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Journal of Microbiology and Biotechnology Research<br />

Scholars Research Library<br />

J. Microbiol. Biotech. Res., 2011, 1 (2): 21-32<br />

(http://scholarsresearchlibrary.com/archive.html)<br />

ISSN : 2231 –3168<br />

CODEN (USA) : JMBRB4<br />

A <strong>review</strong>: <strong>Natural</strong> <strong>products</strong> <strong>from</strong> <strong>plant</strong> <strong>associated</strong> <strong>endophytic</strong> <strong>fungi</strong><br />

Ruby Erach Jalgaonwala* 1 , Bhavna Vishwas Mohite 2 , Raghunath Totaram Mahajan 2<br />

1, 2 Department of Biotechnology Moolji Jaitha College Jalgaon M.S.India<br />

______________________________________________________________________________<br />

ABSTRACT<br />

This <strong>review</strong> describes information on the role of <strong>endophytic</strong> microorganisms and some of<br />

naturally occurring bioactive compounds obtained <strong>from</strong> <strong>endophytic</strong> <strong>fungi</strong> isolated <strong>from</strong> various<br />

host <strong>plant</strong>s. In the recent past years, a great deal of information on the role of endophytes in<br />

nature has been collected. The main topics addressed are isolation of endophytes, host<br />

endophyte relationship, fungal endophyte, their diversity, physiological role of endophyte,<br />

biological activities and chemistry.<br />

Key words: Endophytic microorganisms, biological activity, endophyte diversity, antioxidation<br />

activity.<br />

______________________________________________________________________________<br />

INTRODUCTION<br />

Endophytes are those microorganisms that inhabit interior of <strong>plant</strong>s especially leaves, stems,<br />

roots shows no apparent harm to host [1]. Almost all classes of vascular <strong>plant</strong>s and grasses<br />

examined to date are found to host <strong>endophytic</strong> organisms [2]. Different groups of organisms such<br />

as <strong>fungi</strong>, bacteria, actinomycetes and mycoplasma are reported as endophytes of <strong>plant</strong>s [3]. The<br />

existence of endophytes has been known for over one hundred years. In literal translation, the<br />

word endophyte is derived <strong>from</strong> Greek, ‘endo’ >< ‘endon’ meaning within, and ‘phyte’><<br />

‘phyton’ meaning <strong>plant</strong>. Research of <strong>endophytic</strong> <strong>fungi</strong> has a long history and their diversity<br />

among <strong>plant</strong>s has been found to be considerably large. Each <strong>plant</strong> has been reported to harbor<br />

one or more endophytes [4, 5] (Figure 1). Recently endophytes are viewed as outstanding source<br />

of secondary metabolites bioactive antimicrobial natural <strong>products</strong>. These microorganisms<br />

received considerable attention in last 20 years when their capacity to protect against insect and<br />

pest pathogens was noticed [1]. Botanists have carried out much research into the <strong>plant</strong><br />

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endophyte relation. The aim of this <strong>review</strong> is to provide recent data about fungal endophytes and<br />

their bioprospecting.<br />

Figure 1. Endophytes asexual life Cycle<br />

Host endophyte relationship<br />

Recent studies shows that endophytes are not host specific [6]. Single endophytes can invage a<br />

wide host range. Studies suggest that some strains of the same fungus isolated <strong>from</strong> different<br />

parts of the same host differ in their ability to utilize different substances [7]. So endophytes can<br />

be isolated <strong>from</strong> different <strong>plant</strong>s belongs to the different families and classes and grow under<br />

different ecological and geographical conditions [8]. Host endophyte relationship may be<br />

variable <strong>from</strong> host to host and endophyte. Some research showed that host <strong>plant</strong> and endophyte<br />

relationship are able to balanced pathogen host antagonism not truly symbiotic one [9] (Figure 2)<br />

Plant endophyte interactions affect metabolite production<br />

In 2008 Moricca and Ragazzi [10] showed that the type of interaction between an endophyte and<br />

a <strong>plant</strong> is controlled by the genes of both organisms and modulated by the environment. The<br />

endophyte may present in a metabolically hostile environment and continuously encountering<br />

host defense chemicals [9]. Endophytic <strong>fungi</strong> <strong>from</strong> medicinal <strong>plant</strong>s could be a rich source of<br />

functional metabolites [11-15]. Endophyte <strong>plant</strong> association could be could also be subjugated to<br />

stimulate the production of secondary metabolites by host <strong>plant</strong>. Plants growing in adverse<br />

habitats have to be screened for isolation of endophytes and their metabolites [16,17].The<br />

observation by Baily [18], indicates that endophyte infections alters pattern of gene expression<br />

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in the host <strong>plant</strong>. Endophytes <strong>from</strong> angiosperms and gymnosperms have been studied for novel<br />

secondary metabolites. Suryanarayanan, Zhang and Li emphasis on evaluation of endophytes in<br />

lower <strong>plant</strong>s namely algae, bryophytes, pteridophytes and lichens in addition with higher <strong>plant</strong>s<br />

[19-21].<br />

+ Fungal Benefit +<br />

E<br />

n<br />

d<br />

o<br />

p<br />

h<br />

y<br />

t<br />

Figure 2. Host endophyte relationship<br />

i<br />

s<br />

m<br />

- Fungal harm -<br />

Physiological role of <strong>endophytic</strong> <strong>fungi</strong><br />

Endophytic <strong>fungi</strong> are reported <strong>from</strong> <strong>plant</strong>s occurred in various environment conditions including<br />

tropic [22], temperate [23], Xerophytic [19] and aquatic [24]. Current studies have shown that<br />

tall fescue toxicosis is mediated by a number of compounds including clavinet alkaloids, lysegic<br />

acid amides and ergopeptines [25].<br />

Protection <strong>from</strong> pathogens<br />

Endophytic <strong>fungi</strong> are known to be a rich source of novel antimicrobial substances. The<br />

endophyte <strong>associated</strong> <strong>plant</strong>s produces some metabolites that induces resistance. It was found that<br />

symbiotic <strong>plant</strong> activates defense system more quickly than non symbiotic <strong>plant</strong>s after pathogen<br />

challenge.<br />

Role of endophytes in abiotic stress tolerance<br />

Researchers have shown that some <strong>endophytic</strong> <strong>fungi</strong> are able to protect their host <strong>plant</strong> <strong>from</strong><br />

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drought conditions [26]. Also salt tolerance is observed in <strong>plant</strong>s infected with endophytes [27].<br />

Endophytic <strong>fungi</strong> also increase heat tolerance in their host. So it can be said that endophytes acts<br />

as biological trigger to activate the stress response more rapidly and strongly than non symbiotic<br />

<strong>plant</strong>s [28].<br />

Fungal endophyte diversity<br />

Endophytic fungal symbionts can have profound effects on <strong>plant</strong> ecology, fitness and evolution.<br />

Diverse group of this organism are able to produce number of bioactive agents [29-31]. The<br />

fossil record indicates that <strong>plant</strong> have been <strong>associated</strong> with <strong>endophytic</strong> <strong>fungi</strong>, for >400 Myr and<br />

were likely <strong>associated</strong> when <strong>plant</strong>s colonized land, thus having an important role in driving the<br />

evolution of life on land [32]. Clavicipitaceous endophytes are Class I endophytes represents a<br />

small number of phylogenetically related Clavicipitaceous species that are fastidious in culture<br />

and limited to some cool and warm season grasses[33-34]. Transmission of class I endophytes is<br />

primarily vertical, with maternal <strong>plant</strong>s passing <strong>fungi</strong> on to offspring via seed infections [35].<br />

The benefits conferred by these <strong>fungi</strong> appear to depend on the host species, host genotype and<br />

environmental conditions [36]. Diversity of class 2 endophytes in individual host <strong>plant</strong>s is quite<br />

limited. Class 2 endophyte comprises a diversity of species, all of which are members of the<br />

Dikarya (Ascomycota or Basidiomycota). They have ability to confer habitat specific stress<br />

tolerance to host <strong>plant</strong>s [37]. Researchers proposed that Clavicipitaceous endophytes are<br />

defensive mutualists of host grasses and this hypothesis gets widely accepted on endophytes<br />

natural history, evolution, ecology and physiology and followed by number of researchers [38-<br />

45]. Class 3 endophytes are distinguished on the basis of their occurrence and horizontal<br />

transmission. This includes vascular, nonvascular <strong>plant</strong>s, woody and herbaceous angiosperms in<br />

tropical forest and antarctic communities [46-49]. Class 3 endophytes are especially known for<br />

their great diversity within individual host tissues, <strong>plant</strong>s and populations. Individual leaves may<br />

harbor up to one isolate per 2mm 2 of leaf tissue and contains number of species. Single <strong>plant</strong> may<br />

harbor hundreds of different <strong>endophytic</strong> <strong>fungi</strong>. Class 4 endophytes have darkly melanized septa<br />

and restricted to <strong>plant</strong> roots. They are generally Ascomycetous <strong>fungi</strong> which are conidial or sterile<br />

and that form melanized structures like inter and intracellular hyphae and microsclerotia in the<br />

roots. This class of endophytes found in host <strong>plant</strong>s like nonmycorrhizal <strong>from</strong> antarctic, arctic,<br />

alpine, sub-alpine, temperate zones and tropical ecosystems [50].<br />

Methods for isolation of endophytes<br />

A number of methods for isolation of endophytes are described in literatures. A convenient and<br />

common method accepted by many researchers is to dip the tissues in 70% alcohol for few<br />

seconds or in 0.5-3.5% sodium hypochlorite for 1-2 minutes followed by rinses in sterile double<br />

distilled water before plating it on a nutrient medium for isolation of <strong>endophytic</strong> microorganisms<br />

[29]. Some Isolates require months or more time in culture before they sporulate. Even stop<br />

sporulating after they have been transferred several times. For isolation of fungal endophytes<br />

surface sterilization of tissue requires 70% ethanol for 1-3 minutes, aqueous sodium<br />

hypochloride (4% available chlorine) for 3-5 minutes again rinse with 70% ethanol 2-10 seconds<br />

and final rinse with double distilled water and drying in Laminar air flow [4] also addition of<br />

50mg/l chloramphenicol can be done to suppress bacterial growth [5]. Sterile knife blade is<br />

required to remove outer tissues <strong>from</strong> sample and to excise inner tissues[30].Water agar, Potato<br />

dextrose agar, Yeast extract agar, Rose bengal chloramphenicol agar, Luria bertani agar, Humic<br />

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acid vitamin agar are found to be suitable media for isolation of <strong>endophytic</strong> <strong>fungi</strong>, bacteria and<br />

actinomycetes respectively. Temperature of about 25-30 o C is suitable for their growth.<br />

<strong>Natural</strong> <strong>products</strong> <strong>from</strong> endophytes<br />

Anticancer by endophytes<br />

Taxol is the world’s first billion dollar anticancer drug. In addition it is worthy of note that some<br />

<strong>plant</strong>s generating bioactive natural <strong>products</strong> have <strong>associated</strong> endophytes that produce the same<br />

natural <strong>products</strong> (Figure 3). Such is the case with taxol, a highly functionalized diterpenoid and<br />

famed anticancer agent that is found in each of the world’s yew tree species (Taxus sp) [51]. In<br />

1993, a novel taxol producing fungus Taxomyces andreanae, <strong>from</strong> the yew Taxus brevifolia was<br />

isolated and characterized and biodiversity of some <strong>endophytic</strong> <strong>fungi</strong> <strong>from</strong> 29 traditional<br />

Chinese medicinal <strong>plant</strong>s were studied [52,53].Naphthoquinone Spiroketal isolated <strong>from</strong><br />

<strong>endophytic</strong> fungus Edenia gomezpompae .Six new tetramic acids derivatives Penicillenols<br />

A1,A2,B1,B2,C1,C2 [54] together with citrinin, phenol A acid, phenol A and dihydrocitrinin<br />

were identified <strong>from</strong> Penicillium sp an <strong>endophytic</strong> fungus <strong>associated</strong> with Aegiceras<br />

corniculatum. All the new compounds were evaluated for their cytotoxic effects in four cell lines<br />

by the MTT bioassay Penicillenols A. Endophytic fungus Penicillium sp JP-1 isolated <strong>from</strong><br />

Aegiceras corniculatum showed four polketides, leptusphaerone [55] Penicillenone, 9-demethyl<br />

FR-901235 [14] leptosphaerone C showed cytotoxicity against A-549 cells with an Ic 50 Value<br />

of 1.45 um, while penicillenone showed cytotoxicity against P388 cells with an Ic 50 value of<br />

1.58um and arugosin I [13, 14].<br />

Figure 3.Taxol<br />

Antioxidant compounds produced by endophytes<br />

Antioxidant metabolites are often produced by <strong>endophytic</strong> <strong>fungi</strong>. Pestacin (Figure 6) and<br />

Isopestacin (Figure 5) were isolated <strong>from</strong> Pestalotiopsis microspora <strong>from</strong> <strong>plant</strong> Terminalia<br />

morobensis, native of the Papua New Guinea [56].<br />

Endophytes and their antimicrobial activity<br />

Endophytic <strong>fungi</strong> Aspergillus clavatonanicus isolated <strong>from</strong> Torreya mairei produce clavatol<br />

[40]. Endophytic fungus Phomopsis sp YM 311483 produced four new ten membered lactones.<br />

This lactones shows antifungal activity against Aspergillus niger, Botrytis cinere, and<br />

Fusarium[14] were isolated <strong>from</strong> the extract of <strong>endophytic</strong> fungus Xylaria sp PSU-D14<br />

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exhibiting two compounds Sordaricin [57] (Figure 7) and 2, 3-dihydro-5 hydroxy-2 methyl-4H-<br />

1-banzopyron-4-1,venaceum Penicillium islandicum and Ophiostoma minus. The glucoside<br />

derivatives, Xularosides A [56]. Endophytic Fusarium Sp <strong>from</strong> <strong>plant</strong> Selaginella pollescens<br />

collected in the Guanacaste conservation area of Costa Rica was screened for antifungal activity<br />

shows potent activity against Candida albicans in agar diffusion assay [30]. Jesterone antifungal<br />

compound which was isolated <strong>from</strong> fungus Pestalotiopsis jesteri. Jesterone is only compound<br />

<strong>from</strong> the endophytes in which total synthesis was reported [58].The <strong>endophytic</strong> fungus<br />

Chloridium sp produces Javanicin [4]. This highly functionalized napthaquinone exhibits strong<br />

antibacterial activity against Pseudomonas sp.Three metabolites Phomoenamide [13],<br />

Phomonitroester and Deacetylphomoxanthone B have been reported <strong>from</strong> <strong>endophytic</strong> fungus<br />

Phomopsis sp PSU D [15]. Patulin is a mycotoxin found to be produced by some of <strong>endophytic</strong><br />

<strong>fungi</strong> (Figure 4).<br />

Figure 4. Patulin<br />

Figure 5. Isopestacin<br />

Figure 6. Pestacin<br />

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Figure 7. Sordarcin derivative<br />

Endophytes and some antiviral compounds<br />

Many antiviral agents are reported <strong>from</strong> <strong>endophytic</strong> <strong>fungi</strong>. Two novel compounds cytonic acid A<br />

and B have been isolated <strong>from</strong> the <strong>endophytic</strong> fungus Cytonaema sp. These compounds are<br />

inhibitor of human cytomegalovirus (hCMV) protease [59].<br />

Other natural <strong>products</strong> by endophytes<br />

Gignardic acid was detected in the culture broth of an <strong>endophytic</strong> Gignardia sp. obtained <strong>from</strong><br />

Spondias mombin [60] (Figure 8). The oxidative deamination <strong>products</strong> of L – valine and L –<br />

phenylalanine are biogenetic precursors of this metabolite. A novel compound <strong>from</strong> a Phomopsis<br />

sp <strong>endophytic</strong> in Adenocarpus foliolosus was isolated and identified as phomosine G [61].<br />

Several endophytes are known to have antiinsect properties, nodulisporic acid a novel diterpenes<br />

that exhibits potent insecticidal properties against the larvae of the blowfly. The first<br />

nodulisporium compound was isolated <strong>from</strong> endophytes Nodulispotium sp and Bontia<br />

Daphnoides [4]. Ergot alkaloids consist of different kinds of alkaloids and include ergovaline,<br />

ergotamine, ergosine, ergostine and ergonine. Environmental conditions such as soil temperature<br />

and humidity also are expected to affect the nature and the population of endophytes<br />

Nematicidal/insecticidal metabolites <strong>from</strong> <strong>endophytic</strong> <strong>fungi</strong> Geotrichum sp AL4, isolated <strong>from</strong><br />

leaves of A. indica chlorinated, empimeric compound 1, 3 oxazinane derivatives isolated and<br />

assessed for their nematicidal activity against nematodes. Bursaphelenchus xylophilus and<br />

Panagrellus redivivus showed good bioactivity [62]. Graphislactone A, Botralin and Ulocladol<br />

are acetylcholinesterase (Ach E) inhibitors, isolated <strong>from</strong> an <strong>endophytic</strong> Microsphaeropsis<br />

olivacea, originally obtained <strong>from</strong> Pilgerodendron uviferum a gymnosperm [63]<br />

Figure 8.Gignardic acid<br />

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Figure 9.Abacterial activity against E.coli by <strong>endophytic</strong> <strong>fungi</strong> isolated <strong>from</strong> O.sanctum<br />

Figure 10. Fungal endophyte isolated <strong>from</strong> E. globules<br />

Table 1. Biological activities by endophytes isolated <strong>from</strong> medicinal <strong>plant</strong>s<br />

Plant Family Antimicrobial Antioxidation<br />

Aloe vera Liliaceae +++ +<br />

Azadirachta indica Meliaceae ++ +<br />

Curcuma longa Zingiberaceae +++ +++<br />

Coriandrum sativam Umbelliferae + +<br />

Eucalyptus globules Myrtaceae ++ +<br />

Hibiscusrose sinensis Malvaceae - +<br />

Ixora coccinea Rubiaceae - +<br />

Murrayo koenginii Rutaceae ++ -<br />

Musa paradiasica Musaceae + +<br />

Osimum sanctum Labiataei +++ +<br />

Pongamia glabra Leguminosae +++ +<br />

Sphaeranthus indicus Compositae - +++<br />

Vinca rosea Apocynaceae - +++<br />

Vitex nigundo Vedenaceae + +<br />

Withania somniphera Solanaceae - +<br />

+++, Potent activity, ++, Moderate activity, +, Less activity,-, No activity<br />

We have isolated some fungal endophytes <strong>from</strong> selected fifteen medicinal <strong>plant</strong>s. These are<br />

found to be diverse groups and having potent biological activities (Table 1.) .It was found that<br />

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endophytes are tissue specific .Large number of isolates were found in aerial part than the under<br />

ground parts of selected medicinal <strong>plant</strong>s. It is evident <strong>from</strong> Table 1. That microbial endophytes<br />

<strong>from</strong> medicinal <strong>plant</strong>s namely Aloe vera ,Curcuma longa, Eucalyptus globulus (Figure 10.),<br />

Osimum sanctum ,Pongamia glabra, Vinca rosea , Sphaeranthus indicus have great ability to<br />

synthesize natural <strong>products</strong> as they exhibit excellent antimicrobial (Figure 9.) and antioxidant<br />

activity. Plants such as Azadirachta indica, Coriandrum sativam, Murrayo koenginii, Musa<br />

paradiasica exhibits less potent phytochemicals. However other <strong>plant</strong> endophytes are almost<br />

devoid of such activities.<br />

CONCLUSION<br />

An extensive characterization of different endophytes <strong>plant</strong> associations may also provides<br />

greater insight into the evolution of mutualisms. Scientists are looking for importance and<br />

distribution of functional classes 1, 2, 3 and 4 across the environmental gradients. They<br />

themselves have question in their mind what genomic differences are among functional classes?<br />

Can we predict the outcome of <strong>plant</strong> fungal interactions? Is there any provision to provide new<br />

way of classification? Is it possible to prepare phylogenetic tree and dendiogram?<br />

Endophytic microorganisms are excellent sources of bioactive natural <strong>products</strong> that can be use<br />

to satisfy demand of pharmaceutical, medical agriculture and industries. But question remains as<br />

to how endophytes resides in a <strong>plant</strong> forms a relationship with it? Also how are secondary<br />

metabolite product synthesized? What genetic control exists to control product formation and<br />

synthesis of secondary metabolites? Much more work is essential to understand endophytes<br />

physiology, biochemical pathways, defensive role, secondary metabolite production, motivation<br />

and encouragement of researcher <strong>from</strong> life sciences to contribute research related to endophytes.<br />

Acknowledgment<br />

Its our pleasure to express thanks to Hon , ble Principal, A.G. Rao, Moolji Jaitha College, Jalgaon<br />

for his support, facilitating the window of knowledge, their rich library source during the course<br />

of study on endophytes.<br />

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