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Content uploaded by Pamoda Bhasini Ratnaweera
Author content
All content in this area was uploaded by Pamoda Bhasini Ratnaweera on Mar 09, 2018
Content may be subject to copyright.
A vast majority of fungi are composed of microscopic multicellular hyphae (with a
few unicellular species) and show cryptic lifestyles in soil and dead matter and
become noticeable only when developed fruiting bodies spores in as mushrooms or
molds. Fungi are the principal decomposers of organic matter and perform
important role in nutrient cycling in ecosystems (Cooke 2009). They establish
parasitic relationships with both plants and animals and are known to cause
widespread damage, to certain agricultural crops (Fisher et al. 2012). Endophytic
fungi on the other hand, are symbionts that spend all or part of their life cycle inter
and/or intracellularly colonizing the healthy tissues of a plant without causing any
visible manifestation of symptoms (Tan and Zou 2001). The word “endophyte”
originates from Greek, “endo”denoting within, and ‘phyte’meaning plant and was
first proposed in 1866 (Jalgaonwala et al. 2011; de Bary 1866). In addition to fungi,
bacteria including actinobacteria are reported as the major endophytes of plants
(Bandara et al. 2006).
9.2 Distribution of Endophytic Fungi
The existence of fungi inside the organs of the asymptomatic plants has been known
since nineteenth century (Guerin 1898). The first description of endophytic fungi
was made as far as back in the year 1904, from the seeds of Lolium temulentum
(Freeman 1904). Since then fungi have been found from almost every plant species
examined to date (Guo et al. 2008). Endophytic fungi have a long life history and
their diversity among plants has been found to be one of the largest (Jalgaonwala
et al. 2011).
It is noteworthy that each of the nearly 300,000 existing plant species on Earth is
assumed to host at least one or even several hundred strains of endophytes (Strobel
and Daisy 2003). Fungal endophytes are found in a range of host plants growing in
tropical, temperate, boreal forests to extreme arctic, alphine, and xeric environments
(Zhang et al. 2006). There are as many as 1.5 million different fungal species on our
planet and about 1 million of them are endophytic fungal species (Strobel and Daisy
2003; Radic and Strukelj 2012; Hawksworth 2001). Among them, only about 0.1
million fungal species including endophytic fungi have been described in the past
century (Radic and Strukelj 2012; Ganley et al. 2004). Accordingly, fungal endo-
phytes are a group of mainly undescribed organisms that potentially is a rich and
reliable source of genetic diversity.
Endophytic fungi are known to thrive asymptomatically in the tissues of plants
above ground as well as below ground, including flowers, seeds and ovules, fruits,
stems, leaves, xylem, rachis, bark, tubers, and/or roots (Zhang et al. 2006; Kusari
192 P.B. Ratnaweera and E.D. de Silva
et al. 2012). Recent studies have shown that endophytes are not host-specific
(Cohen 2006). A single species of endophytes can invade a wide range of hosts
while several studies have suggested that same fungus isolated from different parts
of the same host shows diverse abilities to utilize different substances (Carroll and
Petrini 1983), thus host endophyte relationship may vary from host to host and
endophyte in general.
9.3 The Plant-Endophyte Interaction
The relationship between the endophyte and its host may range from mutualistic
symbiosis to phytopathogenesis. Sometimes the endophyte remains latent, with
symptomless nature, inside the host plant until the environmental conditions are
favorable for the fungus or the ontogenetic state of the host changes to the
advantage of the fungus (Rodriguez and Redman 2008; Sieber 2007). Therefore,
with time, endophytic fungi can also be aggressive saprophytes or opportunistic
pathogens as well (Strobel and Daisy 2003; Tan and Zou 2001; Rodriguez and
Redman 2008).
The mutualistic relationship between the fungal endophytes and the host plants
are somewhat complex, but results in fitness benefits for both partners. The plants
provide endophytes with nutrients, protection from desiccation, spatial structure,
and transmission via seed dissemination to the next generation of host (Guo et al.
2008). The plant may also provide important chemical compounds that are essential
for the endophytes’growth and self-defense (Metz et al. 2000; Strobel 2002). On
the other hand, endophytes contribute significant benefits to their host plants by
producing a plethora of bioactive substances required to adapt to abiotic and biotic
stress factors (Guo et al. 2008). Resistance to abiotic stress is enhanced by
increasing tolerance to drought or water stress, high temperature, low pH, high
salinity and presence of heavy metals (Jalgaonwala et al. 2011). In a study done in
Lassen Volcanic and Yellowstone national park, it has been shown that an endo-
phytic Curvularia species isolated from a grass species collected from geothermal
soils gives thermotolerance to the host, probably as a result of production of cell
wall melanin that may disperse heat along fungal hyphae (Gunatilaka 2006).
A study conducted with an endophytic Penicillium minioluteum species and soy-
bean has shown that endophytic association has significantly ameliorated the
negative effects of salinity stress damage and increased the growth and metabolism
of the soybean (Khan et al. 2011).
Plants encounter biotic stress due to bacterial and fungal pathogens, and attack of
insects, nematodes, and mammalian herbivore (Rodriguez et al. 2009). The
bioactive secondary metabolites produced by the endophytes living in these plants
are known to induce resistance to biotic stress factors (Gunatilaka 2006). Previous
researches have reported that in many cases tolerance to biotic stress has been
correlated with fungal natural products (Tan and Zou 2001; Zhang et al. 2006; Aly
9 Endophytic Fungi: A Remarkable Source …193
et al. 2011). There are a number of reports describing various bioactive metabolites
produced by fungal endophytes which help the plant to increase the resistance
against biotic stress (Guo et al. 2008; Suryanarayanan et al. 2009). For example,
production of two macrocyclic alkaloids, pyrrocidines A and B with antibiotic
activity, by the endophytic fungus Acremonium zeae has been implicated in the
protection of its host, maize, against pathogenic and mycotoxin producing fungi
(He et al. 2002). In grasses and herbaceous plants, the endophytes are known to
produce toxic alkaloids that prevent or poison invertebrate and vertebrate herbi-
vores (Rodriguez et al. 2009). Accordingly in symbiotically conferred stress tol-
erance, endophytes act as a biological trigger to activate host defense system more
rapidly and strongly (Rodriguez and Redman 2008). At the same time some
endophytes are capable of enhancing the hosts’allelopathic effects on other species
growing close by, being an opponent for the space and nutrients (Newcombe et al.
2009). Apart from the above benefits, many endophytes are reported to enhance
uptake of phosphorus, and other important elements for plant growth, capable of
fixing nitrogen and producing plant hormones such as auxin, indole acetic acid,
which are essential for regulation of plant growth and development (Guo et al.
2008).
9.4 Biological Rationale in Plant Selection
Due to the vast number of plant species in the world, creative and imaginative
strategies are necessary to quickly narrow down the search for bioactive endo-
phytes. This provides the best opportunities to isolate endophytes prone to produce
novel bioactive products. Plants from distinct environmental settings and/or with an
unconventional biology are considered to be a promising source for isolating novel
endophytes bearing new secondary metabolites (Strobel 2003). Strobel and Daisy
(2003) reported several reasonable hypotheses governing the plant selection for
isolating bioactive endophytes. Selection of plants from a unique environment,
having unusual biology, using novel approaches for survival is one such strategy.
Mangrove environments are an example for hosting such plants. A second tactic is
the selection of plants that have a historic background, which have been exploited
as a source of traditional medicine. Third, plants that are endemic, having an
unusual longevity or that occupy a certain ancient land mass, have the prospect of
lodging such endophytes. Finally, plants growing in areas of high biodiversity, such
as rainforest ecosystems, are potential sources housing novel and bioactive endo-
phytic fungi (Strobel and Daisy 2003).
194 P.B. Ratnaweera and E.D. de Silva
9.5 Bioactive Metabolites from Endophytic Fungal Origin
from Different Ecological Niches
Although the discovery of endophytic fungi dates as far back as the early 1900s,
they did not receive much attention until the recent realization of their pharma-
ceutical and ecological significance (Gunatilaka 2006). Recent developments of
screening technologies have revealed that endophytic fungi are an outstanding
source of biologically active compounds with promising medicinal and agricultural
applications (Aly et al. 2011).
Tropical rainforest ecosystems are the richest ecosystems in the world containing
more than half of the Earth’s biota (Wilson 1988). The extreme biological diversity
of tropical rainforests ultimately implies the chemical diversity resulting from the
constant chemical innovations that exist in such ecosystems (Strobel and Daisy
2003). In tropical rainforests, the resources are limited due to the high species
diversity, therefore competition among species is high, and the selection pressure is
at its peak (Strobel and Daisy 2003). These factors eventually make rainforests a
potentially productive source for the discovery of novel molecular structures and
biologically active metabolites (Redell et al. 2000; Strobel and Daisy 2003).
Specific endophytes may have evolved within endemic plant species in areas of
high plant endemicity with moist, warm, and geographically isolated climates
(Strobel 2003; Strobel and Daisy 2003). This has been reported in rainforests of
Venezuela, Central America, monsoonal areas of Australia, golden triangle of
Thailand, Papua New Guinea, Madagascar, and upper Amazon regions
(Mittermeier et al. 1999). Novel endophytic fungal taxa and series of new bioactive
compounds have been discovered from each of the above areas (Mittermeier et al.
1999).
On the other hand, Strobel (2003) has stated plants growing in extremely moist
conditions or plants growing in rainforests which have a more or less constant 90–
100% relative humidity are prone to attack by certain extremely pathogenic fungi,
thus specialized defensive mechanisms in such plants are necessary for their sur-
vival. Accordingly, such disease defences may have offered by endophytes asso-
ciated with the plant (Strobel 2003). A comparative study using statistical data,
revealed that tropical plant endophytes provide more active natural products and a
larger number of secondary metabolites in comparison to that of temperate plant
endophytes (Bill et al. 2002).
The metabolite demethylasterriquinone B-1, L-783,281 (1), isolated from an
endophytic Pseudomassari sp. collected from an African rainforest tree has acted as
an antidiabetic agent (Strobel et al. 2004; Zhang et al. 1999). Unlike insulin, this
non-peptide secondary metabolite (L-783,281) does not get ineffective in the
digestive tract and thus can be a lead for an orally ingested drug for diabetes.
Similarly, Ambuic acid (2) is an antifungal agent isolated from a common rainforest
9 Endophytic Fungi: A Remarkable Source …195
endophyte Pestalotiopsis microspora (Li et al. 2001). Pestaloside (3), an aromatic
b-glucoside, and two pyrones namely pestalopyrone and hydroxypestalopyrone are
other secondary metabolites isolated from P. microspora with antifungal and
phytotoxic activities (Lee et al. 1995). Antibacterial helvolic acid (4) is a nor-
triterpenoid isolated from Xylaria sp. from an endemic endangered rainforest orchid
Anoectochilus setaceus in Sri Lanka (Ratnaweera et al. 2014). Helvolic acid has
reported for antibacterial activity against Methicillin-resistant Staphylococcus
aureus (MRSA, MIC 4 µg mL
−1
) and Bacillus subtilis (MIC: 2 µg mL
−1
).
HN
OH
NH
CH
3
H
3
C
HO
O
O
H
2
CCH
3
CH
3
(1)
H
3
CCOOH
CH
3
O
OHOH
O
(2)
OH
OH
O
O
HO
HO
HO OH
(3)
OO
C
O
OH
H3C
H3C
O
O
CH3
CH3
O
H
CH3CH3
O
H3C H3C
H
H
(4)
Several important bioactive natural products found in other terrestrial plants are
as follows. Cryptocandin (5), a peptide antifungal agent was isolated and charac-
terized from the endophytic fungus Cryptosporiopsis quercina inhabiting in the
medicinal plant Tripterygium wilfordii (Strobel et al. 1999). This compound has
shown excellent antifungal activity against several human fungal pathogens,
Candida albicans,Trichophyton spp. and number of plant pathogenic fungi,
including Sclerotinia sclerotiorum and Botrytis cinerea. Currently several compa-
nies have tested and developed Cryptocandin to use against a number of fungi
causing skin and nail diseases (Strobel 2003).
196 P.B. Ratnaweera and E.D. de Silva
Enfumafungin (6), is a hemiacetal triterpene glycoside, isolated from
Hormonema sp. comprising in mesophyll tissue of leaves of Juniperus communis L
(Aly et al. 2011). Enfumafungin is a specific inhibitor of fungal cell wall glucan
synthesis. The compound has shown in vitro antifungal activity with 0.07 µM,
EC
50
value against C. albicans (Aly et al. 2011). Extensive structural modifications
of the Enfumafungin resulted in the development of an orally available
semi-synthetic inhibitor derived from this fungal secondary metabolite. This inhi-
bitor, with EC
50
, 0.6 ng mL
−1
against C. albicans and 1.7 ng mL
−1
against
Aspergillus fumigatus, has entered phase I clinical trials as the first oral glucan
synthase inhibitor for fungal infections therapy (Motyl et al. 2010).
OH
N
H
O
N
OH
OH
NH
O
O
NH
(CH
2
)
14
O
OH
OH
HN
O
N
OH
HO
O
HN
O
H
2
N
O
HO
(5)
OHO
O
O
O
O
O
OH
HO
HO
OH HOH
(6)
Highly antibacterial naphthaquinone, javanicin (7), has been isolated from the
endophytic fungus Choridium spp. from root tissues of Azadirachta indica. The
sensitivity to javanicin with MIC value of 2 µg mL
−1
showed antibacterial activity
against Pseudomonas aeruginose and P. fluorescens (Kharwar et al. 2009). Phomol
(8) is a novel polyketide lactone with antibacterial, antifungal, and
anti-inflammatory activities, isolated from an endophytic Phomopsis sp. in
Argentinian medicinal plant, Erythrina crista-galli (Weber et al. 2004). The diter-
penoids guanacastepenes A-O, have been encountered in an unidentified endo-
phytic fungal strain CR115, occurring in Daphnopsis americana. Guanacastepenes
A(9) and I (10) exhibited antibacterial activity against drug resistant strains of
Staphylococcus aureus and Enterococcus faecalis (Brandy et al. 2001). Recent
discovery of two new metabolites, antibacterial active eupenicinicols A and B (11,
12), from an endophytic fungus, Eupenicillium sp. harbored in the roots of a
Chinese medicinal plant, Xanthium sibiricum showed the unfailing potential of
endophytes as probable antimicrobial agents (Li et al. 2014).
9 Endophytic Fungi: A Remarkable Source …197
O
O
O
H3C
OH
OH
CH3
O
CH3
(7)
O
O
HO
HO O
OHO
(8)
HO
OH
CH3
H3C
O
O
H3C O
H3C
H3C
(9)
OH
CH3
H3C
O
H3CO
H3C
H3C
O
OH
(10)
RO
O
H
H
OH
HO
H
(11) R = H
(12) R = CH3
Besides the above-mentioned endophytic fungal antibiotic metabolites, there is a
plethora of endophytes, with no certain compound isolated, but have been reported
to show strong antibiotic activity for tested microorganisms. Methanol extract of a
new endophytic fungus Colletotrichum gloeosporioides from the medicinal plant
Vitex negundo with antimicrobial activity against methicillin-penicillin-and/or
vancomycin-resistant clinical strains of S. aureus, is an example for the former
statement (Arivudainambi et al. 2011).
Cytonic acid A and B (13, 14) are two novel human cytomegalovirus protease
inhibitors isolated from endophytic fungus Cytonaema sp. (Guo et al. 2000). The
absence of appropriate antiviral screening systems in most programs is the main
limitation in this type of compound discovery. Cytochalasins are alkaloids, com-
mon in endophytic Xylaria,Phoma, and Hypoxylon spp. exhibiting antitumor
activities (Wagenaar et al. 2000). Torreyanic acid (15), is a selectively cytotoxic
unusual dimeric quinone isolated from Pestalotiopsis microspora endophytic to the
endangered tree Torrya taxifolia (Lee et al. 1996). Torreyanic acid, in general has
demonstrated 5–10 times more potency to several cancer cell lines that are sensitive
to protein kinase C agonists and caused cell death by apoptosis (Lee et al. 1996).
A recent study has reported a new epitetrathiodioxopiperizine, secoemestrin D (16)
from an endophytic fungal strain Emericella sp., occurred in mesophyll of
Astrgalus lentiginosus. Secoemestrin D exhibited significant cytotoxic activity with
IC
50
values ranging from 0.06 to 0.24 µM and moderate selectivity to human
glioma and metastatic breast adenocarcinoma cell lines (Xu et al. 2013).
198 P.B. Ratnaweera and E.D. de Silva
O
O
O
OR2
R1
OH
O
OH
OH
OHHO
(13)
R1 : (CH2)4CH3
R2: (CH2)2CH3
(14)
R1 : (CH2)2CH3
R2 : (CH2)4CH3
CH3
O
C
O
OH
O
O
H
C
CH3
O
OH
O
CH3
O
O
O
H
CH3
O
(15)
O
O
N
N
O
OH
O
OH
OCH3
H
S4
O
(16)
Apart from the antibiotic activities, endophytic fungi have been a potential
source of various other interesting behaviors. Nodulisporic acid A (17) is an
insecticidal fungal metabolite isolated from endophytic Nodulisporium sp. from the
Hawaiian plant Bontia daphnoides. This compound has shown systemic efficacy
against fleas by modulating an invertebrate-specific glutamate-gated ion channel
and has resulted in identifying a potent and effective oral agent for control of fleas
and ticks in mammals (Ondeyka et al. 1997; Aly et al. 2011). Subglutinols A and B
(18, 19) are immunosuppressive compounds produced by endophytic fungus
Fusarium subglutinans, from Tripterygium wilfordii. Both compounds showed IC
50
value of 0.1 µM in the mixed lymphocyte reaction assay (Lee et al. 1995). Pestacin
(20) and isopestacin (21) are two antioxidants secreted by an endophytic
P. microspora isolated from Timonius morobensis growing on the north coast of
Papua New Guinea (Strobel et al. 2002; Harper et al. 2003). A new alkaloid named
16a-hydroxy-5 N-acetylardeemin (22), demonstrating acetylcholineesterase inhi-
bitory activity (EC
50
: 58.3 µM), has been isolated from a fermented broth of
endophytic A. terreus from stems of Artemisia annua collected from the Zijin
Mountain in China (Ge et al. 2010).
9 Endophytic Fungi: A Remarkable Source …199
N
O
O
OH
HOH
OHO
(17)
OCH3
CH3
O OH
CH3
HO
HHCH3
CH3
H
CH3
(18) *S
(19) *R
*
O
H3C
OH
HO
OH
H
(20)
O
HO
OH
O
OH
H3C
(21)
N
N
N
N
O
H
OH
O
O
H
(22)
Shipunov et al. (2008) have mentioned that in the host’s invaded range, endo-
phytes increase the competitiveness of the host by producing metabolites inhibitory
to evolutionarily native plants. An endophytic fungus Fusarium sp. of the invasive
cactus Opuntia dillenii contained antimicrobial secondary metabolite equisetin (23)
(Ratnaweera et al. 2015a). The production of such biologically active substances
may enhance the competitive ability of the host against microorganisms and per-
haps increase its adaptability to withstand the biotic and harsh abiotic stress factors
that assist in the successful establishment of O. dillenii to the detriment of native
plants in the area.
Various workers have reported grasses and sedges are reservoirs for a number of
endophytic fungi and result in enhancement of the ecological fitness and tolerance
to biotic and abiotic environmental stresses (Gunatilaka 2006; Mukhtar et al. 2010).
In grasses and herbaceous plants, the endophytes are known to produce toxic
alkaloids that prevent or poison invertebrate and vertebrate herbivores (Rodriguez
et al. 2009). A Korean study has shown endophytic fungal isolates of the roots of
Monochoria vaginalis, a weed of rice paddy significantly promote the growth of the
plant mainly due to higher secretions of Gibberellins (Ahmad et al. 2010). Among
the bioactive secondary metabolites, solanioic acid (24) isolated from Rhizoctonia
solani from Cyperus rotundus showed antibacterial activity (Ratnaweera et al.
200 P.B. Ratnaweera and E.D. de Silva
2015b). Solanioic acid has a highly functionalized and rearranged steroidal carbon
skeleton and is a potent antibiotic, active at 1 µg/mL against MRSA. The endo-
phytic Aspergillus strain CY725 and Rhizoctonia sp. strain Cy064 isolated from the
leaves of Cynodon dactylon, have afforded antimicrobial helvolic acid, rhizoctonic
acid, monomethylsulochrin and ergosterol (Li et al. 2005; Ma et al. 2004).
Paspalum conjugatum harbored an endophytic Microthyriaceae sp. which con-
tained a known mycotoxin sterigmatocystin which exhibited antiparasitic activity
against Trypanosoma cruzi, with an IC
50
value of 0.13 µmol L
−1
and a novel
polyketide integrasone B (Almeida et al. 2014).
Mangrove forests are considered as biodiversity ‘hotspots’for marine-derived
fungi (Shearer et al. 2007). This is mainly because, the permanently and inter-
mittently submerged mangrove trunks and aerating roots are host to terrestrial,
marine and an overlap of terrestrial and marine fungi (Sarma and Hyde 2001;
Shearer et al. 2007). According to Schmit and Shearer (2003), 106 fungi have been
reported from mangrove habitats in the Atlantic Ocean, while 173 and 128 are
documented from Pacific and Indian Ocean mangroves, respectively. Among the
mangrove-derived fungal community, the fungal endophytes play an important role
protecting their host against various aggressions (Cheng et al. 2009). According to
reports more than 200 species of endophytic fungi have been isolated and identified
from mangrove plants and dominant among them are species of Alternaria,
Aspergillus,Cladosporium,Colletotrichum,Fusarium,Paecilamyces,Penicillium,
Pestalotiopsis,Phoma,Phomopsis,Phyllosticta, and Trichoderma (Liu et al. 2007).
Mangrove-derived endophytic fungi are believed to contribute to their hosts’ability
to adapt to endure the extreme habitat conditions (Debbab et al. 2013). In addition,
these mangrove endophytic fungi are proven to be a promising source of struc-
turally unique natural products, and drug leads with remarkable bioactivities (Tan
et al. 2008). Cytosporone B (25) is such a novel natural product, isolated from an
endophytic Dothiorella sp. from mangrove plant Avicennia marina at an estuary in
China, with broad antifungal activities against A. niger,Trichoderma sp. and
Fusarium sp. and high activity against human epidermal carcinoma and several
other cell lines (Xu et al. 2005). Recent report of two new antibacterial a-pyrone
derivatives, infectopyrones A (26) and B (27) from the mangrove endophytic
fungus, Stemphylium sp. isolated from a Brguiera sp. also demonstrates the
potential of mangrove endophytes to produce bioactive chemical scaffolds (Zhou
et al. 2014). Two new compounds pinazaphilones B and (±)-penifupyrone with
significant a-glucosidase inhibitory activity have been discovered from a mangrove
endophytic Penicillium sp. isolated from the fresh branches of the mangrove plant
Cerbera manghas (Liu et al. 2015).
9 Endophytic Fungi: A Remarkable Source …201
N
H3C
H
H
H3C
OH
CH3
O
O
HO
CH3
(23)
CHO
CHO
HO
COOH
(24)
n-C7H15
O
HO OH
OEt
O
(25)
HO
O
O
OH
O
O
(26)
O
O
O
OH
O
O
(27)
Mangrove associates are species mainly distributed in terrestrial or aquatic
habitat but also occur in the mangrove ecosystem (Parani et al. 1998). According to
Tomlinson criteria, mangrove associates are also distinguished from true mangroves
by lacking aerial roots, vivipary, and no physiological mechanism for salt exclusion
(Wang et al. 2011). However, mangrove associates growing in the mangrove
habitat also have to face the same extreme ecological conditions as the true man-
groves. Therefore, these mangrove associates also have the potential of producing
bioactive natural products as the true mangroves. This is evident by the recent
report of Ratnaweera et al. (2016), who described the isolation of antimicrobial
gliotoxin (28) and Bisdethiobis (methylthio) gliotoxin (29) from an extract of the
endophytic fungus Hypocrea virens from the plant Premna serratifolia from a
mangrove habitat.
Inland fresh water bodies also are productive ecosystems in the world which
house diverse microorganisms. Aquatic plants highly adapted to its environmental
and ecological conditions also harbor endophytic fungi having bioactive metabo-
lites. A recent investigation of endophytic fungi of Nymphaea nouchali led to the
isolation of the known secondary metabolites chaetoglobosin A and C (30, 31) from
Chaetomium globosum, with chaetoglobisn A showing good antibacterial activities
(Dissanayaka et al. 2016).
202 P.B. Ratnaweera and E.D. de Silva
NN
O
O
OH
CH
3
OH
S
S
H
(28)
NN
O
O
OH
CH
3
OHH
SCH
3
SCH
3
(29)
N
H
HN
O
H
OO
OOH
(30)
N
H
HN
O
H
OO
OO
(31)
Among the vast diversity of marine-derived fungi are endophytic fungi from
macro-algae, sea grasses and other marine plants. Most of these fungi belong to
class Ascomycota and their distribution is governed by plant metabolites, temper-
ature, salinity and pH (Ji and Wang 2016). These fungi are proven to be prolific
producers of huge array of bioactive natural products. Up to date more than 300
natural products have been identified from endophytic fungi of marine macro-algae.
From the published natural products 43% were reported novel compounds with
various biological activities such as antioxidant, anticancer, antiplaspodial, and
antimicrobial (Flewelling et al. 2015). Among the novel antimicrobial metabolites
are Asperamide A, B (32,33), Asporyzin A-C (34–36) and Asperversin A (37),
from endophytic Aspergillus spp., isolated from Colpomenia sinuosa,
Heterosiphonia japonica, and Sargassum thunbergii, respectively (Zhang et al.
2007; Qiao et al. 2010; Miao et al. 2012). Myrocin A (38) and asperwentin A-C
(39–41) are some of the anticancer compounds isolated from Apiospora montagnei
from Polsiphonia violacea and Aspergillus wentii from Sargassum fusiforme
(Klemke et al. 2004; Miao et al. 2014). Three 2-pyridone alkaloids, the known
N-hydroxy-2-pyridone PF1140 (42), and two new 2-pyridones 43 and 44 have been
isolated from a Penicillium species associated with the New Zealand marine brown
algae Xiphophora gladiata (de Silva et al. 2009). PF1140 was active against B.
subtilis and C. albicans and to that of murine leukemia P388 cells. Both 43 and 44
were inactive pointing to the importance of the presence of the N–OH functionality
meant for bioactivity.
9 Endophytic Fungi: A Remarkable Source …203
O
OH
R1
HO
R2
OH
O
HN
OOH
OH
(32) R1: OH, R2: H
(33) R1: H, R2: OH
N
HO
O
O
H
H
H
(34)
N
O
OH
O
H
H
H
(35)
N
HOH
H
H
OH
(36)
OO
O
OH O O
OH
H
O
OO
(37)
O
OH
O
OH
O
OH
(38)
OH
R
(39) R=H2
(40) R=O
O
OH
(41)
N
O
O
R
H
(42) R=OH
(43) R =H
N
H
OH
O
H
OH
(44)
204 P.B. Ratnaweera and E.D. de Silva
9.6 Production of Similar Metabolites by Endophytic
Fungi and Host Plants
The long co-evolution of endophytes with their host plants has resulted a genetic
recombination. It has opened the path for some endophytes to produce the same
bioactive compounds originally characteristic of the host plant (Tan and Zou 2001).
Taxol (45), Berberine (46), Sanguinarine (47) Camptothecin (CPT) (48) producing
endophytic fungi are examples for this phenomenon. Taxol (paclitaxel) (45), the
first billion dollar anticancer drug was discovered initially from Taxus brevifolia
and later from 11 other Taxus species in the world (Stierie et al. 1993). Therefore, as
an alternative source, taxol producing endophytic fungi have been investigated from
these yew plants, and Taxomyces andreanae, was the initially discovered taxol
producing endophytic fungus from host plant Taxus brevifolia (Strobel 2003).
Berberine (46), with diverse pharmacological properties is an isoquinoline
alkaloid isolated from several medicinal plants including Berberis aristata,
Hydrastis canadensis,Coptis chinensis,Coptis rhizome,Coptis japonica,
Phellondendron amurense, and Coscinium fenestratum (Timothy et al. 1997;
Tillhon et al. 2012). This natural product is currently undergoing 10 clinical trials
(Tillhon et al. 2012). Berberine has also been reported from the endophytic fungus,
Fusarium solani, from the roots of Coscinium fenestratum a critically endangered
plant species (Diana and Agastian 2013). Since C. fenestratum also been reported to
produce berberine, it supports the theory that, with the long co-evolution with the
host, an endophyte can adapt to the special microenvironments through genetic
modification which includes uptake of some plant DNA into their own genomes
(Germaine et al. 2004; Diana and Agastian 2013).
Sanguinarine (47) is an antimicrobial benzylisoquinoline alkaloid reported from
several plants belonging to the family Papaveraceae including Macleaya cordata
(Nicoletti and Fiorentino 2015). This compound has also been isolated from the
endophytic fungal strain of Fusarium proliferatum inhabiting the leaves of
Macleaya cordata (Wang et al. 2014). CPT (48) is another anticancer agent first
isolated from the extracts of Camptotheca acuminata, and later from several other
plants (Wall et al. 1966; Asano et al. 2004). The production of CPT in Ophiorrhiza
mungos was first reported by Tafur et al. (1976). Later, Salim et al. (2011) isolated
the CPT producing endophytic fungus Glomerella cingulata from O. mungos
providing an alternative strategy to reduce the need to harvest slow-growing and
possibly rare plants consequently helping to preserve the world’s ever diminishing
biodiversity. In addition, it is easier and more economical to produce a valued
phytochemical by exploiting a microbial source than using a plant, which even-
tually leads to increase availability and low market price (Radic and Strukelj 2012).
9 Endophytic Fungi: A Remarkable Source …205
NH
O
O
OH
HO O
HO
O
OH
O
O
O
O
(45)
N+
O
O
OCH3
OCH3
(46)
N+
O
O
(47)
N
N
O
O
O
OH
(48)
9.7 Factors Influencing the Production of Secondary
Metabolites of Endophytic Fungi
In the natural setting, the climatic conditions, soil, season, location, age and tissue
of the host plant, all affect the endophytes’biology, and consequently considerable
variations in the production of secondary metabolites (Strobel and Daisy 2003).
Therefore, the chemical substances isolated from two endophytic fungi of the same
species may differ from each other. At the same time, the differences also in the
isolation methods and in vitro cultivation conditions can impact the kind and range
of secondary metabolites (Gunatilaka 2006). It has been reported that the size of the
plant tissue fragments used for the isolation, time since harvesting of the tissue,
composition of the culture media and culture conditions such as aeration, temper-
ature, pH, incubation period, agitation, shape of the culturing flask (with respect to
liquid media), all affect the production of secondary metabolites in laboratory (Aly
et al. 2011; Kusari et al. 2012). Even, the production of six new secondary
metabolites by the plant associated fungus Paraphaeosphaeria quadriseptata, only
when the water used to make the media changed from tap water to distilled water is
a good example to prove this fact (Paranagama et al. 2007).
206 P.B. Ratnaweera and E.D. de Silva
9.8 Current Challenges
To achieve a competent endophyte with promising bioactivity is a challenging task
(Scherlach and Hertweck 2009; Kusari et al. 2012). In the traditional way, this
requires screening of a plethora of endophytes from a host. Most of the isolating
endophytes are incompetent, i.e., they do not possess desired potential to produce
bioactivity. However, whole-genome sequencing strategies have revealed that the
incompetent endophytes express only a subset of biosynthetic genes under labo-
ratory culture conditions (Scherlach and Hertweck 2009; Winter et al. 2011).
Therefore there is possibility of utilizing the large reservoir of cryptic natural
metabolites by experimenting with different in vitro culture conditions and under-
standing the chemical ecological interactions of endophytes (Kusari et al. 2012).
Another major challenge is the low yield of the active desirable compound/s
obtained from the cultures (Yu et al. 2010; Aly et al. 2011). This is a major
drawback for bioactive compounds from entering the commercial industry. For an
example, the yield of the anticancer drug paclitaxel obtained from several endo-
phytic fungal cultures are 846.1, 187.6 and 163.4 µg L
−1
, which is too low for
commercial production (Gangadevi and Muthumary 2008; Liu et al. 2009).
Therefore, so far fungal endophytes have not been an industrial source of paclitaxel
(Aly et al. 2011) or any other pharmaceutical. However, genetic engineering
technology and research identifying the regulatory gene/s in the biosynthesis
pathway of the active compound can lead to increase production of the compounds
(Yu et al. 2010; Radic and Strukelj 2012).
The mammalian toxicity of any prospective drug developed has also become a
major concern in the field (Yu et al. 2010). Most of these isolated bioactive
metabolites precluded clinical use due to the toxicities to animals and humans
(Waring and Beaver 1996). However, Strobel (2003) stated that plants as an
eukaryotic system, have naturally served as a selection system for microbes having
bioactive molecules with reduced toxicity toward higher organisms.
Compounds showing moderate biological activity most often cannot be used as
potential chemotherapeutic agents (Yu et al. 2010). Totally or partly unknown
biosynthesis, regulation, and synthesis, of these natural products in endophytes are
other issues in the field (Yu et al. 2010). The rapidly diminishing rainforests, which
is a huge reservoir for novel fungal endophytes and their bioactive products, is one
of the major problems facing the future in this area (Strobel 2003). Therefore,
countries need to establish information repositories of their biodiversity and at the
same time should take conservation measures to protect the biodiversity.
Despite speculation by many authors no endophytic fungal-derived metabolite
has so far become commercially useful (Kusari and Spiteller 2011). However,
interest in the biosynthetic abilities of the endophytic fungi by the scientific com-
munity has not diminished but in fact is on the rise.
9 Endophytic Fungi: A Remarkable Source …207
9.9 Conclusion
Exploration of competent endophyte for only subset of biosynthetic genes
expressed under laboratory culture conditions is not enough to utilize the large
reservoir of natural metabolites produced endophytes. Therefore, incorporation of
genetic manipulation technology so as to advance the research to identify the
regulatory gene/s of several biosynthesis pathways of metabolite production can
lead to increase production of the compounds to be used in human welfare. As such,
innovative approaches are bound to result in the productive utilization of this
important and remarkable resource of much potential in the coming years.
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