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Current Research in Environmental & Applied Mycology Doi 10.5943/cream/2/1/3

31

Biology of Endophytic Fungi

Selim KA1,*

, El-Beih AA1, AbdEl-Rahman TM

2and El-Diwany AI

1

1Chemistry of Natural and Microbial Product Department, National Research Center, 12622 Dokki, Cairo, Egypt.2Botany Department, Faculty of Science, Cairo University, Giza, Egypt.

Selim KA, El-Beih AA, AbdEl-Rahman TM, El-Diwany AI. 2012Biology of Endophytic Fungi.Current Research in Environmental & Applied Mycology 2(1), 3182, Doi 10.5943/cream/2/1/3

Endophytic fungi that are residing asymptomatically in internal tissues of all higher plants are ofgrowing interest as promising sources of biologically active agents. This review focuses on the

biology of endophytic fungi, their discovery, isolation, identification, and diversity and theirbiological activities in environmental and agricultural sustainability. It also considersand theirmedicinal applications especially in the production of anticancer, antimicrobial, antioxidant, and

antiviral compounds. Endophytic fungi are one of the most creative groups of secondary metaboliteproducers that play important biological roles for human life. They are potential sources of novelnatural agents for exploitation in the pharmaceutical industry, agriculture, and in environmentalapplications.

Key words Biological Roles Ecology Endophytic Fungi Identification Isolation Secondary Metabolites

Article InformationReceived 30 January 2012

Accepted 4 May 2012Published online 20 June 2012*Corresponding author: Khaled A. [email protected]

Introduction

Natural Products as Important Sources in

the Drug Discovery Process There is a need to search new

ecological niches for potential sources ofnatural bioactive agents for different pharma-

ceutical, agriculture, and industrial applica-tions; these should be renewable, eco-friendlyand easily obtainable (Liu et al. 2001). Natural

products discovery have played major role inthe search for new drugs, and is the most

potent source for the discovery of novelbioactive molecules. Natural products arechemical compounds derived from livingorganisms. The most prominent producers ofnatural products can be found within differentgroups of organisms including plants, animals,

marine macro-organisms (sponge, corals andalgae), and microorganisms (bacteria,

actinomycetes, and fungi). The discovery ofnatural products involves isolation, structuralelucidation and establishing the bio-synthetic

pathway of the secondary metabolites. This isan area of considerable interest to scientistsdue to the structural diversity, complexity andvarious bioactivities of isolated compounds.

Crude natural products have been used directlyas drugs which were low cost and importantsources of traditional medicines. They also

provided the basic chemical architecture forderiving semi-synthetic natural products(Suryanarayanan et al. 2009).

The role of natural products indiscovery of new therapeutic agents can bedemonstrated by an analysis of the number andsources of bioactive agents. There are at least200,000 natural metabolites with various

bioactive properties (Brdy 2005). Accordingto Cragg et al. (1997) anti-cancer and anti-
mailto:[email protected]:[email protected]


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Fig. 1Distribution of Natural Products as Drugs New Chemical Entities in Time Frame 1981-2006

Adapted from (Newman et al. 2003) (Newman & Cragg 2007)

B Biological; usually a large (> 45 residues) peptide or protein, generally isolated from an organism/cell line orproduced by biotechnological means in a surrogate host; N Natural product; ND Derived from a natural product,usually a semisynthetic modification; S Totally synthetic drug, mostly found by random screening or modification ofan existing agent; S* Made by total synthesis, but the pharmacophore is/was from a natural product; V Vaccine;NM Natural Product mimic.

infective agents that were approved as drugswere more than 60% from natural origin.

Between 1981-2006, about 100 anti-cancer agents have been developed, 25% of

them were natural product derivatives, 18%were natural product mimics, 11% candidateswere derived from a natural product

pharmacophore, and 9% were pure naturalproducts. Actually 47% of total anticancerdrugs and 52% of new chemicals introducedinto the market are of natural origin (Chin et al.2006, Newman & Cragg 2007). In the USAmore than 50% of prescribed drugs are natural

products or semi-synthetic derivatives. In addi-tion, a number of chemicals used in crop prote-

ction are also of natural origin (Schneider et al.2008). Thus natural sources make a very signi-ficant contribution to the health care) Fig.1.

Since, the discovery of potent antibioticagainst Gram-positive bacteria, penicillin fromculture of fungus Penicillium notatum byFleming in 1929, the search for new drugsfrom microbial origin started. Koehn & Carter(2005) and Newman & Cragg (2007) reportedmany of secondary microbial metaboliteswhich show potent pharmaceutical application

against various diseases. This included thetherapeutically used ergotamine, theimmunosuppressive peptide cyclosporine A,

peptidic antibiotic compounds like thepenicillin V and cephalosporin C, thepolyketide lovastatin used in cholesteroltreatment and the antibacterial terpenoid

fusidic acid (Fig 2).Fungi as Producers of Biologically Active

Metabolites

More than 20,000 bioactive metabolitesare of microbial origin (Brdy 2005). Fungi areamong the most important groups of eukaryoticorganisms that are well known for producingmany novel metabolites which are directlyused as drugs or function as lead structures forsynthetic modifications (Kock et al. 2001,

Bode et al. 2002, Donadio et al. 2002, Chin etal. 2006, Gunatilaka 2006, Mitchell et al. 2008,Stadler & Keller 2008). The success of severalmedicinal drugs from microbial origin such asthe antibiotic penicillin from Penicillium sp.,the immunosuppressant cyclosporine fromTolypocladium inflatum and Cylindrocarponlucidum, the antifungal agent griseofulvin from

Penicillium griseofulvum fungus, thecholesterol biosynthesis inhibitor lovastatinfrom Aspergillus terreus fungus, and -lactam

antibiotics from various fungal taxa, has shiftedthe focus of drug discovery from plants tomicroorganisms.


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Fig. 2Structure of some biological active microbial metabolites

Suryanarayanan et al. (2009) discussedmany fungal secondary metabolites withvarious chemical structures and their wideranging biological activities and this reflectsthe high synthetic capability of fungi(Suryanarayanan & Hawksworth, 2005). About1500 fungal metabolites had been reported toshow anti-tumor and antibiotic activity (Pelez2005) and some have been approved as drugs.These include micafungin, an anti-fungalmetabolite from Coleophoma empetri

(Frattarelli et al. 2004), mycophenolate, aproduct ofPenicillium brevicompactum, whichis used for preventing renal transplant rejection(Curran & Keating 2005), rosuvastatin from

Penicillium citrinum and P. brevicompactum,which used for treating dyslipidemias (Scott etal. 2004) and cefditoren pivoxil, a broadspectrum antibiotic derived fromCephalosporiumsp. (Darkes & Plosker 2002).Others include derivatives of fumagillin, anantibiotic produced by Aspergillus fumigates

(Chun et al. 2005), and illudin-S, asesquiterpenoid from Omphalotus illudens(McMorris et al. 1996) which exhibits anti-

cancer activities. Also, fungal metabolites areimportant in agriculture applications (Anke &Thines 2007).

It has been estimated that there may be1.5 million fungal species, while only about100,000 species are presently known(Hawksworth 2004). Only a few taxa havetested for their biological applicationsincluding their ability for drug production and

biological control. Thus it seems that thediscovered percentage of economically

valuable fungal metabolites is small.Soil fungi have been the most studied of

fungi, and typical soil genera such asAcremonium, Aspergillus, Fusarium andPenicillium have shown ability to synthesis adiverse range of bioactive compounds. Morethan 30% of isolated metabolites from fungiare from Aspergillus and Penicillium (Brdy2005). Fungi however were usually obtainedfrom the same ecological niche using the samefungal isolation methods. Therefore the the

same fungal strains were re-isolated and thislead to the re-discovery of known compoundsas the same taxa produce the same metabolites.


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Fig. 3Taxol, anticancer drug produced by many endophytic fungi.Dreyfuss & Chapella (1994) assumed that

different environmental factors includingdifferent physical conditions and different

biological situations in the nature, may changebehavior of microbes and favor the productionof diverse range of secondary metabolites, sothe search for alternatively unexploredecological niches should be targeted. Theinvestigation of fungal isolates from ecological

niches other than soil may lead to investigatingnovel fungal groups with novel and diverse ofsecondary metabolites. Fungi occupy everyliving and non-living niche on earth, thisincludes those in the thermal vents, in deeprock sediments, and in desert as well as marineenvironments (Strobel 2003). Some of unex-

plored fungal groups derived from suchecosystems are endophytic fungi, fresh-waterfungi, and marine-derived fungi (Dreyfuss &Chapella 1994). In this review we will focus

and concentrate on endophytic fungi that residein plants, and the importance of their secondarymetabolites.

EndophytePlants may serve as a reservoir of large

numbers of microorganisms known as endo-phytes (Bacon & White 2000). Endophytes aremicroorganisms (mostly fungi and bacteria)that inhabit plant hosts for all or part of theirlife cycle. They colonize the internal plant

tissues beneath the epidermal cell layerswithout causing any apparent harm or sympto-matic infection to their host, living within the

intercellular spaces of the tissues and its seemsthat they may penetrate the living cells (Strobel2003). Endophytes form inconspicuous infec-tions within tissues of healthy plants for all ornearly all their life cycle and their host tissuesappear symptomless, and theyremain asympto-matic for many years and only become parasi-tic when their hosts arestressed(Firkov et al.2007, Limsuwan et al.2009). Endophytic fungi

are an ecological, polyphyletic group of highlydiverse fungi, mostly belonging to ascomycetesand anamorphic fungi (Huang et al. 2001,Arnold 2007).

Approximately, there are near to300,000 plant species on earth and eachindividual plant is the host to one or moreendophytes, and many of them may colonizecertain hosts. It has been estimated that theremay be as many as one million differentendophytic fungal taxa, thus endophytes may

be hyperdiverse (Petrini 1991, Strobel & Daisy2003, Huang et al. 2007). Endophytesmay

produce a plethora of bioactive metabolites thatmay be involved in the host-endophyterelationship (Strobel 2003), and may serve as

potential sources of novel natural products forexploitation in medicine, agriculture, andindustry (Bacon & White 2000, Strobel &Daisy 2003). The described populations ofendophytic strains are few, which means theopportunity to find new strains and targetingnatural products from endophyticmicroorganisms that colonize plants indifferent niches and ecosystems is great.


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Fig. 4Oocydin A, antifungal agent isolateded from Serratia marcescens.

Discovery the Importance of Endophytic

Fungi

The importance of endophytes had beendemonstrated over a long period as potentialsources of pharmaceutical leads, as many ofendophytic fungi were reported to producenovel bioactive metabolites such asantimicrobial, anticancer, and antiviral agents.The discovery of taxol producing fungiincreased the importance of endophytes andshifted natural products research to endophyticfungi.

Taxol (Fig 3), a highly functionalized

diterpenoid, is found in each yew (Taxus)species, but was originally isolated from Taxusbrevifolia (Suffness 1995, Wani et al. 1971).This compound is the worlds first billiondollar anticancer drug, and it is used fortreatment of ovarian and breast cancers, butnow it is used to treat a number of other humantissue-proliferating diseases as well. Its costmakes it unavailable to the most of worlds

people (Nicolaou et al. 1994). It was suggestedby Stierle et al. (1993) that yew trees mightsupport certain endophytic microorganismsthat may also synthesize taxol.

In the early 1990s, a novel taxol-producing endophytic fungus, Taxomycesandreanae, was isolated from Taxus brevifolia(Strobel et al. 1993). This set the stage for amore comprehensive examination of the abilityof other Taxusspecies and other plants to yieldendophytes producing taxol. An examinationof the endophytes of Taxus wallichianayielded

the endophytic fungus Pestalotiopsismicrospora, and a preliminary screeningindicated that it produced taxol (Strobel et al.

1996). Furthermore, several other P.microspora isolates were obtained from bald

cypress in South Carolina and were also shownto produce taxol (Li et al. 1996). This was thefirst indication that other endophytes than T.andreanae residing in plants other than Taxusspp. were producing taxol. Numerous reportshave shown that many of other endophyticfungi such as Pestalotiopsis guepini and

Periconiasp. also produce taxol (Li et al. 1998,Strobel et al. 1997). Also endophytic Fusarium

solaniisolated from Taxus chinensisand othercommon endophytic genera such as Alternaria

and Aspergillus isolated from Ginkgo bilobaand Podocarpus sp. respectively, had beenreported as producers of taxol (Kim et al. 1999,Sun et al. 2008, Liu et al. 2009).

Thus, the presence of a microbialsource of the drug could eliminate the need toharvest and extract the slow growing andrelatively rare yew trees, and the price for thedrug would also be reduced and the drug will

be available to cancer patients, since taxolcould be produced via fermentation in the sameway that penicillin is fermented (Strobel 2003).However, despite the promised concerning the

production of taxol by endophytes, this has notmaterialized into industrial production. It may

be that the fungi carry residue of taxol overfrom the plant or rapidly lose their abilities to

produce taxol in vitro.

Isolation and Cultivation of Endophytes

Fungi from Plants

Isolation of endophytes is a criticalstep, because it requires sensitivity to recover amaximum number of colonized endophytes and


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should be accurate enough to eliminate theepiphytic microbes which are present on the

plant surface. Endophytes can be isolated fromvarious plant parts such as seeds, leaves andstems. The collected plants for studyingendophytic communities should lookapparently healthy and disease free plant, i.e.

they do not display any visual symptoms ofdiseases, in order to minimize the presence of

plant pathogenic and saprobic species, and toprevent the isolation of localized pathogenicendophytic microorganisms (Strobel 2003,Strobel & Daisy 2003).

Selection of Plant M aterial

It is important to understand the methodsand rationale used to provide the bestopportunities to isolate endophytes, since the

number of plant species in the world is so greatand each individual plant may be host tonumerous endophytes. Creative and imagina-tive strategies must therefore be used to quick-ly narrow the search for the host plants forisolation and target endophytes displaying bio-activity (Mittermeier et al. 1999, Strobel 2003,Strobel & Daisy 2003). Several criteria must beconsidered in plant selection strategy, and theseare as follows (Strobel & Daisy 2003):

1. Plants from a unique ecologicalenvironmental niche, and growing inspecial habitats, especially those with anunusual biology and possessing novelstrategies for survival should seriously beconsidered for study. Strobel et al.(1999a) showed that an aquatic plant,

Rhyncholacis penicillata, which lives inharsh aquatic environment which may beconstantly wounded by passing rocks andother debris, resists infection by common

oomyceteous fungi (water molds that arephytopathogenic) that cause disease. Thepossibility that endophytes associatedwith this aquatic plant may produceantifungal agents that protect the plantfrom attack by pathogenic fungi isfeasible. A novel antioomycetouscompound, oocydin A (Fig 4) wasdiscovered from the endophytic strainSerratia marcescensfrom this plant.

2. Plants that have an ethnobotanicalhistory, and are used for traditionalmedicines should be selected for study, asinhabiting endophytes may be the source

of the medicinal properties of this plant.For example, the endophtic fungus

Fusarium proliferatum possessingantimicrobial activity, was isolated fromtraditional Chinese medicinal plantCelastrus angulatus(Ji et al. 2005).

3. Plants that are endemic, having anunusual longevity, or have occupied acertain ancient land mass, are appropriatefor study. An endophytic fungusChaetomium globosum, isolated from leafof endemic plant Maytenus hookeri,which is only distributed in areas ofYunnan, China, was found to produceChaetoglobosin B which showed anti-tuberculosis activity (Ni et al. 2008).

4. Plants growing in areas of greatbiodiversity also have the potential for

housing endophytes with great diversity.Kumaresan & Suryanarayanan (2001)found that many endophytic fungicolonized trees in mangrove forests.

5. Plants surrounded by pathogen infectedplants, and showing no symptoms aremore likely to lodge endophytes

possessing antimicrobial activity thanother plants. For exampleTuntiwachwuttikul et al. (2008) reportedan endophyte showing antimicrobialactivity against plant pathogenColletotrichum musae.

6. Young plant tissue is more suitable forisolation of endophytic fungi than oldertissues which often contain manyadditional fungi that make isolation ofslow growing fungi difficult to isolate.The collected plant samples are stored at4C until the isolation procedure iscarried out, and isolation should be as

soon as possible after collection to avoidcontamination by air microspora (Bacon& White 1994).

Acquiring endophytes which maydisplay bioactivity, needs selection of plantspecies that may be of interest because of theirunique biology, age, endemism, ethnobotanicalhistory, and/or environmental setting. Yu et al.(2010) showed that medical plants and plantsin special environments were frequently

studied for screening for presence ofendophytes that produce antimicrobial agents(Fig 5).


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Fig. 5 Proportion of biologically active endophytic fungal isolates from different sources withantimicrobial activities. Adapted from: (Yu et al. 2010).

I solation of Endophytic Fungi

The most important step for the

isolation of endophytic fungi that reside inplant tissues is surface sterilization and theplant parts under investigation should be cutinto small pieces to facilitate sterilization andisolation processes. To achieve completesurface sterilization, there are various methodsto eliminate most of the epiphytic fungi fromthe exterior tissues and encourage the growthof the internal mycota, according to the type oftissue as well as its location (Strobel 2003,Strobel & Daisy 2003).

I dentif ication of Endophytic Fungi

Identification of endophytic fungi basedis mainly on morphological methods, usingcharacters of the phenotype of the fungalculture, i.e. colony or hyphae, the characters ofthe spore, or reproductive structure if thesefeatures were discernible (Wei 1979,Carmichael et al. 1980, Barnett & Hunter1998). Most of endophytic fungi belong to theascomycetes and asexual fungi (Huang et al.

2001), but some endophytic isolates may fail toproduce reproductive structures even afterseveral months. These isolates cab beencouraged to sporulate on medium containsstripes or extract of host plant (Matsushima1971). Sterile isolates should be checkedregularly for fruiting bodies over a period of 3-4 months and the isolates that failed tosporulate are referred to as mycelia sterilia, ardivided into different morphotypes accordingto their culture characteristics. These groups offungi are considerably common in endophytesstudies (Lacap et al. 2003). Guo et al. (1998)reported other methods using twigs in conical

flasks over a three months period to promotesporulation of morphospecies (Fig 6).

Endophytes are diverse and often growreadily in culture. With recent technologicaladvances, the use of culture-free methods

promises to discover ever-greater diversity andto expand our understanding of the structure ofthe fungal tree of life. One particularly usefulaspect of culture-free methods may be to showthat particular fungi are present in anenvironment, thus leading endophyteresearchers to optimize culturing conditions asa means to capture those fungi in vitro. Such

efforts are critical for establishing voucherspecimens, which in turn can be used toempirically assess species interactions, as rawmaterials for bioprospecting or biologicalcontrol, and as the basis for future research insystematics or genomics. Because manyendophytes do not sporulate in culture, andthus are classified only as mycelia sterilia, theyare not welcome at most established culturecollections. Depositories are needed to houseand maintain vouchers of these fungi, and to

curate their ecological data (site of origin; hostplant; season; tissue type). Both the specimensthemselves and the data regarding theirrecovery have tremendous intrinsic value,(Arnold 2007).

According to Huang et al. (2008a)sterile mycelia are widely distributed among

plant hosts as they were found in 27 medicinalplants of 29 host plants used for screening forendophytic fungi. High relative frequencies of27% of endophytic fungal isolates from 29medicinal plants were sterile (Fig7). Manyvariable proportions of mycelia sterilia rangingfrom 11% to 54% had been


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Fig. 6Assortment of ascomycetous endophytic fungi recovered from foliage of angiosperms andconifers in North America and Panama, Adapted from: (Arnold 2007).

reported (Petrini et al. 1982; Garcia &Langenheim 1990, Fisher et al. 1994, Taylor etal. 1999, Frhlich et al. 2000, Guo et al. 2000).Thus, the common problem concerningidentification of endophytic fungi are that someof endophytes could not be identified to speciesor genus level (Gamboa & Bayman 2001,Promputtha et al. 2005a & b), and having manynon-identified mycelia sterilia, raises theimportance of use modern molecular

techniques that could be the best alternative toidentify this taxa.

Molecular Characteri zation of Endophytic

Fungi

Molecular approaches have been usedto resolve problems in fungal taxonomy and fordirect detection and identification of fungiwithin natural habitats (Rollo et al. 1995, Ma etal. 1997, Zhang et al. 1997, Liew et al. 1998,Ranghoo et al. 1999). The most frequently

accountered problem in endophytic fungi is thepresence of mycelia sterilia, making theirmorphological identification difficult (Guo etal. 2000). Ribosomal DNA sequence analysisusing specific PCR primers to amplify rDNAfragments of endophytes was used to validatethe morphospecies of different groups ofmycelia sterilia, and to resolve theidentification problem associated withendophytic fungi (Doss & Welty 1995, Lacapet al. 2003) (Fig 8).

According to Huang et al. (2009) rDNAsequence analysis is frequently used to confirmmorphological identification of endophytic

isolates, and to study phylogeny of endophyticfungi, about 24% of endophytic isolates fromthree Attemisia species were sterile, and withaid of molecular techniques the phylogeny of34 endophytic fungi were studied, includingidentification of some sterile species andconfirmation of some morphological identifiedspecies, using amplification of ITS1, 5.8S andITS2 fragments of rDNA.

Molecular techniques can show hidden

diversity and help reveal identities anddiversity of sterile mycelia. However, thecareless use of named GenBank sequenceswithout questioning whether theiridentifications are correct has lead to manyspecies in endophyte studies being wronglynamed (Ko Ko et al. 2011). Extreme cautionmust be taken when using named sequencesfrom GenBank as these are often wronglynamed (Cai et al. 2009, Koko et al. 2011a,b).

Ecology and Biodiversity of EndophytesEndophytic fungi represent an

important and quantified component of fungalbiodiversity, and are known to have an effecton and be affected by plant communitydiversity and structure (Sanders 2004, Gonthieret al. 2006, Krings et al. 2007). Almost allvascular plant species examined to date werefound to harbor endophytes. Endophytes have

been also recorded colonizing marine algae andgrasses, mosses and ferns (Tan & Zou 2001).Endophytes are present in virtually all organsof a given plant host, and some are seed-borne(Hyde & Soytong 2008). Endophytes can be


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Fig. 7Relative frequencies of different endophytic taxa isolated from 29 Chinese medicinal plantsshowing high percent of Sterile Mycelia, Adapted from (Huang et al. 2008a).

transferred from plant to plant via seeds (Aly etal. 2011). The mycelium of the fungus thengrows into the sheath, stem, and leaf tissues,

finally enters the flowering stem and seeds(Firkov et al. 2007). The endophyte is passedto the next generation of plants through theseed, for instance asexual Acremonium grassendophytes are dispersed exclusively throughthe seeds of their hosts (Tan & Zou 2001).

A variety of relationships can coexistbetween endophytes and their host plants,ranging from mutualism or symbiosis toantagonism or slightly pathogenic (Schulz &Boyle 2005, Arnold 2007, Purahong & Hyde

2011). The host-endophyte relationships can bedescribed in terms of host-specificity, host-recurrence, host-selectivity, or host preference(Zhou & Hyde 2001, Cohen 2006).

Host-specificity (a phenomenon inendophytes-plant interaction) is a relationshipin which microorganism is restricted to a singlehost or a group of related species, and suchspecificity implies that complex biochemicalinteraction occur between host and itsassociated endophytes (Holliday 1998, Strobel2003, Strobel & Daisy 2003). Host-specificstrain formation can be interpreted as a form ofecological adaptation. It can be accepted thatmorphologically indistinguishable strains ofthe same species will exhibit different physio-logical traits that may be host-related (Petrini1991). For example Pestalotiopsis microsporais one of the most commonly found endophytesin Taxaspecies (yews). Extracts of 15 isolatesof Pestalotiopsis microspora, obtained from at

least four continents, were examined and it wasobserved that no two chromatograms wereidentical. This is an indication that there is

complex bio-chemical interaction between hostand its associated endophytes, raising enor-mous variability between endophytes, through

mutation, genetic crossing, or by unsubstan-tiated mechanisms such as developing geneticsystem allowing transferring of information

between themselves and host plants (Tan &Zou 2001, Firkov et al. 2007).

Host-recurrence refers to the frequentor predominant occurrence of endophytes on a

particular host or a range of plant hosts, andendophytes can also found infrequently onother host plants in the same habitat (Zhou &Hyde 2001). A single endophytic species may

form relationships with two or many relatedhost plants, but found in a preference for one

particular host, and this phenomenon is definedas host selectivity (Cohen 2006).

The term host-preference is morefrequently used to indicate a common occurre-nce or uniqueness of occurrence of endophytesto particular host, and also used to indicate thedifference in endophytic community composi-tion and relation frequencies from differenthost plants (Suryanarayanan & Kumaresan2000). Endophytes are also able to colonizemultiple host species of the same plant familywithin the same habitat, and the distribution ofsome endophytes can be similar in closelyrelated plant species (Huang et al. 2008a).Colletotrichum, Phoma, Phomopsis and

Phyllostictaendophytes have a wide host rangeand colonize several taxonomically unrelated

plant hosts (Pandey et al. 2003, Jeewon et al.2004, Murali et al. 2006, Sieber 2007, Hyde et

al. 2009, Udagaya et al. 2011, Wikee et al.2011) suggesting that they have developedadaptations to overcome different types of host


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Fig. 8 The phylogenetic context of endophyte symbioses: relative abundance of five classes ofAscomycota among endophytes isolated from three different plant families in southeastern Arizona,USA. Hosts representing the Fagaceae (Quercus spp.; N=44 isolates, dominated by theSordariomycetes), Pinaceae (Pinus ponderosa; N=111 isolates, dominated by the Leotiomycetes),and Cupressaceae (Cupressus arizonica and Platycladus orientalis; N=42 isolates, dominated by theDothideomycetes) differ markedly in the relative abundance and dominance of each class. The

predominant classes listed here also dominate endophyte communities associated with these hostfamilies in other sites, including mesic semideciduous forest (North Carolina; all families) and

boreal forest (Qubec; Pinaceae and Cupressaceae), Adapted from: (Arnold 2007).

defenses.

Effect of Environment on Endophytes

Environmentally, the endophyte may bemetabolically aggressive by affecting hostdefense chemicals (Cabral et al. 1993, Peters etal. 1998, Schulz et al. 1999). Such a hostile

environment may account for the evolution ofthe potentially increased synthetic ability of theendophytes. This perhaps explains the apparentanomaly observed when a species of endophyteisolated from a plant host produces a bioactivecompound but fails to do so when isolatedfrom another plant species (Li et al. 1996). Theherbicidal activity of secondary metabolites ofan endophytic Phyllosticta capitalensisdiffered with the plant host from which theendophyte was isolated. This probably means

that the plant host (and ultimately itsmetabolism) influences the synthetic ability ofan endophyte. This indicates that

bioprospecting for endophyte natural productsshould be host plant based as opposed to fungaltaxon based. In this regard, the endophyte-planthost association could also be exploited inenhancing the production of useful metabolites

by the plant host (Wang et al. 2004).Endophytes residing in the host tissue

in a symptomless state or one that may be

beneficial to its host may turn into a pathogenin response to some environmental cue(Hendry et al. 2002); such a shift in the nature

of the endophyte would also result in a changein its metabolite profile. Also, theenvironmental conditions which effect on host

plant growth, influence the number and varietyof endophytic populations, and affect onmetabolites produced by endophytes.

The difference in endophytes,

difference in their metabolic profile, and hencedifference in their biological activity even ifbetween the same isolates of same species,might be related to the chemical difference ofhost plants (Paulus et al. 2006). This dependson the environment, and shows the importanceof studying host-endophytes relationships, andthe effect of host plants on endophyticmetabolites production. Hence, the importanceof the host plant as well as the ecosystem wereinfluencing endophytes metabolites production,

and affect on biological activitites ofendophytes. More attention should be given tostudying the endophytic biodiversity, thechemistry and bioactivity of endophyticmetabolites, and the relation betweenendophytes and their host plants (Tan & Zou2001, Schulz et al. 2002).

Host - Endophytes interaction

There is a complex relationshipbetween endophytes and their host plants.

Host-endophyte interactions can range frommutualism through commensalism to

parasitism, as the phenotypes of the


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interactions are often plastic, depending on thegenetic dispositions of the two partners, theirdevelopmental stage and nutritional status, butalso on environmental factors (Johnson et al.1997, Redman et al. 2001, Schulz & Boyle2005). Commensalism provides benefit to the

endophyte by enabling an undisturbed existen-ce and nutrient supply without affecting thehost. The mutual relationship benefits theendophytic fungi through provision supply ofenergy, nutrients, shelter as well as protectionfrom environmental stress. On the other handfungal endophytes indirectly benefit plantgrowth by producing special substances mainlysecondary metabolites and enzymes, which areresponsible for the adaptation of plants toabiotic stresses such as light, drought and

biotic stresses, such as herbivore, insect andnematode attack or invading pathogens (Barz etal. 1988, Kogel et al. 2006).

Under certain conditions endophytesmay become parasitic, and become pathogenscausing symptomatic infection (Brown et al.1998) and vice versa (ref) Mutation of patho-genic Colletotrichum magna resulted in theloss of a virulence factor and transformationinto an endophytic fungus (Freeman & Rodri-guez 1993). Hence, parasitism is an exceptionin plant-endophytes interactions; it can beregarded as an unbalanced status of a symbio-sis when the host is stressed and physiologicalor ecological conditions favors virulence(Mller et al. 2005, Schulz & Boyle 2005,Kogel et al. 2006). Endophytes of certain plantcould be a pathogen of other plants, dependingon the balance between pathogenicity andendophytism of the microorganism in thedifferent hosts (Saikkonen et al. 2004).

Schulz & Boyle (2005) proposed thatasymptomatic colonization of endophytes is abalanced antagonistic interaction between hostplant and endophyte (Fig 9), and as long asendophytic virulence and plant defense are

balanced the interaction remains asymptomati.Once the host-endophyte interaction becomesimbalanced either disease results in the host

plant or the plant defense machinery kills thepathogenic endophytic fungus. Whether theinteraction is balanced or imbalanced depends

on the general status of the partners, thevirulence of the fungus, and the defenses of thehost, and both virulence and defense being

variable and influenced by environmentalfactors, nutritional status and developmentalstages of the partners. Hence, commensalismand mutualism require a sophisticated balance

between the defense responses of the plant andthe nutrient demand of the endophyte (Kogel et

al. 2006).Endophytes possess structural similari-ties with pathogens and both possess many ofsame virulence factors, such as production of

phytotoxic metabolites and exoenzymes whichare necessary to infect and colonize the host, soendophytes are object to the hosts non-selfrecognition, i.e. host can respond with the samedefense reactions as to a pathogen (Fig 10).Additionally, cell wall penetration by fungi isnormally accompanied by the release of plant-

elicitor. Hence, endophytes must avoid orovercome non-specific resistance responses toachieve successful penetration by reprogram-ming the invaded cell to accommodate infec-tion structures and to maintain host cell integri-ty for a long-lasting interaction (Kogel et al.2006).

Finally, for mutualistic interactions, it isnot yet clear to what extent friendly recognitionoverbalances unfriendly recognition. The avoi-dance and modification of elicitors circumventsrecognition, or antagonistic pathways are enga-ged to switch off plant defense. Under thisview, mutualistic interactions between endo-

phytic invaders and a host plant are decipheredas a balance, under environmental, physiolo-gical and genetic control, that results in fitness

benefits for both partners, and parasitism is anunbalanced symbiosis (Stracke et al. 2002,Zipfel & Felix 2005, Kogel et al. 2006).Moricca & Ragazzi (2008) indicates that the

type of interaction between an endophyte and aplant is controlled by the genes of bothorganisms and modulated by the environment.

Biological Role of Endophytes

Endophytes play vital roles in variousaspects of life varying from its effects on host

plants to its effects to environmental andhuman life. Endophytes are capable ofsynthesizing bioactive agents that can be used

by plants for defense against pathogens and/or

stimulating plant growth, and other agents havebeen proven useful for novel drug discoveryprocess.


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Fig. 9Hypothesis: a balance of antagonisms between endophytic virulence and plant defenseresponse results in asymptomatic colonization, Adapted from: (Schulz & Boyle 2005).

Roles of Endophytes on Host-Plants

Plants are affected by endophytes invarious ways, and the potential functions ofendophytes have not been clearly defined, butin the most cases, the presences of endophyticmicroorganisms in the host plants are

beneficial to their host plants. Endophytes canactively or passively promote the plant growththrough a variety of mechanisms, asendophytic metabolites provide a variety of

fitness to host plants enhanced by increasingplant resistance to biotic and abiotic stresses, aswell as enhance plant growth (Fig 11). Manyendophytes are reported to be capable ofnitrogen (N) fixation, solubilization of phos-

phate, enhance uptake of phosphorus (P),production of siderophores, ACC deaminase,and plant hormones such as auxin, abscisins,ethylene, gibberellins, and indole acetic acid(IAA), which are important for plant growthand development regulation (Baldani et al.

1986, Goodman et al. 1986, Barraquio et al.1997, Gasoni & Gurfmkel 1997, Malinowski etal. 1999, Zou et al. 1999, Malinowski &Belesky 2000, Boddey et al. 2003, Loiret et al.2004, Sandhiya et al. 2005, Firkov et al.2007).

Role of Endophytes in Host Growth and

Nutrient Uptake

One of the most potential functions ofendophytic fungi, especially root mycorrhizal

fungi is the facilitation of plant nutrient uptakewhich in contrast leads to growth stimulation.Improved nutrition and growth may have

positive indirect effects on the other well-known functions, such as greater stresstolerance or pathogen resistance in plants(Kageyama et al. 2008).

The mechanisms of enhancement ofnutrient uptake by plants colonized byendophytes have remained elusive, but thearguments which are often used in support ofmycorrhizal nutrient uptake may be applied: asextramatrical mycelium extending from thehost roots may increase the surface area andtherefore increase host access to soil nutrients.Barrow & Osuna (2002) present anotherinteresting possibility, in root exclusionexperiment that controlled sources of P in thesubstrate, they showed that Atriplex canescensinoculated with endophytic fungus Aspergillusustus may have gained access to phosphateotherwise it will be unavailable to the host

plant.An endophytic basidiomycet, Piriform-

ospora indica may serve as a smart modelsystem to elucidate the mechanisms of nutrientuptake, host growth and fitness promotion.This Hymenomycete colonizes the roots bothinter- and intracellularly and forms coils orround bodies and branches in the cortexwithout any colonization of the host stele(Verma et al. 1998, Varma et al. 1999). Itseems that P. indica is capable of mobilizing

plant unavailable P by excreting extracellularphosphatases,as well as mediating uptake and

translocation of labeled P via an energydependent process (Singh et al. 2000). It is alsopossible that P. indica is involved in N


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Fig. 10 Symbiotic development of biotrophic endophytes and pathogens a Once Arbuscularmycorrhiza (AM) spores germinate and the germ tube approaches a root, apical dominance isabandoned and the branching of hyphae is triggered by 5-deoxy-strigol (Akiyama et al. 2005).Upon physical contact, the fungus forms an appressorium, which appears to induce the movementof the plant nucleus towards the contact site bCytoskeletal elements and the endoplasmic reticulumform the pre-penetration apparatus along the axis of nuclear movement (Genre et al. 2005) c Thestructure is entered by an infection hypha, from which dcolonization of root cortex begins. Initialinfestation is accompanied by a balanced induction of plant defense genes. e When the fungusfinally reaches the inner cortex, it penetrates the cell wall and builds up a tree-like hyphal structure,the arbuscule. Arbuscule-containing cells have specific cytoskeletal structures and accumulatereactive oxygen species (ROS). While arbuscules develop and decease, the fungus spreads furtherin the root and also colonizes the surrounding soil. There it takes up mineral nutrients, which aretransported into the root and exchanged for carbohydrates f Once a powdery mildew fungusgerminates, it forms an appressorium for host cell wall penetration gAppressoria seem to releasesignals for the formation of membrane domains (yellow) into which host susceptibility factors anddefense factors are recruited (Bhat et al. 2005). In a compatible interaction, the host nucleus

transiently migrates to the site of attempted penetration (not shown) and some action filaments (red)polarize toward this site h During penetration, host cell membrane is formed around the fungalfeeding structure (green), which is closely enveloped by actin filaments and led by a ring of actionaround the growing tip (Opalski et al. 2005) iWhen the haustorium matures, a meshwork of corticalactin is maintained around the haustorial neck, whereas actin polarisation resolvesjEventually, the

parasite establishes secondary haustoria and fulfils its lifecycle by producing a new generation ofconidia, Adapted from: (Kogel et al. 2006).

accumulation in the shoots of Nicotianatobaccum and Arabidopsis thaliana. N contentin N. tobaccum was increased by 22%,

indicatinga transfer of about 60% substrate Ninto the plants. This N content increase wascorrelated with a 50% increase in nitrate

reductase activity, a key enzyme in nitrateassimilation, inN. tobaccum and a similar 30%increase inA. thaliana (Sherameti et al. 2005).

Seed production in N. tobaccum were alsoimproved and increased with inoculation withP. indica as well as in Hordeum vulgare


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(Barazani et al. 2005, Waller et al. 2005). Thisendophyte appears to have a broad host range.It has been shown to colonize and enhancegrowth of, for example, Zea mays, Nicotianatobaccum,Bacopa monniera,Artemisia annua,

Petroselinum crispum,Populus tremula, Oryza

sativa, Sorghum vulgare, Triticum sativum,Glycine max, Cicer arientinum, Solanummelongera, and terrestrial orchids like Dacty-lorhizapurpurella, D. inacrnata, D. majalisandD. fuchsia (Singh et al. 2000, Varma et al.1999).

Role of Endophytes in Production of Phyto-

Hormones

Endophytes may enhance growth byproducing phytohormones without any appa-

rent facilitation of host nutrient uptake orstimulation of host nutrient metabolism. Theendophytic fungi may enhance biomass by

producing growth hormones or inducing thehost hormone production (Petrini 1991, Schulz& Boyle 2005). The use of fungal cultureextracts of endophytes to enhance plantgrowth, indicate that soluble agents in cultureextracts may stimulate host growth similarly tothe actively growing fungi, and this prove thatendophytic fungi produce phytohormones invitroas well as in vivo.

For example, the mycelial extract of P.fortinii induced a similar increase in Larixdecidua shoot and root biomass as did thefungus itself (Rommert et al. 2002), the growth

promotion was attributable to IAA as thefungus synthesized the hormone in vitro. Asimilar effect has also been observed with P.indica. When a fungal filtrate (1% w/v) wasadded to maize seedlings three times a week

for 4 weeks, shoot biomass increase wassimilar to that observed in inoculation experi-ments with living cultures of the fungus(Varma et al. 1999).

Role of Endophytes in Hosts Tolerance to

Stress

Endophytes may help host plants totolerate and withstand environmental stresssuch as drought, salts, and high temperatures(Malinowski & Belesky 2000). In the herbal

plantDichanthelium lanuginosum, which livesin areas where soil temperatures can reach 57C, the presence of the endophytes may increa-se plant fitness as plants with an endophytic

fungus Curvularia sp., survived high soiltemperature and water stress better thanendophyte-free plants (Redman et al., 2002).

Waller et al. (2005) reported thepotential of Piriformospora indica to induceresistance to fungal diseases and tolerance to

salt stress in barley. The beneficial effect onthe defense status was detected in distal leaves,demonstrating a systemic induction oftolerance and resistance by a root endophyticfungus. This systemically alternation wasassociated with increase of anti-oxidativecapacity due to an activation of theglutathioneascorbate cycle and results in anoverall increase in grain yield. Hence, suchsymbioses are of great importance, since theymight help plants to adapt to global climate

change (Rodriguez et al.2004).

Role of Endophytes on Photosynthetic

Capacity of Hosts:Effects of endophytes on photosyn-

thesis have been demonstrated, but they are notalways significant. For example, Colleto-trichum musae in banana decreased photo-chemical capacity compared to endophyte-free

plants (Pinto et al. 2000).

Role of Endophytes in Resistance againstPathogens and Herbivores (Biological

Control)Endophytic fungi can protect their host

plants from pathogens (Fig 11) and from pests(Arnold et al. 2003, Akello et al. 2007). Thesystemic and foliar endophytes can reduceherbivory by producing alkaloids toxic toinsects and vertebrates (Schardl 2001).Endophytic fungi are also capable of inducingresistance to diseases, and a many of

mechanisms have been proposed for thisresistance. The mechanisms of endophyte-induced resistance are related to the nutritionalstatus of the host, and to increase the fitness of

plants by enhancing their tolerance to abioticstress (Aguilar & Barea 1996, Redman et al.2002, Bae et al. 2008).

Protection from Pathogens:There are at least three primary

mechanisms by which endophytes can improve

host resistance to pathogens (Mandyam &Jumpponen 2005).


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Fig. 11 Endophytes and rhizobacteria are promoting plant growth, and acceleratesphytoremediation process though modulation of (a) plant growth promoting parameters, (b) byproviding plants with nutrients, and (c) controlling disease through the production of antifungalmetabolites, Adapted from (Ma et al. 2011).

The first mechanism is based oncompetition between endophyte and pathogenon the same resources (Lockwood 1992). This

is clear in a Fusarium oxysporum system. Anon-pathogenic endophyticF. oxysporum Fo47inhibits the pathogenic F. oxysporum f. sp.radicis-lycopersici and reduces the root rotsymptoms of tomato (Bolwerk et al. 2005).Fo47 spores compete with the pathogen for thesame C source, thereby reducing nutrientavailability to the pathogen. Both of these

Fusarium strains exhibit similar colonizationstrategies, so Fo47 can occupy and reduce thenumber of suitable sites for spore attachmentand subsequent colonization resulting in fewersymptomatic lesions.

The second possible mechanism of

pathogen control may be related to ability ofendophytes to enhance the host to produce

phytoalexins, and/or biocidal compounds, or

ability of the endophyte itself to producefumigants and other antimicrobial agents. As inthe case of Spilanthes calva when inoculatedwithPiriformospora indica, itproduces a rangeof antifungal compounds, as plants inoculatedwith P. indica produced extracts that wereinhibitory to soil-borne pathogens (F.oxysporum and Trichophyton mentagrophytes)suggesting induction of antifungal chemical

production in the host (Rai et al. 2002).The third possible mechanism is

improving host resistance to pathogens byinducing host defense responses by localizedendophytes. This mechanism is often


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encountered in mycorrhizal plants where weakresistance is induced locally or transientlyduring early mycorrhizal colonization.Structural modifications and induction ofdefense signaling can similarly result fromendophyte colonization (Koide & Schreiner

1992, Gianinazzi et al. 1996).An unidentified root-associatedendophyte known as LtVB3 restricted thespread of Verticillium longissima in Brassicacampestris by forming mechanical barriers,cell wall appositions and thickenings(Narisawa et al. 2004). As a result, external andinternal pathogen symptoms were reduced byover 80%. Narisawa et al. (1998) also observedinhibition ofPlasmodiophorabrassicae-causedclubroot in B. campestris by about 5% by

endophytes that were isolated; these endo-phytes were included Heteroconium chaeto-spira, Mortierella elongate, Westerdykella sp.as well as three unknown hyaline andmelanized species. They proposed thatsuperficial (M. elongata), cortical (hyaline andDSE fungi, Westerdykella sp.), or superficialand cortical (H. chaetospira) colonizationcreated a mechanical barrier to the pathogens.

It is likely that many tissue-penetratingendophytes may induce pathogen resistanceand in many cases, more than one of thesethree mechanisms can act simultaneously. Forexample, root colonization by Phialophora

graminicola can pre-emptively reduce thegrowth of the pathogen Gaeumannomyces

graminis by competition for space andresources. However, it can also form mecha-nical barriers resulting from thickening ofendodermis that inhibits colonization of thestele by the pathogen (Speakman & Lewis

1978, Deacon 1981). Similarly, any tissuecolonizing benign organism reduces availablecarbon to pathogens and can occupy likelycolonization sites resulting in fewer possiblesites for pathogen penetration.

Protection from Insects, Worm, Pests and

HerbivoresSome endophytes were found to have

negative effects on insects, inhibiting growth,survivorship or oviposition, especially mycorr-

hizae and systemic and foliar Clavicipetaleangrass endophytes which are widely known toreduce herbivory. Clavicipitaceous fungi

produce toxic alkaloids against insect andvertebrate herbivores, and most of endophyticfungi may similarly play a role in protection ofhosts from pests and herbivores. Mandyam &Jumpponen (2005) suggested three possiblemechanisms by which endophytes can improve

resistance of host plants to herbivores andpests.The first mechanism is based on overall

improvement of plant performance by endo-phytes, which helps plants tolerate herbivoryand sustain damage without visible effects on

productivity (Gehring & Whitham 2002).The second possible mechanism is the

alteration of plant nutritional chemistry bothqualitatively and quantitatively, by altering thecarbohydrate and nitrogen contents, C:N ratio

and phytosterol composition (Jones & Last1991, Bernays 1993, Schulz & Boyle 2005).The endophytes are capable of altering nutrientlevels and content in host plants which coupledwith alteration in carbohydrate metabolism,thus affect the host herbivore susceptibility.

The third possible mechanism of hostherbivore resistance is the production offeeding deterrents by the endophytes them-selves. Toxic alkaloids are produced by foliarendophytes of grasses (Clay 1990, Clay &Holah 1999). Non-pathogenic F. oxysporum, acommon root endophyte in L. esculentum,

produces soluble toxic metabolites that arepresent in culture filtrates (Hallman & Sikora1996). The filtrate has been shown to be toxicto Meloidogyne incognita, a root nematode.These toxic metabolites reduce nematodemobility, inactivate juveniles and are lethalwithin a 24-h exposure. The effects of theendophyte filtrates were reproducible in pot

experiments (Hallman & Sikora 1994), indica-ting that the fungus also produces themetabolites in vivo. Mandyam & Jumpponen(2005) suggest that extensive endophyte colo-nization may also prevent grazing on roots, asmany endophytes produce abundant melani-zed structures, where melanin discouragesmicrobial grazing (Bell & Wheeler 1986,Griffith 1994). Periconia macrospinosaextensively colonizes native grasses in thetallgrass prairie (Mandyam & Jumpponen

2005).Periconia spp. congeneric to those fromnative prairies is known to produce chlorinecontaining compounds that may have antibiotic


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Fig. 12 Importance of soilplantmicrobial interactions in bioremediation for the cleanup of metals andorganics (pesticides, solvents, explosives, crude oil, polyaromatic hydrocarbons), Adapted from (Ma et al.2011).

properties.

Environmental Role of Endophytes

Endophytes are found to play animportant role in the ecological community,with the aim of decreasing the extent ofenvironmental degradation, loss of biodiver-sity, and spoilage of land and water caused byexcessive toxic organic insecticide, industrialsewage, and poisonous gases. Biologicalcontrol using endophytes as a new efficientmethod is becoming widely used in

environmental remediation, and in killinginsects or pathogens (Guo et al. 2008).

A novel application of endophytes inthe area of phytoremediation (plant assistedremoval of xenobiotics and heavy metals fromsoil) has been reported in many reviews (Ma etal. 2011). However, the success of

phytoremediation depends upon microbes andplant ability to tolerate and accumulate highconcentrations of pollutant, while yielding alarge biomass. Due to their importance for

practical applications, pollutant-tolerant plant-microbe associations have been the objective of

particular attention due to the potential of

microorganisms for bioaccumulating heavy

metals and other pollutants from environmentor its enhancing plant growth and pollutantuptake from soil by plant (Fig 12) throughmobilization/ immobilization of pollutant (Maet al. 2011).

Endophytes may play indirect or directrole in phytoremediation process and degra-dation of environmental toxins, indirectlythrough enhancing plant growth having abilityof phytoremediation and this accelerate phyto-remediation process (Fig 11), or directly

through degradation and/or accumulatingpollutants by itself. Van Aken et al. (2004)reported that new endophytic Methylobac-teriumpopulum sp. nov., strain BJ001, wasinvolved in the degradation of energeticcompounds such as 2,4,6-trinitrotoluene, hexa-hydro-1,3,5-trinitro-1,3,5-triazine, and hexa-hydro-1,3,5-trimtro-1,3,5-triazine. Newman &Reynolds (2005) reported that plants inoculatedwith an engineered endophyte strain, had anincreased plant tolerance to toluene and

decrease in the transpiration of toluene to theatmosphere.

Currently bio-insecticides are becoming


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more widely used. Strobel & Daisy (2003)summarized several endophytic insecticidesnaphthalene. Endophytes have a crypticexistence and one of their main role in theecosystem are decomposers, as they are amongthe primary colonizers of dead plant tissues

(Kumaresan & Suryanarayanan 2002, Hyde &Soytong 2008, Oses et al. 2008, Purahong &Hyde 2011).

Bio-Technological Applications of

Endophytes

Endophytes have high ability toproduce various novel and known enzymeswhich could be used in various

biotechnological applications likeenvironmental applications of degradation

enzymes, medical applications, andbiotransformations of organic compounds withmany advantage over other methods (Firkovet al. 2007, Pimentel et al. 2011, Sury et al.2012).

Enzymes Production by EndophytesEndophytes usually produce the

enzymes necessary for the colonization of planttissues. It has been demonstrated that mostendophytes are able to utilize at least in vitro

most plant nutrients and cell components. Mostof investigated endophytes utilize xylan and

pectin, show lipolytic activity and producenon-specific peroxidases and laccases,chitinase and glucanase (Sieber et al. 1991;Leuchtmann et al. 1992, Moy et al. 2002, Li etal. 2004, Promputtha et al. 2011). Endophytesmay be a novel and good producers of xylanaseand the production of extracellular cellulaseand hemicellulases other than xylanases arewidespread but usually limited to organisms

derived from selected hosts or even host tissues(Leuchtmann et al. 1992; Suto et al. 2002).Thermostable amylolytic enzymes are beinginvestigated to improve industrial processes forstarch degradation. Streptosporangium sp. anendophytic actinomycete isolated from leavesof maize (Zea mays L.) showed glucoamylase

production. The isolated enzyme exhibitedthermostable properties (Stamford et al. 2002).The ability of endophytes to produce variousenzymes in vivo and in vitro means that hostsupplies nutrients as well as habitats forendophyte colonization, and could be used for

various biotechnological applications (Tomita2003).

Bio-Transformation Applications of

EndophytesBiotransformation can be defined as the

use of biological systems to produce chemicalchanges to compounds that are not in theirnatural substrates (Borges et al. 2007). Themicrobial growth, sustenance, and reproductiondepends on the availability of a suitable formof reduced carbon source, used as chemicalenergy, which under normal conditions ofculture broth are the common sugars.Microorganisms have high ability to adapt tonew environments and to metabolize variousforeign substrates to carbon and nitrogen

sources (Doble et al. 2004). A molecule can bemodified by transforming functional groups,with or without degradation of carbon skeleton.Such modifications result in the formation ofnovel and useful products not easily prepared

by chemical methods (Borges et al. 2009).Pimentel et al. (2011) reported many of

biotransformation processes by endophytes asfollowing.

Biotransformation is a useful methodfor production of novel compounds withovercoming the problems associated with otherchemical methods (Suresh et al. 2006). For thisreason, the microbial biotransformation usingtheir enzymatic systems has received increasedattention as a method for the conversion oflipids, monoterpenes, diterpenes, steroids,triterpenes, alkaloids, lignans, and somesynthetic chemicals, carrying out stereospecificand stereoselective reactions for the productionof novel bioactive molecules with some

potential for pharmaceutical and foodindustries (Borges et al. 2009, Figueiredo et al.1996).

Endophytic microorganisms are able toproduce many enzymes (Firkov et al. 2007),so they could be used as biocatalysts in thechemical transformation of natural productsand drugs, due to their ability to modifychemical structures with a high degree ofstereospecificity and to produce known ornovel enzymes that facilitates the production of

compounds of interest. The biotransformationof a tetrahydrofuran lignan, (-)-grandisin, bythe endophytic fungus Phomopsis sp. from


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Fig. 13Structure of some Bio-transformable Products

Viguiera arenaria is an example. The processled to the formation of a new compound nameddimethoxyphenyl)-5-methoxy-tetrahydrofuranas 3,4-dimethyl-2-(4-hydroxy-3,5- (Fig 13).

This compound showed trypanocidal activitysimilar to its natural corresponding precursoragainst the causative agent of Chagas disease,the parasite Trypanosoma cruzi (Verza et al.2009).

Zikmundov et al. (2002) reportedisolation of endophytic fungus from the rootsand shoots ofAphelandra tetragona, capable oftransforming benzoxazinones, 2-benzoxazo-linone (BOA) and 2-hydroxy-1,4-benzoxazin-3-one (HBOA), into a different series of

compounds. The use of endophytic fungi in thestereoselective kinetic biotransformation ofthioridazine (THD), a phenothiazineneuroleptic drug (Fig 13), was investigated.Results showed that these microorganisms areable to biomimic mammalian metabolism via

biotransformation reactions (Borges et al.2007). Another study employed endophyticfungus on the biotransformation ofpropranolol (Prop) to obtain 4-OH-Propactivemetabolite in enantiomerically pure form

(Borges et al. 2009).Another interesting biotransformation

process is the use of endophytes in thebiotransformation of terpenes for production ofnovel compounds through enzymatic reactionscarried out by these microbes. Terpenes arelarge class of bioactive secondary metabolitesused in the fragrance and flavor industries, andhave been extensively used in

biotransformation process by microorganismswith focus on the discovery of novel flavorcompounds and on the optimization of the

process condition (Bicas et al. 2009). Microbialtransformations of terpenes were published

recently using R- (+)-limonene, L-menthol, -and -pinene, and -farnesene by diversemicroorganisms (Farooq et al. 2002a & b,Miyazawa et al. 2003, Krings et al. 2006,

Marstica & Pastore 2007, Bicas et al. 2008).Other endophytic microbes were studied forthe capability to biotransform natural productslike taxoids, alkaloids, pigment curcumim,

betulinic, and betulonic acids (Zhang et al.1998, Shibuya et al. 2003, Bastos et al. 2007,Simanjuntak et al. 2010).

Pharmaceutical applications of Endophyte

Secondary Metaboli tes

Endophytes are the chemical

synthesizers inside plants (Owen & Hundley2004). Many of them are capable ofsynthesizing bioactive compounds that can beused as potential sources of pharmaceuticalleads. Endophytic fungi have been provenuseful for novel drug discovery as suggested bythe chemical diversity of their secondarymetabolites. Many endophytic fungi have beenreported to produce novel antibacterial,antifungal, antiviral, anti-inflammatory,antitumor, and other compounds belonging to

the alkaloids, steroid, flavenoid and terpenoidsderivatives and other structure types (Guo et al.2008, Yu et al. 2010). Aly et al. (2011), DeSouza et al. (2011) and Gutierrez et al. (2012)summarized in amazing reviews the up-to-dateand comprehensive information on compoundsfrom endophytes fungi from 1995 to 2011,together with the botany, phytochemistry,

pharmacology and toxicology, and discussedthe possible trends and the scope for futureresearch of endophytes.

The pharmaceutical and medicalconcerns of new drugs are the toxicity of these

prospective drugs to human tissues. Since the


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Fig. 14Structure of Some Important Phytochemical Metabolites Produced by Endophytes

plant tissue where the endophytes exist is aeukaryotic system, it would appear that thesecondary metabolites produced by theendophytes may have reduced cell toxicity;otherwise, death of the host tissue may occur.Thus, the host itself has naturally served as aselection system for microbes having bioactive

molecules with reduced toxicity toward higherorganisms (Strobel 2003).Endophytes are universally present in

all of the worlds higher plants, so it wasreasoned that plants might support certainendophytic microorganisms that could synthe-size important phytochemicals of medicinal

plants as well as the plant itself. Thus, if amicrobial source of the drug was available, itcould eliminate the need to harvest and extractthe slow-growing and relatively rare trees. The

price for the drug would also be reduced, sincethe drugs could be produced via fermentationin such the same way that penicillin isfermented (Strobel 2003).

Many reports supported this idea. Itbegan with the discovery of taxol producingendophytes, the most potent anticancer drug,from the culture of the endophytic fungi,Taxomyces andreanae isolated from Taxusbrevifoliatree, and endophytic fungusPestalo-tiopsis microspora from medicinal plantsTaxus wallichiana andbald cypress Taxodiumdistichum (Stierle et al. 1993, Strobel et al.1993, Li et al. 1996, Strobel et al. 1996). Taxolhas also been found in a number of differentfungal endophytes such as Phyllosticta

spinarum, Bartalinia robillardoides, Pestalo-

tiopsis terminaliae, Botryodiplodia theobro-

mae. Also, other common endophytic generasuch as Alternaria, Aspergillus, Botrytis,Cladosporium,Fusarium, andMucorspp. have

been reported as producers of taxol (Gangadevi& Muthumary 2008, Kumaran et al. 2008,Gangadevi & Muthumary 2009, Pandi et al.2010, Zhao et al. 2010).

Gentiana macrophylla is a traditionalChinese medicinal plant. Its dominant activeconstituents are secoiridoids, mainly gentiopi-crin (Fig14). The biological and pharmaco-logical effects of its active principles includestomachic, choleretic, antihepatotoxic, antinfla-mmatory, antifungal and antihistamine activi-

ties. Endophytic fungal strain QJ18 were foundto produce the bioactive compound gentio-picrin like its host plant G. macrophylla(Yin etal. 2009). Also, the medicinal plant Vincaminorcontains the alkaloid vincamine (Fig14),which is used in the pharmaceutical industry asa cerebral stimulant and vasodilator. An extractfrom endophytic fungus (Vm-J2) were shownto produce the same bioactive ingredient,vincamine, as the host plant (Yin & Sun 2011).Thus, endophyte production of natural metabo-

lites may help to protect the natural resourcesand to satisfy the requirement of drugs viaproduction of plant-derived pharmaceuticalleads by fermentation. Cui et al. (2012)supported this idea by isolation of ginkgolide Bfrom endophytic fungus Fusarium oxysporumisolated from Ginkgo biloba.

Anti Cancer Agents from Endophytes

Cancer is a disease characterized byunregulated cell proliferation, and leads to

spread of abnormal cells and uncontrolledtissue growth (American Cancer Society 2009).It has been considered one of the major causesof death worldwide (about 13% of all deaths)in 2004 (WHO 2009). There are someevidences that bioactive compounds produced

by endophytes could be alternative approachesfor discovery of novel anticancer drugs(Firkov et al. 2007, Guo et al. 2008, Debbabet al. 2011). Chandra (2012) reported many ofendophytic fungi as novel sources of anticancerlead molecules.

The discovery of taxol-producingendophytes opened the way for investigating


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the anticancer properties of secondarymetabolites of fungal endophytes. A selectivelycytotoxic quinone dimmer, torreyanic acid (Fig15), an important anticancer agent, was isolatedfrom the endophytic fungus, Pestalotiopsismicrospora associated with a tree Torreya

taxifolia, and was shown 5 to 10 times morepotent cytotoxicity in cell lines that aresensitive to protein kinase C agonists andcauses cell death by apoptosis (Lee et al.1996). Recently, Mirjalili et al. (2012)identified endophytic Stemphylium sedicolaSBU-16 from the inner bark of Taxus baccatawith Taxustaxadiene synthase (ts) gene, whichencodes the enzyme catalyzing the firstcommitted step of taxol biosynthesis.

The alkaloids are one of most potent

anticancer agents, usually found in endophyticfungi. Wagenaar et al. (2000) reportedidentification of three novel cytochalasinsalkaloid (Fig 15), possessing antitumoractivity, from the endophytic genus

Rhinocladiella. Other important anticanceralkaloids are camptothecin (Fig 15) and itsderivatives, a potent antineoplastic agents;camptothecin is used as a drug for treatment ofskin diseases in China (Guo et al. 2008).Camptothecin and 10-hydroxycamptothecin aretwo important precursors for the synthesis ofthe clinically useful anticancer drugs,topotecan, and irinotecan (Uma et al. 2008).An endophytic fungus isolated from the inner

bark of the plant Nothapodytes foetida, wasfound to produce the anticancer lead compoundcamptothecin when grown in a synthetic liquidmedium (Sabouraud broth) under shake flaskand bench scale fermentation conditions (Puriet al. 2005). The anticancer compounds

Camptothecin and two analogues (9-methoxycamptothecin and 10-hydroxycampto-thecin) were also obtained from the endophyticfungus Fusarium solani isolated fromCamptotheca acuminata (Kusari et al. 2009b).Several reports have described otherCamptothecin and/or analogues producingendophytes (Amna et al. 2006, Rehman et al.2008, Liu et al. 2010b, Shweta et al. 2010).

Lignans are other kinds of anticanceragents originated as secondary metabolites

through the shikimic acid pathway and displaydifferent biological activities that make theminteresting medically (Gordaliza et al. 2004).

Lignans show enormous structural andbiological diversity, especially in cancerchemotherapy (Korkina et al. 2007). Puri et al.(2006) identified a novel fungal endophyte(Trametes hirsuta) that is able to produce aryltetralin lignans podophyllotoxins (Fig 15).

The lignans produced by the microorganismare biologically active, and exhibit potentantioxidant, anticancer, and radioprotective

properties. Derivatives of podophyllotoxins arecurrently used in cancer chemotherapy againstvarious cancer diseases. Also, phenylpro-

panoids have attracted much interest formedicinal applications mainly as anticancerand antioxidant agents, and were reported to be

produced by endophytes (Korkina et al. 2007).The endophytic Penicillium brasilianum,

isolated from root bark of Melia azedarach,promoted the biosynthesis of phenylpropanoidamides (Fill et al. 2010).

The endophytic fungus Curvularialunata isolated from Niphates olemda, wasfound to produce cytoskyrins (Fig 15), whichshow antibacterial activity, and is considered asa potential anticancer agent (Brady et al. 2000,Jadulco et al. 2002). Also, an endophyticfungus Phoma medicaginis associated withmedicinal plants Medicago sativa and Medi-cago lupulina, yielded the antibiotic brefeldineA (Fig 15), which also initiated apoptosis incancer cells (Weber et al. 2004b). Ergoflavin(Fig 15) is a dimeric xanthene, belonging to theclass of ergochromes, and was described as anovel anticancer agent isolated from anendophytic fungi growing in leaves ofmedicinal plant Mimusops elengi (Deshmukhet al. 2009). Secalonic acid D also belonging tothe ergochrome class, and known to have

potent anticancer activities, was isolated fromthe mangrove endophytic fungus and showedhigh cytotoxicity on HL60 and K562 cells byinducing leukemia cell apoptosis (Zhang et al.2009).

Many of endophytes fungal metabolitesposses strong cytotoxicty against differentcancer cell lines, which could be useful for dis-covery of lead anticancer drugs. Endophyticunidentified fungus XG8D isolated from leaftissues of the mangrove plant Xylocarpus

granatum, yielded a new nor-chamigraneendoperoxide, merulin A (Fig 15), and newchamigrane endoperoxides, merulin C (Fig 15).


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Fig. 15Structure of some Anticancer Agents Isolated from Endophytic Fungi


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They showed cytotoxicity against BT474 celllines with IC50 values of 19.60 and 5.57M,respectively, and activity against SW620 celllines with IC50 values of 19.05 and 14.57M,respectively (Chokpaiboon et al. 2010). Thechemical investigation of endophytic fungus

Fusarium sp., isolated from stems of themangrove tree Kandelia candel, lead toisolation of new isoflavone,5-O-methyl-2`-methoxy-3`-methylalpinumisoflavone (Fig 15),it inhibited the growth of HEp-2 and HepG2cancer cell lines with IC50 values of 4 and11M, respectively (Huang et al. 2010). Isakaet al. (2010) isolated three new eremophilane-type sesquiterpenoids (Fig 15) from culture ofendophytic fungus Xylaria sp., obtained fromthe palm Licuala spinosa. The compounds

exhibited moderate cytotoxic activities withIC50values ranging from 3.8 to 21.0M againsthuman cancer cell lines (KB, MCF-7, and NCI-H187) and nonmalignant Vero cells.

Lu et al. (2010) investigated theendophytic fungus Penicillium expansum,isolated from roots of the mangrove plant

Excoecaria agallocha, and found it to producethe new polyphenols, expansols A & B (Fig15). Expansols A exhibited moderate cytotoxicactivity against HL-60 cell line with an IC50value of 15.7 M, while expansols B showed

pronounced activity with IC50 value 1.9 M.The endophytic fungal strainAllantophomopsislycopodina, afforded the new natural productallantopyrone A (Fig 15) and the knownislandic acid-II methyl ester (Fig 15). Bothcompounds exhibited cytotoxic activity againstHL60 cells with IC50 values of 0.32 and 6.55M, respectively, with observed internucleo-somal fragmentation when cells undergo

apoptosis, which indicate induction of apop-tosis by this compounds (Shiono et al. 2010).New depsidone-type metabolites, named paeci-loxocins A (Fig 15) was isolated from endo-

phyticPaecilomycessp., isolated from the barkof mangrove. Its showed significant cytotoxi-city against HepG2 cell line (IC50 2.69 M),and it inhibited the growth of microbial

pathogen Curvularia lunataand Candida albi-cans as well (Wen et al. 2010). Finally, othercompounds with anticancer properties isolated

from endophytic microbes were reported suchas phomoxanthones A-B, and photinides A-F(Isaka et al. 2001, Ding et al. 2009).

The screening of crude extracts ofendophytic fungi of medicinal plants, showed

promising antitumor activity against differentcancer cell lines, as 13.4% of endophyticextracts were cytotoxic on HL-60 cells and6.4% on KB cells (Huang et al. 2001). 9.2% of

other endophytic isolates exhibited antitumouractivity on human gastric tumour cell lineBGC-823 (Li et al. 2005). Another studyshowed that 3.3% of endophytic extractsdisplay potent (IC50


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Control Treated

Fig. 16 Effect of culture extract from endophytic Chaetomium sp. on mouse fibroblast cell line L-929.Note cell enlargement, loss of actin fibres and failure of cell division after nuclear division. (Nucleus stainedwith blue florescent dye and actin with red fluorescent dye). Adapted from: (Suryanarayanan et al. 2009).

(L5178Y mouse lymphoma cell line) with anEC50of 7.0 g/ml (Debbab et al. 2009).

An endophytic Alternaria sp.elaborated several solanapyrones (Fig 15) A,

D, E, F, and G, which are inhibitors of DNApolymerases (Mizushina et al. 2002).Endophytic Nigrospora oryzae producedaphidicolin and several of its derivatives,nigrosporolide, phomalactone, bostrycin andepoxyexserohilone. Aphidicolin is a tetracyclicditerpene-tetraol and an inhibitor of nuclearDNA synthesis in eukaryotes, it inhibited DNAsynthesis by interfering with DNA polymerase enzyme, also this metabolite reported toinhibit the S phase of the cell cycle (Ikegami et

al. 1979, Spadari et al. 1982). Furthermore,endophytic Fusarium sp. was shown toproduce apicidin which is a histone deacetylaseinhibitor that inhibits cell division, andenniatins which are known to function asinhibitors of the yeast transporter protein Pdr5p(Han et al. 2000, Hiraga et al. 2005). From the

previous, certain endophytic genera such asAlternaria, Chaetomium, Colletotrichum,Curvularia, Nigrosporaand Xylariaproduce alarger number of cytotoxic compounds that

could be used in discovery of new anticanceragents (Suryanarayanan et al. 2009).

Antimicrobial Agents from Endophytes

Antimicrobial metabolites (Antibiotics)can be defined as low-molecular-weightorganic compounds made by microorganismsthat are active at low concentrations againstother microorganisms, not required for itsgrowth, produced as an adaptation for specificfunctions in nature, and are the most bioactivenatural products isolated from endophytes(Demain 1981, Strobel & Daisy 2003, Guo etal. 2008). Endophytes are believed to carry out

a resistance mechanism to overcomepathogenic invasion by producing secondarymetabolites bearing antimicrobial activity. It is

believed that screening for antimicrobial

compounds from endophytes is a promisingway to overcome the increasing threat of drugresistant microbes of human and plant

pathogen (Tan & Zou 2001, Yu et al. 2010).The antimicrobial compounds can be used notonly as drugs by humankind but also as food

preservatives in the control of food spoilageand food-borne diseases, a serious concern inthe world food chain (Liu et al. 2008).

The screening of endophytic fungicrude extracts for their antimicrobial activity

indicates that they may possess the steadinessantimicrobial activity against tested pathogenssuch as Staphylococcus aureus, Bacillus

subtilis, Saccharomyces cerevisiae and Alter-naria sp., etc. Li et al. (2005) reported 30% oftested isolates exhibited antifungal activity,also antimicrobial activity was demonstratedfor 8%-92% of endophytic extracts in otherstudies (Banu & Kumar 2009, Hazalin et al.2009, Tong et al. 2011).

Cryptocin (Fig 17.a) and cryptocandin

(Fig 17.b) are antifungal metabolites obtainedfrom the endophytic fungus Cryptosporiopsiscf. quercina. Cryptocandin demonstratedexcellent antifungal activity against someimportant human fungal pathogens, includingCandida albicans and Trichophyton spp., andagainst a number of plant pathogenic fungi,including Sclerotinia sclerotiorum andBotrytiscinerea. Cryptocandin and its related com-

pounds are currently being considered for useagainst a number of fungi causing diseases ofthe skin and nails (Strobel & Daisy 2003).Cryptocin however possesses potent activityagainst plant pathogens only, especially against


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Pyricularia oryzae, the causal organism of oneof the worst plant diseases in the world, withminimum inhibitory concentration 0.39g/ml(Strobel et al. 1999b, Li et al. 2000). Theendophytic fungus Pestalotiopsis microsporawas found to produce number of antifungal

metabolites, like ambuic acid, pestaloside, andpestalotiopsins A and B (Fig 17.a). Theyshowed activity against many of pathogenicfungi, while pestaloside possess also

phytotoxic properties. An endophytic fungiPestalotiopsis jesteri andPestalotiopsis adustawere found to synthesized jesterone (Fig 17.a)and Pestalachlorides A respectively, whichexhibit antifungal activity against a variety of

plant pathogenic fungi (Li et al. 2008a).Pestalachlorides A was proven to display

significant antifungal activity against threeplant pathogenic fungi, Fusarium culmorum,Gibberella zeae, and Verticillium albo-atrum(Lee et al. 1995b, Pulici et al. 1996, Li et al.2001, Li & Strobel 2001, Li et al. 2008a).

Lu et al. (2000) isolated threemetabolites (Fig 17.a) from the culture ofendophytic fungus Colletotrichum sp., residingin the medicinal Artemisia annua. Thesecompounds were shown to have not only haveactivity against human-pathogenic fungi and

bacteria but also be fungistatic to plant-pathogenic fungi. Krohn et al. (2002) reportedfusidikactones (Fig 17.a) with antifungalactivity from endophytic Fusidium species.Preaustinoid A, B (Fig 17.a) isolated from

Penicillium sp., exhibited moderate bacterio-static effect on Escherichia coli, Staphylo-coccus aureus, Pseudomonas aeruginosa,

Bacillus sp. (Dos Santos & Rodrigues-Fo2003). Kim et al. (2004) isolated antibacterial-

periconicins A and B (Fig 17.b) fromendophytic fungus Periconiasp. isolated fromhost plantTaxuscuspidate.Among metabolites

produced by the endophytic fungusAspergillusfumigatus CY018 asperfumoid (Fig 17.a),fumigaclavine C, fumitremorgin C, physcion,and helvolic acid were shown to inhibitCandida albicans (Liu et al. 2004).

Investigation of endophytic fungusRhizoctonia sp. yielded rhizoctonic acid (Fig17.a) with anti-helicobacter pyloriactivity, the

causative bacteria of peptic ulcer (Ma et al.2004). Song et al. (2004) reported isolation ofrubrofusarin B, fonsecinone A, asperpyrone B,

and aurasperone A (Fig 17.a) from Aspergillusniger IFB-E003, an endophyte in Cyndondactylon. The four metabolites exhibitedgrowth inhibitions against the pathogenicmicrobes with minimal inhibitory concentra-tions (MICs) ranging in between 1.9 and 31.2

g/ml. Another novel antibiotic-phomol wasisolated from fermentations of an endophyticfungus Phomopsis species, another twoantimicrobial agents cytosporone B and C (Fig17.b) were isolated, from the same genusPhomopsis sp.; they inhibited two fungiCandida albicans and F. oxysporum with theMIC value ranging from 32 to 64 mg/ml.Investigation of endophytic Phomopsis cassia,ethyl 2,4-dihydroxy-5,6-dimethylbenzoate and

phomopsilactone displayed strong antifungal

activity against two phytopathogenic fungi,Cladosporium cladosporioides, and C.

sphaerospermum (Weber et al. 2004, Silva etal. 2005, Huang et al.2008b).

Chemical investigations of cornendophyte Acremonium zeae led to thediscovery of two antibiotics pyrrocidines A andB (Fig 17.b), which displayed significantantifungal activity against Aspergillus flavusand Fusarium verticillioides (Wicklow et al.2005). More than 50% of endophytic fungalstrains residing in Quercus variabilispossessedgrowth inhibition against at least one

pathogenic fungi or bacteria. Cladosporiumsp., displaying the most active antifungalactivity, was investigated and found to producea secondary metabolite known as brefeldin Awith antibiotic activity (Wang et al. 2007). Theantimicrobial agents Hypericin (Fig 17.a) andEmodin were produced by Hypericum

perforatum. Both compounds possessed

antimicrobial activity against several bacteriaand fungi, including Staphylococcus aureusssp. aureus, Klebsiella pneumoniae ssp.ozaenae,Pseudomonasaeruginosa, Salmonellaenterica ssp.Enteric, andEscherichiacoli, andfungal and candidal pathogens Aspergillusniger and C. albicans (Kusari et al. 2008).Chaetoglobosins A and C with antifungalactivities were characterized from the cultureof an endophytic Chaetomium globosumisolated from leaves of Ginkgo biloba. In agar

diffusion method, these two metabolites wereshown antimicrobial activity against Mucormiehei(Qin et al. 2009).


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The endophytic genus Xylaria wasinvestigated as producers of many antifungalagents; species produce griseofulvin (Fig 17.a)which is used for the treatment of human andveterinary animals mycotic diseases. Sordaricinand multiplolides had antifungal activity

against Candida albicans,7-amino-4-methylcoumarin showed broad-spectruminhibitory activity against several food-borneand food spoilage microorganisms. It wassuggested for use as natural preservatives infood.In vitroand in vivoantifungal activity ofendophyte-produced griseofulvin against plant

pathogenic fungi were effective for controllingeffectively the development of various foodcrops diseases (Boonphong et al. 2001, Cafuet al. 2005, Park et al. 2005, Liu et al. 2008,

Pongcharoen et al. 2008).Curvularide B (Fig 17.a) was isolated

from the endophyte Curvularia geniculataandshowed antifungal activity with increase ininhibition zone in the presence of fluconazole(example of currently used azol drug), whichindicated the synergistic effect of both drugsagainst Candida albicans. The minimuminhibitory concentrations (MIC) values that

produced no visible growth (MIC-0) forfluconazole and curvularide B were 26.1 and782.8M, respectively. While in combination,the MIC-0 values decreased to 3.2 and 48.9M,respectively. Curvularide B did not exhibitcytotoxicity towards ten human cancer celllines even at a concentration of 50g/ml, whichindicates positive results for using it to improveactivity of azol antifungal drugs (Chomcheonet al. 2010). The mangrove derived endophyticfungus Talaromyces sp. produced the antimi-crobial metabolites (7-epiaustdiol, stemphy-

perylenol and secalonic acid A). 7-epiaustdiol(Fig 17.a) displayed significant inhibitoryactivity against Pseudomonas aeruginosa, amultidrug resistant opportunistic pathogen,with MIC value of 26.48M. Stemphy-

perylenol (Fig 17.a) inhibited Sarcinaventriculi with MIC value of 8.86M, which islower than that of ampicillin (35.81M), whilesecalonic acid A (Fig 17.a) exhibited highactivities against all tested organisms.Furthermore, the three compounds showed

moderate to strong cytotoxicity against KB andKBv200 cell lines (Liu et al. 2010c).Fumigants are produced by many of

endophytes. Muscodor is a novel endophyticfungal genus that produces bioactive volatileorganic compounds (VOCs). This fungus, aswell as its VOCs, has enormous potential foruses in agriculture, industry and medicine.Endophytic Muscodor albus and the most

recent discovered Muscodor crispans producea mixture of VOCs that act synergistically tokill a wide variety of plant and human

pathogenic fungi and bacteria. It is alsoeffective against nematodes and certain insects.This mixture of gases consists primarily ofvarious alcohols, acids, esters, ketones andlipids (Fig 17.a). Artificial mixtures of theVOCs mimic the biological effects of thefungal VOCs when tested against a wide rangeof fungal and bacterial pathogens. Potential

applications for mycofumigation by thisgenus are currently used for treating various

plant diseases, buildings, soils, agriculturalproduce and human wastes. Another promisingoption includes its use to replace methyl

bromide fumigation a

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