Endophytes:Proc Indian Natn A Novel Sci Acad Source 74 No.2 for Bioactivepp. 73-86 (2008)Molecules 73

Review Article Endophytes: A Novel Source for Bioactive Molecules

VIJESHWAR VERMA*, PANKAJ SUDAN and AMARDEEP KOUR Dean College of Sciences, Shri Mata Vaishno Devi University (SMVDU), Katra (J&K State) Fast track postal address: PRO Office of SMVDU,15-C (2nd Ext.) Gandhi Nagar, Opp. Bahu Plaza, Jammu 180 003

(Received 15 Feb 2008; Accepted 7 may 2008)

Endophytes inhabit theoretically every on the earth. They have gained increased importance after these have been reported as a novel source of potentially useful bioactive molecules e.g., anticancer, immunosuppressive and anti-viral compounds, alkaloids, antibiotics, anti-oxidants, cytochalacins etc. This has raised the hope that medicinally important might escape the wrath of mankind in their over exploitation for extraction of bioactive molecules from them and might survive the danger of getting extinct in near future. One of the major problems facing the future of endophyte biology is the rapidly diminishing rainforests, which hold the greatest possible resource for acquiring novel microorgan- isms and their products. The present review is to consolidate the data available on the role of endophytes producing various important bioactive molecules of therapeutic importance. Key Words: Endophytic Microorganisma; Bioactive Molecules; Life Cycle; Isolating Endophytes; Eco-friendly; Torreyanic Acid; Cytochalacine; Antibiotics; Alkaloids; Pesticides; Biodiversity.

1 Introduction plant species inhabiting the world, each individual plant The term endophyte refers here to microorganisms is a host to these unique microorganisms (Strobel, mostly fungi inhabiting plant organs without causing unpublished data). Only a few of these plants (grass apparent harm to their host [1]. Plant tissues remain entire species) have been studied relatively complete for their and functional. Since the discovery of fungal endophytes endophytic biology. Consequently, the opportunity to find in conifers [2] numerous investigations on their new and interesting endophytic microorganisms among abundance, species composition and response to various myriad of plants in different settings and ecosystems is environmental factors have been published. Endophytes not only very great but also a thrilling challenge. have one or more varieties of interactions with their host Plants may benefit from the presence of endophytes plant: Some fungi are widespread and are found on many in many ways. Potential plant benefits have been different plant species; others are highly specific to a examined in only a few cases. Rhabdocline parkeri single host in a specific environment. Further, a diverse produces a compound that reduces needle attack by array of interactions between plant and have been borers. Metabolites produced by Phomopsis sp in cotton found. Given that a huge array of fungi may be isolated appear to deter larvae of Helicoverpa from feeding on from any one host, it seems possible that endophytes leaves. Similar properties have been reported with will have one or more arrays of functions, most of which Neotyphodium. In addition, aphids feeding on leaves of are unknown at present [3]. However, in some thoroughly cotton may become colonised by Lecanicillium lecanii, investigated cases, the fungus-plant relationship has when conditions permit. Thus the aphid may be killed turned out to be mutualistic/symbiotic, as the endophyte or it may transfer the fungus to another leaf. In addition, acts antagonistically against insect herbivores [4], [5]. endophytes may upregulate host responses to pathogens Hence, it has been proposed that short-cycle (one or and pests. Chaetomium globosum has been shown to several generations each year) endophytes increase the increase host resistance to rust and tan spot pathogens resistance potential of long-living trees against the effects in wheat. Direct interactions appear to be too small to of short-cycle insects [6]. measure in this case. Presence of Lecanicillium lecanii Endophytic microorganisms are to be found in appears to reduce the feeding by aphids from leaves of virtually every plant on earth. These organisms reside in cotton. The interaction is probably due to induction of a symbiotic relationship with its host plant, obtaining host responses, which is perplexing because plant nutrients from it and in return providing it protection regulatory pathways for responses to insects and against insect pests. Endophytes are invisible to the naked pathogens are not thought to be complementary. eye and must be stained and viewed under a microscope Endophytes appear to have direct and induced effects to be identified. Of the approximately 300,000 higher on plant responses to biotic agents. The interaction with abiotic agents remains largely unexplored. *Corresponding Author: E-mail: [email protected] / [email protected] 74 Vijeshwar Verma et al.

Because of what appears to be their contribution to probably the first researcher who reported an example the host plant, the endophytes may produce ample of protection given by an endophytic fungus to plant by substances of potential use to modern medicine, resisting the growth of insects, in which the endophyte agriculture and industry. Novel antibiotics, antimycotics, Phomopsis oblonga protected elm trees against the beetle immunosuppressant, topoisomerase inhibitors [7] and Physocnemum brevilineum. After four years , Claydon antineoplastic compounds [7], [8] are only a few et al. [15] confirmed it and showed that endophytic fungi examples of what has been found after the isolation, belonging to the Xylariaceae family synthesize culture, purification, and characterization of some novel secondary metabolites in hosts of the genus Fagus and endophytes in the recent studies. The potential prospects that these substances affect the beetle larvae. of finding new drugs that may be effective candidates The best studied endophytic plant/microbes system for treating newly developing diseases in humans, plants, to date is the grass species/Neotyphodium species and animals are great. relationship (Plate 1). This fungal species provides a host Endophytic fungi are an important, yet relatively of biologically active compounds [16] including toxic unstudied group of microbial plant symbionts. alkaloids such as Peramine (deters insect pests), Lolitrem Endophytic fungi live asymptomatically, and sometimes B (deters larvae feeding) and Ergovaline (provides systemically, within plant tissues [6]. Endophytes usually resistance to black beetle). However, enormous inhabit above-ground plant tissues (leaves, stems, bark, opportunities exist for the recovery of novel forms, petioles and reproductive structures), which distinguishes including genera, biotypes and species in the myriad of them from better known mycorrhizal symbionts. The plants yet to be studied. It is now well established that distinction is not firm, because endophytes may also endophytes are a rich and reliable source of genetic inhabit root tissues. Overall, endophytic fungi are diversity and may represent morphological and molecular ubiquitous and extremely diverse in host plants. Every levels, often have been associated with them novel natural plant examined to date harbors at least one species of products [17]. However, there has to be a sound and endophytic fungus and many plants, especially woody specific rationale for the selection of each plant for plants, may contain literally hundreds or thousands of endophyte isolation and natural product/secondary species [9-14]. metabolite discovery. The most accepted such rationale In the past two decades, a great deal of information is the following: on the role of endophytic microorganisms in nature has a) Plants from unique environmental settings, especially been collected. The capability of colonizing internal host those with unusual biology and possessing novel strategies for survival; tissues has made endophytes valuable for agriculture as a tool to improve crop performance. Webber [4] was

The Endophyte Lifecycle

In infected seed, As the seed germinates, endophyte is the endophyte grows found in the into the emerging leaf embryo

Endophyte is When the grass goes concentrated in reproductive, the the base of the endophyte grows up plant, not in the the stem and into the roots seed head

Plate 1: Endophyte life-cycle Endophytes: A Novel Source for Bioactive Molecules 75 b) Plants that have an ethanobotanical history or use Table 1. Sterilization of samples for the isolation of endophytes by the indigenous people that is related to the specific Plant species Minutes in Dilution of Reference uses or applications of interest i.e., through direct NaOCl NaOCl contact with local people or via local literature e.g., Lichens 1 1:5 Petrini (1986) healing powers of a botanical source, in fact, may Mosses 1 1:5 Petrini (1986) Ferns 3 1:5 Dreyfuss & Petrini(1984) have nothing to do with the natural products of the Conifer needles 5 1:2 Carroll & Carroll (1978) plant, but of the endophyte inhabiting the plant; Conifer twigs 7 1:2 Carroll & Carroll (1978) c) Plants that are endemic, having an unusual longevity, Monocots-leaves & 3 1:5 Petrini (1986) culms (Triticum) or that have occupied certain ancient land mass, such Dicots (general) 3 1:5 Petrini (1986) as Gondwanaland, are also more likely to lodge Erica leaves 3 1:5 Petrini (1986) endophytes with active natural products than other Erica stems 5 1:5 Petrini (1986) 3 1:5 Petrini (1985) plants; Vaccinium 3 1:5 Petrini (1985) d) Plants growing in area of great biodiversity also have Isolation of endophtyes: For surface sterilization of the material following prospects of housing endophytes with great sequential steps are followed: A) first dip the sample in 96% EtOH for 1 biodiversity e.g., tropical and temperate rainforests. min (lichen and mosses for only 30 secs.); B) sterilization in NaOCl; C) second dip in 96% EtOH for 30 secs. 2 Isolating Endophytes Endophytes are defined as microorganisms, mostly fungi, Endophytes: Eco Friendly colonizing healthy plant tissue without causing overt A heap of properties of endophytes have been already symptoms in or apparent injury to the host [18]. Many, elaborated, as endophytes may produce substances of if not all woody plant stems harbor neutral stem potential use to modern medicine, agriculture, and endophytes but because they are symptom-less they are industry like antibiotics, antimycotics, immuno- difficult to detect and can only be successfully surveyed suppressant, and anticancer compounds. So by this we by plating out carefully prepared surface-sterilized can use the term eco- friendly for endophytes. Here we tissues. For example, a study on the endophytes isolated have tried to exploit the uses of endophytes fully so that from Alnus xylem and barks tissues in roots and stems more and more consideration to be given to endophytes yielded 85 different fungal taxa [19]. This is considered for the human welfare. a fairly typical taxa for this type of study. Among the Frequently, it has been noticed that many endophytes other woody plants surveyed for endophytes are Ilex, are isolated from the plant species but one or few biotypes Hedera, Ruscus, Ulex, Pinus, Fagus, Juniperus, Fraxinus of a given isolate will produce the desired biological and Quercus. Members of the Ericaceae (Rhododendron, active compound in culture [20]. A great deal of Vaccinium, etc.) also harbor a diverse group of uncertainty also exists between what the endophyte may endophytes that have not been thoroughly investigated. produce in culture and what it may be producing under Endophytes are generally not considered organ-specific, natural endophytic conditions. and it is likely that many of the species isolated from stems also occur in leaves. Most procedures for isolating There are very valid reasons to believe that some endophytes are so simple and can be followed by anyone endophytes produce certain phytochemicals, originally skilled in basic microbiological techniques. One of the characteristic of host plant, which might be related to a critical needs for isolating endophytic fungi is obtaining genetic recombination of the endophyte with the host fresh plant material. The need for preventing desiccation DNA in evolutionary time [21]. This argument was also must be balanced against the need for adequate aeration, produced to explain the production of taxol by Taxomyces the former slows tissue death, whereas the latter andreannae [22]. Thus if the endophyte can produce the minimizes the growth of secondary contaminating fungi same rare or important bioactive compound as their host and bacteria. If plants are to be stored for long periods plants, this would not only reduce the need to harvest of time, especially in frost-free refrigerators, tissue slow growing or possibly rare plants but also help desiccation will occur. However, it is possible to isolate preserve world’s ever diminishing biodiversity. Besides, a surprising number of fungal species even from a microbial source of high value product may be easier desiccated woody tissues after freezer storage of more or economical to produce effectively, thereby reducing than a year 18]. One of the critical needs for isolating its market price. endophytic fungi is obtaining fresh plant material. 4 Endophytes: Source of Bioactive Molecules Isolation procedures with reference to sterilization methods and parts of plants have been given in Tables 1 A number of natural products have been reported to have and 2. been obtained from endophytes with potential in pharmaceutical and agrochemical arenas [23]. One of the important examples is that of Cryptosporiopsis 76 Vijeshwar Verma et al.

Table 2. Selected examples of isolation procedures for endophytes from woody plants Bills et al., 1996

Tissues/Hosts Dissection of Tissue Surface Sterilization Media Ulex-spines, stems Pines and 2 cm stem section 1 min 96% EtOH, 3 min 3.25% NaOCL, MEA 0.5 min 96% EtOH Fagus-bark 2 mm borer Propane torch or none Bark extract, glucose-yeast extract, benomyl MEA Alnus-xylem and bark 1 cm stem sections, bark and xylem 35% peracetic acid Malt extract with 10 mg/l cultured separately cyclosporin Castanea & 5 mm arch punch 0.525% NaOCl —10 min Glucose-yeast Quercus-bark extract medium Picea-mature stems Sawing followed by chisel extraction Only extraction tools sterilized Malt agar slants Picea-roots 1 cm segments sink washing, ultrasound, serial washing MEA Fraxinus, Quercus, 2 cm segments, and xylem bark ethanol flaming MEA Fagus-twigs cultured separately Carpinus-bark 1 cm leather punch 0.525% NaOCl for 3 min., flaming in MEA +benomyl alcohol lamp and surfactants Rhizophora-seedlings 1.1 cm cork borer of sterile sea-water rinse, Mangrove-sea

hypocotyl and radicle 0.1% HgCl2 in 5% EtOH water agar Licuala-leaves 3 mm discs from veins and 1 min 96% EtOH, 10 min 3.25% NaOCl, Cornmeal dextrose interveins 0.5 min 96% EtOH agar, MEA Euterpe-leaves 3 mm discs from veins and interveins 1 min 75% EtOH, 10 min 3.25% NaOCl, Cornmeal dextrose agar 0.5 min 75% EtOH with 5 mg/l cyclosporin A N. foetida-stem Twigs (young& old) 1 min 95% Ethanol Sabouraud agar P.hexandrum- 1cm small pieces 1 min 70% Ethanol Aqueous agar rhizomes 5 min 5%NaOCl

cf.quercina (an imperfect stage of Pezicula cinnamomea) conditions and the original plant source from which the isolated from medicinal plant of Eurasia origin endophytes were isolated. Tripterigeum wilfordii [24-26]. The endophyte produces jesteri is another endophytic fungal a unique peptide antimycotic named cryptocandin isolate obtained from a plant species from Papua New containing a number of peculiar hydroxylated amino Guinea. It produces jesterone and hydroxyjesterone acids and a novel amino acid, 3-hydroxy-4- which exhibit strong antifungal activity against a variety hydroxymethylproline. This antimycotic peptide is of plant pathogenic fungi [31]. Phomopsichalasin is an related to known antimycotics, the echinocandins and important metabolite obtained from an endophte pneumocandins [27]. This peptide is also active against Phomopsis species. This metabolite represents the first the rice blast Pyricularia oryzae, a worst plant pathogen cytochalasin-type compound with three ring system and is being used as a model to synthesise other replacing the cytochalasin macrolide ring. This shows a antifungal compounds. strong antibacterial activity against Bacillus subtilis, Another very important example of endophyte Salmonella gallinarum and Staphylococcus aureus and isolated from the plant source is Pestalotiopsis a moderate activity against Candida tropicalis [32]. A microspora from the bark of Torreya taxifolia producing Colletotrichum species isolated from Artimisia annua, a several compounds having antifungal activities including traditional Chinese herb produces metabolites with pestaloside, an aromatic b-glucoside and two pyrones activity not only against the human pathogenic fungi and (pestalopyrone & hydroxypestalopyrone) having bacteria but also metabolites that were fungistatic to plant phytotoxic properties [28]. Also, antifungal activity of pathogenic fungi [33]. Artemisia annua endophyte against phytopathogenic Besides, there are numerous reports on endophytic fungi is well recognized [29]. Two new caryophyllene bacterial species producing antibiotics and phytotoxic sesquiterpenes (Pstalotiopsins A & B), a new compounds now flooding the literature e.g., ecomycins, sesquiterpene 2a-hydroxy-dimeninol and a highly representing a family of novel lipopeptides with masses functionalized humulane. These caryophyllene of 1153 ND 1181 Da besides, containing unusual amino sesquiterpenes have also been reported from another acids like homoserine and b-hydroxyaspartic acid [34]. endophyte P. microspora isolated from Taxus brevifolia The ecomycins are active against human pathogenic [30]. Variations in the amount of bioactive molecules fungi like Cryptococcus neoformans and Candida found in these fungi depend upon both the culture albicans. Pseudomycins is another group of antifungal Endophytes: A Novel Source for Bioactive Molecules 77 peptides reported from various plant-associated explanation for the wide distribution of endophytic fungi pseudomonads [35], [36]. Pseudomycins are active making taxol seems to be related to the fact that taxol is against a number of important human pathogenic fungi a potent fungicide against world’s known worst plant like Cryptococcus neoformans and Candida albicans and pathogens like Pythium and Phytophthora species[48]. also a variety of plant pathogenic fungi including Thus these endophytic fungi may be producing taxol and Ceratocystis ulmi and Mycoshaerella fijiensis [36]. related taxanes to protect their respective host plants from Pseudomycins also contain several nontraditional amino degradation and disease caused by these pathogens. acids including L-chlorothreonine, L- hydroxyaspartic However, taxol production by these endophytes in culture acid and both D- and L- diaminobutyric acid. conditions is in the range of sub-micrograms to A number of endophytic streptomycetes have been micrograms per litre. A lot of research work is being reported to possess antibiotics e.g., Streptomyces species undertaken the world over to enhance microbial from Lolium perenne producing methylalbonoursin [37] production of taxol by using activator compounds in the Streptomyces species NRRL 30562 from Kennedia culture medium. Besides, search for the new endophytes nigriscans- an important traditional plant used by producing better quantities of microbial taxol/ Australian aborigines to treat cuts and open wounds- intermediates continues. produces a host of extremely potent peptide Endophytes encoding important bioactive molecules antibiotics[38]. The antibiotics produced from this like camptothecin [8],[49] and podophyllotoxin [50] endophyte is called munumbicins possessing wide are reported. The microorganisms involved are novel and variety biological activities. Streptomyces species NRRL have been patented. 30566 from Grevillea pteridifolia produces in culture Taxol, a highly functionalized diterpenoid (Fig.1), novel antibiotics called kakadumycins that are related is found in each of the world’s yew (Taxus) species[50]. to echinomycins [39]. Kakadumycin A significantly In electrospray mass spectroscopy, taxol usually gives inhibited the RNA, protein and cell wall syntheses rates two peaks, one at molecular weight 854 (M + H+), and in B. subtilis [39]. However, the effect was lower in DNA the other at molecular weight 876 (M + Na+) [51]. This synthesis. Recently endophytic streptomycetes have been compound is the world’s first billion-dollar anticancer discovered from the biologically diverse areas like the drug, and it is used to treat a number of other human upper Amazon of Peru yielding streptomycetes species tissue-proliferating diseases. Its cost makes it unavailable possessing outstanding inhibitory activities against to many people worldwide. Therefore, alternative sources pythiaceous fungi as well as malarial parasite are needed, since organic synthesis, while having been Plasmodium falsiparum. The bioactive principle is a accomplished, is not economically feasible [52]. Given mixture of lipopeptides named coronamycin [40]. the fact that endophytes are virtually universally present Endophytic actinomycetes are now being tested regularly in all of the world’s higher plants, it was reasoned that and considered for the use in controlling plant yew trees conceivably might support certain endophytic diseases [41]. microorganisms that also make taxol [51]. Thus, if a Endophytes have also been reported to produce microbial source of the drug were available it could anticancer principles which are highly useful. Taxol and eliminate the need to harvest and extract the slow- its derivatives are the first such major bioactive principles growing and relatively rare yew trees. The price for the reported from an endophytic isolate Taxomyces drug would also be reduced, since taxol could be andreanae from Taxus brevifolia[42]. Pestalotiopsis produced via fermentation in much the same way that microspora from Taxus wallichiana, Tubercularia species O from Chinese yew T. mairei, Sporormia minima, and O Trichothecium species from T. wallichiana have also been O OH 14 shown to produce taxol using C precursors[43-46]. This O lead to the search for taxol producing endophytes residing plants other than Taxus species even in continents not NH O known to have indigenous Taxus species like South America and Australia. Pestalotiopsis guepini was O O H O isolated from Wollemia nobilis (extremely rare and earlier OH O thought to be extinct plant) and it showed to produce OH taxol[47]. Besides, Scimatoantlerium tepuiense isolated from Maguireothamnus speciosus has also been shown to produce taxol[43-44],. Endophytic fungi making taxol has now been shown to be distributed worldwide and in plants other than yews. The ecological and physiological Fig. 1: The structure of taxol 78 Vijeshwar Verma et al. penicillin is fermented. It was also speculated that the novel endophytic fungal species [55]. Simply, it appears ability of any endophyte to make taxol may have arisen that the distribution of those fungi making paclitaxel is from the exchange of genetic material from the yew tree worldwide and not confined to endophytes of yews. to one or more microorganisms living in close association 4.1 As a Source of Torreyanic Acid with it [51]. By the early 1990s, however, no endophytic fungi had been isolated or were even known from any of Torreyanic acid, a selectively cytotoxic quinone dimer the world’s representative yew species. After several (anticancer agent), was isolated from a P. microspora years of effort, a novel taxol-producing endophytic strain. This strain was originally obtained as an fungus, Taxomyces andreanae, was discovered in Taxus endophyte associated with the endangered tree T. brevifolia[42]. The most critical line of evidence for the taxifolia (Florida torreya) as mentioned above[56]. presence of taxol in the culture fluids of this fungus, Torreyanic acid was tested in several cancer cell lines, among others, was the electrospray mass spectrum of and it demonstrated 5 to 10 times more potency in those the putative taxol isolated from T. andreanae as well as lines that are sensitive to protein kinase C agonists and C-14 labelling studies, which irrefutably showed the causes cell death by apoptosis. Recently, a complete presence of fungal-derived taxol in the culture [51]. This synthesis of torreyanic acid has been successfully early work set the stage for a more comprehensive completed using the application of a biomimetic examination of the ability of other Taxus species and oxidation-dimerization cascade [57]. other plants to yield endophytes producing taxol. An 4.2 As a Source of Cytochalacins excellent example of this is the anticancer drug, taxol, which had been previously supposed to occur only in Some of fungal genera such as Xylaria, Phoma, the plant genus Taxus (yew). However, taxol has been Hypoxylon, and Chalara are representative producers of demonstrated to occur in a number of unrelated fungal a relatively large group of substances known as the endophytes including Pestalotia, Pestalotiopsis, cytochalasins (Fig 2). The Cytochalasins (Greek cytos, Fusarium, Alternaria, Pithomyces, Monochaetia and cell; chalasis, relaxation) are a group of fungal others. Thus, this report presents information on the metabolites, related by structure and biological activity. presence of taxol among disparate fungal genera, and Major biological effects include inhibition of the division uses these observations as an additional argument to of cytoplasm [58] reversible inhibition of cell movement support efforts to study fungal endophytes and preserve [59] induction of nuclear extrusion [58],[60], inhibition their associated host plants.. An examination of the of such processes as phagocytosis [61-63] platelet endophytes of Taxus wallichiana yielded P. microspora, aggregation and clot retraction. These compounds and a preliminary monoclonal antibody test indicated possess antitumor and antibiotic activities, but because that it may produce taxol. After preparative thin-layer of their cellular toxicity they have not been developed chromatography, a compound was isolated and shown into pharmaceuticals. Three novel cytochalasins have by spectroscopic techniques to be taxol. This organism recently been reported from a Rhinocladiella sp. as an from several 14C precursors that had been administered endophyte on Tripterygium wilfordii. These compounds to it produced labeled taxol [43-44]. Furthermore, several have antitumor activity and have been identified as 22- other P. microspora isolates were obtained from bald oxa-[12]-cytochalasins [64]. Thus, it is not uncommon cypress in South Carolina and were also shown to to find one or more cytochalasins in endophytic fungi, produce taxol [20]. This was the first indication that and workers in this field need to be alerted to the fact endophytes residing in plants other than Taxus spp. were that redundancy in discovery does occur. Chemical producing taxol. Therefore, a specific search was redundancy (dereplication) usually occurs with certain conducted for taxol-producing endophytes in continents groups of organisms on which previous studies have not known for anyindigenous Taxus spp. already established the chemical identity of major

O CH O CH From the extremely rare, and previously thought to CH CH 2 2 CCCH CC CH CH C=O 3 3 2 CH CH CH be extinct, Wollemi pine (Wollemia nobilis), CH3 CH 3 CH2 CH CH CH CH CH O 2 CH O CH 2 Pestalotiopsis guepini was isolated, which was shown 2 HC CH CH HC O C CH HC O C CH3 HC C=O 3 CH HC HC to produce paclitaxel [53]. Also, quite surprisingly, a O HO O O HO O rubiaceous plant, Maguireothamnus speciosus, yielded HO CH NH NH 2 NH CH CH3 2 a novel fungus, Seimatoantlerium tepuiense, that CH CH 2 2 CH CH CH3 CH produces paclitaxel. This endemic plant grows on the 3 2 2 tops of the tepuis in the Venzuelan-Guyana region in southwestern Venezuela [25],[26]. Furthermore, fungal CYTOCHALASIN A R = O CYTOCHALASIN C CYTOCHALASIN D paclitaxel production has also been noted in a Periconia CYTOCHALASIN B R = H, OH sp., [54] and in Seimatoantlerium nepalense, another Fig.2: Different forms of Cytochalasins Endophytes: A Novel Source for Bioactive Molecules 79 biologically active compounds. For instance, as with the bioassay-guided HPLC. The major components were cytochalasins, they are commonly associated with the four functionalized peptides with masses of 1269·6, xylariaceaous fungi. Since their commercial availability 1298·5, 1312·5 and 1326·5 Da. Various other related in the early 1970’s, this group of compounds has become compounds, having bioactivity, with differing masses and the subject of intense cytological research. lower quantities were also present in the culture broth extract. The munumbicins possessed widely differing 4.3 As a Source of Antibiotics biological activities, depending upon the target organism. Endophytes posses another important property of If citing example, munumbicin B had a minimal producing useful antibiotics. In the Northern Territory inhibitory concentration of 2.5 ¼g/ml against a of Australia, various Aboriginal groups use the ground- methicillin-resistant strain of Staphylococcus aureus, up mass of snakevine (Kennedia nigriscans) to promote whereas munumbicin A was not active against this the healing of skin wounds and infections. The snakevine, organism[42]. However, the most impressive biological known as “mangerrporlo” in Dalabon and Mayali, is activity of any of the munumbicins was that of harvested as a fresh stem piece, placed on some hot coals munumbicin D against the malarial parasite Plasmodium for a short time (10 min), mashed into a pulp, and then falciparum, having an IC50 of 4·5±0·07 ng ml-1 [19]. applied as a sticky paste to a cut, wound, or infection. Another useful antibiotic reported to be produced Due to local and much use of that plant, it was taken as by endophyte is named as Cryptocandin A, which is an to isolate endophytic microorganisms from it with the antifungal peptide and is isolated from endophytic fungus logic that some of the healing properties of the snakevine C. quercina. Cryptosporiopsis quercina is the imperfect may, in fact, be produced as a result of the products of stage of Pezicula cinnamomea, a fungus commonly one or more endophytes. In fact, one of the endophytes associated with hardwood species in Europe. It was isolated from this medicinal plant was a Streptomyces isolated as an endophyte from Tripterigeum wilfordii, a sp. This is of particular interest because, as a culture, it medicinal plant native to Eurasia[24-26]. On petri plates, was extremely bioactive against a number of test C. quercina demonstrated excellent antifungal activity microorganisms. Interest in this endophyte was further against some important human fungal pathogens - piqued because actinomycetes have not been reported Candida albicans and Trichophyton spp. A unique to be endophytic on dicotyledonous plants. However, peptide antimycotic, termed cryptocandin, was isolated later a Streptomyces sp. was reported on an annual plant and characterized from C. quercina[24-26]. This -Lolium perenne [37]. This lolium endophyte also compound contains a number of peculiar hydroxylated produces a weak antibiotic, named methylalbonoursin, amino acids and a novel amino acid: 3-hydroxy-4- which is a diketopiperazine, condensed from leucine and hydroxy methyl proline (Fig. 3). The bioactive compound phenylalanine. Streptomyces spp has also been reported is related to the known antimycotics, the echinocandins to produce novel peptide antibiotics, named and the pneumocandins[27]. munumbicins A, B, C, and D [38]. Munumbicins A, B, C and D are newly described antibiotics with a wide 4.4 As a Source of Alkaloids spectrum of activity against many human as well as plant Many grasses harbor fungal endophytes in the genus pathogenic fungi and bacteria (Table 3), and a Neotyphodium, which enhance host fitness, but some also Plasmodium sp. These compounds were obtained from produce metabolites such as ergovaline believed to cause Streptomyces NRRL 3052, which is endophytic in the medicinal plant snakevine (Kennedia nigriscans), native to the Northern Territory of Australia. This endophyte was cultured, the broth was extracted with an organic solvent and the contents of the residue were purified by

Table 3. Inhibitory activity of the munumbicins against various plant- pathogenic fungi and one human-pathogenic bacterium41

Test organism MIC (µg ml -1) Munum- Munum- Munum- Munum- bicin bicin bicin bicin ABCD Pythium ultimum 2.0 0.2 4.0 0.4 Rhizoctonia solani – 8.0 1.5 15.6 Phytophthora cinnamomi – 6.2 1.5 15.6 Geotrichum candidum – 31.2 15.6 31.2 Sclerotinia sclerotiorum 8.0 0.20 8.0 2.0 Fig. 3: Cryptocandin A, an antifungal peptide obtained from the Pseudomonas syringae 15.6 2.0 15.6 0.2 endophytic fungus C. quercina 80 Vijeshwar Verma et al. livestock toxicoses. In Claviceps species the first step in allograft rejection in transplant patients, and in the future ergot alkaloid biosynthesis is thought to be they could be used to treat autoimmune diseases such a dimethylallyltryptophan (DMAT) synthase, encoded by rheumatoid arthritis and insulin-dependent diabetes. The dmaW, previously cloned from Claviceps fusiformis. The endophytic fungus Fusarium subglutinans, isolated from cloning and characterization of dmaW have been T. wilfordii, produces the immunosuppressive but reported from Neotyphodium sp. isolate Lp1, an noncytotoxic diterpene pyrones subglutinol A and B [28]. endophyte of perennial ryegrass (Lolium perenne). The Subglutinol A and B (Fig. 5) are equipotent in the mixed gene was then disrupted, and the mutant failed to produce lymphocyte reaction assay and thymocyte proliferation any detectable ergovaline or simpler ergot and clavine assay, with a 50% inhibitory concentration of 0.1 µM. alkaloids. The disruption was complemented with the

C. fusiformis gene, which restored ergovaline production. H CH Thus, the biosynthetic role of DMAT synthase was H 3 confirmed, and a mutant was generated for future studies CH3 of the ecological and agricultural importance of ergot H alkaloids in endophytes of grasses [65]. O CH H 4.5 As a Source of Antioxidants 3 Endophyte also plays an important role in the production CH of antioxidants. Two compounds, pestacin and 3 OH isopestacin, have been obtained from culture fluids of P. O microspora, an endophyte isolated from a combretaceaous plant, Terminalia morobensis, growing O CH3 in the Sepik River drainage of Papua New Guinea CH [66],[17]. Both pestacin and isopestacin display 3 antimicrobial as well as antioxidant activity. Isopestacin was suspected of antioxidant activity based on its Fig. 5: Subglutinol A, an immunosuppressant, is produced by an structural similarity to the flavonoids (Fig. 4). Electron endophytic F. subglutinans strain spin resonance spectroscopy measurements confirmed In the same assay systems, the famed immuno- this antioxidant activity; the compound is able to suppressant drug cyclosporine is roughly as potent in scavenge superoxide and hydroxyl free radicals in the mixed lymphocyte reaction assay and 104 more [17] solution . Pestacin was later described from the same potent in the thymocyte proliferation assay. Still, the lack culture fluid, occurring naturally as a racemic mixture of toxicity associated with subglutinols A and B suggests and also possessing potent antioxidant activity [66]. The that they should be explored in greater detail [28]. antioxidant activity of pestacin is believed to have arisen primarily via cleavage of an unusually reactive C—H 4.7 As a Source of Antiviral Compounds bond and to a lesser extent, though O—H abstraction[17]. An endophytic fungus also plays a role in the inhibition The antioxidant activity of pestacin is at least 1 order of of viruses. Two novel human cytomegalovirus protease magnitude greater than that of trolox, a vitamin E inhibitors, cytonic acids A and B, have been isolated from derivative [66]. the solid-state fermentation of the endophytic fungus 4.6 As a Source of Immunosuppressive Compounds Cytonaema sp. Their structures as p-tridepside isomers were elucidated by mass spectrometry and NMR methods Immunosuppressive drugs are used today to prevent [67]. It is apparent that the potential for the discovery of compounds, from endophytes, having antiviral activity OH O 10 HO 9 11 is in its infancy. The fact, however, that some compounds 7 OH have been found is promising. The main limitation in 6 7a 12 O OH compound discovery is probably related to the absence 3a 2 7 13 14 5 9 6 of appropriate antiviral screening systems in most H3C 4 7a 1 10 O OH 5 compound discovery programs. 3a HO 11 13 H3C 4 12 14 4.8 As a Source of Insecticides and Pesticides Isopestacin Pestacin In the early 80’s it was demonstrated that the presence of endophytic microorganisms in their respective hosts Fig. 4: Isopestacin & Pestacin, are antioxidants produced by an could result in the reduction of insect attacks [4]. endophytic P. microspora strain isolated from T. morobensis growing on the north coast of Papua New Guinea demonstrated the existence of plant protection against Endophytes: A Novel Source for Bioactive Molecules 81 insects provided in which the endophyte Phomopsis campestris pv. campestris. Two isolates, Alcaligenes oblonga protected elm trees against the beetle piechaudi and Kluyvera ascorbata were effective in Physocnemum brevilineum. It was suggested that the reducing the disease incidence under greenhouse and endophytic fungus P. oblonga was responsible for field conditions. reducing the spread of the Dutch elm disease causal agent The ability of endophytic fungus to resist insects, Ceratocystis ulmi by controlling its vector, the beetle P. induce weight loss, growth and development reduction brevilineum. The author associated the repellent effect and even to increase pest death rate, was correlated with observed towards the insect to toxic compounds produced toxin production. In several cases, it was shown that the by the fungi. This was confirmed four years later by other mode of action of certain fungi was based on the authors[15], who showed that endophytic fungi capability to render the plant unpalatable to several types belonging to the Xylariaceae family synthesize of pests like aphids, grasshoppers, beetles, etc [6], [73], secondary metabolites in hosts of the genus Fagus and [74]. Endophytic fungi may indirectly affect seed that these substances affect the beetle larvae. dissemination by insects, especially ants. In Festuca, Insect toxins have also been isolated from an seeds infected with certain endophytes are discarded after unidentified endophytic fungus from wintergreen being collected and, therefore, favour plant dissemination (Gaultheria procumbens). The two new compounds, 5- [75]. hydroxy-2-(1'-hydroxy-5'-methyl-4'-hexenyl)benzofuran 5 Molecular Biology and the RDT: Tool of and 5-hydroxy-2-(1'-oxo-5'-methyl-4'-hexenyl) Endophytes benzofuran, both show toxicity to spruce budworm, and the latter is also toxic to the larvae of spruce Endophytes performing a useful role in inhibiting the budworm[68]. Another endophytic fungus, Muscodor insect pests has been shown using molecular biology and vitigenus, isolated from a (Paullina paullinioides), the recombinant DNA technology. More recently, yields naphthalene as its major product. Naphthalene, recombinant DNA technology has been applied to the active ingredient in common mothballs, is a widely improve endophytic microorganisms, aiming to the exploited insect repellant. M. vitigenus shows promising introduction of new characteristics of agronomic interests preliminary results as an insect deterrent and has as biological control of pests [76-77] described the exhibited potent insect repellency against the wheat stem introduction of a heterologous gene in an endophytic sawfly (Cephus cinctus) [69], [70]. As the world becomes microorganism with the purpose of insect control. This wary of ecological damage done by synthetic was achieved through the secretion of an insecticidal insecticides, endophytic research continues for the toxin in the host plant. He used the endophyte C. xyli discovery of powerful, selective, and safe alternatives. subsp. cynodontis, a gram positive, xylem-inhabiting bacterium, capable of colonizing several plant species. The work showing the importance of endophytic The commercial product received the designation fungi to the control of agriculture pests, was curiously INCIDE. This bacterium received a gene from other an indirect demonstration that the endophyte, by blocking bacterium, Bacillus thuringiensis, which is able to insect gall formation, also was able to control a disease produce the d-endotoxin active against insects in nature, caused by a fungus [4-5]. Several other examples were especially against Lepidoptera and Coleoptera. analyzed in which endophytic fungi block or alter insect’s Therefore, the genetically modified bacterium is able to larvae installation and, by doing so, repress potential secrete toxin inside the plant, protecting it against attacks vectors to disseminate diseases. Other interesting effect of target insects. of endophytes over insect galls was seen in oak, where apparently dormant endophytic fungi become active with Endotoxin gene from B. thuringiensis has also been gall formation and, by destroying the leaves, also kill introduced into nitrogen-fixing bacteria from the genus the insect, protecting, therefore, their hosts. This process Bradyrhizobium. In this case, the engineered bacterium was described in three associations between endophytes was introduced into roots of Cajanus cajan, improving and insects, one of them involving the fungus nitrogen fixation and protecting the host against Rivelia Gloeosporium quercinum and the insect Neuroterus angulata larvae [78]. Other reviews on the use of numismalis [71]. genetically modified endophytes may be consulted for more details on the subject [79-81]. Assis et al. [72} isolated endophytic microorganisms from cabbage growing in the State of Pernambuco, Several genes from endophytic fungi and related to Northeast of Brazil. The method applied for endophyte toxin production are now being cloned and studied in isolation was the sonication. Seven isolated species were depth [82] cloned a gene coding for a DMAT Synthase evaluated for antagonistic activities against the causal (dimetilail tryptophane synthase) that is responsible for agent of cabbage black rot disease Xanthomonas the first step in the synthesis of ergot by the fungus 82 Vijeshwar Verma et al.

Claviceps purpurea. Genes related to the latter were cancer. Other camptothecins—such as 9-amino- identified in the endophytic fungi Balansia obtecta and camptothecin (9AC), 9-nitrocamptothecin (9NC), and 7- Neotyphodium spp., known to produce, respectively, (4-methyl piperazino-methylene)-10,11-ethylenedioxy- ergobalansine and ergovaline [83] also cloned a gene camp- tothecin (GG211) have also showed remarkable (DMATS) of C. purpurea involved in the ergot synthetic potential in the treatment of carcinoma [95-98]. pathway in planta [84] found in Epichloë a cluster Camptothecins are lauded as one of the most promising containing genes for the synthesis of mycotoxins like anticancer drugs of the twenty-first century [99]. paxiline and lolitrem B. Nothapodytes foetida is well known to produce an 6 Biodiversity of Endophytes important anticancer alkaloid Camptothecin. The endophytic fungus (Entrophospora infrequens) isolated Tropical and temperate rainforests are the most from it produced Camptothecin under culture conditions. biologically diverse terrestrial ecosystems on earth. The This fungus belongs to the family Phycomycetes [7]. most threatened of these spots cover only 1.44% of the land’s surface, yet they harbor more than 60% of the Lignans are very well known to play an important world’s terrestrial biodiversity [85]. As such, one would role in plant defense. Lignans have antibiotic, antioxidant expect that areas of high plant endemicity also possess and pesticidal properties and therefore confer protection specific endophytes that may have evolved with the against pathogens in plants. Presently, no prokaryotic endemic plant species. cells are able to produce lignans. Tropical rainforests are a remarkable example of this The natural lignan podophyllotoxin is a dimerized type of environment. Competition is great, resources are product of two phenylpropanoid moieties which occur limited, and selection pressure is at its peak. This gives in a few plant species. Since podophyllotoxin is too toxic rise to a high probability that rainforests are a source of for the treatment of neoplastic diseases in humans, it is novel molecular structures and biologically active used as a precursor for the chemical synthesis of semi- compounds [86-87] described a metabolic distinction synthetic antineoplasti drugs, etoposide and teniposide, between tropical and temperate endophytes through which are being successfully applied as antitumor agent statistical data which compares the number of bioactive [100].The availability of this lignan is becoming natural products isolated from endophytes of tropical increasingly limited because of the scarce occurrence of regions to the number of those isolated from endophytes its natural sources and also because synthetic approaches of temperate origin. Not only did they find that tropical for its production are still commercially unacceptable. endophytes provide more active natural products than An endophytic fungus Trametes hirsuta isolated temperate endophytes, but they also noted that a from Podophyllum species, is a novel potential alternative significantly higher number of tropical endophytes source of podophyllotoxin and related aryl tetralin produced a larger number of active secondary metabolites lignans [8]. than did fungi from other tropical substrata. This observation suggests the importance of the host plant in 8 Prospects of Further Research in Endophytes influencing the general metabolism of endophytic A comprehensive study on the endophytes of any microbes. individual rainforest higher plant species has not been done, much less a study on any individual plant in its 7. Present Status of Endophytes at RRL (Jammu) entirety, from its complete root system to its stems, For the last three years, we are working on endophytes petioles, leaves and flowers. The prospects of finding and during this period we isolated two novel endophytic endophytes (fungi and bacteria) that are specific to any fungi, which form an alternative source of two important given higher plant or even occurring only in a local region anticancer drugs i.e, Camptothecin and Podophyllotoxin in a forest seem great, given the paucity of work in this from two different plant species [7], [8], [49]. area. Camptothecin (CPT) is a monoterpenoid indole As endophytes are the source of various bioactive alkaloid originally isolated from Camptotheca acuminata molecules, the production of these molecule can be Decne, a deciduous tree native to south China, that has enhanced by molecular and recombinant DNA gained great attention for its significant antitumor technology tools. New strategies, such as bioconversion activities in experimental studies [88]. Irinotecan (CPT- using precursors to target compounds and stimulation 11) [89-91] and topotecan (TPT) [92-94] two water- of production using elicitors, may be developed to soluble derivatives of CPT, have gained approval by the enhance secondary metabolite production. Food and Drug Administration of the United States of In his in vivo experiments with Pestalotiopsis America (FDA) for treating colorectal and ovarian microspora [101] suggested that the plant might have Endophytes: A Novel Source for Bioactive Molecules 83 acquired the genes for paclitaxel from the fungus. This 5. Carroll G The biology of endophytism in plants with finding has important implications in the biology of P. particular reference to woody perennials In: Microbiology of the Phylloplane. Fokkema NJ and Van der Heavel J (eds). microspora Ne32 since it explains at least one mechanism Cambridge University Press, London UK (1986) 205-222. by which new DNA can be captured by the organisms, 6. Carroll GC Fungal endophytes in stems and leaves: From eventually expressed and replicated. Such a mechanism latent pathogen to mutualistic symbiont Ecology 69 (1988) may begin to explain how the enormous biochemical 2-9. variation may have arisen in this fungus [20]. Also, this 7. Puri SC, Verma, V, Amna, T, Qazi, GN and Spiteller M (2005) initial work represents a framework to aid in the An endophytic fungus from Nothapodytes foetida that understanding of how this fungus may adapt itself to the produces Camptothecin J Nat Prod 68(12): 1717 – 1719. environment of its plant hosts and suggests that the uptake 8. Puri SC, Nazir A, Chawla R, Arora R, Riyaz-ul-Hasan S, Sharma S, Amna T, Ahmed B, Verma V, Singh S, Sagar R, of plant DNA into its own genome may occur. 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