

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metabolic pathways e.g. polyketide, isoprenoid, amino acid derivation [90]. Microbial metabolites seem to be distinctive of certain biotopes, on both an environmental and organism level. Thus, it appears that the search for novel secondary metabolites should core on organisms that inhabit exceptional biotopes. Fungal secondary metabolites produced may vary with the biotope in which it grows and to which it is adapted, e.g., the production of cyclosporin A, enchinocandin B, papulacandins and verrucarins varied with both habitat and substrate [64]. Endophytic fungi are source for screening of natural products, and in optimizing the search for new bioactive secondary metabolites; it is pertinent to consider that the secondary metabolites a fungus synthesizes may correspond with its respective ecological niche and continual metabolic interactions between fungus and plant may enhance the synthesis of secondary metabolites [90]. Recently, several studies have led to the discovery of important plant secondary metabolites from endophytic fungi thus raising the prospect of using such organisms as alternative sources of these metabolites [97]. In addition to being alternative sources for secondary metabolites known from plants, endophytes accumulate a wealth of other biologically active and structurally diverse natural products that are unprecedented in nature [30,35,39,40,98e100] and are of importance for drug discovery or as lead compounds for agriculture [13,101,102]. It is hence now generally accepted that endophytes represent an important and largely untapped reservoir of unique chemical structures that have been modified through evolution and are believed to be involved in host plant protection and communication [99]. Fungal endophytes are known to produce metabolites that mimic the structure and function of host compounds [28] and can produce plant growth hormones such as gibberellins [103]. Plant-associated microorganisms, especially endophytic fungi, are largely underexplored in the discovery of natural products [104]. The prolific endophytes also have a capacity to produce diverse class of plant associated secondary metabolites with a wide variety of biological activities such as antimicrobial agent hypericin [105], acetylcholinesterase inhibitor huperzine A [106], and antitumour agents taxol [21]. Bioprospecting endophytes thus offers tremendous promise to discover natural products with therapeutic value [104], which have attracted increasing attention among microbiologists, ecologists, agronomist and chemists. There has been a great interest in endophytic fungi as potential producers of novel, biologically active products [30,107]. Endophytes are now considered as an important component of biodiversity as the distribution of endophytic mycoflora differs with the host. Due to the world's urgent need for new antibiotics, chemotherapeutic agents and agrochemicals to cope with the growing medicinal and environmental problems facing mankind, growing interest is taken into the research on the chemistry of endophytic fungi. Whereas between 1987 and 2000 approximately 140 new natural products were isolated from endophytic fungi [39], a similar number was subsequently characterised in half of this time span, i.e. between 2000 and 2006 [108]. Many of these exhibit interesting activity profiles. Cryptocin, for example, is a tetramic acid isolated from the endophytic fungus Cryptosporiopsis quercina, an endophyte of Tripterigeum wilfordii, that possesses potent activity against the world's worst plant pests Pyricularia oryzae and other plant pathogenic fungi, advocating it for possible agrochemical usage [109]. From the medicinal plant Erythrina crista-galli the endophyte Phomopsis sp. was isolated, which produced the anti-inflammatory as well as antifungal and antibacterial active polyketide lactone, phomol [110]. An endophytic fungus Fusarium oxysporum was isolated from Rhizophora annamalayana, a mangrove plant and was analysed for taxol production [111]. The secondary metabolites taxol were extracted with ethyl acetate. Taxol extracted was characterised by chromatographic and spectrometric analysis. Thin

Layer Chromatographic plate showed violet-red and IR spectrum values were conformed as group of terpenoid functional groups. Aspergillus fumigatus CY018 was recognized as an endophytic fungus for the first time in the leaf of Cynodon dactylon in a work of Liu et al. [112]. By bioassay-guided fractionation, the ethyl acetate extract of a solid-matrix steady culture of this fungus afforded two new metabolites, named asperfumoid and asperfumin, together with six known bioactive compounds as well as other four known compounds. Through a number of detailed spectroscopicanalyses the structures of asperfumoid and asperfumin were established to be spiro-(3-hydroxyl-2,6-dimethoxyl-2,5-diene-4-cyclohexone(1,30 )-50 -methoxyl-70 -methyl-(10 H, 20 H, 40 H)-quinoline-20 40 -dione) and 5-hydroxyl-2-(6-hydroxyl-2-methoxyl- 4-methylbenzoyl)-3,6dimethoxyl-benzoic methyl ester, respectively. All of the 12 isolates were subjected to in-vitro bioactive assays against three human pathogenic fungi C. albicans, Trichophyton rubrum and A. niger. In a study carried out by Zang et al. [113], three new azaphilone compounds, isochromophilones XeXII (1, 2 and 3), together with two known ones; sclerotioramine (4) and isochromophilone VI (5) were isolated from the cultures of an endophytic fungus Diaporthe sp. The structures were elucidated by extensive HRESIMS and NMR spectroscopic analyses. This was the first report of azaphilones isolated from Diaporthe sp. Three steroids and one nordammarane triterpenoid were isolated for the first time from the endophytic fungus Pichia guilliermondii Ppf9 derived from the medicinal plant Paris polyphylla var. yunnanensis. By means of physicochemical and spectrometric analysis, they were identified as ergosta-5, 7, 22trienol (1), 5a, 8a-epidioxyergosta-6, 22-dien-3b-ol (2), ergosta7,22-dien-3b,5a,6b-triol (3), and helvolic acid (4). Both microdilution-colorimetric and spore germination assays were employed to evaluate their antimicrobial activity. It also showed strong inhibitory activity on the spore germination of Magnaporthe oryzae. Among the three steroids, 5a, 8a-epidioxyergosta-6, 22dien-3b-ol (2) exhibited relatively strong antimicrobial activity [114]. The discovery of the paclitaxel (taxol) producing endophytic fungus T. andreanae from the yew plant T. brevifolia [21,27] set the stage for a more comprehensive examination of other Taxus species and other plants for the presence of paclitaxel producing endophytes, so as to apply it to the industrial production of this pharmacologically important drug. Paclitaxel, the multi-billion dollar anti-cancer compound produced by the yew plant, has activity against a broad band of tumour types, including breast, ovarian, lung, head and neck cancers, as well as advanced forms of Kaposi's sarcoma. Many other endophytic fungi, such as Seimatoantlerium tepuiense, Seimatoantlerium nepalense [115], and Tubercularia sp. strain TF5 [116], have meanwhile been reported to produce paclitaxel. Similarly, the lignan podophyllotoxin, synthesized by Podophyllum species, is highly valued as the precursor to clinically used anti-cancer drugs such as etoposide and teniposide. Further examples of plant secondary metabolites detected in endophytic fungi include naphthodianthrones, such as hypericin, which are well known constituents of St. John's wort (Hypericum perforatum). Hypericum species have been used for centuries against mild forms of depression and anxiety. An endophytic fungus from H. perforatum was found to produce hypericin in culture [105]. Endophyte-infected grasses are known for their ability to produce loline alkaloids which exhibit deterrent and toxic effects towards invertebrate and vertebrate herbivores and are thus possibly involved in protection of endophyte infected grasses against herbivores [117]. Thus, many endophytes are apparently able to synthesize the same natural products that also occur in plants. It is nevertheless assumed that production of these respective compounds in planta does not proceed exclusively by endophytes but is rather the consequence of concomitant plant and fungal

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biosynthesis [105]. In a study carried out by Lu et al. [118], a plant hormone indole-3-acetic acid (IAA) and three new antimicrobial metabolites were characterized from the culture of Colletotrichum sp., an endophyte isolated from inside the stem of Artemisia annua. Also ergosterol (I), 3b,5a,6b-trihydroxyergosta-7, 2, 2-diene (II), 3bhydroxy-ergosta-5-ene (III), 3-oxo-ergosta-4,6,8(14), 2, 2-tetraene (IV), 3b-hydroxy-5a, 8a-epidioxy-ergosta-6, 2, 2-diene (V), 3b-hydroxy-5a, 8a-epidioxy-ergosta-6, 9 (11), 2, 2-triene (VI) and 3-oxoergosta-4-ene (VII) were isolated and characterised from the culture of this endophyte. The structures of the new metabolites were elucidated by a combination of spectroscopic methods (IR, MS, 1H and 13C NMR) as 6-isoprenylindole-3-carboxylic acid (1), 3b, 5adihydroxy-6b-acetoxy-ergosta-7, 2, 2-diene (2) and 3b, 5a-dihydroxy-6b-phenylacetyloxy-ergosta-7, 2, 2-diene (3), respectively. The compounds 1e3 and IIIeV inhibited the growth of all the tested bacteria (Bacillus subtilis, Staphylococcus aureus, Sarcina lutea and Pseudomonas sp.) Eighty-one endophytic fungi isolated from T. media were grouped into 8 genera based on the morphological and molecular identification. Guignardia and Colletotrichum were the dominant genera, whereas the remaining genera were infrequent groups. The genera Glomerella and Gibberella were first reported in Taxus. Three representative species of the distinct genera gave positive hits by molecular marker screening and were capable of producing taxol which was validated by HPLC-MS. Among these 3 taxol-producing fungi, the highest yield of taxol was 720 ng/l by Guignardia mangiferae HAA11 compared with those of Fusarium proliferatum HBA29 (240 ng/l) and C. gloeosporioides TA67 (120 ng/ l). This was the first report of taxol producer from Guignardia [76]. Recently a work was conducted with the aim of isolating an endophytic fungal strain possessing antimicrobial activity. An endophytic fungal strain NFX06 was isolated from leaf of Nothapodytes foetida in Karnataka. The fungal strain was identified as F. oxysporum NFX06 based on its macroscopical and microscopical characteristics. Further confirmation of the species was done by ITS sequencing. In this study, microwave assisted extraction of secondary metabolites from this endophytic fungal strain NFX06 was carried out for the first time. The antimicrobial activity of this fungal isolate was also evaluated using different human pathogens [152]. In another study; taxonomical characterization, fermentation, structural characterization of the secondary metabolites from an endophytic fungi, A.fumigatus were reported together with their antimicrobial and cytotoxic activities. During the research for bioactive secondary metabolites, this endophytic fungal isolate was found to produce a set of promising bioactive compounds (1e10) after its large scale fermentation, working up and purification using a series of chromatographic techniques. Structural elucidation of the yielded compounds using intensive studies of their NMR (1H, 13 C and 2D NMR) and mass (EI MS, ESI MS) spectrometry confirmed them as linoleic acid (1), R()-glycerol monolinoleate (2), bisdethio-(bis-methyl-thio)-gliotoxin (3), fumiquinazoline-F (4), fumiquinazoline-D (5), (Z,Z)-N,N0 -[1-[(4-Hydroxy-phenyl)- methylene]-2-[(4-methoxy-phenyl)-methylene]-1,2-ethanediyl]-bisformamide (6), pyrazoline-3-one trimer (7), Tricho-9-ene2a,3a,11a,16-tetraol (8), 20 -deoxy-thymidine (9), and cerebroside A (10) [153]. 5. Antimicrobial activities of fungal endophytes from different species of medicinal plants During long-time evolution, plants, on which insects, microorganisms and mammals are feeding, usually acquire selfdefending capabilities by producing a variety of secondary metabolites such as alkaloids, terpenoids, steroids and aromatic compounds which are presumably unpleasant or even toxic to the ‘enemy’. Inside the tissue of nearly all the healthy plant, there are a

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lot of micro-organisms called ‘endophytes’. Endophytes are mutualistic to their host; at least some of them are thought to be making returns for the nutrition from the plant by producing special substances such as secondary metabolites to prevent the host from successful attack of fungi, pests and mammals. As a matter of fact, metabolites of endophytes were reported to inhibit a number of micro-organisms [119,120]. Endophytes are believed to carry out a resistance mechanism to overcome pathogenic invasion by producing secondary metabolites [39]. Endophytes are rich source of natural products displaying broad spectrum of biological activities. Many endophytic fungi are known to produce antimicrobial substances. Antimicrobial metabolites can be defined as low molecular weight organic natural substances made by microorganisms that are active at low concentrations against other microorganisms [121]. The crude extracts from culture broths of endophytic fungi have shown antimicrobial activity against pathogenic fungi, bacteria and yeasts, cytotoxic activity on human cell line, anti-Herpes simplex virus type 1 activity (anti-HSV) and antimalarial activity against protozoan Plasmodium falciparum [122]. There are numerous studies on antimicrobial activity of endophytic fungi isolated from different geographical locations [43,60,123e130]. An array of natural products has been characterized from endophytic fungi, which includes anti-cancerous, antioxidants, anti-viral, antiinsecticidal, immunosuppressant, anti-microbial, anti-malarial and anti-mycobacterial [30,93,131]. It seems that most, if not all, of the endophytes examined to date increase host shoot and/or root biomass, possibly as a result of the induction of plant hormones by the host or biosynthesis of plant hormones by the fungi [132]. Many endophytes protect hosts to some extent against fungal pathogens [133e135], reflecting the production of secondary metabolites [136], fungal parasitism [137], or induction of systemic resistance [138]. It is also possible that symbiotically conferred disease protection may be a result of an inability of pathogens to compete with endophtyes for resources or niche space. Antibacterial naphthaquinone Javanicin (C15H14O6) exhibiting activity against Pseudomonas sp. was isolated from Chloridium sp. an endophyte of Neem [139]. Maria et al. [128] studied antifungal and antibacterial activities of 14 endophytic fungi isolated from two mangrove plants, viz. Acanthus ilicifolius and Acrostichum aureum L. and indicated that many endophytes of mangrove plants are likely to possess novel metabolites. Raviraja et al. [129] checked antimicrobial potential of 15 endophytes of leaf, stem and bark from eight medicinally important plant hosts from Western Ghats of India. Partially purified extracts of Alternaria sp., Nigrospora oryzae and Papulospora sp. exhibited considerable antimicrobial activity against selected bacteria and fungi. Tejesvi et al. [130] screened Pestalotiopsis strains from medicinally important plants, A. indica, Holarrhena antidysenterica (Roxb. ex Fleming) Wall., Terminalia arjuna and Terminalia chebula Retz. for their antifungal activity and concluded that species of Pestalotiopsis can be explored for bioactive antifungals for better management of fungal pathogens. Mohanta et al. [140] studied antimicrobial potential of endophytic fungi inhabiting three ethno-medicinal plants of Simplipal Biosphere Reserve, India. In the study, 60 fungal endophytes belonging to 14 genera were tested for their antimicrobial potential. Kharwar et al. [149] reported the production of Javanicin under liquid and solid media culture conditions from the endophyte Chloridium sp. of A. indica. This highly functionalized naphthaquinone exhibits strong antibacterial activity against Pseudomonas spp., representing pathogens of both humans and plants. Similarly, 16 endophytes from Eucalyptus citriodora Hook. were tested for their antagonistic activities against human and plant pathogenic fungi [141]. Mahapatra and Banerjee [142] evaluated endophytes of petiole, bark and leaf isolated from Alstonia scholaris (L.) R.Br. for their antibacterial activity and noted Curvularia sp.,

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Aspergillus sp. and an unidentified fungus shows significant activity. Evaluation of some endophytes was carried for their possible antimicrobial activity from various parts of medicinal plants in Jalgaon Maharashtra (India) by Jalgaonwala et al. [143]. A total of seventy-eight bacterial endophytes and one hundred forty-two fungal endophytes were isolated from the aerial and underground parts of selected medicinal plants. Fourteen positive endophytic fungal isolates possessed antibacterial activity. Endophytic fungal isolates AFR1, AFR4, AFR7 from roots of A. vera possessed strong antibacterial activity against S. typhi in dual culture assay. In another study a total of 22 endophytic fungi isolated from coffee (C. arabica L.) were cultivated in vitro and their crude extracts were screened for their antimicrobial activities by Fernandes et al. [144]. The most effective isolate was A. alternata. Sapindus saponaria L., is a commonly known tree in Brazil which is widely used in folk medicine. Garcia et al. [145] carried out a work to investigate the biotechnological potential of crude extracts of fungal endophytes (Cochliobolus intermedius, Phomopsis sp. and 2 unidentified), isolated from this plant, that were assayed against five pathogenic bacteria. One metabolite, extracted from C. intermedius presented activity for all the tested bacteria. In one more study the discoveries of two antimicrobial and antioxidant compounds from an endophytic fungus Pestalotiopsis microspora isolated from Terminalia morobensis, led to the search for endophytic fungi with antimicrobial and antioxidant potential [146]. Endophytic fungi from three commonly found sea-grasses in southern Thailand were explored for their ability to produce antimicrobial metabolites by Supaphon et al. [147]. One hundred and sixty endophytic fungi derived from Cymodocea serrulata, Halophila ovalis and Thalassia hemprichii were screened for production of antimicrobial compounds against ten human pathogenic microorganisms. 69% of the isolates exhibited antimicrobial activity against at least one test strain. Antifungal activity was more pronounced than antibacterial activity. Among the active fungi, seven isolates exhibited strong antimicrobial activity. The scanning electron microscopy had revealed that the inhibitory extracts at concentrations of 4 times their MIC destroyed the targeted cells. Zeng et al. [148] investigated in vitro antioxidant activities of 49 endophytic fungi isolated from the liverwort Scapania verrucosa. Based on morphological and molecular identification, the endophytic fungi isolated were classified into seven genera (Hypocrea, Penicillium, Tolypocladium, Chaetomium, Xylaria, Nemania, and Creosphaeria), all belonging to one family (Xylariaceae). The ethyl acetate extracts of two endophytic fungal isolates were found to have comparable scavenging abilities on both DPPH-free radicals and hydroxyl radicals when compared with those of the positive controls. These endophytic fungi in S. verrucosa were found to be a potential and novel source of natural antioxidants. The endophytic extracts of 11 fungi associated with asympomatic A. annua Linn., were evaluated for antimicrobial activity against three human pathogenic microbes, Escherichia coli, S. aureus and T. rubrum, and two plant pathogens, Rhizoctonia cerealis and Magnaporthe grisea by Zhang et al. [113]. Among these fungal endophytes, three strains of Aspergillus spp. showed the strongest antimicrobial activities against E. coli, S. aureus, T. rubrum. An endophytic Mucor sp. had the most pronounced effect on R. cerealis. Two strains; Aspergillus sp. and Cephalosporium sp. exhibited the strongest antimicrobial activities against M. grisea. A. annua, well recognized for its production of antimalarial drug artemisinin, is seldom attacked by any of phytopathogenic fungi, which could be partially associated with the presence of endophytes. In an investigation conducted by Liu et al. [123] it was aimed at disclosing whether the endophytes inside A. annua produce antifungal substances. A total of 39 endophytes were isolated and fermented, and the ferment broth was evaluated in vitro for the antifungal activity

against crop-threatening fungi Gaeumannomyces graminis var. tritici, R. cerealis, Helminthosporium sativum, Fusarium graminearum, Gerlachia nivalis and Phytophthora capsici. Out of 39 endophytes investigated, 21 were seen to produce in vitro substances whereas the rest yielded nothing active. Moreover, the most active broth of endophyte IV403 was extracted with ethyl acetate and n-butanol, and comparisons of the antifungal activity of the extracts indicated that the major active metabolites were ethyl acetate-extractable. In another recent study an endophytic fungi DZY16 has been isolated from Eucommia ulmoides Oliv., which was tested for its bioactive components and antimicrobial activities. This endophytic fungal strain was identified by internal transcribed spacer (ITS) sequence as belonging to Nigrospora. The results indicated that the endophytic fungi DZY16 of the plant E. ulmoides Oliv. was a promising source of novel bioactive compounds [157]. 6. Conclusion By the discovery of penicillin from Penicillium notatum, almost 80 years ago in 1928 by Sir Alexander Fleming, made the world to realize for the first time, the significance of fungi as unconventional sources of bioactive compounds. Most of the plants resist invading plant pathogens through the production of antimicrobial compounds; in some cases these compounds may be the product of the plant's associated microflora. Screening such plants for endophytic isolation yielded novel and interesting microbes. This provided a lead-directed approach in screening endophytes from ethnopharmaceutically used plants, as a source for new therapeutic leads. The success of several such medicinal drugs from microbial origin such as the antibiotic penicillin from Penicillium sp., the antifungal agent griseofulvin from fungus Penicillium griseofulvum, b-lactam antibiotics from various fungal taxa, the cholesterol biosynthesis inhibitor lovastatin from A. terreus, and the immunosuppressant cyclosporine from Tolypocladium inflatum and Cylindrocarpon lucidum, has shifted the focus of drug discovery from plants to microorganisms. Thus, in the continuous search for novel drug sources, endophytic fungi have proven to be a promising reservoir of natural products, with great chemical diversity which is largely untapped. Thus, endophytes are the chemical synthesizers inside the plant. Many of these fungal endophytes are capable of synthesizing bioactive compounds that can be used as potential sources of many pharmaceutical leads. These compounds have been optimized by evolutionary, ecological and environmental factors yielding effective bioactive agents. Endophytic fungi have been proven useful for novel drug discovery as suggested by the chemical diversity of their secondary metabolites. Many endophytic fungi have been reported to produce novel antibacterial, antifungal, antiviral, anti-inflammatory, anti-tumour, anti-malarial and other compounds belonging to alkaloids, terpenoids, flavonoids, phenolic compounds, steroid derivatives and other structure types. Thus, endophytic fungi are prospective producers of an abundant and dependable source of bioactive and chemically novel compounds with potential for exploitation in a wide variety of arenas like; medicine, agriculture, and industry. Though, the feasibility of industrial production of bioactive compounds by endophytic fungal sources has still to be proven. To sum it up, isolation of endophytic fungi from medicinal and other plants may result in methods to produce biologically active agents for biological utilization on a large commercial scale as they are easily cultured in laboratory and fermentor instead of harvesting plants and affecting the environmental biodiversity. Despite intensive research, different aspects of the relationship between endophytes and their hosts are yet unclear which forms the basis for future research. Endophytes are a poorly investigated group of

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microorganisms that represent secondary metabolites having an immense impact on modern medicine, since about 40% of prescription drugs are based on them. The described discoveries of a wide range of products and microorganisms already hold inkling for future prospects in cancer, malaria and tuberculosis. In some cases endophytes are found to produce medicinally important natural products originally known exclusively from their host plants, thus raising the prospect of using such organisms as alternative and sustainable sources of these substances.

[28]

Acknowledgement

[29]

The authors are thankful to the Centre of Research for Development, University of Kashmir for providing the laboratory facilities. This review compilation is an inspirational outcome of PhD research work of corresponding author, on fungal endophytes from a folkloric medicinal plant.

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