1
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.