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CHAPTER-1GENERAL INTRODUCTION

1.1 Natural Products and their role in drug discovery: A historical overview

Nature has been a source of medicinal products for millennia, with a number of

useful drugs discovered from plant sources. Following discovery of the penicillin, drug

discovery from microbial sources increased and diving techniques in the 1970s opened

the seas (Cragg and Newman, 2013). Historically, natural products have been used since

ancient times and in folklore for the treatment of many diseases and illnesses (Dias et al.,

2012). Natural products or secondary metabolites of plant and microbial origin from

diversified ecosystem have been the most successful source of potential drug leads.

(Haefner, 2003; Butler, 2004; Cragg and Newman, 2005; Berdy, 2005; Mishra and

Tiwari, 2011).

Some of the ancient examples which report the use of natural product in health

care of mankind date backs to as early as 800 A. D. The benedictine monks were using

many natural medicines, including the Poppy (Papaver somniferum), which was used to

alleviate pain and anaesthetic. Cragg and Newman, (2005) and Dias et al., (2012) has

mentioned the usage of natural product in traditional health care systems. The use of clay

tablets in cuneiform oils from Cupressus sempervirens (Cypress) and Commiphora

species (myrrh) which are still used today to treat cough, cold and inflammation during

2600 B.C. According to Ebers Papyrus, (2900 B.C.) an Egyptian pharmaceutical record

documents over 700 plant-based drugs ranging from gargles, pills, infusions, to

ointments. The Chinese Materia Medica (1100 B.C.) (Wu Shi Er Bing Fang, contains 52

prescriptions), Shennong Herbal (~100 B.C., 365 drugs) and the Tang Herbal (659 A.D.,

850 drugs) also recorded the uses of natural products as drugs.

The Greek physician, Dioscorides (100 A. D.), recorded the collection, storage

and the uses of medicinal herbs, whilst the Greek philosopher and natural scientist,

Theophrastus (~300 B.C.) dealt with medicinal herbs. During the dark and middle ages

the monasteries in England, Ireland, France and Germany preserved this Western

knowledge whilst the Arabs preserved the Greco-Roman knowledge and expanded the

uses of their own resources, together with Chinese and Indian herbs unfamiliar to the

Greco-Roman world. It was the Arabs who were the first to privately own pharmacies

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(8thcentury) with Avicenna, a Persian pharmacist, physician, philosopher and poet,

contributed much to the sciences of pharmacy and medicine through his workCanon

Medicinae (Cragg and Newman, 2013).

Inspite of above long historical track record and from the competition from other

drug discovery methods such as synthetic and combinatorial chemistry, natural products

are still prolific source of new clinical candidates and drugs. Natural products have been

the single most productive source of leads for the development of drugs. Over a 100 new

products in clinical development (Table-1.1), particularly as anti-cancer agents and anti-

infectives, application of molecular biological techniques are increasing the availability

of novel compounds that can be conveniently produced from bacteria andfungi (yeasts).

Combinatorial chemistry approaches are being based on natural product scaffolds to

create screening libraries that closely resemble drug-like compounds. Majority of the

natural product based drugs of plant and microbial origin, which are in the different

stages of clinical development in various projects being studied for therapeutic uses in

many ailments (Table 1.2) (Harvey, 2008).

Table 1.1 Drug based on natural products at different stages of development

Table 1.2 Therapeutic categories of natural product-derived drugs at differentstages of development.

(Source: Harvey, 2008)

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A recent review by Newman and Cragg, (2012) analyzed the number of natural

product derived drugs launchedbetween 1981 and 2010. According to the review, the

utility of natural products as drugs is still alive and well. About 50% of the approved

drugs during the last 30 years are either directly or indirectly derived from natural

products (Fig. 1) and in the area of cancer, over the time frame from around the 1940s to

date, of the 175 small molecules 85 being either natural product or directly derived there

from.

Figure 1: 50% new natural product based drugs approved from 1981-2010

(Source: Newman and Cragg, 2012)

According to the reviews by Newman and Cragg, (2007) and Cragg et al., (2012),

more than half of currently available drugs are natural compounds or are related to

themand only 36% of the 1073 small-molecule approved as drugs for all diseasesare

considered as truly synthetic in origin (S). Approximately 68% of anti-infectives

(antibacterial, antifungal, antiparasitic, and antiviral compounds) are classified as

naturally derived or inspired, whereas 79.8% of compounds in cancer treatment fall in

this category (Fig. 2).

A comprehensive survey conducted by Newman and Cragg (2012), revealed that

among 1130 new chemical entities, 118 were approved as antibacterial drugs. Of which

77 were natural products and their derivatives. Twenty nine chemical entities were

antifungal agents, of which 3 were natural product derivatives. Natural product derived

drugs are well represented in the top 35 worldwide selling ethical drug sales of this

decade.

N (unmodified NP),

NB (NP‘Botanical’ (in general, these have been recently approved),

ND (a modified NP),

S (totally synthetic drug, often found by random screening/modification of an existing

agent),

S* (made by total synthesis, but the pharmacophore is/was from a NP),

S/NM (a synthetic compound with a NP pharmacophore showing competitive inhibition of

the NP substrate),

S*/NM (a synthetic compound with a NP pharmacophore showing competitive inhibition

of the NP substrate)

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Figure 2: Small molecule new chemical entities 1981 to 2010.(Source: Cragg et al., 2012)

Also a total of 15 were launched which included new drug types such an

antimalarial, anti-Alzheimer's drug galantamine (galanthamine) and antibacterial

lipopeptide daptomycin (Newman et al., 2003).

From the review of literature it is clear that, natural products continue to provide

unique structural diversity in comparison to standard combinatorial chemistry, which

presents opportunities for discovering mainly novel low molecular weight lead

compounds. Since less than 10% of the world’s biodiversity has been evaluated for

potential biological activity, many more useful natural lead compounds from untapped

sources await discovery with the challenge being how to access this natural chemical

diversity (Cragg and Newman, 2005). About 80% of the world population primarily in

developing countries depends on traditional system of medicine for their primary health

care needs (Akerele, 1993). Their usage as traditional health remedies has been reported

to have minimal side-effects and is popular among 80% of the population in Asia, Latin

America and Africa (Bibitha et al., 2002; Maghrani et al., 2005).

1.2. Sources of natural products

Medicinal plants provide a good source for isolation of endophytic fungi and as a

alternate source for screening bioactive metabolites produced by the host. Such as in the

case of production of taxol, camptothecin and podophyllotoxin from endophytic fungi

associated with the host plants which is previously used as source of above mentioned

natural product (Stierle et al., 1993; Kusari et al., 2008; Kaur et al., 2008; Kusari et al.,

2009). In this way, the need to sacrifice plants that in some cases are rare or endangered

can be avoided (Tejesvi and Pirttila, 2011). Rapid diminishment of rare and endemic

N (unmodified NP),

NB (NP‘Botanical’ (in general, these have been recently approved),

ND (a modified NP),

S (totally synthetic drug, often found by random screening/modification of an existing

agent),

S* (made by total synthesis, but the pharmacophore is/was from a NP),

S/NM (a synthetic compound with a NP pharmacophore showing competitive

inhibition of the NP substrate),

S*/NM (a synthetic compound with a NP pharmacophore showing competitive

inhibition of the NP substrate)

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plant biodiversity can be avoided by studying their associated endophytic microbes,

which hold the greatest possible resource for acquiring novel microorganisms and

bioactive natural products (Strobel and Daisy, 2003).

1.2.1. Medicinal Plants

Plant kingdom is a rich source of structural biodiversity offering a variety of

natural products. Plants have been utilized to produce various types of medicines for

thousands of years. Plant based medicines were initially used in the form of crude drugs

such as tinctures, teas, poultices, powders and other herbal formulations (Balick and Cox

1997; Samuelsson, 2004). More than 50,000 medicinal plants out of the total of 4,22,000

flowering plants reported worldwide have been used for various medicinal purposes

(Govaerts, 2001; Schippmann et al. 2002). The information on the plants usable for these

purposes and the methods of applying them for a particular ailment were passed down

orally through successive generations.

More recently, the use of plants as medicines has focused on the isolation of

active compounds, for example the isolation of morphine from Papaver somniferum in

the early nineteenth century (Kinghorn, 2001; Samuelsson, 2004). About 80% of the

world’s population is dependent on health-care provided by medicinal plants according to

the World Health Organization (WHO 1991). A wide range of medicinal plant parts are

used as extracts that can be considered raw drugs that possess specific medicinal

properties. The different plant products used to cure various infectious diseases include

leaves, root, stem, flower, fruit, root, twigs, exudates and modified plant organs. Whereas

some of these raw drugs are collected in small quantities for local use by the native

communities and folk healers, many other raw drugs are collected in large quantities and

traded in the market as raw material for herbal industries (Uniyal et al., 2006).

1.2.2. Significance of medicinal plant diversity in India

India is one among the 12 mega diversity countries of the world and has 17,000

flowering plants of the designated 25 hotspots in the world- the Eastern Himalaya and the

Western Ghats (Alagesaboopathi, 2011 and Johsy et al., 2013). India is proud to be rich

in biodiversity possess about 8% of the estimated biodiversity of the world with

around12,600 species (Bosco and Arumugam, 2012). Among 34 hotspots identified, two

are in India such as Indo Burma (earlier Eastern Himalayas) and Western Ghats including

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Sri Lanka. The Western Ghats is considered one of the 34 centers in the world where

mega diversity exists. The Western Ghats also known as Sahyadri hills, a mountain chain

running from the north to the south and isolated by the Arabian seain the west, the arid

Deccan Plateau in the east and the Vindya-Satpura ranges in the north. Of the 15,000

flowering plant species in India, there are an estimated 4,780 species in the Western

Ghats region, been the source of invaluable medicinal plants since man became aware of

the preventive and curative properties of plants and started using them for human health

care(Myers et al., 2000; Amuthavalluvan, 2011; Shanmugam et al., 2012).

Out of the estimated 4,250 species of vascular plants 1,550 endemic plants were

found in the Western Ghats, therefore it represents second largest endemic centre of the

world (Nayar, 1996).The Southern Western Ghats consisting southern Karnataka, Kerala

and part of Tamil nadu are considered as the most species rich region with respect to

endemism. The Western Ghats is an abode of thousands of untapped variety of potential

medicinal plants with excellent curative properties which have been used in different

traditional health care systems. The huge diversity of Western Ghats flora means that we

can expect well diverse chemical structures from their secondary metabolites. As of now

only 10 percent of the world’s biodiversity has been tested for biological activity and

there is a great potential for leads from natural resources (Harvey, 2009). Therefore it

necessitates the detailed studies on the natural products produced by various natural

sources associated with medicinal or endemic plants harbored in rich biodiversity

certainly pave way to the discovery and development of new drug leads according

rationale of plant selection (Strobel and Daisy, 2003).

1.2.3. Historical overview on plant derived natural products

At the dawn of 21st century, 11% of the 252 drugs considered as basic and

essential by the WHO were exclusively of flowering plant origin. The first commercial

pure natural product introduced for therapeutic use is morphine marketed by Merck in

1826 (Veeresham, 2012). Investigation of Papaver somniferum Linn. (Opium poppy),

resulted in the isolation of several alkaloids including morphine (Cragg and Newmann,

2005), a commercially important drug, first reported in 1803. It was in the 1870s that

crude morphine derived from the plant P. somniferum. The well known example to date

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plant derived semi synthetic natural product salicin, isolated from the bark of the willow

tree Salixalba Linn.,was introduced by Bayer in 1899.

Digitalis purpurea L. (foxglove) contain the active constituent digitoxin, a

cardiotonic glycoside was found to enhance cardiac conduction. The anti-malarial drug

quinine isolated from the bark of Cinchona succirubra Pav. Ex Klotsch.had been used for

centuries for the treatment of malaria, fever, indigestion, mouth and throat diseases and

cancer. Pilocarpine found in Pilocarpus jaborandi (Rutaceae) is an L-histidine-derived

alkaloid, which has been used as a clinical drug in the treatment of chronic open-angle

glaucoma and acute angle-closure glaucoma (Marderosian and Beutler, 2002). Paclitaxel

(Taxol®) from Taxus brevifolia and baccatin from Taxusbaccata for lung, ovaria and

breast cancer. Artemisin from traditional Chinese plant Artemisia annua to combat

malaria, silymarin extracted from the seeds of Silybum marianum for the treatment of

liver diseases (Veeresham, 2012).

1. 3. Microbial diversity and their role in drug discovery

Sir Alexander Fleming’s serendipitous discovery of penicillin by filamentous

fungi Penicillium notatum, in 1928 and its subsequent development into a medicine by

Florey and Chain in the 1940s provided the foundation for development of microbial

natural products as a cornerstone of new drug discovery in the 20th century. At the end of

the “Golden Age of Antibiotics” from the 1940s to the 1970s many microbial natural

products had found their way into the clinic as antibacterial, antifungal, antiparasitic,

anticancer and immunosuppressive agents (Challis, 2008). Followed by this discovery,

searching of a huge number of antibiotics from microbes, in particular from members of

the actinomycetes and fungi has enhanced. Many antibiotics discovered until the early

1970s reached the market and their chemical scaffolds were later used as leads to

generate new generations of clinically useful antibiotics by chemical modification. Since

then microbial natural products are the origin of most of the antibiotics currently in the

market (Table 1.3) (Pelaez, 2006).

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Table 1.3 Marketed antibiotics originated from microbial natural products.

(Source: Pelaez, 2006)

Many microbial natural products that have reached the market without any

chemical modifications (Table 1.3) are a testimony to the remarkable ability of

microorganisms to produce drug-like small molecules (Liu et al., 2012). Ganesan, (2008)

analyzed drug-like properties of 24 unique natural products discovered during the period

1970-2006, which were approved as drugs (Table-1.4). Structurally, these 24 leads are

predominantly of polyketide, peptide or terpenoid origin. Microbial secondary

metabolites or natural products have exerted a major impact on the control of infectious

diseases and other medical conditions and the development of pharmaceutical industry.

The most important use of secondary metabolites are as anti-infective drugs. The market

for such anti-infectives was US$55 billion (Table 1.5) and in 2007 it was US$66 billion.

(Barber, 2001; Demain and Sanchez, 2009).

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Table 1.4 Natural products discovered and approved as drugs during 1981-

2006.

(Source: Ganesan, 2008)

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Table 1.5 Anti-infectives in Market 2000

(Source: Barber, 2001)

1.3.1 Diversity and distribution microbial natural products

Antibiotics and similar microbial natural products, being secondary metabolites

can be produced by almost all types of living organisms. They are produced by both

prokaryotic (Prokaryotae, Monera) and eukaryotic organisms. The secondary metabolite

producing ability is very uneven in the species of living world. According to Berdy,

(2005) three major microbial groups such as bacteria, actinomycetes and fungi are

involved in the production of antibiotics among the microorganisms (Table 1.6).

The dramatic technical improvements in screening programs, separation and

isolation techniques contributed to the discovery of over one million natural compounds,

among them 5% are from microbes. Approximately 20–25% of these reported natural

products exhibit biological activity; of these roughly 10% have been obtained from

microbes (Demain and Sanchez, 2009). Excellent survey on microbial metabolites

conducted by Berdy (2005) reveals that among the 22,500 biologically active compounds

those have been obtained so far from microbes, 45% are produced by actinomycetes,

38% and 17% produced by fungi and bacteria respectively (Table.1.6).

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Table 1.6 Bioactive microbial metabolites according to their producers and

bioactivities.

(Source: Berdy, 2005)

A comprehensive survey of microbial natural product as a sources of antibiotics,

from 1950-2001, discovered in United States and Japan reveals that approximately 85%

are produced by actinomycetes, 11% by fungi, and 4.5% by bacteria (Fig. 3). Berdy,

(2005) provided the statistical overview of bioactive metabolites produced by different

groups of microorganisms. Actinomycetes, filamentous fungi and several bacterial

species are the most noteworthy producers in respect of numbers, versatility and diversity

of structures of the produced metabolites and bioactivity among three main groups of

microbial natural product producers (Table 1.7).

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Figure 3: Distribution of the discovered antibiotics according to their origin andperiod (Source: Berdy, 2005)

Table 1.7 Bioactive microbial natural products, according to their producers (up to2002)

(Source: Berdy, 2005)

Secondary metabolites of fungal and bacterial origin such as penicillin,

griseofulvin and gramicidin were in the foreground of the interest, but after the discovery

of streptomycin and later chloramphenicol, tetracyclines and macrolides, the attention

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turned to the Streptomyces species. 70% of antibiotics were discovered from

Streptomyces during 1950-1960, in the next two decades the significance of the non-

Streptomyces actinomycetales species (rare actinos) were increased, up to a 25-30% share

of all antibiotics (Fig.3a). From the early nineties the number of bioactive compounds

isolated from various microscopic forms, filamentous and higher fungal species had

continuously increased up to 50% by the turn of the millennium 2000 (Berdy, 2005).

1.4. Role of fungi in drug discovery

Higher fungi have a long history of use in folk medicine, especially in the Asian

countries and their study has become a matter of great significance in recent decades

(Lindequist, 2010). Since ancient times to treat hepatitis, hypertension,

hypercholesterolemia and gastric cancer the medicinal higher fungus Ganoderma

lucidum and other medicinal mushrooms has been used (Tang and Zhong 2004; Jiang et

al., 2011). Their secondary metabolites are exploited for the development of potential

new lead drugs, product for crop protection (Anke and Thines, 2007).

Since from the discovery of penicillin G from Penicillium notatum by Alexander

Fleming, micro fungal metabolites have had an extraordinary impact on the quality of

human life during the 20th century.Antibiotics such as antibacterial and antifungals

(Penicillins, Cephalosporins, Fusidic acid, Echinocandin and Griseofulvin),

immunosuppressants (Cyclosporine), cholesterol-lowering agents (Mevastatin and

Lovastatin). Echinocandins and Strobilurin derived from fungal compounds have been

used in the clinic during the past 50 years, contributing significantly to the welfare of

mankind and to the spectacular rise in life expectancy observed in the second half of the

century (Aly et al., 2011a). The amazing range of chemical structures observed for fungal

metabolites is derived from a relatively small number of basic metabolic pathways

(mainly polyketides, nonribosomal peptides and terpenoids, plus combinations of these),

which have become extremely diversified during the course of evolution (Pelaez, 2004;

Aly et al., 2011a).

Literature survey with respect to drug discovery during the past few decades

revealed that isolation and characterization of fungal bioactive metabolites with wide

range of biological activities is of prime importance. Besides the fungal-derived

compounds mentioned in Fig.4, other fungal metabolites those are present on the

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pharmaceutical market, such as semi-synthetic or synthetic penicillins and

cephalosporins. Alkaloid ergotamine (Ergo-Kranit®), the antibiotic polyketide

griseofulvin (Likuden M®), the immunosuppressive mixed-biosynthesized compound

mycophenolate mofetil (CellCept®, derivative of mycophenolic acid) used for preventing

renal transplant rejection as well as the antibacterial terpenoid fusidic acid (Fucidine®)

(Sam and Joy, 2010). As the investigations of soil fungi started to show a reduced hit-rate

of novel compounds, attention was drawn to other, alternative sources including marine

microorganisms (Paz et al., 2010; Blunt et al., 2011; Rateb and Ebel 2011) and

endophytic fungi associated with medicinal plants (Zhang et al., 2006; Aly et al., 2010;

Xu et al., 2010; Kharwar et al., 2011).

Figure 4: Biologically active secondary metabolites of fungal origin

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1.5. Need for new sources of natural products

The quest for the discovery of novel natural products that are effective, possess

low toxicity, have a minor environmental impact and with new mode of action against

rapidly developing resistance in infectious microbes such as Staphylococcus,

Mycobacterium, Streptococcus and Pseudomonas to existing drugs and the presence of

naturally resistant organisms causing threat to mankind (Levin and Bonten, 2004;

Mwangi et al., 2007; Hugonnet et al., 2009; Richter et al., 2009; Liu et al., 2012; Wright,

2012).

In addition emerging diseases such as AIDS, SARS, ebola, Legionella, Borrelia,

Cryptosporidium, Bordetella pertussis, Streptococcus pneumoniae, Haemophilus

influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Chlamydia

trachomatis necessitate the discovery and development of new drugs (Ryan and Ray,

2004; Kumarasamy et al., 2010).

The weakened immune system due to AIDS not only requires specific drugs for

treatment but also needs new therapies to combat the secondary problems arisen from it,

and furthermore HIV virus is constantly developing resistance towards the existing drugs

(Richman et al., 2004). Resistance against antifungal drugs of opportunistic pathogens

such as Aspergillus, Cryptococcus and Candida are also virulent in immunocompromised

patients and in patients, who need an organ transplant (Alexander and Perfect, 1997;

Georgopapadakou, 2001; Hoang, 2001; Sing, 2005; Enoch et al., 2006; Ikeda, 2007).

Major problems in many countries is parasitic protozoan and nematodal infections

such as malaria, leishmaniasis, trypanomiasis and filariasis are causing and effective

drugs against them are needed. Malaria is claiming more lives each year than diseases

caused by any other infectious agent, with the exception of AIDS virus and

Mycobacterium tuberculosis (NIH, 2001) enteric infections claim more lives of children

each year than any other disease (Strobel et al., 2004).

Research in antibiotics and natural products has declined significantly during the

last decade as a consequence of diverse factors, among which the lack of interest of

industry in the field and the strong competition from collections of synthetic compounds

as source of drug leads. As a consequence, there is an alarming scarcity of new antibiotic

classes in the pipelines of the pharmaceutical industry. This decline of natural products

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and antibiotic research may be due an expensive, time consuming, cumbersome and

bureaucratic process involving multiple interest groups such as pharmaceutical

manufacturers, governmental regulatory authorities, patent officers, academic, clinical

researchers and trial lawyers along with perception of solved medical need, poor return of

investment.Lack of success stories in developing novel chemical leads, introduction of

new HTS and combinatorial chemistry and technical problem during chemical and

derivatization during lead optimization process (Projan, 2003; Shales et al., 2004; Tulp

and Bohlin, 2004; Koehn and Carter, 2005).

1.6. Strategies for discovering drugs from previously unexplored natural products

High-throughput screening and combinatorial chemistry based drug discovery

efforts which are designed and developed to solve or cope against the major clinical

problems have not led to the expected drug productivity, raising renewed interest in

searching drugs from nature i. e., natural selection found to be superior over the two

methods (Schulz et al., 2002; Li and Vederas, 2009). Recent progress in several aspects

of natural-product research and microbial genomics, suggests that the potential of natural-

product diversity and discovery is vastly underestimated, offering several promising

alternatives to existing methods for the discovery of new natural products to combat

against major global clinical problems (Lanen and Shen, 2006), are as follows:

a) Cloning and characterization of natural-product biosynthetic machinery in the

past two decades has unveiled unprecedented molecular insights into natural-product

biosynthesis.

b) Whole-genome sequencing has revealed that there are far more biosynthetic

gene clusters than currently known metabolites for a given organism.

c) Only 1% of the microbial community is estimated to have been cultivated in

the lab, implying that there is a vast diversity of natural products in microorganisms that

remains to be exploited.

d) Biochemical studies of natural-product biosynthetic enzymes have been

extremely successful in the discovery of new enzyme pathways and unusual chemical

conversions.

These findings have fundamentally changed the landscape of natural-product

research and discovery by enabling the prediction of yet-to-be isolated novel products on

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the basis of gene sequences and biosynthetic potential for natural products in untapped or

non-culturable microorganisms has been greatly under explored than by traditional

methods of natural-product discovery by accessing microbial diversity (Lanen and Shen,

2006).

Natural products are the most consistently successful source of drug leads.

Despite this, their use in drug discovery has fallen out of favor. Natural products continue

to provide greater structural diversity than standard combinatorial chemistry and they

offer major opportunities for finding novel low molecular weight lead structures that are

active against a wide range of assay targets. As less than 10% of the world’s biodiversity

has been tested for biological activity, many more useful natural lead compounds are

awaiting discovery (Harvey, 2000). Renewing natural products research requires

inexhaustible natural resources, as well as new genetic techniques and microbial sources

which can refocus the research on declining trends in microbial metabolite and natural

products (Berdy, 2012).

1.7. Accessing microbial diversity from diverse ecosystem for novel natural products

Natural products remain a consistent source of drug leads with more than 40% of

new chemical entities reported since 1981 being derived from microbial natural products.

Perhaps more astonishing is that more than 60% of the anticancer and 70% of the anti-

infective antibiotics currently in clinical use are natural products or natural product-based

(Newman et al., 2003; Baltz, 2005; Koehn and Carter, 2005; Lanen and Shen, 2006;

Newman and Cragg, 2012). By the impressive track records and successful historical

stories of microbes in drug discovery, microbes not only played pivotal role but also still

remains the sources of novel drug leads (Larsen et al., 2005; Lanen and Shen, 2006).

1.8. Revitalizing microbial drug discovery

A major potential of natural products is the fact that many natural product

resources are largely unexplored and many environmental samples for isolation of

interesting microorganisms have often been collected without a defined strategy (Bull et

al., 2000; Bull et al., 2005).Diverse habitats like tropical forests soils, the deep sea, sites

of extreme temperature, salinity or pHand these habitats often harbor novel

microorganisms and therefore provide the potential for novel metabolic pathways and

compounds (Knight et al., 2003).

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A number of these species have recently been investigated and found to produce

several bioactive cyclic peptides (Dalsgaard et al., 2004). These findings support the

hypothesis that fungi from colder climates may be just as chemically prolific as those

from tropical climates, the latter which are much more often cited as targets for

biodiversity sought in screening programs (Larsen et al., 2005).

Microbial natural products occupy tremendous chemical structural space

unmatched by any other small molecule families. They possess wide range of biological

activities thus remaining the best sources of drugs and drug leads and serving as

outstanding small molecule probes for dissecting fundamental biological processes

(Beutler, 2012). According to Liu et al., (2012), research on microbial natural products

attracts the interest of researchers in natural product chemistry across the globe based on

four general aspects;

a) The biodiversity of microorganisms especially isolated from unexplored or extreme

environments.

b) Structural diversity of secondary metabolites.

c) Broad spectrum of active compounds and

d) Genetic engineering aimed at producing specific secondary metabolites and increasing

the yields of interest.

The resources of novel compounds are undiscovered microbial species inhabiting

unique environments with differing environmental constraints (Bull et al., 1992; Jensen

and Fenical, 1996). The untapped sources from marine and other extremophilic

environment (such as hyper-arid, high temperature, etc.) could provide many novel

chemicals for use in drug discovery assays (Freundlich et al., 2010; Rateb et al., 2011).

The prospect of deriving drugs from untapped species and the effective drug

discovery strategies may be gained from the analysis of approved drugs derived from

previously untapped species, particularly those approved in recent decades. In this

context, Zhu et al., (2012) analyzed the species origins of nature-derived drugs approved

in 1991–2010 with respect to those approved in previous decades (1961–1990) (Table

1.8) to find the exploration trends indicative of future bioprospectingof likely sources of

untapped new drug productive species such as plant and microbes.

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Table 1.8 Historical data of nature-derived approved drugs from previouslyuntapped and previous drug-productive species and drug-productive species during

every five-year period from 1961 to 2010.

(Source: Zhu et al., 2012)

Accessing microbial diversity obtained from diverse habitats and untapped

resources from nature offer microbial metabolites, which represent an unimaginable vast

array of diversified chemical entities, which not only mediate interaction between

microbes but also possess wide range of biological activities. For example exploration of

unculturable microbial species which has been limited by the cost and efficiency of

cultivation technologies (Piel, 2001; Rappe and Giovannoni, 2003). New technologies

that explore cryptic gene-clusters, (Chaing et al., 2011), pathways (Wilkinson and

Micklefield, 2007), inter-species crosstalk (Schroeckh et al., 2009) and high-throughput

fermentation (Baltz, 2008) enable the generation of significantly more diverse groups of

novel microbial natural products which has been anticipated to have some impact on drug

productivity from nature. Recent bioprospecting efforts are based on exploration

microbial species diversity from various untapped sources (Zhu et al., 2012).

The biodiversity of microbes is based on their inhabiting environment. In this

regard the sources for novel secondary metabolites depend on microbial species

inhabiting unique environments conditions and exceptional biotopes (Jensen and Fenical

1996; Bull et al., 1992; Liu et al., 2012; Kaul et al., 2012).

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One such specialized and unique biological niche that supports the growth of

microbes is the intracellular space between cells of higher plants. A growing body of

evidence suggests that plant-associated microorganisms, especially endophytic bacteria

and fungi, represent a huge and largely untapped resource of natural products with

chemical structures that have been optimized by evolution for biological and ecological

relevance (Gunatilaka, 2006).

Endophytic fungi inhabit such a novel biotope and constitutes one of the untapped

bioresources (Schulz et al., 2002; Strobel and Daisy, 2003; Strobel, 2006; Suryanarayana

et al., 2012). Endophytic fungi associated with medicinal plants, have recently attracted

much attention from microbiologists, taxonomists, ecologists, agronomists, chemists and

evolutionary biologists, as a promising sources of secondary metabolites with medical,

industrial use in drug discovery program (Tan and Zou, 2001; Schulz et al., 2002;

Strobel, 2002; Strobel and Daisy, 2003; Strobel et al., 2004; Aly et al., 2011b; Kaul et al.,

2012). This was stimulated by the surprising discovery that certain endophytic fungi

produced the anticancer drug taxol.

Since the discovery of Taxol® is a diterpenoid, first isolated from Taxus

brevifolia was based on hypothesis that endophytes can produce the same rare and

important bioactive compounds as their host plants, such as in the case of a novel

paclitaxel-producing fungus, Taxomyces andreanae, from the yew Taxus brevifolia was

isolated and characterized (Stierle et al., 1993; Strobel et al., 1996; Shrestha et al., 2001).

Later discovery from other member of endophytic fungi, such as Pestalotia spp. and

Pestalotiopsis spp. in their host such as Taxus wallachiana, paves a new way to the

production of the natural product drug (Strobel et al., 1996; Noh et al., 1999).

1.9. Endophytic fungi

1.9.1. Definition

The word endophyte literally means ‘in the plant’ (endon Gr., within; phyton,

plant). The usage of this term is as broad as its literal definition and spectrum of potential

plant hosts and inhabitants (Sculz and Boyle, 2005). The term “endophyte” was

introduced by Anton de Bary and was for some time applied to “any organisms occurring

within plant tissues” (de Bary, 1866). The term “endophyte” was defined in various ways

from researchers. The most common and widely accepted definition is that of Petrini

21

(1991), “All the organisms inhabiting plant organs that at same time in their life can

colonize internal plant tissues without causing apparent harm to the host”. Bacon and

White (2000) gave an inclusive and widely accepted definition of endophytes, “Microbes

that colonize living, internal tissues of plants without causing any immediate, overt

negative effects”. According to Sculz and Boyle, (2005) “Fungi that colonize a plant

without causing visible disease symptoms at any specific moment”. Recently according

to Kusari and Spiteller, (2012) “Endophytism” a unique cost-benefit plant-microbe

association defined by “location” (not “function”) that is transiently symptomless,

unrobtrusive and established entirely inside the living host plant tissues.

1.9.2. Origin and history of endophyte concept

Collectively, more than 100 years of research suggests that most, the early

publications describing an endophytic fungus was by Freeman in 1904 and he makes

reference to four other papers on endophytes that were published in 1898. Freeman found

the fungus in Persian darnel -an annual grass (Schardl et al., 2004). A milestone in the

history of endophyte research was the discovery of the endophytic fungus Neotyphodium

coenophialum as the causative organism of ‘‘fescue toxicosis’’, a syndrome suffered by

cattle fed in pastures of the grass Festuca arundinacea (Bacon et al., 1977). It was later

found that these infected plants contained several toxic alkaloids and the Neotyphodium

species could be beneficial to their plant hosts, increasing their tolerance of biotic and

abiotic stress factors (Schardl et al., 2004).

Grasses are probably the plants that have been most extensively studied as far as

endophytes are concerned and it was discovered that grasses with high endophyte content

were often resistant to attack by certain insects (Azevedo et al., 2000). The best example

of a plant-endophyte association is that of Neotyphodium sp. (fungus) and Lolium sp.

(grass). Toxic alkaloids produced by the fungus protect the plant from grazing cattle and

in turn the endophyte gains shelter and nutrition from host plant (Rodriguez et al., 2009).

1.9.3. Types of endophytes

The ecological significance of these fungi remains poorly characterized despite

more than 100 years of research resulting in thousands of journal articles. Generally

plant-associated fungi are usually divided into five main functional groups: mycorrhizal,

pathogenic, epiphytic, endophytic and saprotrophic fungi. Among them endophytic

22

constitute a part of a mycobiome, have one or multiple functional roles during their life

cycles or in response to plant or environmental factors (Fig. 5) (Alfaro and Bayman,

2011).

Figure 5: Diverse role of endophytic mycobiome

Historically, two endophytic groups clavicipitaceous (C) and nonclavicipitaceous

(NC) have been discriminated based on phylogeny and life history traits. NC-endophytes

represent three distinct functional groups based on host colonization and transmission in

plantbiodiversity and fitness benefits conferred to hosts (Rodriguez et al., 2009). The

criteria and types of two endophytic groups were summarized in Table.1.9.

Table 1.9 The salient feature of clavicipitaceous (C) and nonclavicipitaceous (NC)endophytes

(Source: Rodriguez et al., 2009)

According Schulz and Boyle, (2005) fungal endophytes consist of three basic

ecological groups: the mycrorrhizal fungi, the balansiaceous or “grass endophytes” and

the non-balansiaceous.

23

1.9.3a. Balansiaceous endophytes or Grass endophytes:

The balansiaceous endophytes closely related to clavicipitaceous grass

endophytes. They produce a diverse array of secondary metabolites. They form a unique

group of closely related fungi with ecological requirements and adaptation distinct from

those of other endophytes (Petrini, 1996). The toxic alkaloids include the anti-insect

alkaloids peramine and lolines and the anti-vertebrate alkaloids lolitrem B and

ergovaline. They grow systemically, epicuticularly and intercellularly within all above-

ground plant organs of grasses, resulting in vertical transmission of the endophytes

through the seeds. They belong to the ascomycetous genera Epichloe and Balansia, their

anamorphs are Neotyphodium and Ephelis (Azevedo et al., 2000: Schardl et al., 2004;

Rodiguez et al., 2009).

The primary benefits for the endophytic fungal partner are nutritional, but also

include protection from abiotic stress (Bacon, 1996) such as desiccation and from

competing epiphytic organisms (White et al., 2000). The advantage of the interaction for

plants is protection against herbivory by toxic alkaloids produced by the fungal

endophytes during symbiotic association and they also mediate induced resistance

through activation of the host defense through constitutive and induced resistance

(Bultman and Murphy, 2000).

1.9.3b. Non- balansiaceous endophytes

Studies on this group of fungi produced more than 1000 papers published in

various journals since 1970. Majority of the present data concerning the distribution and

abundance of endophytes in asymptomatic tissues of various plants; the isolation and

analysis of bioactive compounds; their potential use as biocontrol agents; phylogeny-

based identification and systematic are becoming more apparent, engendering growing

enthusiasm from mycologists, ecologists, physiologists and applied scientists (Tan and

Zou, 2001; Schulz et al., 2002; Strobel and Daisy 2003; Selosse et al., 2004; Schulz,

2006; Arnold et al., 2007; Higgins et al., 2007; Aly et al., 2011b; Debbab et al., 2012).

According to Rodriguez et al., (2009) non-clavicipitaceous (NC) endophytes are

phylogentically diverse and often with poorly defined or unknown ecological roles. They

have been recovered from every major lineage of land plants and from all terrestrial

ecosystems, including both agro-ecosystems and biomes ranging from the tropics to the

24

tundra (Arnold and Lutzoni, 2007). The scale of their diversity and ecological

rolesprovide insights into the evolution of various ecological modes in fungi. The ability

of many fungi to switch between endophytic and free-living lifestyle was also explained

by Vasiliauskas et al., (2007); Macia-Vicente et al., (2008) and Selosse et al., (2008).

Most of them belong to the Ascomycota obtained from above ground parts of all

sampled plants. They colonize either inter or intracellular, localized or systemic. Majority

of these isolates belong to ubiquitous genera (e.g. Acremonium, Alternaria,

Cladosporium, Coniothyrium, Epicoccum, Fusarium, Geniculosporium, Phoma and

Pleospora) but some genera are common in both tropical and temperate climates (e. g.

Fusarium, Phomopsis and Phoma) while members of the Xylariaceace, Colletotrichum,

Guignardia, Phyllosticta and Pestalotiopsis predominate as endophytes in the tropics

(Schulz and Boyle, 2005).

1.9.4. Host-endophyte interaction

Fungi are the second largest group of tropical ecosystems throughout the world.

During evolution when plants colonized the land successfully, fungi developed different

types of relationship with them. The group ‘endophytes’ form one of these associations

and their existence have been traced in the fossil records suggesting that endophyte-host

association may have evolved from the time of emergence of first higher plants on earth

(Rodriguez and Redman, 1997; Redecker et al., 2000; Strobel, 2003).

Interaction between host-endophyte in dual culture experiments demonstrates that

chemotactic signaling involved in the interactions with hosts suggest that these

endophytes were not mere incidental opportunists in their hosts and that there has been an

evolutionary adaptation which will help the host to survive under biotic and abiotic stress

conditions by mutual exploitation. Those fungi which are isolated as endophytes have no

set-life history strategy according to Saikkonen et al., (2004), Schulz and Boyle, (2005)

and Rodriguez and Redman, (2008).

The fungus detected as endophytes might be a pathogen in a non-host, latent

pathogen, saprophyte and their spores or virulent pathogens in a latent phase, each of

these represent different life-history strategies (Carroll, 1988). But recent observations

and hypotheses on fungal endophytes suggest that asymptomatic colonization or

endophytism is a balance of antagonisms or synergism between host and endophyte (Fig.

25

6). Because neither of the partner gains the upper hand in the interaction rather than a

‘survival strategy’. The three factors of disease triangle that is innate virulence of

endophytes, host defense response and environmental conditions contribute to the above

interactions (Schulz and Boyle, 2005; Kusari et al., 2012).

Figure 6: Schematic representation of balanced antagonism or synergismbetween host and endophyte

(Source: Kusari et al., 2012)

Endophytes reside within plants and are continuously interacting with their hosts.

Furthermore, expression of the gene cluster for lolitrem biogenesis in endophytic

Neotyphodium lolii resident in perennial ryegrass (Lolium genus) is high in plant, but low

to undetectable in fungal cultures grown in vitro, lending support to the notion that plant

signaling is required to induce expression (Young et al., 2006). It was found that a

camptothecin-producing endophyte, F. solani isolated from C. acuminata (Kusari et al.,

2009), could indigenously produce the precursors of camptothecin. However, a host plant

enzyme absent in the fungus, strictosidine synthase, was employed in planta for the key

stepin producing camptothecin (Kusari et al., 2011). Such plant-fungus interactions

compel reconsidering whether horizontal genetransfer (plant to endophyte genome or

vice versa) is the onlymechanism by virtue of which endophytes produce associated plant

compounds (Kusari and Spiteller, 2011).

26

1.9.5. Endophyte-Endophyte interaction

There are interactions among diverse group of endophytic microbes (bacteria and

fungi) harboring plant. Endophyte-endophyte interspecies or intraspecies cross talk

(Fig.7) mediate through small, diffusible signaling molecules, quorum-sensing signals or

other elicitors, which may trigger silent biosynthetic pathways (Keller and Surette, 2006;

Hughes and Sperandio, 2008; Scherlach and Hertweck, 2009).

For instance, intimate physical interactions between fungi (Aspergillus nidulans)

and bacteria (Streptomyces rapamycinicus) have been observed by Schroeckh et al.,

(2009), which result in an epigenetic regulation involving Saga/Ada-mediated histone

acetylation of fungal secondary metabolism (Nutzmann et al., 2011). This unexpected

interaction led to the production of orsellinic acid-derived polyphenols such as cathepsin

K inhibitors and lecanoric acid. The observation of the latter is intriguing because it is an

archetype lichen metabolite (Schroeckh et al., 2009). To study in more detail the

secondary metabolite function in complex environments as found for endophytes, it

would be intriguing to evaluate the endophyte-endophyte interactions.

Figutre 7: Schematic Representation of Endophyte-Endophyte InterspeciesCrosstalk

(A) Fungus-fungus crosstalk is illustrated. (B) Fungus-bacterial endosymbiont crosstalkis demonstrated. (C) Fungus-bacteria crosstalk (Source: Kusari et al., 2012)

1.9.6. Methods in the study of endophytic fungi from medicinal plants

1.9.6a. Selection of plant material and surface sterilization process plating

The rationale for the host plant selection is crucial to increase the chances of

isolating novel microorganisms and new bioactive compounds. Plants should be selected

mainly on the basis of their unique environmental setting, ethnobotanical history,

endemism, unusual longevity and large areas of biodiversity (Strobel et al., 2004).

27

One of the critical needs for isolating endophytes is the acquisition of fresh

healthy plant material and processed as soon as possible. Subsequently a surfactant such

as ethanol and/or Tween 80 is employed to rinse the plant material, followed by a

sterilizing agent, such as sodium hypochlorite (Schulz and Boyle, 2005) or hydrogen

peroxide (Gao et al., 2005). The surfactant and sterilizing agent concentrations required

for the sterilization varies with the kind of the plant tissue (Table 1.10). The plant tissues

are excised with a sterilized scalpel into pieces of about 5 mm length and plated onto the

culture medium, potato dextrose agar (PDA) (Suryanarayanan et al., 2003) or malt

extract agar (MEA) (Arnold et al., 2000) supplemented or not with antibiotic agents such

as chloramphenicol (Suryanarayanan et al., 2003), streptomycin, tetracycline or penicillin

(Otero et al., 2002) to suppress bacterial growth. Afterward, the plates were incubated at

temperatures ranging from 18 ◦C to 30 ◦C for several days until fungal growth (Table

1.10).

Table 1.10 Recently employed methods for endophytic fungi isolationWashing Rinse with

ethanolsolution

Surfacedisinfection

Rinsed withethanolsolution

Rinsed in steriledistilled water

Incubation(days,temperature)

Reference

Running tap water(RTW)

70% 3%, 3 mina 70% twice 3-15, 28 ◦C Rubini etal.,(2005)

Water and detergentc 70%, 1 min 15%, 1 minb 70%, 1 min ni ni Gao et al.,(2005)

RTW d 70%, 1 min 5%, 5 mina ni twice, 1 min 30 ◦Ce Chomcheon etal.,(2005)

RTW 75%, 1 min 6%, 3 or 5 mina 75%, 0.5 min three times 30, 25 ◦C Raviraja etal.,(2006)

RTW d 70%, 1 min 6%, 5 mina ni twice, 1 min 30 ◦C Chomcheon etal.,(2006)

RTW 95%, 1 min 6%, 5 mina 95%, 0.5 min three times 4-5, 25 ◦C Seena andSridhar, (2004)

a Sodium hypochloride solution; b Hydrogen peroxide solution; c The material was dried with sterile filter paper; d Air-dried; e

Cultivated on banana leaf agar the fungi developed conidia, which permitted their identification; ni: Not informed;

Additionally, aliquots of the water from final rinse solutions can be placed on the

same media employed for the endophyte culture to check the effectiveness of sterilization

procedure (Cao et al., 2004). Fungal outgrowth from the plant tissues is sub cultured on

fresh antibiotic-free medium. Nevertheless, some isolates must be cultured on different

media as banana leaf pieces impregnated on PDA (Tejesvi et al., 2006), oatmeal agar

28

(Bayman et al., 1998), malt extract agar, plant-origin tissue fragments (Strobel et al.,

1999) or several other industrializing media to induce sporulation (Shen et al., 2006).

1.9.6b. Endophytic fungal strain preservation

They can be divided into two main groups based on the continuous or suspended

metabolism of the fungus (Onions, 1983). The first group includes storage in: sterile

water (Castellani, 1963), serial transfer in agar, cool storage in a standard refrigerator at

5-8 ºC, deep freeze at about -20ºC, under mineral or paraffin oil, grain or soil (Douds Jr

and Schenck, 1991) at room temperature. The second group includes drying, silica gel,

freeze drying or lyophilization, liquid nitrogen (Onions, 1983) and prepared cryogenic

freezer beads (Microbank) at -70 ºC (Baker and Jeffries, 2006). Cryopreservation was

considered the ultimate method available for the long-term storage of microbial cultures

due to the stability of secondary metabolite production and the minimized genetic

alterations, though certain fungi have exhibited significant degrees of polymorphism after

revival (Ryan et al., 2001; Borman et al., 2006).

1.9.6c. Endophytic fungal taxonomy

Classification systems of fungi have been historically supported on readily

observable morphological features and their comparison. The most important sets of

characteristics to be observed are the conidia and the process involved in their formation.

Additionally, the pigmentation and shape of hyphae, presence or absence of septa,

occurrence of sclerotia, chlamydospores or any other particular hyphal element may be

very helpful to assist in classification of both anamorph (asexual state) or teleomorph

(sexual state) phases (Bononi and Grandi, 1998). Many attempts have been realized to

classify fungi according to their secondary metabolite production pattern (Stahl and Klug,

1996; Frisvad et al., 1998). Cell wall polysaccharides have also performed a role as traits

for fungal taxonomy and evolution (Leal et al., 2001). Isoenzyme analysis, which is

carried out by eletrophoretic methods, is other really powerful and adaptable technique

that can be used to resolve many problems on fungal genetics, population biology and

taxonomy; specifically it has also determined generic relationship and differentiated

species (Goodwin, 2004).

Molecular identification and phylogenetic studies rely on a large extent on

ribosomal DNA (rDNA) sequence polymorphism. The main reasons for the popularity of

29

rDNA are that it is a multiple-copy, non-protein-coding gene but almost always treated as

a single-locus gene. Additionally, ribosomes are present in all organisms displaying a

common evolutionary origin (Guarro et al., 1999). Regarding PCR amplification, regions

of the molecule are transcribed, generally the 5.8S, 18S or 28S, along with internal and

external transcribed spacers (ITS and ETS). The simultaneous use of highly conserved

LSU rRNA-coding sequence and variable non-coding ITS1 sequence permitted the

connection of genetically indistinguishable species (Campbell et al., 2006).

1.9.6d. Culture conditions

Ultimately, when an endophyte is acquired in pure culture, its ability to grown is

investigated on a number of different media and growth conditions. Subsequently, better

medium and growth conditions are established, the microbe is fermented and extracted

and the extract is submitted to several chromatographic procedures in order to yield the

product of interest.

In most cases, temperature, pH, composition of the medium, length of growth

period of culture and the degree of aeration are some of the factors that can affect the

amount and kinds of compounds that are produced by a particular fungus and can be

manipulated to improve yields and assortment of substances of bioactive significance

(Stahl and Klug, 1996; Strobel et al., 2004; Elias et al., 2006).

1.9.7. Ecological and biological role of endophytes in their hosts.

Endophytes have also been recorded as colonizing in marine algae, grasses,

mosses and ferns (Tan and Zou, 2001). The host-endophyte relationships can be

described in terms of host-specificity, host-recurrence, host-selectivity or host preference

(Zhou and Hyde, 2001; Cohen, 2006).

Host-specificity is a relationship in which microorganism is restricted to a single

host or a group of related species and such specificity implies that complex biochemical

interaction occur between host and its associated endophytes (Strobel, 2003; Strobel and

Daisy, 2003). For example Pestalotiopsis microspora is one of the most commonly found

endophytes in Taxa species (yews).

Host-recurrence refers to the frequent or predominant occurrence of endophytes

on a particular host or a range of plant hosts and endophytes can also found infrequently

on other host plants in the same habitat (Zhou and Hyde, 2001). The term host-preference

30

is more frequently used to indicate a common occurrence or uniqueness of occurrence of

endophytes to particular host and also used to indicate the difference in endophytic

community composition and relation frequencies from different host plants

(Suryanarayanan and Kumaresan, 2000).

Species of Colletotrichum, Phoma, Phomopsis and Phyllosticta endophytes have

a wide host range and colonize several taxonomically unrelated plant hosts suggesting

that they have developed adaptations to overcome different types of host defenses

(Jeewon et al., 2004; Murali et al., 2006).

Apart from producing bioactive novel secondary metabolites, different works

carried out so far regarding the role of endophytes in host plants indicate that they can

stimulate plant growth, increase disease resistance, improve plant's ability to withstand

environmental stress and recycle nutrients (Sturz and Nowak, 2000).

Endophytes can promote the plant growth through a variety of mechanisms, as

endophytic metabolites provide a variety of fitness to host plants enhanced by increasing

plant resistance to biotic and abiotic stresses, as well as enhance plant growth. Many

endophytes are reported to be capable of nitrogen (N) fixation, solubilization of

phosphate, 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 regulation of plant growth and developments

(Singh et al., 2000; Sherameti et al., 2005; Waller et al., 2005; Varma et al., 1999).

Endophytes may help host plants to tolerate and withstand environmental stress

such as drought, salts and high temperatures (Malinowski and Belesky, 2000).

Endophytic fungi can protect their host plants from pathogens and pests (Arnold et al.,

2003; Akello et al., 2007). The systemic and foliar endophytes can reduce herbivory by

producing alkaloids toxic to insects and vertebrates (Schardl, 2001). Endophytes can

protect host by three main mechanisms (a) competition between endophyte and pathogen

(Lockwood et al., 1992) (b) production of biocidal or phytoalexins (Rai et al., 2002) and

(c) through the induction of disease resistance in hosts (Gianinazzi et al., 1996).

1.9.8 Endophytes and fungal diversity

Fungal species have been described so far is less than 100,000, there are probably

many more, perhaps 1,500,000 (Hawksworth and Rossman, 1997; Frohlich and Hyde,

31

1999). Endophytes comprise a large hidden component of fungal biodiversity (Arnold et

al., 2003; Arnold, 2007; Rodriguez et al., 2009). Every plant species harbor endophytes

belong to the different communities dominated by various classes, including

Dothideomycetes, Sordariomycetes, Leotiomycetes, Eurotiomycetes and Pezizomycetes

(Higgins et al., 2007; Jumpponen and Jones, 2009). Zygomycota and Basidiomycota

fungi also occur as endophytes, with agaricales common in grasses (Porras-Alfaro et al.,

2008; Herrera et al., 2010; Khidir et al., 2010) and russulales, polyporales and agaricales

common in woody tissues and roots (Sokolski et al., 2007).

Some endophytes are host-specific. The total number of endophytic species can be

extrapolated from the number of plant species (Bills, 1996; Hawksworth and Rossman,

1997). This indicates the need for more extensive survey of plant organs to evaluate

distribution patterns and diversity of endophytic fungi across large geographical scales.

The mycobiome can vary greatly in a single host species in different sites,

climates, seasons and environments (Rodriguez, 1993; Carroll, 1995; Wilson, 2000;

Gamboa et al., 2002; Lingfei et al., 2005). Mycobiome composition may depend on

multiple factors including plant host, plant density, nutrient availability, environmental

conditions and interactions with external microbiomes (e.g., soil fungi and bacteria).

Differences in endophytic communities in a single host species can increase with distance

(Arnold and Herre, 2003) or show no significant variation (Herrera et al., 2010; Khidir et

al., 2010). Leaves, roots and woody stems of a single plant often differ greatly in the

dominant members of their endophytic communities and may even show functional

differences. Differences in endophytes between roots, stems and leaves may reflect

differences in external environment as much as biological differences among organs and

tissues (Pocasangre et al., 2000; Chaverri and Gazis, 2010; Gazis and Chaverri, 2010;

Herrera et al., 2010).

Diversity of endophytic population is affected by many internal (type of host and

tissue) and external parameters such as seasonal, geographical as well as environmental

conditions. Recently on all these parameters Verma et al., (2012) has surveyed a total of

1,151 endophytic fungal isolates representing 29 taxa from symptom-less, surface

sterilized segments of stem, leaf, petiole and root of Tinospora cordifolia which had been

collected at three locations differing in air pollution in India, (Ramnagar, Banaras Hindu

32

University, Maruadih) during three seasons (summer, monsoon and winter). Endophytes

were most abundant in leaf tissues (29.38% of all isolates), followed by stem (18.16%),

petiole (10.11%) and root segments (6.27%). The frequency of colonization (CF) varied

more strongly among tissue type and season than location. CF was maximal during

monsoon followed by winter and minimal during summer. A species each of Guignardia

and Acremonium could only be isolated from leaves, whereas all other species occurred

in at least two tissue types. Penicillium spp. were dominant (12.62% of all isolates),

followed by Colletotrichum spp. (11.8%), Cladosporium spp. (8.9%), Chaetomium

globosum (8.1%), Curvularia spp. (7.6%) and Alternaria alternata (6.8%). Species

richness, evenness and the Shannon–Wiener diversity index followed the same pattern as

the CF with the tissue type and the season having the greatest effect on these indices,

suggesting that tissue type and season are more influential than geography. Dissimilarity

of endophyte communities in regards to species composition was highest among seasons.

Colletotrichum linicola occurred almost exclusively in winter, Fusarium oxysporum only

in winter and summer but never during monsoon and Curvularia lunata only in winter

and monsoon but never in summer.

Literature review covering the past ten years demonstrated that about 770 fungal

species have been isolated as endophytes. Nevertheless, an impressive number of those

species carry on unidentified. Alternaria sp., A. infectori, Aspergillus sp., Colletrotrichum

sp., C. gloeosporioides, C. musae, Cordana musae, Fusarium sp., Guignardia sp.,

Nigrospora oryzae, N. sphaerica, Penicillium sp., Pestalotiopsis sp., Phomopsis sp.,

Rhizoctonia sp and Xylaria sp., appear among the foremost identified species (Tejesvi et

al., 2006; Arnold and Lutzoni, 2007; Huang et al., 2008; Gazis and Chaverri, 2009;

Tayung and Jha, 2010; Banerjee, 2011; Glenn and Bodri, 2012; Kharwar et al., 2012;

Maheswari and Rajagopal, 2013).

Moreover, some new endophytic fungal species has been found such as

Phialocephala sphaeroides, a dark septate root endophyte from boreal wetland plants of

Canada (Wilson et al., 2004). P. compacta and P. scopiformis, both identified by

Kowalski and Kehr (1995), were isolated from the living branches of German forest

trees. Some ascomycetes isolated from the wild ginger Amomum siamense named as

Gaeumannomyces amomi, Leiosphaerella amomi and Pyricularia sp. (Bussaban et al.,

33

2001), a pyrenomycete Monosporascus ibericus sp. isolated from plants occurring on

Spanish saline soils (Collado et al., 2002). Gonatobotryum sp., a conidial fungus from the

Indian plant Carissa carandas L. (Jacob and Bhat, 2000) and Mycena anoectochila from

the Chinese Orchidaceae Anoectochilus roxburghii (Guo et al., 1997). Muscodor albus, a

novel xylariaceaous fungus described by Strobel et al., (2001) from Cinnamomum

zeylanicum (cinnamon tree).

Majority of the researches concerning endophytic fungifrom temperate plants

(Rodrigues and Petrini, 1997). The research on tropical endophytes has been stimulated

by the role played by these microorganisms on both global fungal diversity (Frohlich and

Hyde, 1999; Hawksworth, 2001) and plant community dynamics (Arnold, 2001).

Endophytes as sources of novel bioactive compounds (Strobel and Long, 1998),

biological control agents for use in tropical agroforestry (Arnold et al., 2001).

Following tropical endophytic studies enlarge and highlight the diversity of

endophytic fungi. From Guyana (Cannon and Simmons, 2002), Panama (Arnold and

Herre, 2003; Suryanarayanan et al., 2003), India (Nair and Bhat, 2002; Suryanarayanan

et al., 2003; Tejesvi et al., 2006; Kharwar et al., 2012), Puerto Rico (Lodge, 1997; Otero

et al., 2002), Brazil (Azevedo et al., 2000; Souza et al., 2004; Campos et al., 2005;Cafeu

et al., 2005; Borges and Pupo, 2006), Australia (Frohlich and Hyde, 1999; Parungao et

al., 2002) and Ecuador (Fisher et al., 1995).

Tropical endophytes have produced bioactive secondary metabolites. For

instance, 3-hydroxypropionic acid, from Phomopsis phaseoli an endophyte of broad-

leaved tree from French Guyana and Melanoconium betulinum an endophyte associated

with Betula pendula and Betula pubescens (Schwarz et al., 2004). Muscodor albus, an

endophyte from the small unidentified vine growing in the Tessa Nile area of Sumatra,

synthesizes antimicrobial volatile antibiotic organic compounds (Atmosukarto et al.,

2005). Li et al., (2001) identified a new and highly functionalized compounds from

tropical endophytes such as ambuic acid, an antifungal compounds isolated from both

Pestalotiopsis sp. and Monochaetia sp.

1.9.9. Importance of secondary metabolites from endophytic fungi:Secondary metabolites, defined as low-molecular-weight compounds not required

for growth in pure culture, are produced as an adaptation for specific functions in nature.

34

They play an important role in vivofor example, important numerous metabolic

interactions between fungi and their plant hosts, such as signaling, defence and regulation

of the symbioses. Microbial metabolites seem to be characteristic of certain biotopes and

specialized ecological niche, both on environmental as well as organism level (Schulz

and Boyle, 2005).

Accordingly, it appears that the search for novel secondary metabolites should

center on organisms that inhabit unique biotopes. Secondary metabolites a fungus

produces may vary with the biotope in which it grows and to which it is adapted. For

example, the production of cyclosporin A, enchinocandin B, papulacandins and

verrucarins varied with both habitat and substrate (Dreyfuss and Chapela, 1994; Liu et

al., 2012).

Since natural products or secondary metabolites are adapted to a specific function

in nature, the search for novel secondary metabolites should concentrate on organisms

that inhabit novel biotopes. Endophytic fungi are one such source for intelligent

screening for the search of novel natural products because they inhabit specialized niche

and unique biotope (Schulz et al., 2002; Aly et al., 2011b; Liu et al., 2012).

Thus bioprospection of endophytic fungi is good source for structurally

unprecedented bioactive secondary metabolites; it is relevant to consider that:

1. The secondary metabolites synthesized by fungus correspond with its respective

ecological niche and

2. Continual metabolic interactions between fungus and plant may enhance the synthesis

of secondary metabolites (Schulz et al., 2002; Strobel and Daisy, 2003).

The advantages of fermentation of endophytic fungi producing bioactive

metabolites include:

(i) Industrial production of bioactive substances requires reproducible, dependable

productivity.

(ii) Microorganisms typically respond favorably to routine culture techniques and tissue

culture or growing plants requires either specialized techniques or months of growth

before harvesting is feasible;

(iii) Product recovery or down streaming is relatively easy in microorganisms.

Optimization in culture conditions and various biosynthetic pathways can be explored,

35

which may lead to even more effective derivatives of lead compounds (Tejesvi et al.,

2007).

1.9.9a. Current scenario of endophyte research with respect to drug discoveryPresumably owing to their specialized niches, no substantial body of work has

accumulated since the first discovery of endophytic fungus in darnel in 1904. In fact,

since the publication of the report by Stierle et al., (1993), there has been a monotonic

increase in the number of US patents filed on endophytic fungi producing important

metabolites with diverse biological activities (Priti et al., 2009).

The research on endophytes is growing enormously, as >650 research articles

covering both bacteria and fungi. When the bibliographic search was restricted to

endophyte and metabolite there were 253 published research articles, which shows that

roughly 40% of the endophyte researchers were looking for secondary metabolites.

More than 650 research articles including bacteria and fungi published during the

period between 1991 and 2010 on endophytes research. More than 253 research articles

in reputed journals, shows that 40% of the endophyte researchers were looking for

secondary metabolites. Above 650 patents were filed and granted for using an endophyte

as a source for new processes or industrial applications on bioactive metabolites, when

searched with the keyword endophyte (Fig. 8) (Tejesvi and Pirttila, 2011).

Figure 8: Publication numbers on endophyte research during 1990-2010.

(Source: Tejesvi and Pirttila, 2011)Interestingly, some useful plant-derived anticancer drugs have also been identified

from endophytic fungal cultures. Among them Paclitaxel (Taxol), one of the most

important drugs available for the treatment of breast and ovarian cancers, was isolated

from Taxomyces andreanae, an endophytic fungus associated with the Pacific yew Taxus

36

brevifolia (Stierle et al., 1993). An endophytic fungus from leaves of Catharanthus

roseus was reported to biosynthesize the potent antileukemia agent vincristine (Yang et

al., 2004). Entrophospora infrequens, an endophyte isolated from Nothapodytes foetida

(Icacinaceae), was able to produce camptothecin (Puri et al., 2005), chemotherapeutic

agent efficient against lung, ovarian and uterine cancer, which was first isolatedfrom

Camptotheca acuminata (Nyssaceae) (Amna et al., 2006). Podophyllotoxin, a natural

product precursor of useful anticancer agents, was found to be synthesized by Trametes

hirsuta, a novel endophyte from Podophyllum hexandrum (Puri et al., 2006) and also by

the endophytic fungus Phialocephala fortinii, associated with Podophyllum peltatum

(Eyberger et al., 2006).

Based on these examples, knowledge of plant-microbe interactions can direct the

research of novel bioactive natural products for pharmaceutical and agrochemical

industries (Tan and Zou, 2001; Rubini et al., 2005; Gunatilaka, 2006; Strobel, 2006).

Inspired by the biosynthesis of anticancer drug paclitaxel (Taxol®) by an

endophytic fungus Taxomyces andreanae from Pacific yew Taxus brevifolia from the

work of Stierle et al., (1993).This discovery laid foundation to speculate that medicinal

plants might constitute alternate source of endophytic fungi with wide range of biological

activity and fungal endophytes residing within these medicinal plants could also produce

metabolites similar to or with more activity than that of their respective hosts (Strobel and

Daisy, 2003; Kaul et al., 2012).

Many scientists hold that plants growing in lush tropical rainforests, where

competition for light and nutrients is severe, are most likely to host the greatest number

of bioactive endophytes and indeed a recent study noted that endophytes from tropical

regions produced significantly more bioactive secondary metabolites than those from

temperate parts of the world (Strobel et al., 2004; Schulz and Boyle, 2005).

Some endophytes produce certain chemical compounds resemble original

characteristic of the host which could be related to a genetic recombination of the

endophyte with the host (horizontal gene transfer) that occurred in evolutionary time

according to concept proposed by Tan and Zou, (2001) to explain possible mechanism of

taxol biosynthesis by an endophytic fungus Taxomyces andreanae from pacific yew

(Stierle, 1993). Thus, if endophytes can produce the same rare and important bioactive

37

compounds as their host plants, this would not only reduce the need to harvest slow-

growing and possibly rare plants but also help to preserve the world’s ever diminishing

biodiversity.

Comprehensive reviews regarding endophytic chemical diversity and biological

activities emphasizing their potential ecological role have been published and confirmed

that fungal endophytes as an outstanding source of new natural products. An array of

natural products has been characterized from endophytes, which includes anti-cancerous,

anti-oxidants, anti-fungal, anti-bacterial, anti-viral, anti-insecticidal and

immunosuppressant (Table 1.11) (Tan and Zou, 2001; Strobel and Daisy, 2003; Strobel et

al., 2004; Gunatilaka, 2006).

The diversity of metabolites that have been isolated from endophytic fungi was

relatively unstudied as potential sources of novel natural products for exploitation in

medicine, agriculture and industry. Recently, several new bioactive products were

isolated and identified with unique core structures and potent biological activities (Table

1.11) (Aly et al., 2010; Aly et al., 2011b; Debabb et al., 2011; Debbab et al., 2012;

Debbab et al., 2013).

Recent reviews by Yu et al., (2010), Radic and Strukelj, (2012) and Mousa and

Raizada, (2013) which emphasize on anti-infective secondary metabolites from

endophytic fungi (Table 1.11). The anti-microbial secondary metabolites, synthesized by

fungal endophytes belong to diverse structural and chemcial classes including alkaloids,

steroids, terpenoids, phenols, quinines, flavonoids, phenylpropanoids, aliphatic

compounds, polyketides and peptides from the interdisciplinary perspectives of

biochemistry, genetics, fungal biology and host plant biology. Terpenoids and

polyketides are the most purified anti-microbial secondary metabolites from endophytes.

Fungal genes which encoded for anti-microbial compounds are clustered on

chromosomes, as different genera of fungi can produce the same metabolite, genetic

clustering may facilitate sharing of anti-microbial secondary metabolites between

endophytic fungi (Mousa and Raizada, 2013).

38

Table 1.11. Some of the bioactive natural products of fungal endophytes

Sl. No. Host plant: place ofcollection

Fungal endophytes Name of the metabolite Nature ofMetabolite

Bioactivity Structure of Metabolite Reference

1. Artemisia annua:Nanjing, China

Colletotrichumspp.

3β, 5ά-dihydroxy-6 β -acetoxy-ergosta-7,22-diene and3β, 5 ά-dihydroxy-6 β –phenylacetyloxy-ergosta-7, 22-diene

(Steroids) Antimicrobial activityagainst human pathogenic

fungi andbacteria,

fungistatic to plantpathogenic fungi 3β, 5ά-dihydroxy-6 β -acetoxy-ergosta-7,22-diene

3β, 5 ά-dihydroxy-6 β –phenyl acetyloxy-ergosta-7,22-diene

[18]Lu et al., (2000)

2. Artemisia mongolica:Zijin mountain,Nanjing,China

Colletotrichumgloeosporioides

Colletotric acid Tridepside(Phenolic nature)

Antibacterial andAntifungal(Helminthosporiumsativum)

Zou et al., (2000)

3. Bontia daphnoidesUK*

Nodulisporiumspp.

Nodulisporic acids Indole diterpenes Anti-insecticidal Polishook et al.,(2001)

4. Cynodon dactylon:Yancheng BiosphereReserve,Jiangsu Province,China

Aspergillusfumigatus CY018

Asperfumoid (1) andAsperfumin (2), sixmonomethylsulochrin,fumigaclavine C, fumitremorginC, physcion, helvolic acid andfour known steroids

3-hydroxyl-2,6-dimethoxyl-2,5-diene-4-cyclohexone-(1,3')-5’-methoxyl-7'-methyl-(1' H, 2'H, 4'H)-quinoline-2',4'-dione) and 5-hydroxyl-2-(6-hydroxyl-2-methoxyl-4-methylbenzoyl)-3,6-dimethoxyl-benzoic methylester.(Steroids)

Antifungal activity(Candida albicans,Trichophyton rubrum andAspergillus niger)

Liu et al., (2004)

39

5. Cynodon dactylon:Seashore near SheyangPort on the YellowSea. China

Aspergillus sp.CY725,

Anti-Helicobacter pylorisecondary metabolites.Helvolic acid,monomethylsulochrin,ergosteroland 3b-hydroxy-5a,8a-epidioxy- ergosta-6,22-diene

(Triterpenes) Antibacterial andantifungal (Helicobacterpylori; Bacillus subtilis,Pseudomonasfluorescens, Escherichiacoli, Staphylococcusaureus and Aspergillusniger, Trichophytonrubrum, Candidaalbicans)

Li et al., (2005)

6. Cinnamomumzeylanicum: Lancetillabotanical garden,La Ceibe, Honduras.

Muscodor albus(xylariaceaousfungus)

Volatile antimicrobials (1-butanol, 3-methyl acetate)

Ester Antimicrobial(Rhizoctonia solani,Ustilago hordei and F.solani(basidiomycetes)Cercospora beticola,Candida spp. &A.fumigatus (human fungalpathogens) Pythiumultimum andPhytophthora cinnamomi(Oomycetes)Antibacterial (E. coli, S.aureus,M. luteus and B.subtilis)

Strobel et al.,(2001)

7. Garcinia dulcis:Songkhla Province,Thailand

Xylaria sp. PSU-D14

Xylaroside A-B,Sordaricin,2,3-dihydro-5-hydroxy-2-methyl-4H-1-benzopyran-4-one

Glucosides,diterpene andcoumarin.

Antifungal (Candidaalbicans)

Pongcharoen et al.,(2008)

40

8. Ginko biloba:Jiangsu andShandong provinces,China

XylariaYX-28. 7-amino-4-methylcoumarin Coumarin Antibacterial ( E. coli,Salmonella thyphia,Salmonellathyphimurium,Salmonella enteritidis,Aeromonas aerophila,Yersinia sp, Shigella sp.Vibrio parahaemolyticus,and antifungal activities (Candida albicans,Aspergillus niger,Penicillium expansum)

Liu et al., (2008)

9. Hopea hainanensis:Hainan Island

Penicllium sp. Monomethylsulochrin (1),Rhizoctonic acid (2),Asperfumoid(3), Physcion (4), 7,8-dimethyl-iso-alloxazine (5) and3,5-dichloro-p-anisic acid (6)

Benzophenones Antifungal ( Candidaalbicans, Trichophytonrubrum and Aspergillusniger) and cytotoxicagainst KB cell line.

Wang et al., (2008)

10. Meliotus dentatus:Coastal area of theBaltic Sea,Ahrenshoop, Germany

UnidentifiedAscomycetousfungus ( Sterilefungus)

7-hydroxyphthalide (1) , 4-hydroxyphthalide (2) , 5-methoxy-7-hydroxyphthalide (3), 5,7-dihydroxyphthalide (4),(3R,4R)-cis-4-hydroxymellein(5) , (3R,4R)-cis-4-hydroxy-5-methylmellein (6), ergosterol (7)and 5α,8α-epidioxyergosterol

(8)

Pthalides (1–4), twoisocoumarin (5,6)and twosteroids (7 and 8)

Antibacterial andantifungal (Escherichiacoli andBacillus megaterium andMicrobotryumviolaceum)

Hussain et al.,(2009)

11. Quercus variabilis:Southern hill side ofthe Zijin Mountain inthe eastern suburb ofNanjing, China.

Fusarium sp. 2S,2'R,3R,3'E,4E,8E,10E)-1-O-ß-D-glucopyranosyl-2-N-(2'-hydroxy-3'-octadecenoyl)-3-hydroxy-9-methyl-4,8,10-sphingatrienine (1)and (2S,2'R,3R,3'E,4E,8E)-1-ß-D-glucopyranosyl-2-N-(2'-hydroxy-3'octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine (2)

Cerebrosides 1-2 Antibacterial activityagainst Bacillus subtilis,E. coli and Pseudomonasfluorescens

Shu et al., (2004)

41

12. Taxus brevifolia: UK* Taxomycesandreanae

Taxol Diterpenoid Anti-carcinogenic (P-388,P-1534,α-1210 murineleukaemia, Walker256 carcinoma, sarcoma180

Stierle et al., (1993)

13. Taxus wallachiana:Foothills of Himalyas.

Pestalotiopsismicrospora

Taxol Diterpenoid Anti-carcinogenic Strobel et al.,(1996)

14. Terminaliamorobensis:Sepik river drainagesystem,Papua, New Guinea

Pestalotiopsismicrospora

Isopestacin, Pestacin Isobenzofuranone,1, 3, dihydroisobenzofuran

Antioxidant, antifungal(Pythium ultimum)

Strobel et al.,(2002); Harper et

al., (2003)

15. Torreya taxifolia:UK*

Pestalotiopsismicrospora

Torreyanic acid Quinone dimmer Anticancerous andantibiotic

Lee et al., (1996)

16. Tripterygium wilfordii:UK*

(a) Fusariumsubglutinans(b)Cryptosporiopsisquercina

(a) Subglutinols A and B(b) Cryptocin

(a) Diterpene(b) Tetramic acid(Peptides)

(a) Immunosuppresive(b) Antimycotic(Pyricularia oryzaeand other plantpathogenic fungi)

(a)

(a) Lee et al.,(1995)(b) Li et al., (2000)

42

(b)17. UK*:

Xinglong, HainanProvince,People’s Republic ofChina,

Pestalotiopsisadusta

(A1)2,4-dichloro-5-methoxy-3-methylphenol,(B1), (C1)7,11b-dihydrobenz[b]indeno[1,2-d]pyran

Pestalachlorides A–C (1-3)(Amines orAmides)

antifungal activity(Fusarium culmorum,Gibberella zeae andVerticillium aibo-atrum).

Li et al., (2008)

18. Taxua baccata,Torreya taxifolia,Taxodiumdisticum,Wollemianobellis, DendrobiumSpeciosum,Taxumwallichiana:Tropical rainforestplants

Pestalotiopsismicrospora ,Pestalotiopsisgupenii andMonochaetia sp.

Ambuic acid cyclohexenone Antimycotic (Pyriculariaoryzae, Rhizoctoniasolani, Botrytis cinerea,Fusarium solani,Fusarium cubense,Helminthosporiumsativum,Diploida natelensis,CephalosporiumGramineium andPhythium ultimum)

Li et al., (2001)

19. UK*:Dongzai,Hainan Province,

Pestalotiopsisfoedan

Pestafolide A (1) andpestaphthalides A (2) and B (3),

New reduced spiroazaphilonederivative andisobenzofuranones

Antimycotic activity(Candida albicans,Geotrichum candidum,Aspergilllus fumigatus)

Ding et al., (2008)

43

20. Pinus sp.:UK*

Microdiplodiasp.KS75-1

8a-Acetoxyphomadecalin C Eremophilanesesquiterpenes

Pseudomonas aeruginosa(ATCC 15442)

Hatakeyama et al.,(2010)

21. Urospermum picroidesEgypt

Ampelomyces sp. 6-O-MethylalaterninAltersolanol A

Tetrahydroanthraquinones(Polyketides)

AntibacterialEnterococcus faecalis, S.aureus and S. epidermidis

6-O-Methylalaternin Altersolanol A

Aly et al., (2008)

22. UK*:Hangzhou China

Pestalotiopsis fici Pestalofones(C and E)

Cyclo hexanoneLignan

AntifungalAspergillus fumigatus

Pestalofone C Pestalofone E

Liu et al., 2009

23. Excoecaria agallocha:Mangrove

Phomopsis sp. Cytosporone B and C Aliphaticcompounds

AntifungalCandida albicans andFusarium oxysporum

Cytosporone BCytosporone C

Huang et al., 2008

24. Moringaoleifera : UK Nigrospora sp. Griseofulvin (1),Dechlorogriseofulvin (2),and mellein (4)

Polyketides Antifungal activityagainst plant pathogenicfungi (Botrytis cinerea,Colletotrichumorbiculare, Fusariumoxysporum f. sp.cucumerinum, Fusariumoxysporum f.sp. melonis,Pestalotia diospyri,Pythium ultimum,Rhizoctonia solani,Sclerotinia sclerotiorum)

Zhao et al., (2012)

44

* UK: Unknown

25. Cistus monspeliensis:Germany

Phomopsis sp. Phomochromone A and B (1 and2) phomotenone (3)

Two newchromones andone cyclopentenonederivative,

Antibacterial(Escherichia coli andBacillus megaterium) andantifungal(Microbotryumviolaceum)

Ahmed et al.,(2011)

45

1.10. Aims and scope of the study

The present study was aimed at isolation and identification of antimicrobial

metabolite from endophytic fungi isolated from plant sources and their seasonal variation

with respect to distribution and diversity. Mirabilis jalapa Linn. and Ficus pumila Linn.

were selected as plant source based on their medicinal properties. Endophytic mycoflora

isolated were evaluated for antimicrobial activity. The study also focus on a functional

gene-based molecular screening strategy was used to target type I polyketide synthase

(PKS) gene bioactive endophytic mycoflora.

The objectives of the present investigation are

1. Screening, isolation and identification of fungal endophytes.

2. Bioactivity screening of endophytic fungal extracts against important human

and phytopathogenic bacteria and fungi.

3. Molecular characterization and amplification of ketosynthase domain

sequence from selected bioactive endophytic fungal polyketide synthase gene.

4. Isolation, identification and characterization of bioactive compound from

potential fungal endophytes.