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MICROBIOLOGY RESEARCH ADVANCES
ENDOPHYTIC FUNGI
DIVERSITY, CHARACTERIZATION
AND BIOCONTROL
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ENDOPHYTIC FUNGI
DIVERSITY, CHARACTERIZATION
AND BIOCONTROL
EVELYN HUGHES
EDITOR
New York

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Library of Congress Cataloging-in-Publication Data
Names: Hughes, Evelyn, editor.

Title: Endophytic fungi : diversity, characterization and biocontrol /
editor, Evelyn Hughes.
Description: Hauppauge, New York : Nova Science Publisher's, Inc., [2016] |
Series: Microbiology research advances | Includes bibliographical
references and index.
Identifiers: LCCN 2016045425 (print) | LCCN 2016048986 (ebook) | ISBN
9781536103410 (hardcover) | ISBN 9781536103588 (ebook) | ISBN
9781536103588
Subjects: LCSH: Endophytic fungi. | Metabolites
Classification: LCC QK604.2.E53 E52 2016 (print) | LCC QK604.2.E53 (ebook) |
DDC 579.5/1785--dc23
LC record available at https://lccn.loc.gov/2016045425
Published by Nova Science Publishers, Inc. † New York

CONTENTS
Preface vii
Chapter 1 Endophytic Fungi: Occurrence, Classification,
Function and Natural Products 1
Afra Khiralla, Rosella Spina, Sakina Yagi,
Ietidal Mohamed and Dominique Laurain-Mattar
Chapter 2 Endophytic Fungi Are Multifunctional
Biosynthesizers: Ecological Role
and Chemical Diversity 39
Khaled A. Selim, Mohamed M. S. Nagia
and Dina E. El. Ghwas
Chapter 3 Endophytic Fungi Isolated from Vochysia divergens
in the Pantanal, Mato Grosso Do Sul: Diversity,
Phylogeny and Biocontrol of Phyllosticta citricarpa 93
Y. M. Hokama, D. C. Savi, B. Assad, R. Aluizio,
J. A. Gomes-Figueiredo, D. M. Adamoski,
Y. M. Possiede and C. Glienke
Chapter 4 Dark Septate Endophytes (DSE) in Polluted Areas 125
Elena Fernández-Miranda Cagigal
Index 147

PREFACE
Endophytic fungi are important biotechnological tools because they

produce many secondary metabolites. However, to access this important
source of bioactive molecules, it is essential to explore the diversity of
endophytic fungi and catalog their species richness in different ecosystems.
This book reviews the diversity, characterization and biocontrol of endophytic
fungi.
Chapter 1 – Introduction: Researches on endophytic fungi have proven
they are a promising source of biocontrol agents. These organisms are present
in the internal healthy plant tissues during a part or/all of their life cycle
without causing apparent harm to their hosts. They influence greatly the
physiological activities of their host plants. Fungal endophytes enhance their
host resistance against abiotic stress, disease, insects and mammalian
herbivores by producing a broad range of fungal metabolites. Indeed several
interesting metabolites isolated from endophytic fungi belong to diverse
chemical classes, including: alkaloids, steroids, flavonoids, terpenoids,
quinones and phenols. Since the isolation of paclitaxel in 1993 from an
endophytic fungus of Pacific Yew, fungal endophytes took a consider attention
as alternative source of active compounds produced by their host plants,
however they could be an alternative source of novel natural products for
exploitation in modern medicine, agriculture and industry. Conclusion: The
chapter sets out to present general overview of endophytic fungi and focus on
their occurrence, classification, functions and several classes of their
secondary metabolites. Finally examples are given concerning natural products
isolated from fungal endophytes with potent biological activity.
Chapter 2 – Symbiosis is a widespread phenomenon in nature. Endophytes
are defined as all microorganisms that colonize asymptomatically within living

viii Evelyn Hughes
healthy tissues. In general, endophytes are considered as commensalistic

symbionts, where they receive nutrients and shelter from the host. In return,
they are thought to provide the host with chemical constituents that can be
used in the growth or defense mechanisms. Fungal endophytes have attracted a
great interest to microbiologists, chemists and ecologists as a treasure of
biological resource, because they play diverse indispensable roles in the
ecosystem for stress tolerance, eco-adaptation, and promoting growth and
development. Recently, endophytic fungi have drawn a particular attention,
due to their considerable biodiversity, unparalleled metabolic pathways and
unique habitats. Therefore, they were considered as an unusual source of novel
secondary metabolites, exhibiting a variety of biological activity, which are in
use in modern agriculture, pharmaceutical and biotechnological industry. In
the last two decades, the extensive discovery of endophytic secondary
metabolites reflected the tremendous chemical diversity of different natural
compounds classes with incredible bioactivity, but still the chemistry of
endophytes needs to be comprehensively studied. On the other hand, the
search for alternative sources of fuels is becoming increasingly important and
biodiesel has been shown to be one of the most promising alternatives. Fungal
endophytes have been reported to produce volatile low molecular mass
hydrocarbons such as alcohols, alkenes and trepenoidal mycodisel. In this
review, the authors will focus on characterization and diversity of endophytic
fungi, with highlighting their ecological role in nature. Besides, they will
emphasis on the variety of chemical classes and the wide spectrum biological
functions of endophytic metabolites as well as their potential as an energy
source for biofuel production.
Chapter 3 – Endophytic fungi are important biotechnological tools
because they produce many secondary metabolites. However, to access this
important source of bioactive molecules, it is essential to explore the diversity
of endophytic fungi and catalog their species richness in different ecosystems.
Tropical regions are recognized as areas of high diversity, although many
areas remain unexplored, such as the Pantanal of Mato Grosso do Sul, Brazil.
This study is the first to explore the diversity of endophytic fungi in the
medicinal plant Vochysia divergens, found in the Pantanal. In total, 77 isolates
were identified by ITS1–5.8S–ITS2 rDNA sequencing and phylogenetic
analysis as belonging to the genera Antrodia, Irpex, Peniophora, Phyllosticta,
Neofusicoccum, Pseudofusicoccum, Polyporus, Daldinia, Nigrospora,
Colletotrichum, Diaporthe, Lanceispora, Cladosporium, Phaeosphaeria, and
Annellosympodiella. Nineteen isolates were identified as belonging to the
Xylariaceae family, and the data indicate that these isolates are members of a

Preface ix
new genus in this family. The authors also explored the antifungal activity of
three isolates, two of which belong to the family Xylariaceae (LGMF1119 and
LGMF1133) and one belongs to the genus Nigrospora (LGMF1121) that
inhibited Phyllosticta citricarpa mycelium growth and pycnidia formation in
vitro assays.
Chapter 4 – Dark septate endophytes (DSE) constitute a very
heterogeneous group of Ascomycetes characterized by a septate and melanized
mycelium. Inside, tissues show intra- and intercellular development and are
able not only to generate mantle and Hartig net but also to produce typical
intracellular structures (microsclerotia), all without causing apparent damage
to the plant. DSE were previously thought to be restricted to infertile boreal or
alpine habitats, where arbuscular mycorrhizal fungi cannot persist. However,
in recent years DSE have been found extensively distributed in polluted areas
around the world, supporting a growing body of evidence that points to a
prominent ecological role, even when these organisms have not been studied
from the physiological role of a host-fungi perspective. It has been
hypothesized that DSE dominance as root endophytes might relate to their
melanised cell walls, known to play an important function in heavy metal
immobilization by sequestration. In addition to the improved nutritional
performance associated with mycorrhizal fungi, this capacity provides the
plant with an extra feature. Due to the promising role on ecological
reforestation of the DSE, further research is needed, including new approaches
(molecular, histological and physiological) that will allow to better
characterize the relationship between these fungi and plants growing in
polluted areas.

In: Endophytic Fungi ISBN: 978-1-53610-341-0
Editor: Evelyn Hughes © 2017 Nova Science Publishers, Inc.
Chapter 1
ENDOPHYTIC FUNGI: OCCURRENCE,

CLASSIFICATION, FUNCTION
AND NATURAL PRODUCTS
Afra Khiralla1,2, Rosella Spina1,2, Sakina Yagi3,

Ietidal Mohamed3 and Dominique Laurain-Mattar1,2,
1
Université de Lorraine, SRSMC, Vandœuvre-lès-Nancy, France
2
CNRS, SRSMC, Vandœuvre-lès-Nancy, France
3
Botany Department, Faculty of Science,
University of Khartoum, Khartoum, Sudan
ABSTRACT
Introduction: Researches on endophytic fungi have proven they are a
promising source of biocontrol agents. These organisms are present in the
internal healthy plant tissues during a part or/all of their life cycle without
causing apparent harm to their hosts. They influence greatly the
physiological activities of their host plants. Fungal endophytes enhance
their host resistance against abiotic stress, disease, insects and
mammalian herbivores by producing a broad range of fungal metabolites.
Indeed several interesting metabolites isolated from endophytic fungi
belong to diverse chemical classes, including: alkaloids, steroids,
flavonoids, terpenoids, quinones and phenols. Since the isolation of
paclitaxel in 1993 from an endophytic fungus of Pacific Yew, fungal

Corresponding author E-mail address: dominique.mattar@univ-lorraine.fr.

2 Afra Khiralla, Rosella Spina, Sakina Yagi et al.
endophytes took a consider attention as alternative source of active

compounds produced by their host plants, however they could be an
alternative source of novel natural products for exploitation in modern
medicine, agriculture and industry.
Conclusion: The chapter sets out to present general overview of
endophytic fungi and focus on their occurrence, classification, functions
and several classes of their secondary metabolites. Finally examples are
given concerning natural products isolated from fungal endophytes with
potent biological activity.
1. INTRODUCTION
The term “endophyte” is derived from the Greek, endon = within and
phyte = plant. It was first introduced in 1866 by de Bary. It was used broadly
to refer to any organism found within tissues of living plants; including
everything from virulent foliar pathogens to mycorrhizal root sombionts;
subsequent re-definitions led to confusion regarding the meaning of the term.
Modern mycologists generally agree that endophytes are organisms that
colonize internal plant tissues without causing apparent harm to their host.
Different groups of organisms such as fungi, bacteria, actinomycetes and
mycoplasma are reported as endophytes of plants (Arnold, 2007).
Collectively, more than 100 years of research suggest that most, if not all,
plants in natural ecosystems are symbiotic with mycorrhizal fungi and/or
fungal endophytes (Petrini, 1986). Unlike mycorrhizal fungi that colonize
plant roots and grow into the rhizosphere, endophytes reside entirely within
plant tissues and may grow within roots, stems and/or leaves, emerging to
often occur sparsely as hypha in the intercellular fluids and wall spaces of their
plant hosts, sporulate at plant or host-tissue senescence (Bacon and White,
2000). Studies of endophytic fungi were initiated nearly 200 years ago, when
Person in 1772 described the species Sphaeria typhena, now known as
Epichloe typhina (Pers.) Tul. (Khan, 2007). Fossils, in a 400-million-year-old,
indicated that plants have been associated with endophytes. Krings et al.,
(2007) studied petrographic thin sections of the Rhynie chert plant Nothia
aphylla, they found that three fungal endophytes occur in prostrata axes of this
plant.

Endophytic Fungi 3
2. RELATIONSHIPS AND OCCURRENCE OF ENDOPHYTES

WITHIN THE HOST PLANT TISSUES
Endophytic fungi have been recovered from plants in hot deserts, Arctic
tundra, mangroves, temperate and tropical forests, grasslands and savannas,
and croplands. They are known from mosses and other nonvascular plants,
ferns and other seedless plants, conifers, and flowering plants. Their biological
diversity is enormous, especially in temperate and tropical rainforests. The
fungi are hosted in nearly 300,000 land plant species, with each plant hosting
one or more of these fungi (Arnold, 2008).
A variety of relationships exist between fungal endophytes and their host
plants, ranging from mutualistic or symbiotic to antagonistic or slightly
pathogenic (Arnold, 2007). Results from grass-endophyte systems suggest that
endophytes are herbivore antagonists and enhance plant growth (Clay, 1990).
Correspondingly, mutualistic antagonism towards insects and pathogens has
been claimed also for forest endophytes (Faeth, 2002). Furthermore, a
significant number of fungi exhibit multiple ecological roles, such as the
human pathogen and soil saprotroph Coccidioides posadasii. Similarly, fungi
such as Chaetomium globosum are known as endophytes, saprotrophs, and
pathogens (Arnold and Engelbrecht, 2007). Although it is not yet clear
whether the same genotypes can play each of these roles with equal success,
the ecological lability of these species is remarkable. Understanding the
mechanisms behind that lability represents one among many frontiers in
endophyte biology (Arnold, 2007).
Figure 1. Endophytic fungi hyphae and conidia within a healthy plant tissues
(Photos by Afra Khiralla).

Host-specificity is the relationship in which a fungus is restricted to a

single host or a group of related species, but does not occur in other unrelated
plants in the same habitat (Holliday, 1998). Petrini (1991) used two different
terms, establishment specificity and expression specificity, to identify the
relationship. Establishment specificity was defined when an endophyte
colonizes only selected plant species, while expression specificity is
colonization of several hosts by a given fungus, but forming specific structures
(usually fruiting bodies) on a limited number of plant taxa. However, some
researchers found no or very little evidence of host-specificity in endophytes
(Umali et al., 1999, Andrew, 2000; Khiralla, 2015).
Added to that, some endophytes revealed tissue specificity. Bagchi and
Banerjee (2013) studied the tissue specificity symbiosis; they isolated
endophytic fungi from leaf, petiole and stem of Bauhinia vahlii. They found
that, the colonization frequency of endophytic fungi is much higher in petiole
(86.67%) in comparison to stem (77.33%) and leaf (70.67%). Whereas, some
researchers reported that endophytic fungal colonization is higher in leaf
segments rather than stem segments of some tropical medicinal plants
(Raviraja, 2005; Banerjee and Mahapatra, 2010).
Besides, some researchers stated other host relationship phenomenons
such as host-selectivity and host-preference. Host-selectivity is described
when one endophytic fungal species may form relationships with two related
plant species, but demonstrate a preference for one particular host (Cohen,
2006). The term host-preference, however, is more frequently used by
mycologists to indicate a common occurrence or uniqueness of the occurrence
of a fungus on a particular host. The differences in endophyte assemble from
different hosts might be related to the chemical differences of the host (Paulus
et al., 2006).
3. DOES CLIMATE EFFECTING FUNGAL

ENDOPHYTE COMMUNITIES?
Environmental factors, such as rainfall and atmospheric humidity might
influence the occurrence of some fungal endophyte species (Petrini, 1991;
Selvanathan et al., 2011). In the Sudan Khiralla et al., (2015; 2016) attempted
to analyze the diversity of the culturable fungal endophytes in 5 plant species
that are used in the traditional medicine. Only between three and six strains
were recovered from the leaves, stems and/or seeds per plant, however the

Endophytic Fungi 5
isolation of the endophtyes was performed during the dry months (October to
January). Chareprasert et al., (2006) investigated the seasonal variation effects
on endophyte communities into leaves of two different plants, the sampling
was done during one year (January to December). They found that the lower
number of isolates recovered from trees during the dry season. Rodrigues
(1994) suggested that, the lower number of isolates recovered during the dry
season could be related to the effects of water stress. It is known that under
water deficit, some plants may accumulate non-structural carbohydrates. This
accumulation generally leads to build up of carbon-based defences such as
tannins, making the plant less susceptible to fungal endophyte colonization
during the dry season. However, a copious studies indicated the enhancement
effect of endophytes on the host plants, these effects could be summerized by
increase biomass production, decrease stomatal conductance, and reduce
overall water loss (Elmi and West, 1995; Kannadan and Rudgers, 2008;
Rodriguez et al., 2008; Kane, 2011).
4. CLASSIFICATION OF ENDOPHYTIC FUNGI

Schaechter (2011) stated that endophytic fungi have frequently been
divided into two major groups based on differences in taxonomy, host range,
colonization transmission patterns, tissue specificity and ecological function.
Group one is the Clavicipitaceous endophytes (C-endophytes) which infect
some grasses. Group two is the Nonclavicipitaceous endophytes (NC-
endophytes). While Rodriguez et al., (2009) stated another point of view of
fungal endophytes classification, they classified them into four classes. Two
major endophytic groups (Clavicipitaceous and Nonclavicipitaceous) based on
phylogeny data and life history traits. However, they classified
nonclavicipitaceous endophytes into three functional groups based on host
colonization and transmission, in planta biodiversity and fitness benefits
conferred to hosts.
4.1. Clavicipitaceous Endophytes (Class I)
The Clavicipitaceae is a family of fungi (Hypocreales; Ascomycota)

including free living and symbiotic species associated with insects and fungi
or grasses, rushes and sedges (Bancon and White, 2000). Many of its members
produce alkaloids which are toxic to animals and humans. Clavicipitaceous

endophytes of grasses were first noted by European investigators in the late

19th century in seeds of Lolium temulentum, L. arvense, L. linicolum, and L.
remotum (Guerin, 1898; Vogl, 1898). From their earliest discovery,
investigators hypothesized a link to toxic syndromes experienced by animals
that consume infected tissues. And these hypotheses were tested when Bacon
et al., (1977) linked the endophyte Neotyphodium coenophialum to the
widespread occurrence of ‘summer syndrome’ toxicosis in cattle grazing tall
fescue pastures (Festuca arundinacea).
Mycelium of clavicipitaceous endophytes occurs in intercellular spaces of
leaf sheaths, culms, and rhizomes, and may also be present, if sparsely, on the
surface of leaf blades (White et al.,1996; Moy et al., 2000; Dugan et al., 2002;
Tadych et al., 2007).
The effects of clavicipitaceous endophytes on host plant are listed below:
 Insects deterrence
Most clavicipitaceous endophytes enhance resistance of hosts to insect

feeding; the benefits arise in part from the production of alkaloidic mycotoxins
loline and peramine which are generally associated with resistance to insects
(Rowan and Gaynor, 1986; Clay, 1990; Patterson et al., 1991; Riedell et al.,
1991).
 Mammalian herbivores deterrence
Some clavicipitaceous endophytes have been reported to deter feeding by

mammalian herbivores, because they produced mycotoxins such like ergot and
lolitrem alkaloids (White, 1987; Gentile et al., 1999).
 Reduction of nematodes
Also some studies indicated that clavicipitaceous endophytes had anti-

nematode activity; Kimmons et al., (1990) stated that infection of tall fescus
Festuca arundinacea with an endophytic fungus Acremonium coenophialum
has been shown to reduce nematode population’s in field soils.

Endophytic Fungi 7
 Increase resistance of host disease
Some studies indicated that clavicipitaceous endophytes produced indole

derivative compounds, a sesquiterpene, and a diacetamide from Epichloë
festucae that inhibit the growth of other pathogenic fungi (Lee, 2010).
 Enhance the ecophysiology of host plants
Clavicipitaceous endophytes enhance the ecophysiology of host plants and

enable plants to counter abiotic stresses such as drought (Arechavaleta et al.,
1989) and metal contamination. For example, Neothyphodium coenophialum
infection leads to the development of extensive root systems that enable plants
to better acquire soil moisture and absorb nutrients, resulting in drought
avoidance and faster recovery from water stress. In some cases, endophytes
stimulate longer root hairs and enhance exudation of ‘phenolic-like
compounds’ into the rhizosphere, resulting in more efficient absorption of soil
phosphorus and enhanced aluminum tolerance via chelation (Malinowski and
Belesky, 2000).
4.2. Nonclavicipitaceous Endophytes (Class II)
Traditionally NC-endophytes treated as a single functional group, while

Rodriguez et al., (2009), who showed that NC-endophytes represent three
distinct functional groups.
Class II endophytes include the hyperdiverse endophytic fungi associated
with leaves of tropical trees (Lodge et al., 1996; Fröhlich and Hyde, 1999;
Arnold, et al., 2000; Gamboa and Bayman, 2001), as well as the highly diverse
associates of above-ground tissues of nonvascular plants, seedless vascular
plants, conifers, and woody and herbaceous angiosperms in biomes ranging
from tropical forests to boreal and Arctic/Antarctic communities (Carroll and
Carroll, 1978; Petrini, 1986; Stone, 1988).
Most fungal endophytes species belong to Ascomycetes, with a minority
of Basidiomycetes. Fungal group ‘dark septate endophytes’ (DSE) are
distinguished as a functional group based on the presence of darkly melanized
septa.
The effects of these endophytes on host plant are listed below:

 Avoiding abiotic stress
One attribute that appears unique to Class II NC-endophytes is the ability

of individual isolates to asymptomatically colonize and confer habitat-adapted,
fitness benefits on genetically distant host species representing monocots and
eudicots (Rodriguez et al., 2009). This phenomenon was discovered by
comparing fitness benefits conferred by Class II endophytes in plants growing
in geothermal soils Curvularia protuberate, coastal beaches Fusarium
culmorum and agricultural fields Colletotrichum spp. (Redman et al., 2002;
Márquez et al., 2007).
 Increase of biomass
Most of class II endophytes examined have increased host shoot and/or

root biomass. Tudzynski and Sharon (2002) stated that this was a result of the
induction of plant hormones by the host or biosynthesis of plant hormones by
the fungi.
 Protection from fungal pathogens
Many endophytes of class II protect hosts to some extent against fungal

pathogens (Danielsen and Jensen, 1999; Narisawa et al., 2002; Campanile et
al., 2007) by different strategies like production of secondary metabolites
(Schulz et al., 1999). Few studies revealed interactions with host defenses;
fungal parasitism (Samuels et al., 2000); induction of systemic resistance (Vu
et al., 2006); or competition with endophytes for resources or niche space.
5. ENDOPHYTES VERSUS EPIPHYTES

Endophytes are often contrasted with epiphytes, which live on external
plant surfaces (Santamaria and Bayman, 2005). In practice, the distinction is
that epiphytes can be washed of plant surfaces or be inactivated by surface
disinfection, usually with sodium hypochlorite and ethanol to break surface
tension, whereas endophytes cannot. Thus, an epiphyte that survives surface
disinfection and grows in culture might be assumed to be an endophyte
(Arnold and Lutzoni, 2007). Although there are few studies comparing
phylloplane and endophytic fungal communities of the same leaves,

Endophytic Fungi 9
comparisons within pine and coffee leaves indicate that endophytic

communities are distinct from epiphytic ones, even though they may live less
than a millimeter apart (Santamaria and Bayman, 2005). Temporally as well as
practically, the distinction between endophytes and epiphytes is often
arbitrary. Many horizontally transmitted endophytes presumably start growing
on the surface of the leaf before penetration. Also, endophytes may become
epiphytes when internal tissues are exposed, and may protect the exposed
tissues from the environment. In shoot tip–derived tissue cultures of Pinus
sylvestris, calli were found to be covered by hyphae of the endophytes
Hormonema dematioides, Rhodotorula minuta, and associated biofilms
(Pirttila et al., 2002). How such endophytes coordinate function, interact with
other microbiome biofilm components, and affect plant fitness needs further
exploration.
6. FUNGI AS SOURCE OF BIOACTIVE COMPOUNDS

6.1. Fungal Metabolites
Fungal metabolites are diverse including those associated with proteins

synthesis and respiration. Several secondary metabolites have been isolated
and frequently, chemically defined. Some of these are waste products while
others such as pigments, toxins, and antibiotics clearly have biological
functions. Because of their synthetic abilities, fungi are used in industry for the
production of alcohol, citric acid and other organic acids, various enzymes,
riboflavin, (Kirk et al., 2008).
Schulz et al., (2002) emphasis on fungal endophytes secondary
metabolites. Through 12 years studying endophyte metabolites, they found a
correlation between biological activity of fungal metabolites and biotope. They
reported that a higher proportion of the endophytic fungi exhibited biological
activity than the soil isolates did; whereas 83% of the algal isolates and 80% of
endophytic fungi from plants inhabited at least one of the test organisms for
antibacterial, fungicidal, algicidal or herbicidal activities and only 64% of
those from soil did. Also they had isolated compounds belonged to different
structural groups: terpenoids, steroids, xanthones, chinones, phenols,
isocoumarines, benzopyranones, tetralones, cytochalasines and enniatines.

6.2. Diverse Classes of Isolated Natural Products

from Endophytic Fungi
6.2.1. Alkaloids
Alkaloids are naturally occurring chemical compounds containing basic
nitrogen atoms. Alkaloids are quite common secondary metabolites in
endophytes, and some of them showed also anti-microbial activities (Souza et
al., 2004). Chaetoglobosins A, G, V, Vb, and C were characterized from the
culture of an endophytic Chaetomium globosum isolated from Ginkgo biloba.
Some of them revealed cytotoxicity (Li et al., 2014). A great interest that some
of the most potent of these plant-derived antitumor alkaloids have also been
reported as isolates from endophytic fungi. These endophytes have usually
been associated with a host organism that has also been reported to produce
the compound of interest. Camptothecin (CPT) was isolated in 2005 from a
fungal endophyte isolated from the inner bark of Nothapodytes foetida
identified as Entrophosphora infrequens (Puri et al., 2005). Vincristine
(Oncovin®), also known as leurocristine, is a vinca alkaloid originally isolated
from Catharanthus roseus. It has been isolated by different researchers from
the Catharanthus roseus endophyte Fusarium oxysporum (Zhang et al., 2000).
Chaetoglobosin U is a cytochalasin-based alkaloid isolated from Chaetomium
globosum, an endophytic fungus residing within the stem of healthy Imperata
cylindrical (Ding et al., 2006).
6.2.2. Phenols
Phenols and phenolic acids have often been isolated from some endophyte
cultures originating from a variety of the host plants (Yu et al., 2010).
Pestalachloride A and B had a significant anti-fungal activity against three
plant pathogens (Li et al., 2008a). Pestalachloride C and D showed moderate
antibacterial activity (Li et al., 2008b). Furthermore, two isomeric novel
tridepsides cytonic acids A and B were reported as human cytomegalovirus
(an ubiquitous opportunistic pathogen) protease inhibitors from the culture of
the endophytic fungus Cytonaema sp. isolated from Quercus sp (Guo et al.,
2000). Tricin and related flavone glycosides, toxic to mosquito larvae, have
been isolated from endophyte-infected blue grass Poa ampla (Tan and Zou,
2001). Two antimicrobial flavonoids were isolated from the culture extract of
endophytic fungus Nodulisporium sp. from Juniperus cedre on Gomera Island
(Dai et al., 2006).

Endophytic Fungi 11
6.2.3. Steroids
Many steroids are produced by endophytes, but most of the isolated
compounds showed moderate antimicrobial activities. Along with ergosterol,
3β,5α,6β-trihydroxyergosta-7,22-diene; 3β-hydroxyergosta-5-ene; 3-
oxoergosta-4,6,8,22-tetraene; 3β-hydroxy-5α,8α-epidioxyergosta-6,22-diene;
3β-hydroxy-5α,8α-epidioxyergosta-6,9,22-triene and 3-oxoergosta-4-ene, two
new steroids, 3β,5α-dihydroxy-6β-acetoxyergosta-7,22-diene and 3β,5α-
dihydroxy-6β-phenyl- acetoxyergosta-7,22-diene were characterized from the
liquid culture of an fungal endophyte Colletotrichum sp. of Artemisia annua.
Some of these metabolites were shown to be antifungal against some crop
pathogens Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis,
Helminthosporium sativum and Phytophthora capisici (Lu et al., 2000; Yu et
al., 2010).
6.2.4. Terpenoids
Sesquiterpenes, diterpenoids and triterpenoids are the major terpenoids
isolated from endophytes (Yu et al., 2010). In period of 2006-2010, sixty five
sesquiterpenes, fourty five diterpenes, five monoterpenes and twelve other
terpenes, amounting to 127 terpenoids were isolated from endophytic fungi
and all have biological activity such as anti-microbial, anti-cancer and anti-
protozoa (Souza et al., 2011).
Three novel eremophilane-type sesquiterpenes were isolated from the
endophyte Xylaria sp. associated with Licuala spinosa. The three compounds,
eremophilanolide 1, 2 and 3 exhibited moderate cytotoxic activity with IC50
values of 3.8–21 µM against cancer cell lines KB, MCF-7, and NCI-H187
(Isaka et al., 2010). Two ent-eudesmane sesquiterpenes, ent-4(15)-eudesmen-
11-ol-1-one and ent-4(15)-eudesmen-1R, 11-diol were isolated from the
endophytic fungus Eutypella sp. BCC 13199 from the plant Etlingera littoralis
(Earth ginger) (Isaka et al., 2009). Four cytotoxic sesquiterpene compounds, 8-
deoxytrichothecin, trichothecolone, 7α-hydroxytrichodermol and 7α-
hydroxyscirpene, were isolated from fungal isolate KLAR 5, a mitosporic
Hypocreales found in a healthy twig of the Thai medicinal plant Knema
laurina. Also, tauranin, merulin A and C, sesquiterpene compounds, were
isolated from endophytic fungi with cytotoxic activity (Kharwar et al., 2011).
Two insect toxins of a pimarane diterpene framework were isolated from
the broth of an unidentified endophyte from a needle of the balsam fir Abies
balsamea (Tan and Zou, 2001). Subglutinol A and B, immunosuppressive but
noncytotoxic, were produced by Fusarium subglutinans, an endophytic fungus
from the perennial twining vine Tripterygium wilfordii (Lee et al., 1995).

Guanacastepene, a novel diterpenoid produced by an unidentified fungus from

the branch of Daphnopsis americana growing in Guanacaste, Costa Rica, was
shown to be antibacterial against methicillin-resistant Staphylococcus aureus
and vancomycin-resistant Enterococcus faecium (Tan and Zou, 2001). Taxol
originally characterized from the inner bark of the Pacific yew, Taxus
brevifolia, is an efficacious anticancer diterpene found in extremely small
quantities in slowly growing Taxus species. Taxol has been isolated from
Taxomyces andreanae an endophytic fungus recovered from T. brevifolia
(Stirele et al., 1993).
6.2.5. Quinones
Some endophytes produced quinones displaying significant growth
inhibition against phytopathogens such as spiroketals from Edenia
gomzpompae (Wiyakrutta et al., 2004). Torreyanic acid is an unusual dimeric
quinone isolated from Pestalotiopsis microspora, an endophyte of Torreya
taxifolia (Lee et al., 1996). Insecticidal rugulosin was characterized from
Hormonema dematioides, an endophytic fungus of balsam fir. from cultures of
an unidentified endophyte obtained from an eastern larch (Larix laricina)
needle, 8,1’,5’-trihydroxy-3’,4’dihydro-1’H-[2,4’]binaphthalenyl-1,4,2’-trione
was characterized as a toxin to spruce budworm larvae (Findlay et al., 1997).
Preussomerin N1, palmarumycin CP4a, and palmarumycin CP5 were new ras
farnesyl-protein transferase inhibitors produced by an endophytic
Coniothyrium sp. (Tan and Zou, 2001). A highly hydroxylated quinone
altersolanol A, characterized from phytopathogenic Alternaria spp., was
reisolated from an endophytic Phoma multirostrata with its antibacterial
activity disclosed (Yang et al., 1994).
6.2.6. Peptides
Many peptides produced by endophytes displayed significant
antimicrobial activities, such as leucinostatin A produced by Acremonium sp.
(Strobel et al., 1997a). Leucinostatin A was isolated almost forty years ago
from cultures of Penicillium lilacum. It has received much attention over the
years, because of its potent biological activity against several different cell
lines. Acremonium sp., isolated from Taxus baccata, was also shown to
produce Leucinostatin A and leucinostatin A di-O-b-glucoside when grown in
liquid culture (Kharwar et al., 2011). The cyclopeptides echinocandins A, B, D
and H, were produced by Aspergillus rugulosus and A. nidulans var.
echinulatus. Further they were isolated from endophytes Cryptosporiopsis sp.
and Pezicula sp. in Pinus sylvestris and Fagus sylvatica respectively, and

Endophytic Fungi 13
shown to be antimicrobial. Cryptocandin, a cyclopeptide with potent

antifungal activities, is a metabolite of endophyte Cryptosporiopsis cf.
quercina of red wood (Tan and Zou, 2001). Cryptocandin exhibited resistant
against multiple human pathogens including Candida albicans and
Histoplasma capsulatum causal agent of the lung disease Histoplasmosis, in
addition to Trichophyton rubrum and Trichophyton mentagrophytes.
Cryptocandin is also active against a number of plant-pathogenic fungi
including Sclerotinia sclerotiorum and Botrytis cinerea (Strobel et al., 1999b).
Two new cyclic pentapeptides, cyclo-(L-Phe-L-Leu1-L-Leu2-L-Leu3-L-Ile)
and cyclo-(Phe-Val-Leu–Leu-Leu) were purified from the culture of
endophytic fungus strain (No. 2524) which was recovered from Avicennia
marina (Forsk.) Vierh. seeds. They demonstrated inhibitory activity against
the human cancer cell line Bel-7402. Cellular viability was 67 % at a dose of
15 µg mL-1, whereas no dose related effects were detected for dosages
between 15 and 500 µg mL-1 (Li et al., 2004; Li et al., 2005). Five hybrid
peptide-polyketides, curvularides A–E, were obtained from the endophytic
fungus Curvularia geniculata, isolated from the limbs of Catunaregam
tomentosa. Curvularide B demonstrated antifungal activity against C. albicans,
and it also exhibited synergistic activity with a fluconazole drug (Chomcheon
et al., 2010). A detailed review concluding several isolated endophytic fungi
peptides was presented by Abdalla and Matasyoh (2014).
6.2.7. Polyketides
A new polyketide synthase−nonribosomal peptide synthetase hybrid
pericoannosin B, was isolated from the endophytic fungus Periconia sp. F-31
which grew inside the medicinal plant Annona muricata (Zhang et al, 2016).
Codinaeopsin, a tryptophan−polyketide hybrid, was isolated from an
endophytic fungus CR127A that was collected from a white yemeri tree
Vochysia guatemalensis in Costa Rica. Codinaeopsin revealed activity against
Plasmodium falciparum, the causative agent of the most lethal form of
malaria, with IC50 = 2.3 μg mL-1 or 4.7 μM (Kontnik and Clardy, 2008).
Chaetomugilin D, together with three known metabolites, chaetomugilin A,
chaetoglobosins A and C, has been isolated from the EtOAc extract of the
cultures of Chaetomium globosum, an endophytic fungus found in the leaves
of Ginkgo biloba. These compounds displayed significant growth inhibitory
activity against the brine shrimp Artemia salina and Mucor miehei (Qin et al.,
2009). Six major heterodimeric polyketides, acremoxanthone derivative:
Acremoxanthones A, B, C and acremonidins A and B were obtained from the
culture of endophytic fungus Acremonium camptosporum isolated from the

leaves of Bursera simaruba (Gonzalez et al., 2015). An endophytic fungus

Epicoccum sp. CAFTBO, obtained from Theobroma cacao was found to
produce three polyoxygenated polyketides, namely epicolactone, epicoccolides
A and B. These compounds showed potent antimicrobial activities and
significant inhibitory effects on the mycelia growth of two peronosporomycete
phytopathogens, Pythium ultimum and Aphanomyces cochlioides, and the
basidiomycetous fungus Rhizoctonia solani (Talontsi et al., 2013).
6.2.8. Acids
Li et al., (2016) stated that an endophytic fungus from Salvia miltiorrhiza
produces salvianolic acid C as its host plant. This fungal endophyte identified
as Phoma glomerata D14. Khiralla (2015) isolated a new acid 3,7,11,15-
Tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethyl- hexacosa-
4E,8E,12E,16,18-pentaenoic acid; Khair acid from the solid culture of the
endophyte Curvularia papendorfii isolated from Vernonia amygdalina. This
acid revealed an average antibacterial effect against methicillin-resistant
Staphylococcus aureus with MIC value of 62.5 μg mL-1. Zhao et al., (2012)
reported three endophytic fungi Fusarium solani, F. oxysporum and F.
proliferatum from pigeon pea Cajanus cajan producing cajaninstilbene acid
(CSA). This acid is one of the major stilbenes found in pigeon pea. However,
cajaninstilbene acid revealed hypotriglycerimic, hypoglycemic, anti-
inflammatory, analgesic and antioxidant activities. Cytonic acids A and B have
been isolated from the solid-state fermentation of the endophytic fungi
Cytonaema sp. Cytonic acids A and B showed potential anti-viral effect
against human cytomegalovirus (hCMV) protease with values of IC50 = 43µM
and IC50 =11µM respectively (Guo et al., 2000). Two new 10-oxo-10H-
phenaleno[1,2,3-de]chromene-2-carboxylic acids, xanalteric acids I and II
were purified from extracts of the endophytic fungus Alternaria sp., isolated
from the mangrove plant Sonneratia alba collected in China. The two acids
exhibited weak antibiotic activity with MIC values of 125 and 250 μg mL-1
respectively against multidrug-resistant Staphylococcus aureus (Kjer et al.,
2009). The endophytic fungus Cryptosporiopsis cf. quercina produces
cryptocin in culture. This unique tetramic acid displays powerful antimycotic
activity against several plant pathogenic fungal strains including Pythium
ultimum, Pyricularia oryzae, with MIC values 0.39 and 0.78 μg mL-1
respectively (Li et al., 2000). Two new acids were isolated from Mangrove
endophytic fungus (No. ZZF13) (Xia et al., 2008).

Endophytic Fungi 15
Several other compounds belonging to different chemical classes have

been reported such as: aldehydes, chromones, cyclohexanones, esters,
lactones, xanthones (Kharwar et al., 2011).
7. SIGNIFICANCE OF ENDOPHYTES ASSOCIATED

WITH MEDICINAL PLANTS
Many of the pharmaceuticals currently available to physicians have a long

history of use as herbal remedies, including opium, aspirin, digitalin, and
quinine. According to World Health Organization (WHO) estimation, 80% of
the population of some Asian and African countries presently use herbal
medicine for some aspect of primary health care (Alves and Rosa, 2007). In
recent years a lot of efforts have been employed to identify novel molecules
derived from natural sources that exhibit a range of clinical and
pharmacological activities. This thus led to an extensive research on organic
substances synthesized by various plants and microorganisms, growing in
diverse habitats and displaying a range of habit. Endophytic fungi of medicinal
plants are considered as an attractive source of novel bioactive compounds.
This ability is of great importance, it provides an alternative strategy for
reducing the need to harvest slow growing and possibly rare plants, also help
to preserve the world’s ever diminishing biodiversity. Moreover, the
production of a high value phytochemical by exploiting a microbial source is
easier and more economical and it leads to increased availability and the
reduced market price of the product (Strobel et al., 2004). The significance of
endophytic fungi associated with medicinal plants spouts from different
approaches:
1) Some endophytes produce the same natural products as their host

plants (Tan and Zou, 2001; Strobel and Daisy, 2003). Such is the case
of paclitaxel-producing fungus, Taxomyces andreanae, from the yew
Taxus brevifolia (Stierle et al., 1993). At least nineteen genera of
endophytic fungi (Alternaria, Aspergillus, Botryodiplodia, Botrytis,
Cladosporium, Ectostroma, Fusarium, Metarhizium, Monochaetia,
Mucor, Ozonium, Papulaspora, Periconia, Pestalotia, Pestalotiopsis,
Phyllosticta, Pithomyces, Taxomyces, Tubercularia) were screened to
have the ability to produce paclitaxel and its analogues (Zhao et al.,
2010). Alternaria sp. an endophyte vinblastine-producing was isolated
from Catharanthus roseus (Guo et al., 1998). Camptothecin was

isolated from at least three endophytes genera including Fusarium,

Nothapodytes, Neurospora (Zhao et al., 2010; Amna et al., 2006).
However, around six genera of endophytic fungi: Alternaria,
Fusarium, Monilia, Penicillium, Phialocephala, Trametes were found
to be capable to produce podophyllotoxin, the potent compound with
anticancer, antiviral, antioxidant, antibacterial, immunostimulation
and anti-rheumatic activities. In addition, this compound has been
used as a precursor for chemical synthesis of the anticancer drugs like
etoposide, teniposide and etopophose phosphate (Zhao et al., 2010).
2) Endophytes from plants with ethnobotanical history might have
interesting biological activities. Yu et al., (2010) screened the
presence of endophytes and revealed antimicrobial agents into
medicinal plants and different plants in special environments
frequently. They found that 35% of endophytes which were isolated
from the medicinal plants had an antimicrobial activity, 29% from
crops, while 18% from plants in special environment. However,
Strobel and Daisy (2003) pointed out also the significance of plants in
special environments. For example, Strobel et al., (1999a) studied an
aquatic plant Rhyncholacis penicillat collected from a river system in
Southwest Venezuela. They postulated that, aquatic environment
created many portals through which common phytopathogenic
oomycetes could enter into the plant. Still, the plant population
appeared to be healthy, possibly due to protection from an endophytic
product. Eventually, an endophytic fungus Serratia marcescens which
recovered from R. penicillat founded to produce oocydin A a potent
antioomycetous compounds.
3) The diversity of the biological activities could be obtained by the
same fungal endophyte strain isolated from different medicinal plants,
increasing the opportunities to isolate a plenty of new compounds.
Added to that, the various natural products produced by endophytic
fungi possess unique structures, thus representing a huge reservoir
which offers an enormous potential for exploitation in agricultural and
industrial areas (Tan and Zou, 2001).
4) Bioprospecting from endophytic fungi and their natural products is
one avenue for the discovery of novel pharmaceuticals. A great
number of novel compounds were isolated from endophytes
possessing several potent properties including antioxidant, anticancer,
antimicrobial, antifungal and antiviral activities (Strobel and Daisy,
2003; Huang et al., 2007; Kharwar et al., 2011; Yu et al., 2010).
Table 1 presents a summary of some isolated bioactive agents from
endophytic fungi associated with medicinal plants.

Table 1. Some isolated compounds with anti-oxidant, anti-bacterial, anti-fungal, anti-viral and anti-cancer activities
from endophytic fungi
Compounds Biological activity Fungal endophytes Host plants References

Khair acid Anti-bacterial Curvularia papendorfii Vernonia amygdalina (Khiralla, 2015)
Conidiogenone B Anti-bacterial Penicillium chrysogenum Laurencia sp. (Gao et al., 2012)
Conidiogenol
Diversonol Anti-bacterial Microdiplodia sp. Lycium intricatum (Siddiqui et al., 2011)
Ergosterol
Microdiplodiasol
Microdiplodiasone
Microdiplodiasolol
8 α-Acetoxyphomadecalin Anti-bacterial Microdiplodia sp. Pinus sp. (Hatakeyama et al.,
C Phomadecalin E 2010)
Helvolic acid Anti-bacterial Pichia guilliermondii Paris polyphylla (Zhao et al., 2010)
Diepoxin κ Anti-bacterial Dzf12 Dioscorea (Cai et al., 2009)
zingiberensis
Alterporriol N and E Anti-bacterial Stemphylium globuliferuman Mentha pulegium (Debbab et al., 2009)
Xananteric aids I and II Anti-bacterial Alternaria sp. Sonneratia alba (Kjer et al., 2009)
Javanicin Anti-bacterial Chloridium sp. Azadirachta indica (Kharwar et al., 2008)
Pestalone Anti-bacterial Pestalotia sp. Rosenvingea sp. (Cueto et al., 2001)
Colletotric acid Anti-bacterial Colletotrichum Artemisia mongolica (Zou et al., 2000)
gloeosporioides
Guanacastepene Anti-bacterial CR115 Daphnopsis americana (Singh et al., 2000)
Phomosines A–C Anti-bacterial Phomopsis sp. Teucrium scorodonia (Krohn et al., 1995)
Chaetoglobosin A Anti-cancer Chaetomium globosum Ginkgo biloba (Li et al., 2014)
Brefeldin A Anti-cancer Aspergillus clavatus Taxus mairei (Kharwar et al., 2011)

Table 1. (Continued)

Brefeldin A Anti-cancer Paecilomyces sp. Torreya grandis (Kharwar et al., 2011)
Merulin A and C Anti-cancer XG8D (Basidiomycete) Xylocarpus grantum (Chokpaiboon et al.,
2010)
Anthracenedione Anti-cancer Halorosellinia sp. Mangrove plant (Zhang et al., 2010)
Guignardia sp.
Pestaloficiol I Anti-cancer Pestalotiopsis fici Camellia sinensis (Liu et al., 2009)
Pestaloficiol J
Pestaloficiol K
Pestaloficiol L
Cochliodinol Anti-cancer Chaetomium sp. Salvia officinalis (Debbab et al., 2009)
Isocochliodinol
Tauranin Anti-cancer Phyllosticta spinarum Platyclocarpus (Wijeratne et al., 2008)
granatum
Camptothecin Anti-cancer Neurospora crassa Camptotheca (Rehman et al., 2008)
acuminata
Alternariol Anti-cancer Alternaria sp. Polygonum (Aly et al., 2008)
Alternusin senegalense
Daldinone C Anti-cancer Hypoxylon truncatum Artemisia annua (Gu et al., 2007)
Daldinone D
Beauvericin Anti-cancer Fusarium oxysporium Ephedra fasciculata (Zhan et al., 2007)
Podophyllotoxin Anti-cancer Trametes hirsuta Podophyllum (Puri et al., 2006)
hexandrum
Chaetopyanin Anti-cancer Chaetomium globosum Polysiphonia (Wang et al., 2006)
urceolata

Chaetoglobosin U Anti-cancer Chaetomium globosum Imperata cylindrica (Ding et al., 2006)
Chaetoglobosin C
Chaetoglobosin F
Chaetoglobosin E
Penochalasin A
Camptothecin Anti-cancer Entrophospora infrequens Nothapodytes foetida (Puri et al., 2005)
Two cyclic pentapeptides Anti-cancer Strain 2524 Avicennia marina (Li et al., 2004; 2005)
Globosumone A Anti-cancer Chaetomium globosum Ephedra fasciculata (Bashyal et al., 2005)
Globosumone B
Dicerandrol A Anti-cancer Phomopsis longicolla Dicerandra frutescens (Wagenaar and Clardy,
2001)
Phomoxanthone Anti-cancer Phomopsis sp. Tectona grandis (Isaka et al., 2001)
Phomoxanthone
Vincristine Anti-cancer Fusarium oxysporum Catharanthus roseus (Zhang et al., 2000)
Sequoiatones A Anti-cancer Aspergillus parasiticus Sequoia sempervirens (Stierle et al., 1999)
Sequoiatones B
Leucinostatin A Anti-cancer, Acremonium sp. Taxus baccata (Strobel et al., 1997a)
Anti-fungal
Torreyanic acid Anti-cancer Pestalotiopsis microspora Torreya taxifolia (Lee et al., 1996)
Cytochalasin 1 Anti-cancer Rhinocladiella sp. Tripterygium wilfordii (Lee et al., 1995)
Cytochalasin 2
Cytochalasin 3
Cytochalasin E
Paclitaxel Anti-cancer Taxomyces andreanae Taxus brevifolia (Stierle et al., 1993)
Periconicin Anti-cancer Periconia atropurpurea Xylopia aromatica (Stierle et al., 1993)
Epicolactone, Anti-fungal Epicoccum sp. Theobroma cacao (Talontsi et al, 2013)
Epicoccolides A and B


Phomenone Anti-fungal Xylaria sp. Piper aduncum (Silva et al., 2010)
Curvularides A–E Anti-fungal Curvularia geniculata Catunaregam (Chomcheon et al., 2010)
tomentosa
Cycloepoxylactone Anti-fungal Phomopsis sp. Laurus azorica (Hussain et al., 2009)
Cycloepoxytriol B Anti-bacterial
Pestalachloride A Anti-fungal Pestalotiopsis adusta Unknown Chinese tree (Li et al., 2008a)
Isofusidienol A, B, C, and Anti-fungal Chalara sp. (strain 6661) Artemisia vulgaris (Lösgen et al., 2008)
D
Isofusidienol A–D Anti-fungal Chalara sp. Artemisia vulgaris (Lösgen et al., 2008)
Altenusin Anti-fungal Alternaria sp Trixis vauthieri (Cota et al., 2008;
Anti-parasitic Johann et al., 2012)
Trichodermin Anti-fungal Trichoderma harzianum Ilex cornuta (Chen et al., 2007)
Nodulisporins A-C Anti-fungal Nodulisporium sp. Juniperus cedrus (Dai et al., 2006; 2009 )
Nodulisporins D-F Anti-bacterial Erica arborea
Anti-algal
3,12-Dihydroxycadalene Anti-fungal Phomopsis cassiae Cassia spectabilis (Silva et al., 2006)
Pyrrocidines A and B Anti-fungal Acremonium zeae Zea mays (Wicklow et al., 2005)
Cryptocin Anti-fungal Cryptosporiopsis cf. quercina Tripterygium wilfordii (Li et al., 2000)
Cryptocandin Anti-fungal Cryptosporiopsis cf. quercina Tvipterigeum wilfordii (Strobel et al., 1999b)
Oocydin A Anti-fungal Serratia marcescens Rhyncholacis (Strobel et al., 1999a)
penicillata
Mullein Anti-fungal Pezicula livida Fagus sylvatica (Schulz et al., 1995)
Anti-bacterial

Gamahonolide A and B Anti-fungal Epichloe typhina Phleum pretense (Hiroyuki et al., 1992)
Cinnamic acid Anti-oxidant Strain M7226 Curcuma wenyujin (Yan et al., 2014)
Kaempferol Anti-oxidant
Flavipin Anti-oxidant Chaetomium globosum Ginkgo biloba (Ye et al., 2013)
4,6-dihydroxy-5-meth- Anti-oxidant Cephalosporium sp. Sinarundinaria nitida (Huang et al., 2012)
oxy-7-methylphthalide.
Cajaninstilbene acid Anti-oxidant Fusarium solani Cajanus cajan (Zhao et al., 2012)
Hypotriglycerimic, F. oxysporum
Hypoglycemic, F. proliferatum
Anti-inflammatory,
Analgesic
Monocerin Anti-oxidant Colletotrichum sp. Piper ornatum (Tianpanich et al., 2011)
Fusarentin
A pure compound
Chlorogenic acid Anti-oxidant Sordariomycete sp. Eucommia ulmoides (Chen et al., 2010)
Corynesidones A, B Anti-oxidant Corynespora cassiicola Lindenbergia (Chomcheon et al., 2009)

philippensis
Chaetopyranin Anti-oxidant, Chaetomium globosum Polysiphonia (Wang et al., 2006)
Cytotoxic urceolata
Graphislactone A Anti-oxidant Microsphaeropsis olivacea Pilgerodendron (Hormazabal et al., 2005;
Cephalosporium sp. uviferum Song et al., 2005)
Trachelospermum
jasminoides
Pestacin Anti-oxidant Pestalotiopsis microspora Terminalia morobensis (Harper et al., 2003)
Isopestacin


Pestalotheols C Anti-viral Pestalotiopsis theae Unidentified tree (Li et al., 2008a)
Pestalotheol C Anti-viral Pestalotiopsis theae Unidentified tree (Li et al., 2008b)
Brefeldin A Anti-viral, Paecilomyces sp. Taxus mairei (Wang et al., 2007;
Anti-bacterial, Aspergillus clavatus Torreya grandis Betina, 1992)
Anti-fungal,
Anti-nematode
Cytonic acids A and B Anti-viral Cytonaema sp. Quercus sp. (Guo et al., 2000)
Palmarumycin CP17 Anti-leishmanial Edenia sp. Petrea volubilis (Martínez-Luis et al.,
Palmarumycin CP18 2008)
Codinaeopsin Anti-malarial CR127A Vochysia (Kontnik and Clardy,
guatemalensis 2008)
Subglutinol A Immunosuppressive Fusarium subglutinans Tripterygium wilfordii (Lim et al., 2015)

Endophytic Fungi 23
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BIOGRAPHICAL SKETCH
Dr. Afra Ahmed Isamil Khiralla
Affiliation: Université de Lorraine, SRSMC, UMR 7565, BP 70239, F-

54506 Vandœuvre-lès-Nancy, France

Education:
2015, PhD in Chemistry (Phytochemistry), Doctoral School SESAMES

SRSMC UMR 7565, Faculty of Sciences and Technologies, University of
Lorraine, Nancy, France.
2008, Master in Botany, Botany Department, Faculty of Sciences,
University of Khartoum, Khartoum, Sudan.
2004, Bachelor of Sciences (Honors) Second class-Division (I), Faculty of
Sciences, Botany department, University of Khartoum, Khartoum, Sudan.
Professional Experience:
2015 - 2016 University of Lorraine

UMR 1121/ INRA, Laboratory of Agronomy and environment. University
of Lorraine. Nancy, France.
Position: Assistant researcher
Duties: Work on project between University of Lorraine and Syngenta
Company on the ecophysological effect of two fungicides on wheat.
2010 - 2015 Shendi University

Botany Department. Faculty of Sciences and Technologies. Shendi
University. Shendi, Sudan.
Position: lecturer
Duties: Taught different botany courses include: Virology, Bacteriology,
Mycology and Plant physiology. Supervision researches.
2008 - 2010 Omdurman Islamic University

Department of Cognosy, Faculty of Pharmacy, Omdurman Islamic
University. Khartoum, Sudan
Position: lecturer (part-time)
Duties: Taught Botany and Pharmacognosy practical lessons.
2004- 2007 University of Khartoum

Department of Botany, Faculty of Sciences, University of Khartoum.
Khartoum, Sudan.
Position: Teaching assistant (part-time)
Duties: Taught different botany courses include: Mycology and Plant
pathology practical lessons.

Endophytic Fungi 37
Researches:
 Endophytic fungi associated with medicinal plants from Sudan.

 A Study on the ecological group coprophilous (Dung) fungi in
Khartoum.
 Banana Post harvest diseases in Khartoum.
Publications Last Three Years
Scientific Papers
Khiralla A, Mohamed I, Tzanova T, Schohn H, Slezack-Deschaumes S, Hehn

A, André P, Carre G, Spina R, Lobstein A, Yagi S, Laurain-Mattar D.
Endophytic fungi associated with Sudanese medicinal plants show
cytotoxic and antibiotic potential. FMES microbiol. Lett. DOI:
http://dx.doi.org/10.1093/femsle/fnw089. 2016.
Khiralla A, Mohamed I, Thomas J, Mignard B, Spina R, Yagi S & Laurain-
Mattar D A pilot study of antioxidant potential of endophytic fungi from
some Sudanese medicinal plants. Asian Pac. J. Trop. Med. 2015. 8 (9):
701-704.
Posters:
Khiralla A, Mohamed I, Spina R, Boisbrun M, Lemiere P, Slezack-

Deschaumes S, Hehn A, Tzvetomira T, Schohn H, André P, Muller CD,
Lobstein A, Yagi, S, Laurain-Mattar D. Future perspectives on the
endophyte Curvularia papendorfii: a source of cytotoxic and antibacterial
agents. Focused Meeting 2016: The Dynamic Fungus. Exeter, UK. 2016.
Khiralla A, Fadl Almoulah N , Mohamed I, Yagi S, Babikr R, Tzanova T,
Slezack-Deschaumes S, Hehn A, André P, Carre G, Lobstein A, Schohn
H, Spina R, Laurain-Mattar D. Aromatic and medicinal plants from
Sudan: a source of promising bioactive compounds. Phytoday. University
of Lorraine. Nancy, France. 2016.
Khiralla A, Mohmed I, Thomas J, Mignard B , Spina R, Yagi S, Laurain-
Mattar D. Potential antioxidant resource of endophytic fungi from some
Sudanese Medicinal plants. Phytoday. University of Strasbourg.
Strasbourg, France. 2015.

Communications:
Yagi S, Mohamed I, Khiralla A, Mahdi T, Laurain-Mattar D. Biological

activity of endophytic fungi associated with medicinal plants from Sudan.
The 16th NAPRECA Symposium on Natural Products, Arusha, Tanzania.
2015.
Khiralla A, Yagi S, Laurain-Mattar D. Antibacterial activity of endophytic
fungus isolated from Vernonia amygdalina (Asteraceae). Journée de
rentrée. Ecole Doctorale SESAMES. University of Lorraine. Metz,
France. 2014.
Khiralla A, Yagi S, Laurain-Mattar D. Anti-oxidant and anti-cancer
metabolites from endophytic fungi isolated from Sudanese Tropical
plants. Journée de Printemps. Ecole Doctorale SESAMES. University of
Lorraine. Nancy, France. 2013.

Chapter 2
ENDOPHYTIC FUNGI ARE

MULTIFUNCTIONAL BIOSYNTHESIZERS:
ECOLOGICAL ROLE
AND CHEMICAL DIVERSITY
Khaled A. Selim1,*, Mohamed M. S. Nagia2

and Dina E. El. Ghwas3
1
Interfaculty Institute of Microbiology and Infection Medicine,
Eberhard Karls Universität Tübingn, Tübingn, Germany
2
Institute of Pharmaceutical Biology,
Technische Universität Braunschweig, Braunschweig, Germany
3
Chemistry of Natural and Microbial Products Department,
Pharmaceutical and Drug Industries Research Division,
National Research Centre, Cairo, Egypt
ABSTRACT
Symbiosis is a widespread phenomenon in nature. Endophytes are
defined as all microorganisms that colonize asymptomatically within
living healthy tissues. In general, endophytes are considered as
commensalistic symbionts, where they receive nutrients and shelter from
the host. In return, they are thought to provide the host with chemical
*
Corresponding Author address: Email: khaled.selim@uni-tuebingen.de.

40 Khaled A. Selim, Mohamed M. S. Nagia and Dina E. El. Ghwas
constituents that can be used in the growth or defense mechanisms.

Fungal endophytes have attracted a great interest to microbiologists,
chemists and ecologists as a treasure of biological resource, because they
play diverse indispensable roles in the ecosystem for stress tolerance,
eco-adaptation, and promoting growth and development. Recently,
endophytic fungi have drawn a particular attention, due to their
considerable biodiversity, unparalleled metabolic pathways and unique
habitats. Therefore, they were considered as an unusual source of novel
secondary metabolites, exhibiting a variety of biological activity, which
are in use in modern agriculture, pharmaceutical and biotechnological
industry. In the last two decades, the extensive discovery of endophytic
secondary metabolites reflected the tremendous chemical diversity of
different natural compounds classes with incredible bioactivity, but still
the chemistry of endophytes needs to be comprehensively studied. On the
other hand, the search for alternative sources of fuels is becoming
increasingly important and biodiesel has been shown to be one of the
most promising alternatives. Fungal endophytes have been reported to
produce volatile low molecular mass hydrocarbons such as alcohols,
alkenes and trepenoidal mycodisel. In this review, we will focus on
characterization and diversity of endophytic fungi, with highlighting their
ecological role in nature. Besides, we will emphasis on the variety of
chemical classes and the wide spectrum biological functions of
endophytic metabolites as well as their potential as an energy source for
biofuel production.
Keywords: endophytic fungi, ecological role, biofuel and hydrocarbon

production, chemical diversity, secondary metabolites, pharmaceutical
applications
INTRODUCTION
Almost all plants studied to date in the natural ecosystems are infected by
fungi and/or bacteria, some with no visible effect on their host plant in
phenomena called endophytes and others infect their hosts and significantly
impact their capacity and survival. Endophytic microorganisms are a group of
microorganisms linked with different vascular tissue of some aquatic and
terrestrial plants (Stone et al., 2000). Many groups of mycoplasma, bacteria,
actinomycetes and fungi are proved to be endophytes. Endophytic
microorganisms are invisible in plant tissue, but they have important
modulations for biodiversity, natural communities and agriculture.
Furthermore, endophytes are superb system for studying associations between

Endophytic Fungi Are Multifunctional Biosynthesizers 41
microorganisms. The importance of endophytes remained hidden until 1975,

when Bacon et al., (1975) proved that, cattle toxic syndrome caused by an
endophyte of pasture grasses.
The most important group of eukaryotic microorganisms is fungi. The
structure of fungi is branching colorless hypha which extends through the
substrate where the fungus grows (Madigan et al., 2008) then; a vast number
of the hyphae of each fungus are twisted to form a tangled web named
mycelium. They have been isolated from dust, sand, soil, fresh and sea water,
marine sediment, vertebrates, marine invertebrates and tissues of terrestrial
and marine plants. They play important roles in nature, biotechnology,
agriculture and with many living organisms. Endophytic fungi research has a
long history and its diversity among plant is large. Each plant has been
colonized by one or more endophytes (Verma et al., 2007 and Kharwar et al.,
2008). Lately, endophytes are discovered to be a source of bioactive
antimicrobial natural products and secondary metabolites. Thus, endophytes
received attention in the last 20 years when the research discovered that, they
protect against pest pathogens and insect.
1. DEFINITION OF ENDOPHYTES
The endophytic word means “in the plant” (endon Gr. = within, phyton =
plant). The utilization of this term is as wide as its exacting definition and
range of potential hosts and inhabitants, e.g. fungi (Stone et al., 2000), bacteria
(Kobayashi and Palumbo, 2000), algae (Peters, 1991), insects in plants (Feller,
1995) and plants (Marler et al., 1999). This means that, any organ of the host
can be colonized. The endophytic term is also used for parasitic endophytic
plants (Marler et al., 1999), fungi (Sieber, 2002 and Schulz and Boyle, 2005),
pathogenic endophytic algae (Bouarab et al., 1999) and mutualistic endophytic
bacteria (Adhikari et al., 2001 and Bai et al., 2002). In addition, the endophyte
was named for the first time in 1866 by de Bary, to illustrate the fungi that
found in the internal tissues of stems and leaves (Wilson, 1995). From this
time this identification has been modified. Two widely accepted definitions
have been followed:
1) Endophytes colonize symptomless the internal tissues of their living

host, and after a period of times they may or may not cause diseases
(Petrini, 1991).

2) The endophytes fungi or bacteria are living a part or all of their life
cycle and causing asymptomatic infections inside plant tissues
(Wilson, 1995).
Both of these definitions (but not de Bary’s) include endophytes of roots,

which have been found in everywhere (Schulz et al., 2006). In addation, the
final stages of infections by pathogens are inclusive in theses identification,
but excluded from them mycorrhizal fungi because they are partly external and
often symptomatic (Saikkonen et al., 1998). The term endophytes are often
joined with alternate to refer to a specific host, the type of tissue occupied or a
taxonomic group of hosts (e.g., bark endophytes and systemic grass
endophytes). This mean that, the identification of endophytes are depend
primarily on location rather than symbiotic interaction type. Recent
applications of the terms of endophytes never consistent and not accepted by
all researchers but in 1991 Petrini showed an identification of endophytes that
has been accepted widely. Endophytic are important group of microorganisms
linked with different organs and tissues of aquatic and terrestrial plant, whose
infections are unclear and the infected host tissue are symptomless (Stone
et al., 2000) rather than parasites which caused disease and reduce fitness of
their host plant. Therefore, the defined endophytes are wide enough to contain
any microorganisms (cyanobacteria, fungi and bacteria) that living in the
internal tissues of plants. On the other hand, the word endophyte for grass
hosts (primarily Poaceae) was specially used for this type of systemic
nonpathogenic symbiosis. These grasses endophyte support their hosts with
many advantage like protection against herbivory and pathogens (Saikkonen
et al., 1998). Taxonomically theses fungi begin related to Neotyphodium
anamorphs of Balansiae (Clavicipitaceae); they colonize root tissues and culm
of species of leaf and cool season grasses. Host and fungus are working
together as a single organism and sporulation on the host is completely
suppressed. Also, endophytes of Festuca, Lolium and other genera of grasses
which derived from Epichloë species are inter specific hybrid strains and lead
to partial or completely host sterility (Moon et al., 2000).
2. DISTRIBUTION OF ENDOPHYTIC FUNGI IN NATURE

In the last two decades, researchers proved that, endophytic fungi colonize
land plants everywhere on the earth. Endophytes are isolated from plants
growing in in tropical temperature, boreal forest, aquatic and extreme arctic

(Suryanarayanan et al., 2000; Mohali et al., 2005 and Šraj-Kržič et al., 2006).
Additionally, they isolated from various habitats such as xeric environments,
mesic temperature, and extreme arctic and tropical forests. Endophytic fungi
were found in hepatics and mosses (Mohali et al., 2005). Moreover, a lot of
them wrer found in gymnosperms and angiosperms including tropical palms
and the estuarine plants Spartina alterniflora, Salicornia perennis and Suada
fruticosa (Fröhlich and Hyde, 2000 and Hyde et al., 2000). Furthermore, they
exist in ferns and fern allies and broad-leaved trees (Amatangelo and Vitousek,
2008) as illustrated in Table 1.
Table 1. Examples of endophytic mycobiota in various host

plants worldwide
Host Tissue or No. of Location References

organ species
Abies alba Branch 44 Germany,Poland Kowalski and Kehr,
(1992)
A. rubra Leaves bases 25 British, Sieber et al., (1991)
Columbia
Betula pendula Branch bases 23 Poland Germany, Kowalski and Kehr,
(1992)
Cuscuta reflexa Stems 45 India Suryanarayanan et al.,
(2000)
Euterpe Leaves 62 Brazil Rodrigues, (1994)
oleracea
Heisteria Leaves 242 Panama Arnold et al., (2000)
concinna
Licuala ramsayi Leaves 11 Australia Rodrigues and
Samuels, (1992)
Musa leaves 24 Hong Kong Brown et al., (1998)
acuminata
Ouratea lucens Leaves 259 Panama Arnold et al., (2000)
P. mariana Roots 97 Ontario Summerbell, (1989)
Quercus ilex Twigs, 149 Spain Collado et al., (2000)
Leaves
Salicornia Stems 31 United Kingdom Petrini and Fisher,
perennis (1986)
Tilia cordata Leaves 17 Germany Pehl and Butin, (1994)
Vitis vinifera Leaves, 46 South Africa Mostert et al., (2000)
Stems
Zea mays Leaves, 23 United Kingdom Fisher et al., (1992)
Stems

On the other hand, large woody perennials assistance a parasite likes

dodders and mistletoes, may have endophytic fungi (Suryanarayanan
et al., 2000). Due to the unclarity of endophytes infections, the diversity of the
internal mycobiota is relatively high and a small potential hosts have been
investigated till now (Arnold et al., 2000). Examination of economically
important plants for endophytic fungi till now, every now and again yields
new taxa. Investigations of endophytic fungi are expected to give us a lot of
information for their distribution and diversity. Typically the endophytic
micro-fungi exist as unseen, internal and microscopic hyphae; their existence
proved that, they sporulate seasonally and transient. Detection and isolation of
endophytic fungi need selective methods.
Identification includes always host microscopical examination and usually
need a high degree of taxonomic experience. This is right for isolates that
cannot produce spores or cannot identify their structure therefore, finding the
growth conditions that stimulate sporulation are very important. Furthermore,
the ribosomal DNA (rDNA) gene sequences are used for endoptytic fungi that
neither grow nor sporulate, which can illustrate the phylogenetic position (Guo
et al., 2000). The major block of endophytes ecological studies is the lack of
basic taxonomic information. The issue can be overcome by coordinating the
present databases (nomenclature and host indices); however major biological
overview work is required.
3. ROLE OF ENDOPHYTES IN NATURE

Previously, endophytic fungi have been known as mutualists and it is
much related to virulent pathogens; however their pathogenicty is limited and
developed directly from plant pathogenic fungi. The symbiosis of mutualistic
containing the absences of most cells or tissues, nutrient and chemical cycling
between hosts and fungus, promoted pathosynthetic capacity, induce longevity
of tissue and promoted living of fungus. It is always difficult to distinguish
between pathogen and endophyte as many plant pathogens submit an extended
phase of asymptomatic latent infection before the occurrence of disease
symptoms, also the mutation can alter a pathogenic to a nonpathogenic
endophyte with no influence on their host specify. Latent infection means that
the host infected by the pathogen, however does not show any persists and
symptoms until symptoms are induce to appear as an environmental or
nutritional conditions (Brader et al., 2014).

The endophytic term is always difficult to be classified as special species

and it is bears a close relationship to the term pathogen. Other scientists make
a compromise between latent infection and endophyte colonization by fungi
and proved that, they are completely different (Shearer, 2002). Too, many
scientists found that, endophytic fungi are mutualistic and asymptomatic,
whereas infectious fungi cannot be mutualistic but they are parasitic.
Moreover, the high attention that has been paid to endophytes of grass which
gives rise to the idea that, all endophytes must be mutualists (Stanley, 2002).
Also, now there is a great idea that mutualism in the fungal species may be
termed to those which are transmitted by seeds due to transmission will
increase as a host survival.
The endophytes correlated with grasses paid more attention because they
proved to produce physiologically active alkaloids that make their host toxic to
insect herbivores and mammals (Cheplick and Clay, 1988). As well,
endophytes in other plant hosts and in the grasses have been demonstrated to
promote plant growth, increase nitrogen uptake in nitrogen deficit-soils,
increase stress tolerance and reduce infection by nematodes (Bultman and
Murphy, 2000). Many of studies are available on secondary metabolite
production by endophytes (Petrini et al., 1992). Endophytic fungi produce
biologically active compounds containing many of paxilline, alkaloids,
tertaenone steroids and loliterms, proved plant growth factors and antibiotics
production. Therefore, endophytes are being identified as a group of
microorganisms that could produce secondary metabolites for agricultural and
biotechnological use (Thalavaipandian et al., 2011).
4. ECOLOGICAL ROLE OF ENDOPHYTES

Biology of endophytes is having a lot of work in research with multitude
of objectives that can be broadly classified in two classes: plant microbe
symbiosis and bioprospecting as shown in Figure 1. The bioprospecting aspect
of endophytes has been widely studies (Aly et al., 2011; Porras-Alfaro and
Bayman, 2011 and Strobel, 2015). While, the plant microbe symbiosis aspects
at molecular level has been badly understood, plant microbe interaction are
found everywhere in nature (Redman et al., 2002; Kuldau and Bacon 2008 and
Mitter et al., 2013). Actually, every plant is a complex community and this is
related to its connection in various heterospecific associations (Kiers and
Denison 2008 and Rodriguez et al., 2009). Ecophysiology like growth rate,
resistance to biotic and abiotic stress conditions, plant nutrition as well as

distribution and plant survival is affected by the common complexity of verity

of microbial communities with the host plant (Iqbal et al., 2013). The unity of
microbes with plants dates back to more than 400 million years ago therefore,
the existence of symbiotic microorganisms in the internal parts of plant tissues
look to be the rule and not exception (Strobel et al., 2004 and Partida-Martínez
and Heil, 2011).
Figure 1. Endophytic biology is studied with the aim of bioprospection of genuine

microbial products, potential host metabolites and industrially important volatile
organic compounds (VOCs) or to understand the principles of endophytism and its
consequences on the secondary metabolism of the partners as well as adaption of the
plant host to biotic and abiotic stress conditions.
The ecological roles of endophytic fungi are various and multiple.

Endophytic have been qualified as mutualists that prevent both conifers and
grasses from attack by insect. Besides, a lot of these fungi release biologically
active secondary metabolites. Other researches proved that, there are more
than 30% of compounds produced by endophytic fungi have antifungal and

antibacterial activities. Further, isolates of endophytes such as

Cryptosporiopsis species, Abies Alba and Pleurophomopsis species showed to
have antibiotic activity. From several deciduous and coniferous tree hosts, the
investigators isolated strains of the endophytic Pezicula species that found to
produce many bioactive secondary metabolites in culture media (Schulz et al.,
1995). On the other hand, endophytic species from Xylariaceae thought to
produce several compounds with high biological activity such as indole
diterpenes and cytochalasins (Hensens et al., 1999). However, there is a wide
variety of toxins produce by endophytic fungi which are difficult to be
detected in plant host tissue.
No grass endophytes produce insecticidal compounds (Hensens et al.,
1999) as well as antibacterial substances and antifungal (Peláez et al., 2000). It
is not clear whether the metabolites are produced in the plant during the period
of clam infection of endophytes in host plant or when endophytes produce a
perfect amount of metabolites that is important to the host in protective
mutualism.
One endophyte can infect a wide host range. Besides, several scientists
showed that, the same strain isolated from different parts of the same host
differs in its ability to utilize different substance. Rainforests and tropical
regions are the most important as diverse terrestrial ecosystems on earth. It is
cover about 1.44% of the land surface. Therefore, we could expect that, high
plant endemicity contain specific endophytes. Finally, biological variety
means chemical variety due to constant chemical renewal that present in the
ecosystems where the development race to survive is the most active. A
wonderful example of this type of environment is tropical rainforests.
Selection is at its peak, resources are limited and competition is great. This
motivates a lot of scientist to conclude that the rainforests are a high source of
new biologically active compound and molecular structures (Redell and
Gordon, 2000).
5. CLASSIFICATION OF ENDOPHYTES
Endophytic fungi have a great effect on the plant fitness, ecology and
evolution. Various groups of these microorganisms are able to produce
number of bioactive agents (Brundrett et al., 2006). In the past, endophytic
fungi were classified into two groups: - Clavicipitaceous and non-
Clavicipitaceous according to their taxonomy, evolution, ecological function
and host specificity. After that, Rodriguez et al., 2009 illustrated that, there are

four special groups depending on six properties which are tissue(s) colonized,
plant colonization pattern, host range, plant biodiversity levels, ecological
functions and mechanism of transmission between host generations. Class (1)
is Clavicipitaceous endophytes while; non-Clavicipitaceous endophytes are
divided into three special groups class (2), class (3) and class (4) (Rodriguez et
al., 2009). We will summarize each class briefly:
European investigators in 19th century were the first who noted the class
(1) (Clavicipitaceous endophytes) was found in the seeds of Lolium linicolum,
Lolium arvense, Lolium remotum and Lolium temulentum (Vogl, 1898). After
that, Clay, 1988 demonstrated that they are defensive mutualists of host
grasses. From this time, investigations on endophyte natural history, ecology,
physiology and evolution has been followed (Schardl and Moon, 2003; Rao
et al., 2005 and Koulman et al., 2007). Usually these endophytes found within
plant shoots and make systemic intercellular infections. Colonized plants
always harbor one controlling fungal isolate/ genotype. According to Clay and
Schardl, 2002 there are three types of Clavicipitaceous endophytes extended
from pathogenic and symptomatic species (Type I) to mixed combination and
asymptomatic endophytes (Types II and III) respectively. The class (1)
endophytes are primarily transmitted vertically and horizontally on offspring
via seed infections (Saikkonen et al., 2002). Class (1) endophytes confer
drought tolerance, increase plant biomass and produced chemicals that toxic to
herbivory and animals (Clay, 1988). However, the importance of these fungi is
due to the host genotype, environmental conditions and host species (Faeth
and Sullivan, 2003).
On the other hand, class (2) (non-Clavicipitaceous endophytes) are highly
various, symbolizing a polyphyletic aggregation of primarily ascomycete's
fungi with diverse and always bad unknown ecological role. Non-
Clavicipitaceous endophytes have been isolated from everywhere of land plant
and from all terrestrial ecosystems, containing both biomes and ecosystems
ranging from the tundra to the tropics (Arnold and Lutzoni, 2007). The
capacity of lot of fungi to switch between free-living lifestyles and endophytic,
the prudence they supply into the evolution of different ecological modes in
fungi, their ecological roles, their potential applications and the scale of their
diversity are attractive to ecologists, mycologists, applied scientists and
physiologists (Vasiliauskas et al., 2007and Selosse et al., 2008). Non-
Clavicipitaceous endophytes can be distinguish into three classes depend on
mechanism of transmission between host generations, host colonization
patterns, ecological function and planta biodiversity levels as shown in Table
(2).

Table 2. Symbiotic criteria used to characterize fungal endophytic classes
Criteria Clavicipitaceous Non-clavicipitaceous

Class (1) Class (2) Class (3) Class (4)
Host range Narrow Broad Broad Broad
Tissue(s) Shoot and Shoot, Root Shoot Root
colonized rhizome and rhizome
In planta Extensive Extensive Limited Extensive
colonization
In planta Low Low High Unknown
biodiversity
Transmission Vertical and Vertical and Horizontal Horizontal
horizontal horizontal
Fitness NHA NHA and HA NHA NHA
benefits*
*Nonhabitat-adapted (NHA) benefits like drought tolerance and growth enhancement
are common among endophytes regardless of the habitat of origin. Habitat-
adapted (HA) benefits result from habitat-specific selective pressures such as pH,
temperature and salinity.
Class (2) endophytes (non-Clavicipitaceous endophytes) that can grow in

both below and above ground tissues can widely colonize tissues. Endophytes
of class 2 containing different species are all organs of the Dikarya
(Basidiomycota or Ascomycota). Actually, this group of endophytes is totally
limited in single host plant. They can transfer vertically and horizontally by
seeds, seed coats or rhizomes. One superb aspect of class 2 endophytes is their
capacity to give habitat-specific stress possibility to host plants (Rodriguez et
al., 2008) which, are identified as habitat-adapted if the benefits are a result of
habitat-specific selective pressures like temperature, salinity and pH or as
nonhabitat-adapted if the useful are joint between endophytes regardless of
habitat.
Class (3) endophytes (non-clavicipitaceous endophytes) are particularly
outstanding for their high verity into individual host plant, tissues and
populations. Class (3) endophytes containing the hyper diverse endophytic
fungi connected with leaves of tropical trees (Arnold et al., 2000), as well as
the highly different mate of ground-above tissues of nonvascular plants,
conifers, woody, seedless vascular plants and herbaceous angiosperms in
biomes extended from arctic and boreal/Antarctic society to forests (Murali
et al., 2007 and Davis and Shaw, 2008). Particularly leaves may accommodate
up to one isolate per 2 mm2 of leaf tissue and have a lot of species. Also, to

occurring within herbaceous and photosynthetic tissues, class (3) endophytes

are present in fruits and flowers, as well as in inner bark and asymptomatic
wood (Tejesvi et al., 2005). Fungi with identical life recorded to class (3)
endophytes also found inside asymptomatic lichens and this case are called
‘endolichenic’ fungi (Arnold et al., 2003). It forms highly localized infections.
The variety of class (3) endophytes inside a host tissue or plant can be much
high (like > 20 species listed from a single tropical leaf (Arnold et al., 2003).
Merlin, 1922, while isolating and studying ectomycorrhizal fungi had
spotted a brown to blackish pigmented fungus related to terrestrial plant roots.
After that it was identified as class (4) endophytes (non-clavicipitaceous
endophytes) which are initially ascomycete’s fungi that are sterile or conidial
and that make melanized structure like as intra- and intercellular hyphae and
microsclerotia in the roots. These root-associated fungi and sterile called
“mycelium radicus astrovirens (MRA)”. The different type of class (4)
endophytes inside individual plants has not been enough evaluated. Also, it is
not confirmed to now if it increases fitness to hosts, rhizosphere competence
and mode of transmission or not. This class of endophytes is present in host
plants such as non-mycorrhizal from arctic, temperature zones, alpine, tropical
ecosystems, Antarctic and sub-alpine (Jumpponen, 2001).
6. BIOTECHNOLOGICAL APPLICATIONS OF ENDOPHYTES

Endophytes are producers of various types of bioactive secondary
metabolites containing derivatives of benzopyranones, phenolic acids,
alkaloids, flavonoids, terpenoids, xanthones, quinones, steroids, tetralones,
xanthones and others (Tan and Zou, 2001). These bioactive metabolites have
wide applications such as antibiotic, antiparasitics, agrochemicals, anticancer
agents, immunosuppressants and antioxidants (Strobel, 2003). In addition, they
play essential roles in nature.
6.1. In Agriculture Fields
About 300.000 plant species growing in different area on the earth are
becoming a host to one or more endophytes (Strobel and Daisy, 2003). Also,
the existence of big number of different endophytes plays important role on
ecosystems with a big difference, for example the temperature rainforests and
tropical area which, are found in Brazil containing about 20% of its

biotechnological sources (Strobel, 2003). Endophytes play important role as

safeguard to the plant host from pathogens such as insect pests and cattle and
predators (Azevedo et al., 2000). Moreover, endophytes increase the resistance
of plants versus abiotic and biotic stresses. Furthermore, they effect plant
growth directly or indirectly and can support the hosts with compound that are
released by fungi for accelerate the process of nutrient uptake in the
environment. The fungus Piriformospora indica increase the growth of
different hosts and which demonstrated its benefit for promotion for plant
growth (Varma et al., 1999). Besides, several endophytes isolated from
Eucalyptus were able to enhance growth of seedlings by preventing diseases in
the premature stages of plant development (Procopio, 2004). Moreover,
Romao et al., (2011) illustrated that, G. citricarpa release large amounts of
enzymes like pectinases, endoglucanases and amylases in contrast to G.
mangiferae which produces enzymes considered as the key in the
improvement of citrus black spot, such as pectin-lyases that degrade pectin
more effectively in the pathogenic strains.
On the other hand, a lot of studies proved the ability of endophytes to
control disease vectors and pests (Azevedo et al., 2000). The Basidiomycete
Moniliophtora perniciosa which caused witches (broom disease) of cacao is
considered the most important pathogen of this crop. Therefore, several
scientists have isolated and studied some fungi for their ability to inhibit it and
they found Gliocladium catenulatum which decrease the infection of disease in
cacao seedling to 70% (Rubini et al., 2005). Between the isolated fungi from
cacao, M. perniciosa was set colonizing healthy parenchymatic tissues and for
the first time proved that this fungus may be conducted as an endophyte (Lana
et al., 2011). Other scientists isolated Beauveria bassiana from maize (Zea
mays), which used to control the European corn borer (Ostrinia nubilalis). In
addation, a lot of laboratories isolated endophytic fungi that controlling
nematode and insects, these were isolated from plants hosts such as soybean,
maize and sugarcane (Stuart et al., 2010). Furthermore, from maize many of
isolated Beauveria strains were used against the insect pest Spodoptera
frugiperda. These endophytic Beauveria, related to the Beauveria bassiana
species; also, Beauveria amorpha able to control the bovine tick
Rhipicephalus microplus which are ectoparasite that make economic losses
because it caused toxicity and anemia to their host (Campos et al., 2010).
Moreover, from petrol-contaminated mangroves many endophytes were found
to be able to decrease oil contaminations.

6.2. As a Hydrocarbons Producers
The search for other sources of energy is becoming important because the
sources of liquid fuel are decreasing. The microorganisms in unique ecological
niches are the most effective ways to search for unusually metabolically active
secondary metabolites. The endophytic fungi have been recently discovered to
produce fuel related compound. These compounds are compatible with the
presence motive infrastructure and renewable which, depend on drop fuel
technology. Beside, these microorganisms have the abilities of growing and
releasing gaseous products on cellulosic wastes and agricultural instead of
agriculture products that may act as feed and food sources. These endophytic
fungi produce bioactive volatile compounds and a lot of them are fuel related.
Reviews on the endophytic microorganisms that producing these volatile
compounds have been appeared recently (Strobel, 2006, 2011 and 2012).
This encouraged researchers to directly plan their researches for the
discovery of new endophytic fungi that could produce hydrocarbons like
Muscodor albus (Worapong et al., 2001). Till now, all members of genus
Muscodor albus release volatile compound that kill and/or inhibit pathogenic
bacteria and fungi and some are detrimental to insects too (Strobel, 2011 and
2012). Due to the effective nature and the broad range of its volatile organic
compounds (VOCs), this fungal genus has been studies and promoted for use
as biological control (Strobel, 2011 and 2012). M. vitigenus, M. crispans, M.
sutura, and M. roseus all are identified as new species of this fungus, which
were isolated from different parts of the earth such as China, India and
Thailand (Strobel, 2012).
Furthermore, screening the chemistry of the volatile compounds released
by these endophytes, detected the presence of alkenes, benzene derivatives,
alkylesters, alkanes, polyaromatic hydrocarbons, terpenoids and other
compounds that either similar to or are linked to the chemical compound
families of diesel fuel. Therefore VOCs were called mycodiesel (Strobel et al.,
2008). Some samples acquired from stems of Eucryphia cordifolia in
rainforest of Chile. These patterns contain an organism that grow in the
appearance of the VOCs of M. albus and also release VOCs with antimicrobial
activities (Stinson et al., 2003a). This organism was identified morphologically
as Gliocladium roseum, however through taxonomic studies the organism was
strange deficient stage of the ascomycetous fungus-Ascocoryne sarcoides
(Strobel et al., 2010). This fungus is important due to its ability to create a
wide range of the acetate esters of straight chained alkanes containing heptyl
and sec-octyl alcohols (Strobel et al., 2008). After that, the scientist knew that

the alkyl alcohol side chains of these acetate esters act the basic free alkanes of
diesel as they specially have been detected in every diesel sample that had
been analyzed. Moreover, breakages of the ester bond go after by a reduction
of the alcohol to a methyl group and resulting in alkane which similar to that
found in diesel.
Moreover, further work has indicated the presence of decyl esters and
nonyl in the components of fungal VOCs (Griffin et al., 2010). However,
diesel fuels have series of straight chained hydrocarbons; also there they have
cyclic alkanes, branched alkanes and benzene derivatives along with a lot of
polyaromatic hydrocarbons (Griffin et al., 2010 and Mallette et al., 2014). In
addition, the analysis of VOCs of this fungus is resulted in an array of ketones,
alcohols, esters and different other hydrocarbons such as benzenes derivatives,
cycloalkenes and cycloalkanes (Mallette et al., 2014).
7. ENDOPHYTES AND CONCERN OF MAJOR

HEALTH PROBLEMS
The expanding of world health problems caused by various deadly
diseases such as drug-resistant microbes, viruses, and cancers are considered
as an alarm. Although thousands of diseases have been described, less than
one-third of them can be treated symptomatically and only a few can be cured
(Strobel 2003 and Selim et al., 2012). Cancer is the largest single cause of
death, claiming over hundred million lives each year. To date, a lot of
anticancer drugs are discovered and clinically in use. However, resistance to
anticancer medications was discovered recently. Therefore, an intensive search
for new, potent and effective agents to compact with these serious disease
problems is now under way and the endophytes was introduced as a novel and
rich source of functional metabolites which have potentially useful medicinal
applications (Selim et al., 2011 and 2012).
8. ENDOPHYTES AS A PROMISING SOURCE

FOR DRUG DISCOVERY
In the past few decades, scientists have begun to realize that almost all
higher plants may serve as a reservoir of indescribable numbers of endophytic
microbes (Bacon and White 2000). These endophytic microbes are fungi and

bacteria, and live in the intercellular spaces of their hosts and this also true for
marine plants including algae or seagrasses and also for invertebrate animals
including soft corals and sponges. Almost all endophytes release bioactive
secondary metabolites which may be implicated in a host-endophyte
relationship (Selim et al., 2012). As a direct consequence of the natural role of
endophytic secondary metabolites, they may ultimately to have applicability in
medical field for improving the drug discovery (Selim et al., 2011 and 2012).
A worldwide scientific effort is to isolate endophytes and to study their natural
products is now under way (Strobel, 2003).
Overall, the rational responses for studying endophytes as promising
sources of new therapeutic agents is the fact that this field of the science is an
unexplored area of biochemical diversity. Additionally, the focus on
endophytes as a potential source for pharmaceutical industry is due to the
reality that the endophytes contribute to the host by providing protection to
them with a variety of secondary metabolites (volatiles, insecticidal,
antimicrobial, hormones-like compounds and …. etc.). Some of endophytic
metabolites may be of interest clinically since they possess antibacterial,
antifungal, antimalarial and other interesting biological activities (Verma et al.,
2009 and Selim et al., 2012). Finally, one of major concern to the
pharmaceutical companies is the toxicity of any potential probable drug to
human tissues. Apparently, plants as a pool of endophytic microbes are a
eukaryotic system in which the endophyte exists. The metabolites synthesized
by the endophytes may have less cell toxicity; otherwise, they could destroy
the target tissues of the host. Thus, the host itself has naturally served as a
selection system for microbes producing bioactive molecules with reduced
toxicity toward eukaryotes. Therefore, the endophytes consider being an
important source of novel natural compounds and a notable growing trend is
the characterization of endophytic secondary metabolites in drug discovery
(Strobel, 2003; Verma et al., 2009 and Selim et al., 2012).
9. PHARMACEUTICAL ACTIVATES OF ENDOPHYTES

Endophytic variability must be acquired during long term of interaction
with the host, by genetic crossing, or gene transfer, or simply with mutations
or by a set of unsubstantiated mechanisms yet such as genetic exchange with
its hosts. It appears that, endophytes represent microbial factories of bioactive
secondary metabolites. For this reason, extensive investigations have been
carried out for chemical screening of novel metabolites that may have

potential use for pharmaceutical applications (Strobel, 2003; and Selim et al.,
2012). A background understanding that involves some specific examples and
rationale of the presence of endophytic microorganisms in higher plants will
aid in the development of a drug discovery program involving these
organisms.
9.1. Endophytes Producing Anticancer Compounds
A search for a rare and a costly product such as taxol may be facilitated by
investigating the endophytic microbes of plants that are able to synthesize this
compound. Taxol is found in all Taxus sp., (yew trees) and it is belong to
diterpenoid compounds (Stierle et al., 1993).
This compound is the world’s first billion dollar anticancer drug and it is
used to treat a number of cancer diseases. Due to its high price it is not readily
available to worldwide. Thus, alternative sources are required for producing
reasonable amounts of such important drug (Stierle et al., 1993 and Strobel,
2006). In fact, all world’s higher plants contain certain endophytic species,
thus, it may be possible that yew trees might be a shelter for a certain
endophytes that can synthesize taxol. Thus, with the availability of microbial
source that is able to produce this drug, the need to harvest the slow-growing
and relatively rare yew trees is eliminated (Strobel, 2006). The taxol price
would be much cheaper, since it can be produced via fermentation like
penicillin production. It was speculated that the ability of any endophyte to
make taxol may have arisen with a gene transfer from the yew tree to one or
more of endophytes living in close association with it. Until early 1990s, no
endophytic fungi had been recorded from any of the world’s representative
yew species. After several years of effort a novel taxol-producing endophytic
fungus Taxomyces andreanae was identified from Taxus brevifolia (Strobel et
al., 1993 and Selim et al., 2012).
9.2. Endophytes Producing Antibiotics
The imperfect stage of Pezicula cinnamomea is a fungus identified as

Cryptosporiopsis cf. quercina, which is frequently associated with hard wood
species. This fungus and related species are always found worldwide as
endophytes. In 1999, it was isolated as an endophyte from a medicinal plant
Tripterigeum wilfordii (Strobel et al., 1999).

C. quercina showed brilliant antifungal activity toward serious human

pathogens Candida albicans and Trichophyton spp. Since fungal infections are
a growing health problem especially among immunocompromised patients,
new antifungals are required to struggle this problem. A cryptocandin is a
unique antimycotic peptide that was isolated from C. quercina (Figure 2). This
compound contains a novel amino acid, 3-hydroxy-4-hydroxy methyl proline,
and a number of peculiar hydroxylated amino acids. It was active against a
huge number of plant pathogen as well, including Botrytis cinerea and
Sclerotinia sclerotiorum. Cryptocandin is currently being verified and
industrialized by several pharmaceutical companies for usage against different
skin and nails pathogenic fungi (Strobel, 2003).
9.3. Endophytes Producing Antiviral Agents
(R)- (-)-mellein (Figure 2) is a naturally occurring di-hydroiso coumarin

which was first isolated as a metabolite of Aspergillus melleus (Nishikawa,
1993) and later from a number of endophytic fungi such as Pezicula livida,
Plectophomella sp. and Cryptosporiopsis malicoticis (Krohn et al., 1997).
Recently, (R)-(-)-mellein isolated from Geniculosporium sp., a fungus that is
associated with the red alga Polysiphonia sp. and isolated from the Baltic Sea
at Ahren shoop. It shows a number of interesting biological activities such as
fungicidal, antibacterial and algicidal activity in agar diffusion tests (Holler
et al., 1999) and it inhibits HCV protease with an IC50 value of 35 mM (Florke
et al., 2006).
9.4. Endophytes with other Important Biological Activities
A surprise was the realization that endophytes produce substances that can
influence the immune system of animals. Subglutinols A and B (Figure 2) are
immunosuppressive compounds produced by the marine-derived Fusarium
subglutinans, and another endophyte from T. wilfordii as illustrated in (Lee et
al., 1995). Both compounds have IC50 values of 0.1 μM in the mixed
lymphocyte reaction assay. In the same assay, cyclosporin was found roughly
as potent as the sub-glutinols. These compounds are being examined more
thoroughly as immunosuppressive agents. Their role in the endophyte and its
relationship to the host are unknown.

Figure 2. Chemical structures of endophytic metabolites: Cryptocandin, (R)-(-)-

mellein, Subglutinols A, and Cytochalasin, displaying antibiotic, antiviral,
immunosuppressive, and antitumor activities respectively.
The alkaloids are commonly found in endophytic fungi. Fungal genera

such as Xylaria, Phoma, Hypoxylon and Chalara are representative producers
of a relatively large group of substances known as the cytochalasins of which
over 20 are now known (Figure 2) (Wagenaar et al., 2000). Many of these
compounds possess antitumor and antibiotic activities but, because of their
cellular toxicity they have not been developed into pharmaceuticals. Three
novel cytochalasins have recently been reported from Rhinocladiella sp. as an
endophyte on T. wilfordii (Wagenaar et al., 2000). These compounds have
antitumor activity and have been identified as 22-oxa-(12)-cytochalasins.
Thus, it is not uncommon to find the cytochalasins in endophytic fungi and
workers in this field need to be alert to the fact that redundancy in discovery
does occur. Chemical redundancy usually occurs with certain groups of
organisms in which previous studies have already established the chemical
identity of major biologically active compounds. For instance, as with the
cytochalasins they are commonly associated with the Xylariaceaous fungi.
Moreover, endophytes were reported for production of antioxidants and
butyryl cholinesterase inhibitors, one of neuro hydrolase that is involved in

development of Alzheimer’s disease (Selim et al., 2014). Compounds isolated

from the endophytic fungus Chaetomium globosum isolated from Egyptian
medicinal plant showed promising butyryl cholinesterase inhibitory activities,
beside strong antioxidant activity with EC50 11.5 µg/ml (Selim et al., 2014 and
2016).
10. CHEMICAL VARIETY OF METABOLITES ISOLATED

FROM ENDOPHYTIC FUNGI
Endophytic fungi are considered as a promising source of active

compounds or an alternative source of metabolites produced by higher plants
(Kusari and Spiteller, 2011). Actually, a lot of bioactive compounds produced
by endophytes fungi contain antimycotic, anticancer agents, antibiotics,
insecticides and antiviral compound (Strobel and Daisy, 2003 and Verma et
al., 2009). These compounds are different in both their complexity and their
chemical class like peptides and their substituted derivatives, glycosides,
phenolics, completely odd and complex lipids and unanticipated structure as
(3- carbamoyl- quinoxalinium) chloride and tetramic acids. The focus of this
part is to present the wide chemical variety of entities produced by endophytes
with an emphasis on the different biological activates of those compounds and
highlights the recent updates of new compounds. In this respect, the structures
of endophytic compounds were identified based on different sophisticated
chemical methods mainly (IR, UV and HRESIMS, 1D and 2D NMR, NOE,
CD, X-ray crystallography and chiral HPLC).
10.1. Alkaloids
Alkaloids are a large group of basic nitrogenous organic compounds.

Commonly, they have a bitter taste. The most common examples are caffeine,
morphine and nicotine. Since long the plants were the sole source of alkaloids
in this section some alkaloids isolated from endophytic fungi will be
represented.
Based on the analysis and correlative interpretation of HRESIMS, NMR
and CD spectral data two novel diketopiprazine alkaloids were obtained from
Fusarium sp. Separated from a Marine Alga, fusaperazine A (1) and
fusaperazine B (2) which is an O-prenylated derivative (Usami et al., 2002).

Fusapyridons A (3) and B (4) produced by the endophyte Fusarium sp.

YG-45 are two 3, 4, 5-trisubstituted N-methyl-2-pyridone alkaloids.
Fusapyridons A displayed antibacterial activity against Staphylococcus aureus
and Pseudomonas aeruginosa (Tsuchinari et al., 2007).
Penicidones A-C (5-7) were isolated from Penicillium sp. IFB-E022, an
endophytic fungus isolated from the stem of Quercus variabilis (Fagaceae).
Their structures were elucidated by comprehensive interpretation of spectral
data obtained from IR, UV, HRESIMS and analysis of 1D and 2D NMR
spectra. The stereochemistry of 5 and 6 was identified by comparing the
optical rotation data with those of vermistatin. Penicidones A–C (5-7) were the
first group of natural products possessing a penicidone framework.
Compounds (5-7) exhibited moderate cytotoxicity against four cancer cell
lines (Ge et al., 2008).
Novel azaphilone alkaloid dimers namely chaetofusins A (8) and B (9)
from the fungus Chaetomium fusiforme isolated from liverwort Scapania
verrucosa Heeg were identified. The evaluation of the antifungal properties
reveled a strong activities against C. albicans and A. fumigates (Peng et al.,
2012). The dimer structure of those compounds were confirmed by analysis of
their HRESIMS and 13C NMR spectra (Peng et al., 2012). Azaphilone dimer
alkaloids were first reported in 2008 from the fungus Chaetomium globosum
(Ming et al., 2008). Another three related new chlorinated azaphilone alkaloids
were reported from the culture broth of C. globosum in 2013 named as
chaetomugilides A-C (10-12) (Li et al., 2013) which reflects the role of
metabolite profiling in the chemotaxonomy of endophytes.
Chemical investigations of the cultures of fungus Penicillium vinaceum
isolated from the corm of Crocus sativus gave rise to a unique quinazoline
alkaloid (13), identified as (-)-(1 R,4R)-1,4-(2,3)-indolmethane-1-methyl-2,4-
dihydro-1 H-pyrazino-(2,1-b)-quinazoline-3,6-dione (Zheng et al., 2012).
Antifungal and cytotoxic properties were then assayed (Zheng et al., 2012). X-
ray crystallography confirmed the structure and absolute configuration of
Fusarimine (14) which is a new polyketide isoquinoline alkaloid isolated at
Prof. H. Laatsch group from Fusarium sp. LN12, isolated from Melia
azedarach (Yang et al., 2012).
In 2013 two new alkaloids, Mycoleptodiscins A (15) and B (16), with
naturally uncommon indol-terpene scaffold were identified from endophytic
fungus isolated from Desmotes incomparabilis identified as Mycoleptodiscus
sp. (Ortega et al., 2013).
A metabolite isolated from the Chinese mangrove Bruguiera gymnorrhiza
associated endophytic fungus Penicillium sp. GD6 was identified as a new

pyrrolizidine alkaloid, namely Penibruguieramine A (17) which possesses an

unusual 1-alkenyl-2-methyl-8-hydroxymethylpyrrolizidin-3-one skeleton
(Zhou et al., 2014a). Determination of its absolute configuration was
according to its experimental and Time-Dependent Density Functional Theory
(TDDFT) calculated electronic circulardichroism (ECD) spectra. This method
is considered a strong method in assigning the absolute configuration of
natural compounds (Zhou et al., 2014a). Figure (3) summarize the diversity of
alkaloid metabolites isolated from endophytic fungi (structures 1-17).
Figure 3. (Continued).

Figure 3. Chemical structures (1-17) show the diversity of alkaloid metabolites isolated
from endophytic fungi.
10.2. Coumarins and Isocoumarins Derivatives
Coumarins are bicyclic aromatic compound contain 2H-chromen-2-one or

any of its derivatives while isocoumarins are isomers of coumarins in which
the orientation of the lactone is reversed to be 1H-isochromen-1-one.
Dihydroisocoumarin is a reduced form of isocoumarin where the double bond
between carbon atoms no. 3 and 4 is saturated.
The ethyl acetate extract of an endophytic fungus identified as Periconia
atropurpurea collected from Xylopia aromatica which is an indigenous plant
of Brazilian Cerradocontained 6,8-Dimethoxy-3-(2'-oxo-propyl)-coumarin
(18) (Teles et al., 2006). A novel coumarin series, pestalasins A–E (19) was
identified from the endophytic fungus Pestalotiopsis sp., associated with the
leaves of the Chinese mangrove Rhizophora mucronata in the work group of

professor Peter Proksch (Xu et al., 2009). Recently a new coumarine, 4, 6-

dihydroxy-7-formyl-3-methylcoumarin (20) was isolated from the endophyte
Pestalotiopsis versicolor along with other known compounds (Yang et al.,
2015).
Novel metabolite has the isocoumarin nucleus with uncommon
substitution pattern, namely (4S)-(+)-ascochin (21) from Ascochyta sp was
isolated in 2007. Associated with Meliotus dentatus its absolute configuration
was confirmed by integration of X-ray diffraction and solid state TDDFT CD
(Krohn et al., 2007).
From the herbaceous plant Fagonia cretica associated endophyte
Microdochium bolleyi three Monocerin related isocoumarins were isolated
compounds (22-24). Compound (24) is considered as an open ring analogue
(Zhang et al., 2008). The absolute configuration of these compounds was
identified by modified Mosher’s method (Ohtani and Mosher, 1991).
A number of isocoumarins and isocoumarin derivatives had been isolated
from endophytic fungi associated with mangrove plants. (3R*, 4S*)-6, 8-
dihydroxy-3, 4, 7-trimethylisocoumarin (25) was isolated from Penicillium sp.
091402 in 2009 (Han et al., 2009). From Cephalosporium sp., a novel
prenylated dihydroisocoumarin was identified as 5-(3'-methylbut-2'-enyloxy)-
3, 4-dihydro 8-hydroxy-3-methylisochromen-1-one (26) (Wei et al., 2010).
Recently six new isocoumarin derivatives representative compounds are
shown (27-28) were isolated from Talaromyces amestolkiae YX1 along with
another nine previously reported compounds. Two of these compounds
showed antibacterial effects against different strains of bacteria and four
exhibited α-glucosidase inhibitory activity (Chen et al., 2016). Their absolute
configuration was assigned using modified Mosher’s method (Ohtani and
Narisada, 1991).
The dihydroisocoumarin (3R, 4R)-3,4-dihydro-4,6-dihydroxy-3-methyl-1-
oxo-1H-isochromene-5-carboxylic acid (29) was isolated from Xylaria sp., a
fungus associated with Piper aduncum (Piperaceae) was proved to have
antifungal and acetyl cholinesterase (AChE) inhibitory activities in vitro
(Oliveira et al., 2011). Tenuissimasatin (30) is a new dihydroisocoumarin
produced by the endophytic fungus Alternaria tenuissima Wiltshire isolated
from the bark of Erythrophleum fordii Oliver (Fang et al., 2012). Figure (4)
summarize the diversity of coumarins and isocoumarins derivatives isolated
from endophytic fungi (structures from 18-30).

Figure 4. Chemical structures (18-30) show the diversity of coumarins and

isocoumarins derivatives isolated from endophytic fungi.

10.3. Glycosides
Since decades the search for potentially antioxidant compounds from

natural sources has drawn the attention of scientists as they may contribute in
the treatment of diseases attributed to oxidative stress (Rice-Evans and
Diplock, 1993). The culture broth of the endophytic fungi Eurotium rubrum
which was derived from the stems of marine mangrove plant Hibiscus
tiliaceus and from which a novel anthraquinone glycoside designated 3-O-(α-
Dribofuranosyl)-questin (31) together with three known related glycosides
were tested for DPPH radical scavenging activity (Li et al., 2009). Another
Eurotium sp. identified as Eurotium cristatum isolated from the marine algae
Sargassum thunbergii produce a new anthraquinone glycoside, 3-O-(α-D-
ribofuranosyl) questinol (32) (Du et al., 2014). In 2015 the antioxidant activity
of a steroidal saponin isolated from an endophyte Lasiodiplodia theobromae
identified from Saraca asoca was investigated (Jinu et al., 2015).
Marine derived endophytic fungi provide us with an everlasting resource
for chemically and structurally different and varying novel natural products
(Bugni and Ireland, 2004). From a marine mangrove Scyphiphora
hydrophyllacea derived fungi a novel fatty acid glycoside was isolated whose
structure was identified by combining spectroscopic and chemical methods as
R-3-hydroxyundecanoic acid methylester-3-O-α-L-rhamnopyranoside (33)
(Zeng et al., 2012). As an emphasis on the importance of marine mangrove
plants one Penicillium sp. was derived from the plant Avicennia marina that
produced a novel aurone glycoside. Its structure identified as (Z)-7, 4'-
dimethoxy-6-hydroxyaurone-4-O-β-glucopyranoside (Song et al., 2015). The
endophytic fungus Phomopsis sp. (ZH76) isolated from the stem of the
mangrove tree Excoecaria agallocha found in Sea cost at south chine
produced a xanthone O-glycoside for the first time namely 3-O-(6-O-α-L-
arabinopyranosyl)-β-D-glucopyranosyl-1,4-dimethoxyxanthone (34) (Huang
et al., 2013).
Various types of glycosides have been identified from different genera of
endophytic fungi. A macrolacton glycoside was identified from the endophytic
Lecythophora sp. derived from the Indonesian plant Alyxia reinwardtii. It was
designated 23-methyl-3-(1-O-mannosyl)-oxacyclotetracosan-1 –one and it
exhibited antifungal activities against Aspergillus and Candida (Sugijanto
et al., 2011). Figure (5) summarize the diversity of glycosides compounds
isolated from endophytic fungi (structures from 31-34).

Figure 5. Chemical structures (31-34) show the diversity of glycosides compounds

isolated from endophytic fungi.
10.4. Peptides
Xylaria sp. as a seed-endophye of an angiosperm tree found in the South

China Sea produced a new cyclic peptide (35) with an unusual allenic ether
linkage of an N-(p-hydroxycinnamoyl) amide. The molecular formula of the
compound was determined by combining the data obtained from elemental
analysis. The allenic ether linkage identified by IR (Lin et al., 2001).
Epichlicin (36), a new cyclic peptide, was isolated from the endophytic fungus
Epichloe typhina derived from Phleum pratense L. Through the combination
between advanced Marfey method and chemical investigation the
stereochemical configuration of its atoms was identified (Seto et al., 2007).
Advanced Marfey’s method that involve acid hydrolysis (Fujii et al.,
1997) is used widely to assign the absolute configuration of peptides. This
method was used for the determination of the absolute configuration of
pullularins E (37) and F (38), two novel cyclic peptides identified from the
extracts of the fungus Bionectriaochroleuca isolated from the leaves of the
Sonneratia caseolaris (Ebrahim et al., 2012). Another example of using
advanced Marfey’s for peptides is the isolation and structure elucidation of
two new cyclic peptides, namely talaromins A (39) and B (40). Their ring

system contains six α-amino acid residues connected to a β-amino acid from
the metabolites of Talaromyces wortmannii an Aloe vera derived endophyt
(Bara et al., 2013). Very recently this year this method was used to identify the
absolute configuration of a novel cyclic hepta-peptide unguisin F (41) obtained
from the Mucor irregularis that was associated with the medicinal plant
Moringa stenopetala found in Cameroon (Akone et al., 2016).
By means of spectectral, chiral HPLC and X-ray crystallographic data two
new cyclic peptides were identified from the broth of mangrove endophytic
fungus isolated from the leaves of Kandelia candel growing in Hong Kong.
Their structures were assigned as cyclo-(D-Leu-Gly-L-Tyr-L-Val-Gly-S-
OLeu) and cyclo-(D-Leu-Gly-L-Phe-L-Val-Gly-S-O-Leu) (Huang et al.,
2007). Cycloaspeptide A (42) is a cyclic peptide isolated from the extracts of
the fermentation broth of Penicillium janczewskii isolated from the phloem of
the Chileangymnosperm Prumnopitys andina (Schmeda-Hirschmann et al.,
2008). The bioassay guided fractionation of a plant associated fungus
identified as Aspergillus tubingensis isolated from Brucea javanica lead to the
isolation of a cyclic penta-peptide namely malformin A1 (43) which showed a
high anti-Tobacco mosaic virus (TMV) activities (Tan et al., 2015). Figure (6)
summarize the diversity of peptides metabolites isolated from endophytic
fungi (structures from 35-43).
10.5. Steroids
Aspergillus sp. is extensively found to be associated with marine algae. In

addition, many steroids have been isolated from different Aspergillus sp., 7-
Nor-ergosterolide (44) was characterized from Aspergillus ochraceus EN-31
an endophytic fungus isolated from the marine brown alga Sargassum
kjellmanianum. It was the first report of a natural 7-nor-ergosteroid possessing
a pentalactone B-ring system and it displayed cytotoxic activities against
different cell lines (Cui et al., 2010). Another steroid, Asporyergosterol (45)
was identified from the broth extract of marine red algae Heterosiphonia
japonica derived Aspergillus oryzae. This novel steroid is characterized by
having an E double bond between C-17 and C-20 (Qiao et al., 2010).
Aspergillus flavus isolated from another marine red alga Corallina officinalis
produced a new steroid, namely 3β, 4α-dihydroxy-26-methoxyergosta-7, 24
(28)-dien-6-one (46) together with other previously reported steroids (Qiao et
al., 2011).

Figure 6. Chemical structures (35-43) show the diversity of peptides metabolites


Penicillium sp. as endophytic fungi found commensalistic with higher

plants and marine algae are considered an interesting source of new natural
compound and metabolites belonging to different chemical classes among
which steroids are of great importance. Penicisteroids A (47) and B (48) are
two novel highly oxygenated steroids with uncommon structure contains
tetrahydroxy and C-16-acetoxy groups. They were isolated from the
Penicillium chrysogenum isolated from red algae (Gao et al., 2011). From the
same species found associated to the traditional Chinese herb Huperzia serrata
three new steroids possess a unique C25 steroid with a bisnor C-atom side
chain, namely norcyclocitrinol A (49), erythro-11α-hydroxyneocyclocitrinol
(50) and pseudocyclocitrinol A (51) (Ying et al., 2014). Related structures
were isolated very recently in 2016 from another Penicillium sp. from
Saccharum arundinaceum for example, erythro-23-O-Methylneocyclocitrinol
(52) and its threo- isomer (Deng et al., 2016). Penicillium sp. GD6 derived
from Chinese mangrove Bruguiera gymnorrhiza produced a novel acetylated
steroid (53) (Zhou et al., 2014b). Very recently in 2016, Penicillium sp. from
Saccharum arundinaceum produced novel steroids with the unprecedented C25
structure.
Other species contribute also to the production of steroids from
endophytes. Globosterol (54) is a polyhydroxylated C29 steroid. It was firstly
reported from Chaetomium globosum isolated from Ginkgo biloba along with
already identified ergosterol derivative (Qin et al., 2009). Three novel steroids
designated as ergosta-5, 7, 22-trienol (55), 5α, 8α-epidioxyergosta-6, 22-dien-
3β-ol (56) and ergosta-7, 22-dien-3β,5α,6β-triol (57) were identified from the
culture extracts of Pichia guilliermondii derived from the medicinal plant
Paris polyphylla along with one nordammarane triterpenoid (Zhao et al.,
2010). Culture broth of Phomopsis sp. isolated from Aconitum carmichaeli
contained two novel steroids, namely (14β, 22E)-9, 14-dihydroxyergosta-4, 7,
22-triene-3, 6-dione (58) and (5α, 6β, 15β, 22E)-6-ethoxy-5, 1 5-
dihydroxyergosta-7, 22-dien-3-one (59) their antifungal activity was screened
(Wu et al., 2013). Figure (7) summarizes the diversity of steroidal metabolites
isolated from endophytic fungi (structures from 44-59).
10.6. Terpenoids
Terpenoids are a large group of organic natural products formed by the

assembly of a number of C5 isoprene units. Arisugacins I (60) and J (61) are
two novel compounds belonging to the subgroup meroterpenoids that were

isolated for the first time from Penicillium sp. SXH-65 in 2014 together with
five analouges compounds (Sun et al., 2014). Based on the interpretation of
extensive 1D and 2D NMR, mass spectroscopic and x-ray crystallographic
data, four novel merterpenoids compounds (62-65) were isolated from the
mangrove plant Acanthus ilicifolius associated-endophyte Aspergillus flavipes
(Bai et al., 2015).

Figure 7. Chemical structures (44-59) show the diversity of steroidal metabolites

Another four merterpenoids compounds were identified from the extracts

of Guignardia mangiferae isolated from the medicinal plant Smilax glabra,
namely guignardones P-S (66-69) (Sun et al., 2015). All the compounds were
screened for cytotoxic activities against various cell lines. Recently in 2016
four meroterpenoids, designated chermesins A-D (70-73) whose crystal
structure was assigned to have drimane-type sesquiterpene nucleus with an
unusual cyclohexa-2,5-dienone unit were firstly reported from the extracts of
the broth of Penicillium chermesinum EN-480 isolated from the marine red
alga Pterocladiella tenuis (Liu et al., 2016).

Sesquiterpenoids were repeatedly reported from endophytes associated

with plants from different habitats. Brasilamides (74-75) are tricyclic
sesquiterpens derived from the endophytic fungus Paraconiothyrium
brasiliense isolated from the branches of Acer truncatum (Liu et al., 2010). An
endophyte identified as Guignardia mangiferae was isolated from the leaves
of toxic plant Gelsemium elegans. The chemical investigation of the cultures
of this fungus yielded five novel Eremophilane sesquiterpenes (76-80) along
with known related terpenes (Liu et al., 2015).

Figure 8. Chemical structures (60-84) show the diversity of terpenoidal metabolites

An endophytic fungus identified as Aspergillus sp. YXf3 isolated from

Ginkgo biloba produced a C18 norditerpenoid, aspergiloid I (81). Its structure
is characterized by having a novel carbon scaffold containing a rare 6/5/6
tricyclic ring system holding α, β- unsaturated spirolactone unit in ring B. It
was the first report of a new subclass of norditerpenoid named aspergilane.
The biosynthetic pathway of compound (81) is proposed to start by the
attachment of dimethylallyl pyrophosphate unit (DMAPP) with three
isopentenyl pyrophosphate (IPP) units to form geranyl geranyl pyrophosphate
then an intermediate pimarane (Guo et al., 2014). Chemical examination of the
extracts of an endophytic fungus belongs to Ulocladium sp. isolated from the
lichen Everniastrum sp. from china resulted in the isolation of three novel
mixed terpenoids, tricycloalternarenes F–H (82-84) with another ten
previously reported terpenoidal compounds (Wang et al., 2013). Figure (8)

summarize the diversity of terpenoidal metabolites isolated from endophytic

fungi (structures from 60-84).
10.7. Others (New Chemical Classes)
Examples of compounds isolated and characterized from Endophytic fungi

belong to various chemical groups will be displayed in this section. Taxol (85)
the world wide famous anticancer drug is a densely function-groups
substituted diterpene derivative was firstly reported from endophytic fungi in
1993 from Taxomyces andreanae isolated from the yew Taxus brevifolia
(Huang et al., 2008). From culture extract of Curvularia sp. obtained from the
leaves of the Brazilian plant Ocotea corymbosa two new benzopyranes were
obtained together with two other related compounds. Depending on their
spectral data their structures were (2`S)-2-(propan-2`-ol)-5-hydroxy-
benzopyran-4-one (86) and 2, 3-dihydro-2-methyl-benzopyran-4, 5-diol (87).
The antifungal and cytotoxic activities against different cell lines were tested
(Teles et al., 2005). An isolate of an endophytic fungus from the marine brown
algae defined as Aspergillus niger EN-13 produced a novel antifungal
naphthoquinoneimine derivative named 5,7-dihydroxy-2-(1-(4-methoxy-6-
oxo-6H-pyran-2-yl)-2-phenylethylamino)(1,4)naphthoquinone (88) (Zhang et
al., 2007). Three chlorinated benzophenones were characterized from the
culture extracts of the plant endophytic fungi Pestalotiopsis adusta, namely
Pestalachlorides A–C (89-91), X-ray crystallography was used to confirm the
structures of compounds (89-91) Pestalachlorides A was obtained as a racemic
mixture (Li et al., 2008).
In 2012 four butyrolactone derivatives were isolated from the endophytic
fungus Aspergillus flavipes MM2 associated with Egyptian rice hulls were
reported, namely butyrolactone I (92), aspulvinone H (93), butyrolactone-V
(94) and 4, 4’-diydroxypulvinone (95). The antimicrobial activity of these
compounds against various microorganisms and their cytotoxicity was
screened using brine shrimp (Nagia et al., 2012). Recently the chemical
investigation of the broth extract of endophytic fungus Aspergillus terreus
from isolated from the roots of Carthamus lanatus resulted in the isolation of
two novel butyrolactones, Aspernolides F (96) and G (97). The antimicrobial
and antileishmanial activity of these compounds were tested (Ibrahim et al.,
2015). Figure (9) summarize the diversity of new classes of endophytic
metabolites (structures from 85-97).

Figure 9. Chemical structures (85-97) show the diversity of new classes of endophytic
metabolites.

11. FUTURE PERSPECTIVES AND NEW PROMISING

ECOLOGICAL NICHES
An intensive search for new and more effective agents to deal with
increasing disease problems is now under way and this including the search in
new ecological niches for potential new sources. The marine environment was
introduced novel and rich source of functional metabolites which have
potentially useful medicinal applications (Debbab et al., 2010 and Ebada et al.,
2010).
In the last decades it was shown that marine natural products are of great
importance in the drug discovery process, particularly in the areas of deadly
diseases such as neoplasia and infections by multi-drug resistant pathogen.
Recently, much attention has been given to marine organisms due to their
considerable biodiversity that has been found in the wide spread oceans that
cover over 70% of the world. Consider the fact that many marine
microorganisms have a chemical system of defense, bioactive natural products
from marine microorganisms are released into the water and therefore are
rapidly diluted and accordingly they must be very potent materials to have the
desired end effect (Blunt et al., 2016; Debbab et al., 2010 and Ebada et al.,
2010).
A wider range of compounds were the predominantly reported compound
classes has been reported from soft coral, algae (green, brown and red) and
sponge. The cyclic depsipeptide kahalalide F (Figure 10) originally isolated
from the dietary source the green alga Bryopsis sp., and was introduced into
Phase I trials as a lead compound against prostate cancer (Hamann et al.,
1993), while other isolated compounds, like brominated diterpenes (prevezols
C–E) from red algae, Laurencia sp. (Figure 10) which displayed significant
cytotoxicity against the human tumors cell lines (Iliopoulou et al., 2003). A
new norsesterterpene acid named muqubilone were isolated from the Red Sea
sponge Diacarnus erythraeanus and display in vitro antiviral activity and
antimalarial with potent activity against Toxoplasma gondii without significant
toxicity (El Sayed et al., 2001). Therefore, the marine environment especially
sponges, soft corals and algae continue to be a noteworthy source of novel
metabolites and an extraordinary growing tendency for the characterization of
compounds from endophytic microbes that have been isolated from marine
sources (Blunt et al., 2016; Debbab et al., 2010 and Ebada et al., 2010).

Figure 10. Chemical structures of promising compounds isolated from marine

macrorganisms.
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Chapter 3
ENDOPHYTIC FUNGI ISOLATED

FROM VOCHYSIA DIVERGENS IN
THE PANTANAL, MATO GROSSO DO SUL:
DIVERSITY, PHYLOGENY AND BIOCONTROL
OF PHYLLOSTICTA CITRICARPA
Y. M. Hokama1, D. C. Savi1, B. Assad1, R. Aluizio1,

J. A. Gomes-Figueiredo1, D. M. Adamoski1,
Y. M. Possiede2 and C. Glienke1,*
1
Federal University of Paraná, Department of Genetics,
Curitiba, PR, Brazil
2
Federal University of Mato Grosso do Sul,
Department of Biology, Campo Grande, MS, Brazil
ABSTRACT
Endophytic fungi are important biotechnological tools because they
produce many secondary metabolites. However, to access this important
source of bioactive molecules, it is essential to explore the diversity of
endophytic fungi and catalog their species richness in different
ecosystems. Tropical regions are recognized as areas of high diversity,
although many areas remain unexplored, such as the Pantanal of Mato
*
Corresponding author: Dr. Chirlei Glienke, ch.glienke@gmail.com, Phone: (55) 4133611562.

94 Y. M. Hokama, D. C. Savi, B. Assad et al.
Grosso do Sul, Brazil. This study is the first to explore the diversity of
endophytic fungi in the medicinal plant Vochysia divergens, found in the
Pantanal. In total, 77 isolates were identified by ITS1–5.8S–ITS2 rDNA
sequencing and phylogenetic analysis as belonging to the genera
Antrodia, Irpex, Peniophora, Phyllosticta, Neofusicoccum,
Pseudofusicoccum, Polyporus, Daldinia, Nigrospora, Colletotrichum,
Diaporthe, Lanceispora, Cladosporium, Phaeosphaeria, and
Annellosympodiella. Nineteen isolates were identified as belonging to the
Xylariaceae family, and our data indicate that these isolates are members
of a new genus in this family. We also explored the antifungal activity of
three isolates, two of which belong to the family Xylariaceae
(LGMF1119 and LGMF1133) and one belongs to the genus Nigrospora
(LGMF1121) that inhibited Phyllosticta citricarpa mycelium growth and
pycnidia formation in vitro assays.
Keywords: Vochysia divergens, pantanal, biodiversity, endophytes,

Phyllosticta citricarpa, biological control
INTRODUCTION
Endophytic fungi inhabit a unique biological niche, because of their
ability to asymptomatically colonize plant tissues (Jia et al. 2016). It is
estimated that there are more than 420,000 plant species in nature; however,
the endophytic community has only been catalogued in a few of these.
According to Chowdhary et al. (2015) the diversity of fungal endophytes is
7% out of total of 1.5 million fungi on earth. Endophytic fungi are important
because of their capacity to produce structurally and biologically unique
natural products (Strobel et al. 2004; Gunatilaka 2006; Glienke et al. 2012;
Chowdhary et al. 2015). To increase our knowledge of fungal diversity, as
well as to describe new species, plants that live in peculiar habitats are a
promising source for the exploration of endophytes.
Our group is mainly interested in exploring the endophytic communities
of medicinal plants located in the Brazilian Pantanal. The Pantanal is a
floodplain of more than 140,000 km², located in central South America,
mainly in Brazil with some extensions into Paraguay and Bolivia (Arruda et al.
2016). The Pantanal harbors diverse flora and fauna, resulting from its unusual

Endophytic Fungi Isolated from Vochysia divergens … 95
variety of habitats and its hydrological cycle, with seasonal inundation

enduring for more than 200 days a year (Junk et al. 2006). Because of the
dynamism of the Pantanal, a limited number of plants are able to tolerate the
long periods of flooding. Among them is Vochysia divergens Pohl (Arieira et
al. 2006), a medicinal plant found in large communities in areas of the
Pantanal that are both periodically and permanently flooded (Arieira et al.
2006). This plant, used in folk medicine to treat respiratory and gastric
diseases (Pott et al. 1994) is a source of various substances, some with
antimicrobial potential (Honda et al. 1995) and others with pharmacological
importance (Hess et al. 1999).
This study aimed to assess the biodiversity of the endophytic community
in the medicinal plant V. divergens Pantanal/Brazil, as well as the capacity of
three isolates to control the phytopathogen Phyllosticta citricarpa, the
etiological agent of citrus black spot (CBS) disease, which depreciates the
commercial value of fruit and increases production cost considerably in citrus-
producing regions worldwide (Wulandari et al. 2009, Glienke et al. 2011).
MATERIAL AND METHODS

Sample Collection and Endophyte Isolation
Leaves of V. divergens Pohl (Cambará) were collected from Santa Emilia

Farm (Rio Negro Pantanal sub-region), Mato Grosso do Sul, Brazil (Figure 1).
Nine plants were collected between the years 2008 and 2010 (Table 1).
V. divergens leaves without any stains or bruises caused by insects,
mechanical damage, or pathogens were collected. All plant materials were
placed in sterile plastic bags and transported to laboratory under refrigeration
within 72 hours of collection. The leaves were submitted to a five-step
disinfection protocol to eliminate epiphytic microorganisms as described by
Petrini et al. (1993). Leaves were cut, aseptically, into 0.5-cm pieces and
seeded in petri dishes containing potato dextrose agar (PDA), pH 6.8, with
antibiotic tetracycline (100 μg/mL). Petri plates were incubated at 28°C with a
12-h photoperiod for 30 days. Fungi growing from leaf segments were
subsequently transferred to fresh PDA plates.

Table 1. Isolates, identification, position of individually sampled Vochysia

divergens, soil characteristic and GenBank access number
Filo Class Identification Fungal Tree Soil GenBank

isolate Location Characteristic access
number
ITS
Basidiomycota Agaricomycetes Antrodia sp. LGMF1145 E Dry JX559566
Irpex lacteus LGMF1153 E Dry JX559574
Peniophora laxitexta LGMF1159 C Dry JX559574
Polyporus sp. LGMF1191 A Flooded JX559609
Ascomycota Sordariomycetes Daldinia sp. LGMF1131 2 Flooded JX559552
Xylariaceae LGMF1126 2 Flooded JX559547
LGMF1134 1 Dry JX559555
LGMF1115 4 Flooded JX559537
Nigrospora sp. LGMF1156 D Flooded JX559577
LGMF1133 1 Dry JX559554
LGMF1138 1 Dry JX559559
Colletotrichum sp. LGMF1173 A Flooded JX559593
LGMF1137 1 Dry JX559558
LGMF1135 1 Dry JX559556
Diaporthe sp. LGMF1144 E Dry JX559565
LGMF1157 C Dry JX559578
LGMF1165 B Dry JX559585
LGMF1141 1 Dry JX559562
LGMF1136 1 Dry JX559557
Lanceispora sp. LGMF1142 E Dry JX559563
LGMF1152 E Dry JX559573
Dothideomycetes Phyllosticta LGMF1139 1 Dry JX559560
capitalensis LGMF1177 A Flooded JX559597
LGMF1174 A Flooded JX559594
LGMF1140 1 Dry JX559561

Filo Class Identification Fungal Tree Soil GenBank

isolate Location Characteristic access
number
Ascomycota Dothideomycetes Phyllosticta LGMF1175 A Flooded JX559595
capitalensis LGMF1155 D Flooded JX559576
LGMF1132 1 Dry JX559553
LGMF1154 D Flooded JX559575
Phyllosticta sp. LGMF1193 A Flooded JX559611
Phaeosphaeria sp. LGMF1195 A Dry JX559613
Neofusicoccum LGMF1116 4 Flooded JX559538
grevilleae
Pseudofusicoccum LGMF1122 2 Flooded JX559543
stromaticum LGMF1118 3 Flooded JX559540
Cladosporium sp. LGMF1129 2 Flooded JX559550
Annellosympodiella LGMF1117 3 Flooded JX559539
sp.
Note: Nine plant samples were collected on different occasions between 2008-2011. The collection points were
identified by letters and numbers: 1(19.501944ºS/55.601389ºW), 2(19.500833ºS/55.600556ºW),
3(19.500556ºS/55.600278ºW), 4(19.501111ºS/55.600556ºW), A(19.508548ºS/55.628775ºW),
B(19.506286°S/55.610172ºW), C(19.509930ºS/55.627039ºW), D(19.509038ºS/55.626354ºW) and
E(19.507243ºS/55.615815ºW).
DNA Extraction, Amplification, Sequencing, and Phylogenetic

Analysis
To extract DNA, the isolates were cultured in PDA for 3 days at 25°C and
then subjected to a MOBIO Ultraclean Microbial DNA Isolation Kit (MO
Bio, Carlsbad, US), following the manufacturer’s instructions.

Figure 1. Map showing the Pantanal colored in gray and the collection site is in the
magnification box, with the points at Santa Emilia Farm illustrating the nine sampled
trees. The collection points were identified by letters and numbers:
1(19.501944ºS/55.601389ºW), 2(19.500833ºS/55.600556ºW),
3(19.500556ºS/55.600278ºW), 4(19.501111ºS/55.600556ºW),
A(19.508548ºS/55.628775ºW), B(19.506286°S/55.610172ºW),
C(19.509930ºS/55.627039ºW), D(19.509038ºS/55.626354ºW) and
E(19.507243ºS/55.615815ºW).
Amplification of the internal transcribed spacer (ITS) region was

performed under the conditions described by White Jr. and Morrow (1990)
with the primers V9G (De Hoog et al. 1998) and ITS4 (White and Morrow
1990). The reactions were performed in an Eppendorf MasterCycler Gradient
Thermal Cycler. PCR amplification products were visualized and quantified
by electrophoresis in a 1.5% agarose gel (w/v), stained with GelRed Nucleic
Acid Gel Stain (Biotium Inc., Hayward, US) and using a Ludwig Biotec
(Ludwig Biotec, Alvorada, BR) molecular weight marker. PCR products were
purified by ethanol precipitation (Fredricks et al. 2005) and directly sequenced
using a DYEnamic ET Dye Terminator Kit (Amersham Biosciences),
following instructions from the manufacturer. The sequencing reactions were
purified using Sephadex gel G-50 column (GE Healthcare) in ELISA wells,

and subjected to electrophoresis on automated DNA sequencer MegaBACE

(Amersham Biosciences).
DNA sequences were compared with sequences available in the National
Center for Biotechnology Information database (http://blast.ncbi.
nlm.nih.gov/Blast.cgi) using the BLAST tool (Altschul, 2005). Sequences of
type strains were obtained from MycoBank (http://www.mycobank.org) and
GenBank (http://www.ncbi.nlm.nih.gov/genbank). Alignments of DNA
sequences were performed using the BioEdit version 7.2.5 (Hall, 2013) and
ClustalW (Thompson et al. 1994) in MEGA version 6 (Tamura et al. 2013).
Bayesian inference of the phylogeny was performed in MrBayes version 3.2.1
(Ronquist et al. 2011), with permutations allowed until a frequency of division
≤0.01 was reached. The general time-reversible (GTR) substitution model was
used. FigTree version 1.4.2 (Rambaut, 2012) was used to edit the phylogenetic
trees that were constructed. Sequences obtained in this study were deposited in
GenBank with the accession numbers listed in Table 1.
Antifungal Activity against the Phytopathogen

Phyllosticta citricarpa
Based on previous results, isolates LGMF1133, LGMF1121, and

LGMF119 were selected to evaluate their antifungal activity against the
phytopathogen Phyllosticta citricarpa. The fungal isolates were examined for
the production of non-volatile or volatile metabolites in a randomized design
with five replicates, using methods outlined by Morris et al. (2010). Each
sample was cultured in a petri dish and evaluated after 14, 21 and 28 days. The
negative control received only Phyllosticta citricarpa, and the positive control
received fungicide glyphosate (1 mg/mL). To determine the inhibition
percentage (IP), the diameters of colonies were measured, and the IP was
calculated according to the following formula: IP = mycelial growth in the
control − mycelial growth in the treated sample/mycelial growth in the control
× 100. The endophytes that produced non-volatile active metabolites were
selected for the production of extracts.
Extract Production
Extracts from the endophytes were obtained by fermenting three mycelial

discs (8 mm) in 250 mL of potato dextrose (PD) medium (200 g of potato, 20

g of glucose, and 1000 mL distillated water) under agitation for 28 days (120
rpm, 28°C). After fermentation, mycelia were separated from the fermented
liquid by filtration through Whatman nº4 filter paper. Metabolites were
extracted with ethyl acetate (3 × v) to obtain the liquid fraction, and the
mycelia were extracted using another 30 mL of ethyl acetate (Merck,
Germany, PA). The solvent was evaporated using a rotaevaporator at 45°C.
The final extracts were diluted in methanol at a concentration of 10 mg/mL.
Extract Activity against the Phytopathogen

Antagonistic activity of the endophyte extracts against Phyllosticta

citricarpa was evaluated by two approaches: determining the growth of P.
citricarpa and inhibition of pycnidia formation.
Growth Inhibition Analysis
The inhibition of mycelial growth was evaluated by measuring colony

diameters on PDA medium, where one central disc with the phytopathogen (12
mm) was deposited in the center of each petri dish, and its growth on the plate
surface was compared with that of the phytopathogen treated with 1 mg of
extract. Measurements were recorded after 14, 21, and 28 days of incubation,
with 10 experimental replicates. Glyphosate 1.0 mg/mL (Bayer Leverkusen)
was used as a positive control.
Pycnidia Formation Analysis
To analyze pycnidia formation, Citrus limonia Osbeck leaves were

washed in water, cut into fragments (10 mm), and autoclaved in distilled
water. Three leaf fragments were placed in petri dishes with water-agar (1.5%
w/v) culture medium. Four 2-mm-thick discs of Phyllosticta citricarpa
mycelia were inoculated close to each leaf fragment, 100 µg of extract was
added to each dish, except for that of the control, in which just the
phytopathogen was cultured. The petri dishes were sealed and maintained
at 28°C with a 12-h photoperiod for 21 days. After this period, the pycnidia of
P. citricarpa that formed above the leaves were counted under a

stereomicroscope. For this experiment, we used a randomized design with

three replications.
RESULTS AND DISCUSSION

Medicinal plants are considered a repository of endophytic fungi, and the
quest to identify novel bioactives has led to the exploration of plants located in
places with unique ecological adaptations (Somaiah et al. 2014). To explore
biodiversity in the medicinal plant V. divergens found in the Pantanal, a
peculiar wetland in Brazil, 77 endophytic fungal strains were isolated. These
isolates were identified by phylogenetic analysis of the ITS region as strains
belonging to the phyla Basidiomycota (4%) and Ascomycota (96%).
Figure 2. Bayesian phylogenetic tree based on ITS sequence, showing the relationships
between Polyporus, Peniophora, Irpex and Antrodia genera. The tree is rooted with
Geastrum campestre sequence obtained from GenBank. Scale bar shows 8 changes and
Bayesian posterior probability values are indicated at the nodes.

between Xylariaceae family species. The tree is rooted with Neurospora crassa
sequence obtained from GenBank. Scale bar shows 2 changes and Bayesian posterior
probability values are indicated at the nodes.
Isolates identified by ITS phylogenetic analysis as Basidiomycota (Figure

2) were found to belong to class Agaricomycetes, genera Antrodia (Antrodia

sp. LGMF1145), Irpex (Irpex lacteus LGMF1153), Peniophora (Peniophora

laxitexta LGMF1159), and Polyporus (Polyporus sp. LGMF1191). Endophytic
fungi in this phylum are unusual, as Basidiomycota are usually isolated as
mycorrhizal fungi from plants in the family Orchidaceae or directly from the
environment (Rungjindamai et al. 2008). We report here, for the first time,
endophytic strains belonging to the genera Antrodia and Irpex. Antrodia and
Irpex species are normally associated with diseases in roots (Kim et al. 2001;
Tavčar et al. 2006). Peniophora, however, is an endophyte that was previously
isolated from Pinus tabulaeformis (Pinaceae) in northeast China (Wang et al.
2005), and Polyporus is an endophyte that was isolated from branches and
bark of Taxus globosa (Rivera-Orduña et al. 2011). Polyporus species are
recognized as producers of numerous metabolites that are effective in treating
effluents from the textile industry (Sinegani et al. 2011).
Ascomycota strains represented 96% of the isolates, and these isolates
were classified as belonging to the classes Dothideomycetes and
Sordariomycetes.
Sordariomycetes
The phylogenetic trees (Figures 3 to 8) show that the Sordariomycetes

strains segregate into five distinct clades, residing in the genera Daldinia
(n = 1), Colletotrichum (n = 3), Lanceispora (n = 4), Nigrospora (n = 5) and
Diaporthe (n = 5), and one other clade (Xylariaceae) which genus remain to be
elucidated (n = 11).
Xylariaceae
In the Xylariaceae family, twelve isolates were attributed, but using ITS
sequences (Figure 3). The isolate LGMF1131 was identified as Daldinia sp.
and the species was not clarified (Figure 3 and 4). However, the strains
LGMF1114, LGMF1115, LGMF1119, LGMF1120, LGMF1123, LGMF1124,
LGMF1126, LGMF1127, LGMF1128, LGMF1130, and LGMF1134 in the
ITS tree clustered on two isolated branch and were characterized as
Xylariaceae 1 and Xylariaceae 2 (Figure 3). To verify whether these strains
constitute to a new genus, we sequenced the LSU region of strains
LGMF1119, LGMF1131 and LGMF1134 (Figure 5). Strain LGMF1131
clustered with the Daldinia genus in the LSU analysis, whereas strains
LGMF1119 (Xylariaceae 1) and LGMF1134 (Xylariaceae 2) formed a clade
distinct from other genera in the Xylariaceae family. These data suggest that

these 11 strains are species of new genera inside Xylariaceae, however,

additional studies combining morphological and multigene sequences analysis
are necessary to describe these genera.
between Daldinia species. The tree is rooted with Annulohypoxylon minutellum
probability values are indicated at the nodes.

Figure 5. Bayesian phylogenetic tree based on LSU sequence, showing the

relationships between Xylariacea family. The tree is rooted with Lulworthia
grandispora sequence obtained from GenBank. Scale bar shows 10 changes and
Bayesian posterior probability values are indicated at the nodes.

Figure 6. A. Bayesian phylogenetic tree based on ITS sequence, showing the

relationships between Nigrospora species. The tree is rooted with Neurospora crassa
probability values are indicated at the nodes. B. Bayesian phylogenetic tree based on
LSU sequence, showing the relationships between Nigrospora species. The tree is
rooted with Neurospora terricola sequence obtained from GenBank. Scale bar shows 4
changes and Bayesian posterior probability values are indicated at the nodes.
Nigrospora
In the phylogenetic analysis (Figure 6A and 6B), isolates LGMF1121,
LGMF1133, LGMF1138, and LGMF1156 were found to be closely related to
the species Nigrospora oryzae, whereas isolate LGMF1125 was most closely
related to strain Nigrospora sphaerica (Figure 6 A–B). Strains LGMF1121
and LGMF1133 did not cluster with any type species in this genus; however,
there are few sequences available for this genus (Figure 6). In contrast,
Nigrospora is a genus that has been studied widely because of its secondary
metabolites (Kim et al. 2001; Zhang et al. 2009), and strains in this genus have
been isolated as endophytes from a large number of tropical plant species,
especially medicinal plants (Martinez-Luis et al. 2011).
Colletotrichum
Based on ITS sequence analysis, strains LGMF1135, LGMF1137, and
LGMF1173 were classified as species belonging to the Colletotrichum
gloeosporioides complex. However, as expected, ITS sequences are not
enough to identify the species in this complex (Figure 7). Species of the genus
Colletotrichum are pathogenic (Huang et al. 2009) as well as endophytic in

several plant species, mainly from tropical regions (Refaei et al. 2011; Vega et
al. 2010).
between species of Colletotrichum gloeosporioides complex. The tree is rooted with
Colletotrichum boninense type strain sequence obtained from GenBank. Scale bar
shows 5 changes and Bayesian posterior probability values are indicated at the nodes.
T indicates type strain sequence.

between Diaporthe species. The tree is rooted with Diaporthella corylina sequence
obtained from GenBank. Scale bar shows 8 changes and Bayesian posterior probability
values are indicated at the nodes.

between Phyllosticta species. The tree is rooted with Botryosphaeria obtusa sequence
obtained from GenBank. Scale bar shows 10 changes and Bayesian posterior
probability values are indicated at the nodes. T indicates the type strain sequence.
Diaporthe
Strains LGMF1136, LGMF1141, LGMF1157, and LGMF1165 were
identified as belonging to the genus Diaporthe (Figure 8). Strains LGMF1136

and LGMF141 are clustered with strains named as Diaporthe sp. 1 from Brazil
(Gomes et al. 2013), showing 100% of similarity. However, strains
LGMF1144 and LGMF1157 formed a clade with long branch suggesting these
isolates belong to a new species of Diaporthe. To more precisely assign strains
LGMF1144 and LGMF1157 to a taxon, multilocus sequencing is suggested, as
used previously for endophytic strains from Brazilian medicinal plants (Gomes
et al. 2013). Diaporthe species are frequently isolated endophytes found in
several plants (Hakizimana et al. 2011; Singh et al. 2011; Gomes et al. 2013).
Lanceispora
Isolates LGMF1142, LGMF1143, LGMF151, and LGMF1152 showed
high sequence similarity with the species Lanceispora amphibia (family
Amphisphaeriaceae; GenBank accession number LC146743). Even though the
genus Lanceispora was described by Nakagiri et al. in 1997 and was found to
comprise two species, L. amphibia and L. phyllophila, there are no L.
phyllophila sequences available in public databases, thus complicating the
identification of our isolates at the species level. Both L. amphibia and L.
phyllophila were identified as endophytes, first isolated from Bruguiera
gymnorrhiza in mangrove forests in the Southwest Islands of Japan and from
unknown dicotyledonous leaves in Singapore (Nakagiri et al. 1997; Sarma et
al. 2001). It is interesting that we isolated strains of this genus from medicinal
plants in Brazil, showing the high level of biodiversity in Pantanal of Mato
Grosso do Sul.
Dothideomycetes
Dothideomycetes order are commonly isolated as endophytes from a wide
number of plants (Crous et al. 2006). In our study, based on the phylogenetic
trees (Figures 9 to 12) the endophytic strains belonging to Dothideomycetes
were attributed to six genera: Phyllosticta (n = 38), Pseudofusicoccum (n = 2),
Neofusicoccum (n = 2), Cladosporium (n = 1), Annellosympodiella (n = 1) and
Phaeosphaeria (n = 1).
Phyllosticta
Among the isolates belonging to the genus Phyllosticta genus (Table 1,
Figure 9), strains LGMF1172 and LGMF1193 are most closely related to the
species Phyllosticta podocarpi, P. pseudotsudae, P. owaniana, and P.
bifrenariae, however with high genetic distance when compared with others
Phyllosticta species in the phylogenetic tree (Figure 9). The remaining
Phyllosticta isolates were identified as Phyllosticta capitalensis, supported by

a 100% bootstrap value and low intraspecific diversity (Figure 9). Phyllosticta
capitalensis is a common endophyte found in a large number of plants
(presently described in >70 plant families) distributed worldwide (Glienke et
al. 2011).
Figure 10. Bayesian phylogenetic tree based on ITS sequence, showing the
relationships between the species of Neofusicoccum and Pseudofusicoccum genera.
The tree is rooted with Botryosphaeria dothidea sequence obtained from GenBank.
Scale bar shows 2 changes and Bayesian posterior probability values are indicated at
the nodes.
Botryosphaeriaceae
In the phylogenetic analysis strains LGMF1122 and LGMF1118 grouped
with Pseudofusicoccum stromaticum, whereas the isolate LGMF1116
clustered with Neofusicoccum brasiliense type strain (Figure 10). Both
Pseudofusicoccum and Neofusicoccum belong to Botryosphaeriaceae family,
and they were recently separated from the genus Fusicoccum based on
morphological differences in their conidia and molecular sequences (Crous

et al. 2006). Species in these genera are normally found as endophytes and
pathogens of woody plant species such as Eucalyptus and Acacia (Mohali et
al. 2007; Pérez et al. 2010), and they have caused serious economic losses in
Pinus cultures in South America (Burgess et al. 2007).
relationships between species of Cladosporium genus. The tree is rooted with
Cercospora beticola sequence obtained from GenBank. Scale bar shows 2 changes and
Bayesian posterior probability values are indicated at the nodes. T indicates type strain
sequence.

relationships between Phaeosphaeria species. The tree is rooted with Stogonospora
foliicola type strain sequence obtained from GenBank. Scale bar shows 1 change and
Bayesian posterior probability values are indicated at the nodes. T indicates type strain
sequence.
Isolate LGMF1129, found to belong to the genus Cladosporium, with high

similarity with the type strain Cladosporium tenuissimum (Figure 11). This
genus includes endophytes that are commonly isolated from various plants, as
well as from atmospheric air and soil samples (Bensch et al. 2010; Nair et al.
2014).
Isolate LGMF1117 was identified as Annellosympodiela juniper, the type
species from the genus that was described in 2014 by Crous et al. Isolate
LGMF1117 showed 97% similarity in ITS sequence with A. juniper type strain
and probably represents a new species of the Annellosympodiela genus.
Strain LGMF1195 was found to belong to the genus Phaeosphaeria, and
are closely related to type strain P. oryzae, P. musae and P. papayae (Figure
12) and a multilocus analysis is necessary to identify the species.
Phaeosphaeria species are common endophytes found in a large number of
plants, including plants found in peculiar areas, such as Antarctic (Rosa et al.
2009).

ENDOPHYTES BIODIVERSITY IN FLOODING

AND DRY SESSION
To evaluate the influence of an inundation pulse on the endophytic

community in V. divergens, we analyzed the rates of isolation in the flooding
period (November–June) and dry period (July–October). Out of 77 isolates, 33
(42.8%) were obtained in the dry period and 44 (57.2%) were obtained in the
flooding period. Isolates belonging to genera Polyporus (Basidiomycota,
Agaricomycete); Neofusicoccum, Pseudofusicoccum, and Cladosporium
(Ascomycetes, Dothideomycetes) were obtained exclusively in the flooding
period, whereas isolates belonging to the genera Antrodia, Irpex, Peniophora
(Basidiomycota, Agaricomycete); Diaporthe (Ascomycetes, Diaporthaceae),
Lanceispora (Ascomycetes, Amphisphaeriaceae); Annellosympodiela and
Phaeosphaeria (Ascomycetes, Dothideomycetes) were isolated exclusively in
the dry period. Other genera were isolated from V. divergens in both periods
(Table 1). Based on species number and species diversity, our data suggest
preferential colonization during the dry period (Table 1, Figure 2–12).
Preferential endophytic colonization with varying water availabilities has been
reported previously in the literature. For example, Osmorhiza depauperata,
Agrobacterium tumefaciens, and Sinorhizobium meliloti bacteria were found
to be more abundant in sites with higher precipitation and higher annual
temperatures, whereas Paenibacillus strains were more common at sites in
higher latitudes and with lower precipitation (Li et al. 2012). In addition, rice
endophytic nitrogen-fixing Azoarcus spp. are more abundant with greater
water availability (Gaiero et al. 2013).
Antifungal Activity against the Phytopathogen

Endophytic microorganisms are explored because of their capacity to

reduce herbivory or phytopathogen settling, which promotes plant growth and
induces systemic resistance in plants (Miller et al. 2012). Thus, endophytic
protection can provide an alternative to traditional pesticide treatments (Niu et
al. 2014). Our group is interested in biological control of citrus diseases.
Among them, citrus black spot (CBS) disease is of great importance, because
it is associated with large economic losses that result from phytosanitary
restrictions, the lack of an effective treatment, and restrictions on the presence

of fungicide residues in fruits (Schreiber et al. 2012). Therefore, we explored

the endophytic community in the medicinal plant V. divergens to identify
strains that could be used to control CBS disease caused by the fungus
Phyllosticta citricarpa (Hokama, 2012).
Three strains were selected for an exploration of their capacity to control
Phyllosticta citricarpa based in previous results from paired cultures
(Hokama, 2012). Strains LGMF1133 and LGMF1121, both belonging to the
genus Nigrospora, and LBMF1119, belonging to family Xylariaceae,
completely inhibit growth of the phytopathogen Phyllosticta citricarpa (Table
2 and Figure 13). In addition, these strains were examined for their production
of volatile compounds and activity of less than 20% was observed (Table 2),
suggesting that the activity against P. citricarpa was due to the production of
non-volatile metabolites. For the extraction of secondary metabolites, the three
strains were cultivated in PD liquid medium. The liquid phases of cultures
were separated from mycelia by filtration, and both were subjected to
extraction by ethyl acetate. The solvent was evaporated, and metabolites were
weighed and diluted to a final concentration of 10 mg/mL. The extract from
strains LGMF1133, LGMF1121, and LBMF1119 showed strong inhibition of
mycelial growth (>80%), except for the extract from strain LGMF1121 (Table
3 and Figure 14).
We also examined inhibition of Phyllosticta citricarpa pycnidia formation
by these extracts. High inhibitory activity (65.3%) was observed in the extract
from the liquid phase of strain LGMF1121 (Table 3 and Figure 15); however,
all extracts inhibited pycnidia formation by more than 15%. Minimization of
pycnidia in plants is important in terms of disrupting the CBS disease cycle,
because the asexual stage of fungal growth aggravates disease in the plant and
surrounding areas (Perryman et al. 2014). Therefore, strategies to minimize the
formation of P. citricarpa pycnidia in citrus plants are of fundamental
importance and may offer a breakthrough in disease control research. To
confirm the biological control activities of these strains, studies of interactions
in plants as well as chemical identification of the secondary metabolites will
be needed. Strains belonging to the genus Nigrospora and family Xylariaceae
are promising as disease control agents, because these taxa are associated with
the production of large numbers of secondary metabolites with diverse
biological activities (Buchanan et al. 1995, Pongcharoen et al. 2008; Zhan et
al. 2009; Zhang et al. 2016).

Figure 13. Activity of non-volatile metabolites produced by endophytic strains against

Phyllosticta citricarpa 14 days after inoculation with a) 5 mg of fungicide glyphosate
(Positive control); the strains b) LGMF1133 (Nigrospora sp.), c) LGMF1121
(Nigrospora sp.) and d) LGMF1119 (Xylariaceae 1); e) Water (Negative control).
Table 2. Inhibition of Phyllosticta citricarpa LGMF06 mycelia growth by

non-volatile and volatile metabolites produced by three endophytic
isolates from Vochysia divergens
Treatment Non-volatile Volatile

Mycelium growth Mycelium growth
Inhibition % Inhibition %
NC 6,24 ± 1,9 0 11,69 ± 0,9 0
PC 0 ± 0 100 0 ± 0 100
LGMF1133 (Nigrospora sp.) 0 ± 0 100 9,7 ± 1,6 17
LGMF1121 (Nigrospora sp.) 0 ± 0 100 9,57 ± 2,9 18
LGMF1119 (Xylariaceae) 0 ± 0 100 11,27 ± 0,6 4
Note: NC: negative control, just the phytopathogen LGMF06; PC: positive control, the
phytopathogen LGMF06 plus the fungicide glyphosate (50 mg/mL).
Table 3. Inhibition of Phyllosticta citricarpa mycelia and pycnidia growth

by different extracts from endophytic isolates from Vochysia divergens
Extract Mycelia Pycnidia

Inhibition % Inhibition %
LGMF06 (PC) 0 ± 0 100 - - - -
LGMF1119 1,02 ± 0,10 83 134,4 ± 42,2 49,5
LGMF1119-mc 1,04 ± 0,12 83 122,75 ± 38,85 53,9
LGMF1133-mc 1,07 ± 0,09 82 226,3 ± 82,1 15,0
LGMF1121-mc 1,08 ± 0,23 82 159,55 ± 40,89 40,1
LGMF1133 1,17 ± 0,40 80 165,8 ± 91,3 37,7
LGMF1121 1,24 ± 0,17 79 92,5 ± 31,37 65,3
LGMF06 (NC) 5,95 ± 2,2 0 266,2 ± 83,9 0
Note: PC: positive control, the phytopathogen plus the fungicide glyphosate (50 mg/mL);
mc: extract from mycelium; NC: negative control, just the phytopathogen.

Figure 14. Activity of extract from strain LGMF1119 (Xylariaceae sp.1) against
Phyllosticta citricarpa mycelial growth. a) Methanol (Negative control); b) 1 mg of
Extract; c) 5 mg of with fungicide glyphosate (Positive control).
Figure 15. Activity of extract from strain LGMF1121 (Nigrospora sp.) against
Phyllosticta citricarpa pycnidia formation. a) 100 µg of Extract; b. Methanol
(Negative control).
In conclusion, we explored the biodiversity of the endophytic community

in the medicinal plant V. divergens, found in a unique wetland in Brazil, the
Pantanal. Based on the number and richness of isolates, these organisms
showed preferential colonization in the dry period. We report, for the first
time, the identification of isolates of the genera Antrodia and Irpex as
endophytes, as well as describe many isolates that need to be better
characterized, with the potential for new species. In addition, three isolates
belonging to the family Xylariaceae and genus Nigrospora showed promising
results in the biological control of CBS. Studies on the biodiversity of
endophytic microorganisms are important to increase our knowledge of the
biodiversity in Brazil, as well as to discover new species for environmental
and industrial purposes.

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Fitoterapia, 2016, 112, 85–89.

Chapter 4
DARK SEPTATE ENDOPHYTES (DSE)

IN POLLUTED AREAS
Elena Fernández-Miranda Cagigal *
Faculty of Biology, Department of Biology of Organism and Systems,

Area of Plant Physiology University of Oviedo, Oviedo, Spain
ABSTRACT
Dark septate endophytes (DSE) constitute a very heterogeneous
group of Ascomycetes characterized by a septate and melanized
mycelium. Inside, tissues show intra- and intercellular development and
are able not only to generate mantle and Hartig net but also to produce
typical intracellular structures (microsclerotia), all without causing
apparent damage to the plant. DSE were previously thought to be
restricted to infertile boreal or alpine habitats, where arbuscular
mycorrhizal fungi cannot persist. However, in recent years DSE have
been found extensively distributed in polluted areas around the world,
supporting a growing body of evidence that points to a prominent
ecological role, even when these organisms have not been studied from
the physiological role of a host-fungi perspective. It has been
hypothesized that DSE dominance as root endophytes might relate to
their melanised cell walls, known to play an important function in heavy
metal immobilization by sequestration. In addition to the improved
nutritional performance associated with mycorrhizal fungi, this capacity
*
Corresponding Author: Email: cagigal_11@hotmail.com, Tel.: +34985104835, Fax:
+34985104867.

126 Elena Fernández-Miranda Cagigal
provides the plant with an extra feature. Due to the promising role on
ecological reforestation of the DSE, further research is needed, including
new approaches (molecular, histological and physiological) that will
allow to better characterize the relationship between these fungi and
plants growing in polluted areas.
INTRODUCTION
Soil is one of the most sensitive and vulnerable natural resources to
pollution and degradation. According to the Global Soil Partnership (GSP), an
organism belonging to the Food and Agriculture Organization of the United
Nations (FAO), soil is defined as a finite natural resource, non-renewable, as
well as a fundamental basis for agricultural and sustainable development. Soil
provides the foundation for food, fuel, fibre, water availability, nutrient
cycling, organic carbon stocks and biodiversity. The surface of fertile soil is
limited and is increasingly under pressure due to climate change and
competing, unsuitable land uses, resulting in increasing degradation, so much
so that currently 46% of the world’s lands are considered degraded (GSP,
2011).
Industrial activity, mining, intensive farming systems and infrastructures
generate emissions and pollutant discharges in soil produce that are among,
heavy metals the most dangerous elements derived from such activities,
causing serious problems in many areas around the world (Gadd, 2007) due
the quality reduction of the physical and chemical properties of the soil (Simon
et al., 2000).
The European Environment Agency (EEA, 1999) defines heavy metals as
stable metal or metalloid materials with a density greater than 4.5 g/cm3.
According to the report of the Effects Coordination Center (Posch et al.,
2005), the distribution and magnitude of the deposition of these elements
constitute a serious risk to large areas of European ecosystems. This fact is
reflected in the Geochemical Atlas of Europe (de Vos & Tarvainen, 2006) and
in the results of Lado et al., (2008), which performed a geostatistical study
based on data from the 26 countries that make up the Forum of European
Geological Surveys (FOREGS), demonstrating the high extent of affected land
and clear correlation between high concentrations of cadmium (Cd), cupper
(Cu), mercury (Hg), lead (Pb) and zinc (Zn), and industrial activity and/or
intensive agriculture.

Dark Septate Endophytes (DSE) in Polluted Areas 127
Given the stable nature of these compounds, heavy metals are a group of
very persistent pollutants in the soil. Of the 118 elements of the periodic table,
49 are considered as heavy metals. Not all these high-density metals are
particularly toxic in normal concentrations; for instance, iron (Fe),
molybdenum (Mo), Zn and manganese (Mn) are essential micronutrients.
However, others are toxic at low concentrations and have no known function,
such as silver (Ag), arsenic (As), Hg, Cd, Pb and antimony (Sb) (Niess, 1999).
The risk of heavy metals resides in the fact that they can be chemically or
biologically degraded (Kabata-Pendias, 2000), and in addition, they tend to
bioaccumulate provoking diverse toxic effects. High concentrations of these
metals (essential or not) in the soil can produce symptoms of toxicity in plants,
since they displace the essential elements and disable multifaceted activity
causing, for example, inhibition of growth (Van Assche & Clijster, 1990).
Among its negative effects on plant biology, it can result in growth inhibition
or in oxidative stress-related damage, due to the formation of reactive oxygen
species (ROS) (Mudipalli, 2008). Moreover, these metals can also bind to
organic molecules, such as pigments or enzymes, replacing some essential
metals, altering their specific function (Malayeri et al., 2008). In this regard,
heavy metals show a remarkably high affinity for the sulfhydryl, amino,
phosphate, carboxyl and hydroxyl groups. For example in the carbonic
anhydrase enzyme, the Zn atom present in its active center can be replaced by
a heavy metal, thereby reducing Calvin cycle efficiency (Navarro-Aviñó et al.,
2007).
A wealth of physical (cancer, hurts in the kidney, autoimmunity, etc) and
psychical problems (anxiety, passiveness, etc) have been described for
humans. Hence, their persistence, progressive accumulation and the
transference likelihood to other systems pose a threat both to human health and
ecosystems (Becerril et al., 2007). For these reasons, the adoption of necessary
measures to enable the restoration of soil becomes essential.
SOIL (BIO)REMEDIATION
Soil remediation represents a technological challenge both for industries
and governmental institutions. Contaminated soil can be remediated by
chemical, physical or biological techniques (Mc Eldeowney et al., 1993). In
turn, technologies can be classified in two categories: (a) ex-situ, which
requires removal of the polluted soil to treat it (inside/outside) and (b) in situ,
in which remediation is performed without removing the contaminated soil.

This latter technique is more convenient given its lower cost and impact on the
ecosystem. In contrast, ex situ techniques such as excavation, washing and soil
storage together with the removal of chemical or physical contaminants causes
a physicochemical alteration leading to loss of soil functionality due to the
elimination of its biological activity (including nitrogen-fixing bacteria,
mycorrhizal fungi and fauna) (Ghosh & Singh, 2005).
For all the above reasons, it is necessary to develop new techniques, more
safe and secure and with less associated costs. Among the most widely
accepted in situ biological techniques of recent years for decontamination of
polluted soils stand phytoremediation. This technique is based on the use of
plants that accumulate high concentrations of heavy metals in their tissues
(Miransari, 2011) or at least tolerate it, allowing for reforestation of these
polluted areas.
Considering the plants tolerance/intolerance to growth on contaminated
soil heavy metal, we can classify them as metallophyte or intolerant.
Metallophytes are those plants, which have developed physiological
mechanisms to resist, tolerate and survive in soils with high levels of metals
(Becerril et al., 2007). And inside of the metallophytes we can distinguish
between “strict metallophytes,” restricted entirely to metal-rich soils and
“facultative metallophytes,” with populations on both metalliferous and
nonmetalliferous soils. Among them, some support the presence of metals but
do not allow the entry into the root and therefore the translocation to other
parts of the plant. Conversely, others may accumulate metals in their aerial
tissues. This accumulation depends on type of metal and plant species. These
plants are called hyperaccumulators and are used in phytoextraction.
Not all heavy metals are considered harmful to plant development.
Toxicity varies depending on concentration, persistence and origin. Therefore,
phytoavailability of metals can be defined as the ratio of metals found in the
soil that can be absorbed by a given plant genotype (Prasad, 2004).
There are several biochemical and genetic mechanisms that allow plants
develop in habitats a priori harmful to their development. These mechanisms
can be summarized as follows (Navarro-Aviñó et al., 2007):
 Cell wall: translocation of metals can be prevented by attachment to

it.
 Plasma membrane: tolerant plants have a number of mechanisms that
confer protection from heavy metals to their plasma membrane.
 Chelation: inside cells, plants use complexation mechanisms to
cushion the effect of heavy metals, joining with ligands to form more

complex compounds. However, these compounds can still take part on

the cell’s metabolism and thus remain potentially toxic.
 Vacuole compartmentalization: transport inside vacuoles reduces the
concentration of certain toxic metals by retention inside the vacuole.
 Biotransformation: set of decomposition, conjugation or synthesis
reactions involved in the proper processing of the contaminant.
 Cellular repair mechanisms: mechanisms in response to the
deterioration suffered aiming to re-establish the plant native
characteristics.
 Presence of mycorrhizas: plants with mycorrhizal fungi have a higher
tolerance to heavy metals, mainly due to their ability to immobilize
them at the root, preventing them from reaching the aerial part.
The latter point opens the door to the possible use of several fungal
species in what has been called mycoremediation programs.
FUNGAL ROLE
Some of the most paramount interactions between plants and the
environment occur within and around soils, such as nutrient and element
uptake, environmental toxicity due to pollution, root diseases and soil
formation. In most cases, fungi mediate these interactions. There are many
studies on metal tolerance in plants and metal-tolerant cultivars. However,
only few have taken into consideration the importance of mycorrhizal fungi in
plants from polluted areas, and even less in forest species.
Numerous studies have shown the accumulating capacity of heavy metals
by the fruiting bodies of different species of mycorrhizal and saprophytic fungi
(Garcia et al., 2009; Michelot et al., 1998; Kalač, 2010). This capability is
known as bio-absorption and has been defined by Shumate and Strandberg
(1985) as a series of undirected physicochemical mechanisms that can occur
between different species of metals and cellular components of various
biological species. Biological uptake by fungi can be divided into three
categories (Danesh et al., 2013): (1) Capture by binding to specific sites on the
cell structure, (2) intracellular uptake and (3) chemical transformation. The last
two are made by living cells and involve an active uptake, so it could be called
bioaccumulation (Kapoor et al., 1999).

It has been demonstrated that several fungi, including mycorrhizal fungi,

are involved in metal immobilization and accumulation within biomass by
binding metals to cell walls or pigments, by intracellular uptake or by
extracellular precipitation of mycogenic toxic metal oxalates and carbonates
(Fomina et al., 2005). Leading in this case to the occasional generation of
precipitation or crystallization in the cell wall of evaporable minerals such as
gypsum (CaSO4 2H2O) and iron oxides (magnetite, Fe3O4) (Gorbushina et
al., 2002). Furthermore, several fungal species are capable of mobilizing heavy
metals by means of chelation, the release of the metabolites, siderophores,
methylation of metals and organometallics or redox reactions that can result in
volatilization (Gadd, 2007; Harms et al., 2011).
Each species of fungus could use one or more of these mechanisms, with
varying responses to metal exposure. Thus, while species such as Candida
glabata or Schizosaccharomyces pombe in the Cd presence produce
phytochelatines and metallothionein (Ow et al., 1994), in Paxillus involutus
these molecules rarely appear, while and glutathione is abruptly increased
(Courbot et al., 2004).
Moreover, mycorrhizae can increase phytoextraction, either directly or
indirectly, increasing metal accumulation on the ground, or through increased
biomass production (Fernández et al., 2008). Despite the capacity for tolerance
and accumulation of different species of fungi seems to be an intrinsic
character, in many cases it has been observed that species or ecotypes isolated
from contaminated areas respond better to the presence of heavy metals (Gadd,
2007).
Therefore, an extensive knowledge on the diversity of fungal communities
existing in the roots and rhizosphere of plants colonizing highly polluted areas
is paramount for their potential use in restoration programs.
FUNGAL ASSOCIATIONS
The dependency of trees from mycorrhizal fungi has been long
established, especially where soil characteristics are adverse. Numerous papers
have demonstrated that if degraded areas are reforested using mycorrhized
trees, the survival and development rates achieved are superior to those
achieved with trees without mycorrhizal fungi (Duñabeitia et al., 2004;
Meharg & Cairney, 2000).
The two most widespread types of mycorrhizal association are arbuscular
mycorrhizal (AM) and ectomycorrhizal (ECM). It is known that AM establish

symbiosis with nearly 75% of all families of vascular plants from natural
ecosystems, (Brundrett, 2009) as well as in degraded and contaminated areas
(Jeffries et al., 2003). These associations improve nutrition, tolerance and
accumulation of heavy metals in the plants (Tonin et al., 2001). In case of
ECM, estimated point towards only 4.5% of plants with this type of
association (Brundrett, 2009). Despite its great importance in plants with
agroforestry interest (Marx & Cordell, 1989). ECM fungi are able to mobilize
soil nutrients by secreting low molecular weight organic compounds (oxalates,
siderophores or citrates), and are involved in several biogeochemical cycles
such as the carbon (C) and nitrogen (N).
Nonetheless, other fungi that can be found abundantly and with worldwide
distribution in these stressful environmental conditions (Cevnik et al., 2000;
Vrålstad et al., 2002a; Wilberforce et al., 2003; Li et al., 2012; Fernández-
Miranda, 2014) are dark septate endophytes (DSE). Jumpponen (2001) defined
them as conidial or sterile ascomycetes fungi mostly belonging to the order
Helotiales with darkly pigmented and septate hyphae that colonise living plant
roots without causing apparent negative effect. Colonization by DSE produces
typical intracellular structures called microsclerotia (Figure 1) inside root cells.
Figure 1. Intracellular microsclerotia formed by Cadophora finlandica inside of root of

Salix atrocinerea.
The role of these fungi on host plant is controversial because while some
authors observe his influence as a promoter of plant hormones (Schulz, 2006)
and positive effects, as would a mycorrhizal fungus (Jumpponen, 2001;
Newsham, 2011) others observe negative effects (Wilcox & Wang, 1987;
Tellenbach et al., 2011). The predominance of DSE as endophytes in heavy

metal contaminated areas can relate to their melanised cell walls, which can
sequester heavy metals and therefore confer protective effects to the plant.
However, despite this evidence, a limited number of studies have focused on
DSE function.
DISTRIBUTION
DSE fungi were previously thought to be restricted to infertile boreal or
cold-stressed habitats (Jumpponen & Trappe, 1998), where AM fungi, the
typical mutualists of herbaceous plant roots at lower latitudes and altitudes,
cannot persist or occur only sporadically (Upson et al., 2007; Newsham et al.,
2011). However, in recent years there have been reports of more than 600
plant species spanning 100 plant families worldwide (Barrow & Aaltonen,
2001; Addy et al., 2005). They appear in diverse habitats such as arid, arctic,
boreal, alpine, temperate forest, or tropical ecosystems (Jumpponen & Trappe,
1998; Mandyam & Jumpponen, 2005). Moreover, latest studies have shown
that DSE are the dominant fungi in metal contaminated sites (Cevnik et al.,
2000; Vrålstad et al., 2002a; Wilberforce et al., 2003; Li et al., 2012;
Fernández-Miranda, 2014). They colonize both herbaceous and ligneous
plants, and particularly roots of metal hyperaccumulators, halophytes, orchids,
or marine macrophytes (Mandyam & Jumpponen, 2005). And recent studies
demonstrated that DSE reported that DSE are the dominant fungi from healthy
fine roots of Erica herbacea (Cevnik et al., 2000) Betula pubescens, Populus
tremula, Picea abies, Pinus sylvestris, Calluna vulgaris, Vaccinium myrtillus,
V. vitis-idaea, Deschampsia flexuosa (Vrålstad et al., 2002a) Arabis hirsuta,
Acacia decurrens, Symplocos paniculata, Rabbosia eriocalyx, Arenaria
serpyllifolia, Rosa longicuspis (Li et al., 2012) B. celtiberica and Salix
atrocinerea (Fernández-Miranda, 2014) in polluted soils in around the world.
With all these data, Mandyam & Jumpponen (2005) analyzed the reports
of present of DSE in alpine, arctic, antarctic, boreal, temperate and tropical
habitats and conclude that DSE may be as abundant as mycorrhizal fungi, are
ubiquitous in occurrence, co-occur with different types of mycorrhizae c) are
most prevalent in stressed environments. So what is the function of these fungi
in a natural ecosystem?

TAXONOMY OF DSE
These fungi belong to a wide range of ascomycete taxa, but are often
members of the order Helotiales, such as Phialocephala fortinii,
Leptodontidium orchidicola or Meliniomyces bicolor (Jumpponen & Trappe,
1998; Addy et al., 2005; Vrålstad et al., 2002a). Belonging to this order we can
find an ecologically diverse and complex group of pathogenic fungi,
saprophytes, DSE, parasites of others fungi and ectomycorrhizal and ericoid
mycorrhizal fungi (Vrålstad et al., 2002a; Tedersoo et al., 2009).
Teleomorphic species of the Helotiales are characterised by inoperculate asci
and discoid, turbinate or clavate ascocarps ranging in size from the hardly
visible members of the Hyaloscyphaceae to more prominent members of the
Geoglossaceae and Sclerotiniaceae. The Helotiales was erected by Nannfeldt
(1932), and was replaced with Leotiales (Carpenter, 1988). However, the
Helotiales sensu str. and Leotiales sensu str. are currently recognised as two
separate orders, the latter only comprising the Leotiaceae sensu str. (Korf &
Lizon, 2001).
As shown in Figure 2 (Fernández-Miranda, 2014), the position of many
species within the order Helotiales is very complex, and there are species with
an uncertain position as seen in the last reorganization of the
Hyaloscypaheceae family (Han et al., 2014).
Based on rDNA ITS sequence analysis, Tedersoo et al., (2009)
differentiated 6 complexes. Several Helotiales subgroups such as the
Phialocephala–Acephala and Rhizoscyphus–Meliniomyces complexes and
Lachnum spp.
FORM INDICATES FUNCTION?

But DSE not only forms microsclerotia. Several studies demonstrated
(Fernández-Miranda, 2014; Hrynkiewick et al., 2009; Vrålstad et al., 2002a)
that these fungi are able to generate layers of fungal hyphae covering the root
surface (Figure 3 A), which could be defined as mantle, and a labyrinthine
network of hyphae among root cells that could be a Hartig net (Figure 3 B).
Therefore, these organisms are capable of generating structures that could fit
into the definition of ectomycorrhizas, at least from a structural point of view.
Moreover, in the same root system of Salix glauca, Fernando & Currah
(1996) observed that the fungi Phialocephala fortinii is capable of forming

ectomycorrhizas in some roots and penetrate intracellularly in others giving

rise to a typical DSE microsclerotia. A similar result was described by
Fernández-Miranda (2014) in Betula celtiberica and Salix atrocinerea, in this
case due to the action of the fungus Heliotial sp. or Cadophora finlandica,
respectively. But, can we consider DSE as mycorrhizas?
Figure 2. Maximum likelihood (ML) consensus tree derived from the ITS data set. On
each branch, the percentages (%) of 10,000 bootstrapping replicates supported by ML
are shown and the Bayesian PPs. The sequence of our isolation is represented in bold.
The tree was rooted using a sequence from Bulgaria inquinans. The scale shows the
expected number of changes per nucleotide. Only Bayesian posterior probability
values above 50% are indicated.

The term mycorrhiza was coined by the German botanist Albert Berhhard
Frank in 1885, who defined it as a symbiotic association between the roots
(rhizos) of a plant and certain groups of soil fungi (mycos), where both
members of the association benefit and actively involved in the transport and
absorption of nutrients (Mykorhizen). Brundrett (2004) redefined the term
mycorrhizas as symbiotic associations between a fungus (specialised for life in
soils and plants) and a root (or other substrate-contacting organ) of a living
plant that is primarily responsible for nutrient transfer, and that could be
essential for one or both partners. Mycorrhizas take place in a specialised plant
organ where intimate contact results from synchronised plant-fungus
development. This last definition is based on developmental and functional
characteristics that can summarized on 5 criteria (Brundrett, 2009):
1. The structure and development of mycorrhizal fungus hyphae is

substantially altered in the presence of roots of host plants. These
root-borne hyphae are distinct from hyphae, which are specialised for
growth in soil.
2. All mycorrhizas have intimate contact between hyphae and plant cells
in an interface where nutrient exchange occurs.
3. The primary role of mycorrhizas is the transfer of mineral nutrients
from fungus to plant. In most cases, there is also substantial
metabolite transfer from plant to fungus.
4. Mycorrhizas require synchronised plant-fungus development, since
hyphae only colonise young roots.
5. Plants control the intensity of mycorrhizas by root growth, digestion
of old interface hyphae in plant cells, or altered root system form.
It is clear that DSE establish an intimate contact with the host plant and
could generate modified root systems, but the crucial point of nutrient and
metabolite exchange is until unclear. For example, Peterson et al., (2008)
described that both DSE’s hyphae and microsclerotia in root cells lack a host-
derived perifungal membrane and interfacial matrix material, and hence cannot
be regarded as specialized interfaces for nutrient transfer between plant and
fungus. On the other hand, however, it is been widely reported that
Rhizoscyphus ericae (Current Name Pezoloma ericae), a member of the order
Helotiales and the typical mycorrhizal associate of ericaceous plant species,
enhances the uptake of organic N from the acidic heathland soils that its hosts
inhabit (Smith & Read, 2008). DSE’s effect on host plants is controversial,
with plant responses to experimental inoculation ranging from negative

(Wilcox & Wang, 1987 Phialocephala fortinii in association with Pinus

resinosa and Picea rubens; Stoyke & Currah, 1993 P. fortinii in association
with Menziesia ferruginea; Tellenbach et al., 2011 P. fortinii in association
with Picea abies) to positive (Newsham, 1999 P. graminicola in association
with Vulpia ciliata spp. ambigua; Usuki & Narisawa, 2007 Heteroconium
chaetospira in association with Brassica rapa). Furthermore, it has also been
suggested that the effect of dark septate endophyte colonization can vary along
a continuum from parasitism to mutualism, much as the effect of mycorrhizal
symbioses (Jumpponen, 2001).
Figure 3. A) Ectomycorrhiza caused by Helotial sp. in Betula celtiberica, B) Cross-

Section stained with blue cotton.
Several hypotheses have been put forward to explain the observed positive
responses to root endophyte colonization, being the two most prominent: 1)
modulation of plant growth via nutrient mineralization (as in mycorrhizae)
(Jumpponen, 2001; Mandyam & Jumpponen 2005; Upson et al., 2009;
Newsham, 2011) and 2) production of plant growth promoting phytohormones
(Mucciarelli et al., 2002; Schulz & Boyle, 2005; Schulz, 2006).
In view of the above evidence, a meta-analysis performed by Mayerhofer
et al., (2013) showed that identity of the inoculated endophyte affects plant
response, as was the case for plants inoculated with Phialocephala, which tend
to have smaller biomass than controls (Tellenbach et al., 2011; Reininger et
al., 2012). Not to mention differences in experimental conditions, which
undoubtedly contribute to the high levels of variability in plant response seen
in the literature. In conclusion, it appears that the effects depend on the host-
symbiont combination, and therefore more studies are necessary to clarify
which species inside this genus could be considered mycorrhizal fungi and
which not.

ROLE IN POLLUTED AREAS

Despite apparent toxicity, DSE survive, grow and flourish in metal-
polluted locations and a variety of mechanisms, both active and incidental,
contribute to their tolerance. All metals exert toxicity when present above
certain threshold concentrations in bio-available forms (Gadd, 1993).
Mechanisms of metal tolerance in fungi include reduction of metal uptake
and/or increased efflux, metal immobilization by cell-wall adsorption or
extracellular binding by polysaccharides, and intracellular sequestration by
metallothioneins and phytochelatins or vacuolar localization (Collin-Hansen et
al., 2003, 2007; Gadd, 2000, 2007). Besides, the establishment of a particular
organism may directly and/or indirectly rely on several survival strategies and
in the case of DSE, Zhang et al., (2011) indicated that the mechanism of heavy
metal tolerance of strains isolated from metal soil would be a complex process.
In the case of DSE one of these mechanisms appears to be the presence of
melanins in the hyphae. Melanins develop in large quantities in organisms that
live in unfavourable environments (Bell & Wheeler, 1986). The melanins are
known to provide rigidity to the cell wall, resistance to microbial grazing and
protection from desiccation and radiation damage (Kuo & Alexander, 1967;
Bell & Wheeler, 1986; Griffith, 1994). A variety of heavy metals might induce
or accelerate the production of melanin pigmentation in certain fungi (Zhan et
al., 2011; Gadd, 1984) like showing the work of Ban et al., (2012) where they
found that melanin content in Gaeumannomyces cylindrosporus increased
when it was exposed to 0.2 and 0.3 mg/ml Pb(II) and decreased slightly at
higher concentrations.
Redman et al., (2002) suggested that the fungal melanin could play a role
in heat dissipation or form complexes with oxygen radicals generated during
stress. If this is true, then the DSE that produce highly melanised hyphae and
microsclerotia, could perform similar functions, which may be essential to
plant survival and growth in those stressing environments. Martino et al.,
(2000) reported that a pigmented ericoid mycorrhizal fungus to tolerate heavy
metals on is deposition of melanin on the cell wall and secretion to the media.
Moreover, Vrålstad et al., (2002b) suggest that the melanized hyphae of the
outer mantle layer and extramatrical mycelium of DSE may function as
protective barriers between the external environment and the active fungus-
plant symbiosis inside the root been a key-factor to their apparent successful
colonization of burnt and metal polluted habitats.
Antioxidant enzymes such as glutathione, superoxide dismutase and
catalase are other important heavy metal tolerance agents. Glutathione is the

most abundant cellular thiol-rich heavy metal binding peptide in fungi (Singh
et al., 1997) and some works tend to consider the soluble tripeptide as the first
line of defence against heavy metal cytotoxicity (Viarengo & Nott, 1993).
Superoxide dismutase and catalase are crucial for cellular detoxification,
controlling the levels of superoxide anion radical and hydrogen peroxide
(Pócsi et al., 2004; Bai et al., 2003). Zhan et al., (2011) found that superoxide
dismutase and catalase activities in the hyphae of Exophiala pisciphila had
positive correlations with Pb(II) and Cd(II) concentrations, and Ban et al.,
(2012) confirmed these results observing that these enzymes play an effective
role in protecting G. cylindrosporus against oxidative stress induced by Pb(II).
Moreover superoxide dismutase appears to act as the primary defence against
acute Pb(II) stress.
The presence of melanins and antioxidant enzymes are not the only
mechanisms that these fungi possess. It has been demonstrated, the
relationship between toxic metal mineral solubilisation and metal in Cu-
tolerant isolates of P. ericae demonstrated a much higher ability to solubilize
Cd, Cu and Zn phosphates than isolates from non-polluted areas (Fomina &
Gadd, 2007). Bartholdy et al., (2001) revealed that P. fortinii excreted
hydroxamate siderophore for Fe(III) mobilization (ferricrocin, ferrirubin and
ferrichrome C). All these results, further and more in-depth knowledge relating
to these metal tolerance mechanisms in fungal endophytes is necessary and
their potential positive effects on the survival of their plant hosts in metal-
enriched soils will ultimately lead to powerful applications in bioremediation.
CONCLUSION
Based on the studies available, we can conclude that DSE fungi are
prevalent in various habitats and colonise a high rage of plant species. This
group of fungi cannot be overlooked while assessing the fungal communities
of any ecosystem. Even in the absence of clear consensus about positive
impacts on host fitness, growth or performance, DSE can be said to be likely
to perform functions similar to those attributed to mycorrhizal fungi. Although
experimental evidence is limited and experimental results may conflict, DSE
are seems to be involved in host nutrient uptake. And finally, the presence of
melanins may indicate a function of altering environmental tolerances.
Further and more in-depth studies on the metal tolerance mechanisms in
DSE and their effects on host plant survival in metal-enriched soils, but the
possibility of pre-inoculation of seedling with DSE as a strategy for achieving

more rapid re-vegetation of polluted sites open a new an interesting field to

their applications in bioremediation.
ACKNOWLEDGMENTS
Sincere thanks for Abelardo Casares PhD, Carolina Fernández, Marcos
Viejo PhD, Norma Alas and Rosa García-Verdugo PhD for their help and
suggestions to this manuscript.
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INDEX
Ascomycetes, ix, 7, 80, 114, 125, 131, 145,

# 146
Ascomycota, 5, 49, 96, 97, 101, 103
10-oxo-10H-phenaleno[1,2,3-de]chromene-
Aspergillus, 12, 15, 17, 19, 22, 34, 56, 64,
2-carboxylic acids, 14
66, 69, 72, 73, 77, 78, 80, 83, 85, 89, 91,
3,7,11,15-Tetrahydroxy-18-hydroxymethyl-
139, 141, 142
14,16,20,22,24-pentamethyl- hexacosa-
Aspergillus rugulosus, 12
4E,8E,12E,16,18-pentaenoic acid, 14
aspirin, 15
7α-hydroxyscirpene, 11
Avicennia marina, 13, 19, 64
7α-hydroxytrichodermol, 11
8,1’,5’-trihydroxy-3’,4’dihydro-1’H-
[2,4’]binaphthalenyl-1,4,2’-trione, 12 B
Basidiomycetes, 7
A Bauhinia vahlii, 4, 23
biofuel, viii, 40
A. nidulans var. echinulatus, 12
Botryodiplodia, 15
Abies balsamea, 11
Botrytis, 13, 15, 56
Acremonium camptosporum, 13, 26
Botrytis cinerea, 13, 56
Acremonium coenophialum, 6
Bursera simaruba, 14
Acremonium sp., 12, 19, 32
Acremoxanthones A, B, C, 13
alkaloids, vii, 1, 5, 6, 10, 30, 45, 50, 57, 58, C
59, 81, 82, 83, 84, 89
Alternaria, 12, 14, 15, 17, 18, 20, 25, 27, cajaninstilbene acid, 14, 21, 35
28, 62, 79 Cajanus cajan, 14, 21
Alternaria sp., 14, 15, 17, 18, 25, 27, 28 Camptothecin, 10, 15, 18, 19, 23, 30, 31
altersolanol A, 12 Candida albicans, 13, 56
Annona muricata, 13 Catharanthus roseus, 10, 15, 19, 35, 81
Aphanomyces cochlioides, 14 Catunaregam tomentosa, 13, 20
Artemia salina, 13 chaetoglobosin U, 10, 19, 25
Artemisia annua, 11, 18, 26, 29 chaetoglobosins A and C, 13

148 Index
chaetoglobosins A, G, V, Vb, and C, 10 Enterococcus faecium, 12

Chaetomium globosum, 3, 10, 13, 17, 18, ent-eudesmane sesquiterpenes, 11, 27
19, 21, 24, 25, 29, 30, 33, 34, 58, 59, 68, Entrophosphora infrequens, 10
82, 83, 85, 86, 87 Epichloë festucae, 7
Chaetomugilin D, 13 Epichloe typhina, 2, 21, 23, 26, 65, 87
citric acid, 9 epicoccolides A and B, 19
Cladosporium, viii, 15, 33, 94, 97, 110, 112, Epicoccum sp., 14, 19, 33
113, 114, 118 epicolactone, 14, 19
Clavicipitaceous, 5, 6, 7, 30, 47, 48, 49, 50, epiphytes, 8, 31
81 eremophilanolide 1, 2 and 3, 11
Coccidioides posadasii, 3 ergosterol, 11, 17, 68
Codinaeopsin, 13, 22, 28 ergot, 6
Colletotrichum sp., 11, 21, 29, 33, 96 etopophose phosphate, 16
Colletotrichum spp., 8 etoposide, 16
Coniothyrium sp., 12
Coumarins, 61, 62, 63, 90
Cryptocandin, 13, 20, 32, 56, 57, 88 F
Cryptosporiopsis cf. quercina, 13, 14, 20,
F. oxysporum, 14, 21
29, 32, 55, 88
F. proliferatum, 14, 21
Cryptosporiopsis sp., 12
Fagus sylvatica, 12, 20
Curvularia geniculata, 13, 20, 24
Festuca arundinacea, 6
Curvularia papendorfii, 14, 17, 37
Fusarium, 8, 10, 11, 14, 15, 18, 19, 21, 22,
Curvularia protuberate, 8
25, 28, 29, 33, 34, 56, 58, 59, 82, 89, 91
curvularides A–E, 20
Fusarium culmorum, 8
Cytonaema sp., 10, 14, 22
Fusarium oxysporum, 10, 19, 33, 34
Cytonic acids A and B, 14, 22
Fusarium solani, 14, 21
Fusarium subglutinans, 11, 22, 28, 29, 56,
D 82
Daphnopsis americana, 12, 17

digitalin, 15 G
Gaeumannomyces graminis var. tritici, 11

E ginkgo biloba, 10, 13, 17, 21, 29, 30, 34, 68,
72, 85
Ectostroma, 15 glycosides, 10, 58, 64, 65
Edenia gomzpompae, 12 guanacastepene, 12, 17, 32
endophytic fungi, vii, viii, 1, 2, 3, 4, 5, 7, 9,
10, 11, 13, 14, 15, 16, 17, 23, 24, 25, 26,
28, 29, 31, 32, 33, 34, 35, 37, 38, 40, 41, H
42, 44, 45, 46, 47, 49, 51, 52, 55, 56, 57,
Helminthosporium sativum, 11
58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70,
Histoplasma capsulatum, 13
72, 73, 77, 80, 83, 84, 87, 89, 90, 93, 94,
Hormonema dematioides, 9, 12
101, 103, 120, 121, 122, 142, 143
hypocreales, 5, 11
ent-4(15)-eudesmen-11-ol-1-one, 11

Index 149
Nonclavicipitaceous, 5, 7
I Nothapodytes, 10, 16, 19, 30, 31
Nothapodytes foetida, 10, 19, 30, 31
Imperata cylindrical, 10
Nothia aphylla, 2
isocoumarins, 27, 33, 61, 62, 63, 78, 91
O
J
Oncovin®, 10
Juniperus cedre, 10, 25
opium, 15
Ozonium, 15
K
P
Khair acid, 14, 17
Knema laurina, 11
paclitaxel, vii, 1, 15, 19
Papulaspora, 15
L Penicillium, 12, 16, 17, 59, 62, 64, 66, 68,
69, 70, 78, 79, 80, 82, 84, 86, 87, 88, 91
L. arvense, 6 Penicillium lilacum, 12
L. linicolum, 6 peptides, 12, 23, 29, 58, 65, 66, 67, 77, 79,
L. remotum, 6 80
Larix laricina, 12, 25 peramine, 6, 81
leucinostatin A, 12, 19 Periconia, 13, 15, 19, 34, 61, 89
Licuala spinosa, 11 Periconia sp., 13, 34
loline, 6, 31 peronosporomycete, 14
lolitrem, 6 pestalachloride A and B, 10
Lolium temulentum, 6, 48 pestalachloride C and D, 10
Pestalotia, 15, 17
Pestalotiopsis, 12, 15, 18, 19, 20, 21, 22, 26,
M 28, 29, 33, 61, 73, 82, 90, 91
Pestalotiopsis microspora, 12, 19, 21, 26,
merulin A and C, 18 28
Metarhizium, 15 Pezicula sp., 12
methicillin-resistant, 12, 14 Phialocephala, 16, 133, 136, 139, 141, 145
Monilia,, 16 Phoma glomerata, 14, 29
Monochaetia, 15 Phoma multirostrata, 12, 34
Mucor, 13, 15, 66, 76 phylloplane, 8
Mucor miehei, 13 Phyllosticta, v, viii, 15, 18, 34, 93, 94, 95,
96, 97, 99, 100, 109, 110, 114, 115, 116,
N 117, 119, 121, 123
Phytophthora capisici, 11
Neothyphodium coenophialum, 7 Pinus sylvestris, 9, 12, 30, 83, 132
Neotyphodium coenophialum, 6 Pithomyces, 15
Neurospora, 16, 18, 31, 102, 106 Plasmodium falciparum, 13
Nodulisporium sp., 10, 20, 25, 80 Poa ampla, 10, 33

150 Index
podophyllotoxin, 16, 18, 30

Preussomerin N1, 12
T
Pyricularia oryzae, 14
T. brevifolia, 12
Pythium ultimum, 14
tauranin, 11, 18
Taxomyces, 12, 15, 19, 32, 55, 73, 87, 88
Q Taxomyces andreanae, 12, 15, 19, 32, 55,
73, 87, 88
Quercus sp, 10, 22 Taxus baccata, 12, 19, 32
quinine, 15 Taxus brevifolia, 12, 15, 19, 55, 73
teniposide, 16
terpenoids, vii, 1, 9, 11, 32, 50, 52, 68, 72,
R 90
Theobroma cacao, 14, 19, 33
Rhizoctonia cerealis, 11 Torreya taxifolia, 12, 19
Rhizoctonia solani, 14 torreyanic acid, 12, 19, 28
Rhodotorula minuta, 9 Trametes, 16, 18, 30
Rhyncholacis penicillat, 16, 20 Trichophyton mentagrophytes, 13
riboflavin, 9 Trichophyton rubrum, 13
tricin, 10
S Tripterygium wilfordii, 11, 19, 20, 22
Tubercularia, 15
Salvia miltiorrhiza, 14, 29
Sclerotinia sclerotiorum, 13, 56 V
secondary metabolites, vii, viii, 2, 8, 9, 10,
31, 34, 35, 40, 41, 45, 46, 50, 52, 54, 82, vancomycin-resistant, 12
83, 86, 93, 106, 115 Vernonia amygdalina, 14, 17, 38
Serratia marcescens, 16, 20, 32 vincristine, 10, 19
Sonneratia alba, 14, 17, 28 Vochysia guatemalensis, 13, 22
Sphaeria typhena, 2
spiroketals, 12
Staphylococcus aureus, 12, 14, 59 X
steroids, vii, 1, 9, 11, 45, 50, 66, 68, 78, 79,
85, 90, 91 xanalteric acids I and II, 28
symbiosis, vii, 4, 25, 27, 30, 31, 39, 42, 44, Xylaria sp., 11, 20, 27, 31, 62, 65, 82, 84,
45, 78, 85, 131, 137, 144, 145 121
The author has requested enhancement of the downloaded file. All in-text references underlined in blue are linked to publications on ResearchGate.

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NOTICE TO THE READER

Library of Congress Cataloging-in-Publication Data

Names: Hughes, Evelyn, editor.

Published by Nova Science Publishers, Inc. † New York

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Endophytic fungi are important biotechnological tools because they

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healthy tissues. In general, endophytes are considered as commensalistic

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ENDOPHYTIC FUNGI: OCCURRENCE,

Afra Khiralla1,2, Rosella Spina1,2, Sakina Yagi3,

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endophytes took a consider attention as alternative source of active

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2. RELATIONSHIPS AND OCCURRENCE OF ENDOPHYTES

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Host-specificity is the relationship in which a fungus is restricted to a

3. DOES CLIMATE EFFECTING FUNGAL

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4. CLASSIFICATION OF ENDOPHYTIC FUNGI

4.1. Clavicipitaceous Endophytes (Class I)

The Clavicipitaceae is a family of fungi (Hypocreales; Ascomycota)

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endophytes of grasses were first noted by European investigators in the late

The effects of clavicipitaceous endophytes on host plant are listed below:

Most clavicipitaceous endophytes enhance resistance of hosts to insect

 Mammalian herbivores deterrence

Some clavicipitaceous endophytes have been reported to deter feeding by

Also some studies indicated that clavicipitaceous endophytes had anti-

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 Increase resistance of host disease

Some studies indicated that clavicipitaceous endophytes produced indole

 Enhance the ecophysiology of host plants

Clavicipitaceous endophytes enhance the ecophysiology of host plants and

4.2. Nonclavicipitaceous Endophytes (Class II)

Traditionally NC-endophytes treated as a single functional group, while

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 Avoiding abiotic stress

One attribute that appears unique to Class II NC-endophytes is the ability

Most of class II endophytes examined have increased host shoot and/or

 Protection from fungal pathogens

Many endophytes of class II protect hosts to some extent against fungal

5. ENDOPHYTES VERSUS EPIPHYTES

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comparisons within pine and coffee leaves indicate that endophytic

6. FUNGI AS SOURCE OF BIOACTIVE COMPOUNDS

Fungal metabolites are diverse including those associated with proteins

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6.2. Diverse Classes of Isolated Natural Products

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Guanacastepene, a novel diterpenoid produced by an unidentified fungus from

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shown to be antimicrobial. Cryptocandin, a cyclopeptide with potent