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MICROBIOLOGY RESEARCH ADVANCES

ENDOPHYTIC FUNGI DIVERSITY, CHARACTERIZATION AND BIOCONTROL

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MICROBIOLOGY RESEARCH ADVANCES

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 Chapter 1

Chapter 2

Chapter 3

Chapter 4

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

Index

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39

93

125 147



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


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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


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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 Editor: Evelyn Hughes

ISBN: 978-1-53610-341-0 © 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: [email protected].


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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.


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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).


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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


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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 (NCendophytes). 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


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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 antinematode 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.


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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:


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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,


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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.


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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).


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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; 3oxoergosta-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,5αdihydroxy-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 antiprotozoa (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)-eudesmen11-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, 8deoxytrichothecin, trichothecolone, 7α-hydroxytrichodermol and 7αhydroxyscirpene, 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).


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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


14


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 3,7,11,15Tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethylhexacosa4E,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, antiinflammatory, 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-10Hphenaleno[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


16

Afra Khiralla, Rosella Spina, Sakina Yagi et al. 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 Khair acid Conidiogenone B Conidiogenol Diversonol Ergosterol Microdiplodiasol Microdiplodiasone Microdiplodiasolol 8 α-Acetoxyphomadecalin C Phomadecalin E Helvolic acid Diepoxin κ

Biological activity Anti-bacterial Anti-bacterial

Fungal endophytes Curvularia papendorfii Penicillium chrysogenum

Host plants Vernonia amygdalina Laurencia sp.

References (Khiralla, 2015) (Gao et al., 2012)

Anti-bacterial

Microdiplodia sp.

Lycium intricatum

(Siddiqui et al., 2011)

Anti-bacterial

Microdiplodia sp.

Pinus sp.

Anti-bacterial Anti-bacterial

Pichia guilliermondii Dzf12

Alterporriol N and E Xananteric aids I and II Javanicin Pestalone Colletotric acid

Anti-bacterial Anti-bacterial Anti-bacterial Anti-bacterial Anti-bacterial

Guanacastepene Phomosines A–C Chaetoglobosin A Brefeldin A

Anti-bacterial Anti-bacterial Anti-cancer Anti-cancer

Stemphylium globuliferuman Alternaria sp. Chloridium sp. Pestalotia sp. Colletotrichum gloeosporioides CR115 Phomopsis sp. Chaetomium globosum Aspergillus clavatus

Paris polyphylla Dioscorea zingiberensis Mentha pulegium Sonneratia alba Azadirachta indica Rosenvingea sp. Artemisia mongolica

(Hatakeyama et al., 2010) (Zhao et al., 2010) (Cai et al., 2009)

Daphnopsis americana Teucrium scorodonia Ginkgo biloba Taxus mairei


(Debbab et al., 2009) (Kjer et al., 2009) (Kharwar et al., 2008) (Cueto et al., 2001) (Zou et al., 2000) (Singh et al., 2000) (Krohn et al., 1995) (Li et al., 2014) (Kharwar et al., 2011)

Table 1. (Continued) Compounds Brefeldin A Merulin A and C

Biological activity Anti-cancer Anti-cancer

Fungal endophytes Paecilomyces sp. XG8D (Basidiomycete)

Host plants Torreya grandis Xylocarpus grantum

Anthracenedione

Anti-cancer

Mangrove plant

Pestaloficiol I Pestaloficiol J Pestaloficiol K Pestaloficiol L Cochliodinol Isocochliodinol Tauranin

Anti-cancer

Halorosellinia sp. Guignardia sp. Pestalotiopsis fici

References (Kharwar et al., 2011) (Chokpaiboon et al., 2010) (Zhang et al., 2010)

Camellia sinensis

(Liu et al., 2009)

Anti-cancer

Chaetomium sp.

Salvia officinalis

(Debbab et al., 2009)

Anti-cancer

Phyllosticta spinarum

(Wijeratne et al., 2008)

Camptothecin

Anti-cancer

Neurospora crassa

Alternariol Alternusin Daldinone C Daldinone D Beauvericin Podophyllotoxin

Anti-cancer

Alternaria sp.

Anti-cancer

Hypoxylon truncatum

Platyclocarpus granatum Camptotheca acuminata Polygonum senegalense Artemisia annua

Anti-cancer Anti-cancer

Fusarium oxysporium Trametes hirsuta

(Zhan et al., 2007) (Puri et al., 2006)

Chaetopyanin

Anti-cancer

Chaetomium globosum

Ephedra fasciculata Podophyllum hexandrum Polysiphonia urceolata


(Rehman et al., 2008) (Aly et al., 2008) (Gu et al., 2007)

(Wang et al., 2006)

Compounds Chaetoglobosin U Chaetoglobosin C Chaetoglobosin F Chaetoglobosin E Penochalasin A Camptothecin Two cyclic pentapeptides Globosumone A Globosumone B Dicerandrol A

Biological activity Anti-cancer

Fungal endophytes Chaetomium globosum

Host plants Imperata cylindrica

References (Ding et al., 2006)

Anti-cancer Anti-cancer Anti-cancer

Entrophospora infrequens Strain 2524 Chaetomium globosum

Nothapodytes foetida Avicennia marina Ephedra fasciculata

(Puri et al., 2005) (Li et al., 2004; 2005) (Bashyal et al., 2005)

Anti-cancer

Phomopsis longicolla

Dicerandra frutescens

Phomoxanthone Phomoxanthone Vincristine Sequoiatones A Sequoiatones B Leucinostatin A

Anti-cancer

Phomopsis sp.

Tectona grandis

(Wagenaar and Clardy, 2001) (Isaka et al., 2001)

Anti-cancer Anti-cancer

Fusarium oxysporum Aspergillus parasiticus

Catharanthus roseus Sequoia sempervirens

(Zhang et al., 2000) (Stierle et al., 1999)

Anti-cancer, Anti-fungal Anti-cancer Anti-cancer

Acremonium sp.

Taxus baccata

(Strobel et al., 1997a)

Pestalotiopsis microspora Rhinocladiella sp.

Torreya taxifolia Tripterygium wilfordii

(Lee et al., 1996) (Lee et al., 1995)

Anti-cancer Anti-cancer Anti-fungal

Taxomyces andreanae Periconia atropurpurea Epicoccum sp.

Taxus brevifolia Xylopia aromatica Theobroma cacao

(Stierle et al., 1993) (Stierle et al., 1993) (Talontsi et al, 2013)

Torreyanic acid Cytochalasin 1 Cytochalasin 2 Cytochalasin 3 Cytochalasin E Paclitaxel Periconicin Epicolactone, Epicoccolides A and B


Table 1. (Continued) Compounds Phomenone Curvularides A–E

Biological activity Anti-fungal Anti-fungal

Fungal endophytes Xylaria sp. Curvularia geniculata

Cycloepoxylactone Cycloepoxytriol B Pestalachloride A Isofusidienol A, B, C, and D Isofusidienol A–D Altenusin

Anti-fungal Anti-bacterial Anti-fungal Anti-fungal

Trichodermin Nodulisporins A-C Nodulisporins D-F 3,12-Dihydroxycadalene Pyrrocidines A and B Cryptocin Cryptocandin Oocydin A Mullein

References (Silva et al., 2010) (Chomcheon et al., 2010)

Phomopsis sp.

Host plants Piper aduncum Catunaregam tomentosa Laurus azorica

Pestalotiopsis adusta Chalara sp. (strain 6661)

Unknown Chinese tree Artemisia vulgaris

(Li et al., 2008a) (Lösgen et al., 2008)

Anti-fungal Anti-fungal Anti-parasitic Anti-fungal Anti-fungal Anti-bacterial Anti-algal Anti-fungal Anti-fungal Anti-fungal Anti-fungal Anti-fungal

Chalara sp. Alternaria sp

Artemisia vulgaris Trixis vauthieri

Trichoderma harzianum Nodulisporium sp.

Ilex cornuta Juniperus cedrus Erica arborea

(Lösgen et al., 2008) (Cota et al., 2008; Johann et al., 2012) (Chen et al., 2007) (Dai et al., 2006; 2009 )

Phomopsis cassiae Acremonium zeae Cryptosporiopsis cf. quercina Cryptosporiopsis cf. quercina Serratia marcescens

Anti-fungal Anti-bacterial

Pezicula livida

Cassia spectabilis Zea mays Tripterygium wilfordii Tvipterigeum wilfordii Rhyncholacis penicillata Fagus sylvatica


(Hussain et al., 2009)

(Silva et al., 2006) (Wicklow et al., 2005) (Li et al., 2000) (Strobel et al., 1999b) (Strobel et al., 1999a) (Schulz et al., 1995)

Compounds Gamahonolide A and B Cinnamic acid Kaempferol Flavipin 4,6-dihydroxy-5-methoxy-7-methylphthalide. Cajaninstilbene acid

Biological activity Anti-fungal Anti-oxidant Anti-oxidant Anti-oxidant Anti-oxidant

Fungal endophytes Epichloe typhina Strain M7226

Host plants Phleum pretense Curcuma wenyujin

References (Hiroyuki et al., 1992) (Yan et al., 2014)

Chaetomium globosum Cephalosporium sp.

Ginkgo biloba Sinarundinaria nitida

(Ye et al., 2013) (Huang et al., 2012)

Anti-oxidant Hypotriglycerimic, Hypoglycemic, Anti-inflammatory, Analgesic Anti-oxidant

Fusarium solani F. oxysporum F. proliferatum

Cajanus cajan

(Zhao et al., 2012)

Colletotrichum sp.

Piper ornatum

(Tianpanich et al., 2011)

Anti-oxidant

Sordariomycete sp.

Eucommia ulmoides

(Chen et al., 2010)

Corynesidones A, B

Anti-oxidant

Corynespora cassiicola

(Chomcheon et al., 2009)

Chaetopyranin

Anti-oxidant, Cytotoxic Anti-oxidant

Chaetomium globosum

Anti-oxidant

Pestalotiopsis microspora

Lindenbergia philippensis Polysiphonia urceolata Pilgerodendron uviferum Trachelospermum jasminoides Terminalia morobensis

Monocerin Fusarentin A pure compound Chlorogenic acid

Graphislactone A

Pestacin Isopestacin

Microsphaeropsis olivacea Cephalosporium sp.


(Wang et al., 2006) (Hormazabal et al., 2005; Song et al., 2005)

(Harper et al., 2003)

Table 1. (Continued) Compounds Pestalotheols C Pestalotheol C Brefeldin A

Cytonic acids A and B Palmarumycin CP17 Palmarumycin CP18 Codinaeopsin Subglutinol A

Biological activity Anti-viral Anti-viral Anti-viral, Anti-bacterial, Anti-fungal, Anti-nematode Anti-viral Anti-leishmanial

Fungal endophytes Pestalotiopsis theae Pestalotiopsis theae Paecilomyces sp. Aspergillus clavatus

Host plants Unidentified tree Unidentified tree Taxus mairei Torreya grandis

References (Li et al., 2008a) (Li et al., 2008b) (Wang et al., 2007; Betina, 1992)

Cytonaema sp. Edenia sp.

Quercus sp. Petrea volubilis

Anti-malarial

CR127A

Immunosuppressive

Fusarium subglutinans

Vochysia guatemalensis Tripterygium wilfordii

(Guo et al., 2000) (Martínez-Luis et al., 2008) (Kontnik and Clardy, 2008) (Lim et al., 2015)


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Strobel, G. A; Hess, W. M.; Li, J. Y.; et al., Pestalotiopsis guepinii, a taxolproducing endophyte of the wollemi pine, Wollemia nobilis. Aust. J. Bot. 1997a, 45(6), 1073–1082. Strobel, G.; Daisy, B. Bioprospecting for microbial endophytes and their natural products. Microbiol. Mol. Biol. Rev. 2003, 67, 491–502. Tadych, M.; Bergen, M.; Dugan, F. M.; et al., Evaluation of the potential role of water in the spread of conidia of the Neotyphodium endophyte of Poa ampla. Mycol. Res. 2007, 111, 466–472. Talontsi, F. M.; Dittrich, B.; Schüffler, A.; et al., Epicoccolides: Antimicrobial and antifungal polyketides from an endophytic fungus Epicoccum sp. associated with Theobroma cacao. Eur. J. Org. Chem. 2013, 15, 3174– 3180. Tan, R. X.; Zou, W. X. Endophytes: a rich source of functional metabolites. Nat. Prod. Rep. 2001, 18, 448–459. Tianpanich, K.; Prachya, S.; Wiyakrutta S.; et al., Radical scavenging and antioxidant activities of isocoumarins and a phthalide from the endophytic fungus Colletotrichum sp. J. Nat. Prod. 2011, 74(1), 79–81. Tudzynski, B.; Sharon, A. In Biosynthesis, Biological Role and Application of fungal phyto-hormones. Osiewacz, H. D.; Ed.; The Mycota X. Industrial Applications; Springer-Verlag: Berlin, 2002. Vogl, A. Mehl und die anderen mehlprodukte der cerealien und leguminosen. Zeitschrift Nahrungsmittle Untersuchung Hyg. Warenkunde. 1898, 12, 25– 29.[Flour and the other flour products of thecereals and legumes.Zeitschrit Nahrungsmittle Untersuchung Hyg. Warenkunde. 1898, 12, 25–29.] Vu, T.; Hauschild, R.; Sikora, R. A. Fusarium oxysporum endophytes induced systemic resistance against Radopholus similis on banana. Nematol. 2006, 8, 847–852. Wagenaar, M. M.; Clardy, J. Dicerandrols, new antibiotic and cytotoxic dimers produced by the fungus Phomopsis longicolla isolated from an endangered mint. J. Nat. Prod. 2001, 64, 1006–1009. Wang, F.; Jiao, R.; Cheng, A.; et al., Antimicrobial potentials of endophytic fungi residing in Quercus variabilis and brefeldin A obtained from Cladosporium sp. World J. Microbiol. Biotechnol. 2007, 23, 79–83. Wang, S.; Li, X. M.; Teuscher, F.; et al., Chaetopyranin, a benzaldehyde derivative, and other related metabolites from Chaetomium globosum, an endophytic fungus derived from the marine red alga Polysiphonia urceolata. J. Nat. Prod. 2006, 69, 1622–1625.


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White, J. F. J. The widespread distribution of endophytes in the Poaceae. Plant Dis. 1987, 71, 340–342. White, J. F. J.; Martin, T. I.; Cabral, D. Endophyte-host associations in grasses. III. Conidia formation by Acremonium endophytes in thephylloplanes of Agrostis hiemalis and Poa rigidifolia. Mycologia. 1996, 88, 174–178. Wicklow, D. T.; Roth, S.; Deyrup, S. T.; et al., A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides. Mycol. Res., 2005, 109, 610–618. Wijeratne, E. M. K.; Paranagama, P. A.; Burns, A. M.; et al., Sesquiterpene quinones and related metabolites from Phyllosticta spinarum, a fungal strain endophytic in Platycladus orientalis of the sonoran desert. J. Nat. Prod. 2008, 71, 218–222. Wiyakrutta, S.; Sriubolmas, N.; Panphut, W.; et al., Endophytic fungi with anti-microbial, anti-cancer and anti-malarial activities isolated from Thai medicinal plants. World J. Microbiol. Biotechnol. 2004, 20, 265–272. Xia, X. K.; Yang, L. G.; She, Z. G.; et al., Two new acids from mangrove endophytic fungus (no. zzf13). Chem. Nat. Compd. 2008, 44(4), 416–418. Yan, J.; Qi, N.; Wang, S.; et al., Characterization of secondary metabolites of an endophytic fungus from Curcuma wenyujin. Curr. Microbiol. 2014, 69(5), 740–744. Yang, X.; Strobel, G. A.; Stierle, A.; et al., A fungal endophyte - tree relationship - Phoma multirostrata in Taxus wallachiana. Plant Sci. 1994, 102, 1–9. Ye, Y.; Xiao, Y.; Ma, L.; et al., Flavipin in Chaetomium globosum CDW7, an endophytic fungus from Ginkgo biloba, contributes to antioxidant activity. Appl. Microbiol. Biotechnol. 2013, 97, 7131. Yu, H.; Zhang, L.; Li, L.; et al., Recent developments and future prospects of antimicrobial metabolites produced by endophytes. Microbiol. Res. 2010, 165(6), 437–449. Zhan, J.; Burns, A. M.; Liu, M. X.; et al., Search for cell motility and angiogenesis inhibitors with potential anticancer activity: beauvericin and other constituents of two endophytic strains of Fusarium oxysporum. J. Nat. Prod. 2007, 70(2), 227–232. Zhang, D. W.; Tao, X. Y.; Liu, J. M.; et al., A new polyketide synthase−nonribosomal peptide synthetase hybrid metabolite from plant endophytic fungus Periconia sp. Chin. Chem. Lett. 2016, 27(5), 640–642.


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Zhang, J. Y.; Tao, L. Y.; Liang, Y. J.; et al., Anthracenedione derivatives as anticancer agents isolated from secondary metabolites of the mangrove endophytic fungi. Mar. Drugs. 2010, 8, 1469–1481. Zhang, L. B.; Gou, L. H.; Zeng, S. V. Preliminary study on the isolation of endophytic fungus of Catharanthus roseus and its fermentation to produce product of therapeutic value. Chin. Tradit. Herbal Drugs. 2000, 11, 805– 807. Zhao, J. T.; Fu, Y. J.; Luo, M.; et al., Endophytic fungi from Pigeon Pea [Cajanuscajan (L.) Millsp.] produce antioxidant Cajaninstilbene acid. J. Agric. Food. Chem. 2012, 60, 4314−4319. Zhao, J.; Mou, Y.; Shan, T.; et al., Antimicrobial metabolites from the endophytic fungus Pichia guilliermondii isolated from Paris polyphylla var. Yunnanensis. Molecules. 2010, 15, 7961–7970. Zhou, D. Q.; Hyde, K. D. Host-specificity, host-exclusivity, and hostrecurrence in saprobic fungi. Mycol. Res. 2001, 105, 1449–1457. Zou, W. X.; Meng, J. C.; Lu, H.; et al., Metabolites of Colletotrichum gloeosporioides, an endophytic fungus in Artemisia mongolica. J. Nat. Prod. 2000, 63, 1529–1530.

BIOGRAPHICAL SKETCH

Dr. Afra Ahmed Isamil Khiralla Affiliation: Université de Lorraine, SRSMC, UMR 7565, BP 70239, F54506 Vandœuvre-lès-Nancy, France


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Afra Khiralla, Rosella Spina, Sakina Yagi et al. 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

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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 & LaurainMattar 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, SlezackDeschaumes 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, LaurainMattar D. Potential antioxidant resource of endophytic fungi from some Sudanese Medicinal plants. Phytoday. University of Strasbourg. Strasbourg, France. 2015.


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Afra Khiralla, Rosella Spina, Sakina Yagi et al. 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: [email protected].


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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).


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Khaled A. Selim, Mohamed M. S. Nagia and Dina E. El. Ghwas 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



43

(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

No. of species 44

Location

References

Abies alba

Tissue or organ Branch

Germany,Poland

A. rubra

Leaves bases

25

Betula pendula

Branch bases

23

British, Columbia Poland Germany,

Kowalski and Kehr, (1992) Sieber et al., (1991)

Cuscuta reflexa

Stems

45

India

Euterpe oleracea Heisteria concinna Licuala ramsayi

Leaves

62

Brazil

Kowalski and Kehr, (1992) Suryanarayanan et al., (2000) Rodrigues, (1994)

Leaves

242

Panama

Arnold et al., (2000)

Leaves

11

Australia

Musa acuminata Ouratea lucens P. mariana Quercus ilex

leaves

24

Hong Kong

Rodrigues and Samuels, (1992) Brown et al., (1998)

Leaves Roots Twigs, Leaves Stems

259 97 149

Panama Ontario Spain

Arnold et al., (2000) Summerbell, (1989) Collado et al., (2000)

31

United Kingdom

Leaves Leaves, Stems Leaves, Stems

17 46

Germany South Africa

Petrini and Fisher, (1986) Pehl and Butin, (1994) Mostert et al., (2000)

23

United Kingdom

Fisher et al., (1992)

Salicornia perennis Tilia cordata Vitis vinifera Zea mays


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Khaled A. Selim, Mohamed M. S. Nagia and Dina E. El. Ghwas

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).



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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


46


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



47

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 nonClavicipitaceous according to their taxonomy, evolution, ecological function and host specificity. After that, Rodriguez et al., 2009 illustrated that, there are


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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. NonClavicipitaceous 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). NonClavicipitaceous 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).


49


Table 2. Symbiotic criteria used to characterize fungal endophytic classes Criteria Host range Tissue(s) colonized In planta colonization In planta biodiversity Transmission

Clavicipitaceous Class (1) Narrow Shoot and rhizome Extensive

Non-clavicipitaceous Class (2) Class (3) Broad Broad Shoot, Root Shoot and rhizome Extensive Limited

Low

Low

High

Unknown

Vertical and horizontal NHA

Vertical and horizontal NHA and HA

Horizontal

Horizontal

Class (4) Broad Root Extensive

Fitness NHA NHA benefits* *Nonhabitat-adapted (NHA) benefits like drought tolerance and growth enhancement are common among endophytes regardless of the habitat of origin. Habitatadapted (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


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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



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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.


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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



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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


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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



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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).


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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.



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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


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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).



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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-2,4dihydro-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). Xray 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


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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).



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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


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professor Peter Proksch (Xu et al., 2009). Recently a new coumarine, 4, 6dihydroxy-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, 8dihydroxy-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-1oxo-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).



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Figure 4. Chemical structures (18-30) show the diversity of coumarins and isocoumarins derivatives isolated from endophytic fungi.


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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-(αDribofuranosyl)-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-Dribofuranosyl) 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-Larabinopyranosyl)-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).



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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


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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-SOLeu) 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., 7Nor-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).



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


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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-dien3β-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 5dihydroxyergosta-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



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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).



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

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).



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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).



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

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)



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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-hydroxybenzopyran-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-6oxo-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).


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



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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).


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Figure 10. Chemical structures of promising compounds isolated from marine macrorganisms.

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Yang, S. X., Xiao, J., Laatsch, H., Holstein, J. J., Dittrich, B., Zhang, Q., Gao, J. M. (2012). Fusarimine, a novel polyketide isoquinoline alkaloid from the endophytic fungus Fusarium sp. LN12 isolated from Melia azedarach. Tetrahedron Lett. 53(47), 6372-6375. Yang, X.-L., L. Huang, H.-Y. Li, D.-F. Yang and Z.-Z. Li (2015). Two new compounds from the plant endophytic fungus Pestalotiopsis versicolor. J. Asian Nat. Prod. Res. 17(4): 333-337. Ying, Y. M., Zheng, Z., Zhang, Z. L. W., Shan, W. G., Wang, J. W., Zhan, Z. J. (2014). Rare C25 steroids produced by Penicillium chrysogenum P1X, a fungal endophyte of Huperzia serrata. Helv. Chim. Acta 97(1), 95-101. Zeng, Y. B., Wang, H., Zuo, W. J., Zheng, B., Yang, T., Dai, H. F., Mei, W. L. (2012). A fatty acid glycoside from a marine-derived fungus isolated from mangrove plant Scyphiphora hydrophyllacea. Mar. Drugs 10, 598-603. Zhang, W., K. Krohn, S. Draeger and B. Schulz (2008). Bioactive isocoumarins isolated from the endophytic fungus Microdochium bolleyi. J. Nat. Prod. 71(6), 1078-1081. Zhang, Y., Li, X. M., Wang, C. Y., Wang, B. G. (2007). A new naphthoquinoneimine derivative from the marine algal-derived endophytic fungus Aspergillus niger EN-13. Chinese Chemical Letters 18(8), 951953. Zhao, J., Mou, Y., Shan, T., Li, Y., Zhou, L., Wang, M., Wang, J. (2010). Antimicrobial metabolites from the endophytic fungus Pichia guilliermondii isolated from Paris polyphylla var. yunnanensis. Molecules 15, 7961-7970. Zheng, C.-J., L. Li, J.-p. Zou, T. Han and L.-P. Qin (2012). Identification of a quinazoline alkaloid produced by Penicillium vinaceum, an endophytic fungus from Crocus sativus. Pharm. Biol. 50(2), 129-133. Zhou, Z. F., Kurtan, T., Yang, X.-H., Mandi, A., Geng, M.-Y., Ye, B.-P., Taglialatela-Scafati O., and Guo Y.-W. (2014a). Penibruguieramine A, a novel pyrrolizidine alkaloid from the endophytic fungus Penicillium sp. GD6 associated with Chinese mangrove Bruguiera gymnorrhiza. Org. Lett. 16(5), 1390-1393. Zhou, Z. F., Yang, X. H., Liu, H. L., Gu, Y. C., Ye, B. P., Guo, Y. W. (2014b). A New Cyclic Peptide and a New Steroid from the Fungus Penicillium sp. GD6 isolated from the Mangrove Bruguiera gymnorrhiza. Helv. Chim. Acta 97(11), 1564-1570.





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, [email protected], Phone: (55) 4133611562.


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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, Phyllosticta citricarpa, biological control

biodiversity,

endophytes,

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 …

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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 citrusproducing 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.


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Y. M. Hokama, D. C. Savi, B. Assad et al.

Table 1. Isolates, identification, position of individually sampled Vochysia divergens, soil characteristic and GenBank access number Filo

Class

Basidiomycota Agaricomycetes

Ascomycota

Sordariomycetes

Identification

Fungal isolate

Tree Location

Soil Characteristic

Antrodia sp. Irpex lacteus Peniophora laxitexta Polyporus sp. Daldinia sp. Xylariaceae

LGMF1145 LGMF1153 LGMF1159 LGMF1191 LGMF1131 LGMF1126 LGMF1134 LGMF1115 LGMF1130 LGMF1114 LGMF1120 LGMF1128 LGMF1127 LGMF1124 LGMF1123 LGMF1119 LGMF1156 LGMF1121 LGMF1125 LGMF1133 LGMF1138 LGMF1173 LGMF1137 LGMF1135 LGMF1144 LGMF1157 LGMF1165 LGMF1141 LGMF1136 LGMF1142 LGMF1152 LGMF1143 LGMF1151 LGMF1139 LGMF1177 LGMF1161 LGMF1174 LGMF1140 LGMF1181 LGMF1194 LGMF1183 LGMF1148 LGMF1179 LGMF1150 LGMF1186 LGMF1182 LGMF1178

E E C A 2 2 1 4 2 4 3 2 2 2 2 3 D 2 2 1 1 A 1 1 E C B 1 1 E E E E 1 A C A 1 A A A E A E A A A

Dry Dry Dry Flooded Flooded Flooded Dry Flooded Flooded Flooded Flooded Flooded Flooded Flooded Flooded Flooded Flooded Flooded Flooded Dry Dry Flooded Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Flooded Dry Flooded Dry Flooded Flooded Flooded Dry Flooded Dry Flooded Flooded Flooded

Nigrospora sp.

Colletotrichum sp.

Diaporthe sp.

Lanceispora sp.

Dothideomycetes

Phyllosticta capitalensis


GenBank access number ITS JX559566 JX559574 JX559574 JX559609 JX559552 JX559547 JX559555 JX559537 JX559551 JX559536 JX559542 JX559549 JX559548 JX559545 JX559544 JX559541 JX559577 JX559577 JX559546 JX559554 JX559559 JX559593 JX559558 JX559556 JX559565 JX559578 JX559585 JX559562 JX559557 JX559563 JX559573 JX559564 JX559572 JX559560 JX559597 JX559582 JX559594 JX559561 JX559601 JX559612 JX559603 JX559569 JX559599 JX559571 JX559606 JX559602 JX559598

Endophytic Fungi Isolated from Vochysia divergens … Filo

Class

Identification

Fungal isolate

Tree Location

Soil Characteristic

Ascomycota

Dothideomycetes

Phyllosticta capitalensis

LGMF1175 LGMF1155 LGMF1132 LGMF1180 LGMF1189 LGMF1163 LGMF1160 LGMF1149 LGMF1176 LGMF1158 LGMF1167 LGMF1185 LGMF1170 LGMF1162 LGMF1192 LGMF1184 LGMF1147 LGMF1190 LGMF1171 LGMF1169 LGMF1146 LGMF1168 LGMF1154 LGMF1193 LGMF1172 LGMF1195 LGMF1116

A D 1 A A C C E A C B A A C A A E A A B E B D A A A 4

Flooded Flooded Dry Flooded Flooded Dry Dry Dry Flooded Dry Dry Flooded Flooded Dry Flooded Flooded Dry Flooded Flooded Dry Dry Dry Flooded Flooded Flooded Dry Flooded

Phyllosticta sp.

97 GenBank access number JX559595 JX559576 JX559553 JX559600 JX559607 JX559584 JX559581 JX559570 JX559596 JX559579 JX559587 JX559605 JX559590 JX559583 JX559610 JX559604 JX559568 JX559608 JX559591 JX559589 JX559567 JX559588 JX559575 JX559611 JX559592 JX559613 JX559538

Phaeosphaeria sp. Neofusicoccum 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.


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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,



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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


100


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 Phyllosticta citricarpa 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



101

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.


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Figure 3. Bayesian phylogenetic tree based on ITS sequence, showing the relationships 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



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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


104


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.

Figure 4. Bayesian phylogenetic tree based on ITS sequence, showing the relationships between Daldinia species. The tree is rooted with Annulohypoxylon minutellum sequence obtained from GenBank. Scale bar shows 2 changes and Bayesian posterior 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.


105

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



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several plant species, mainly from tropical regions (Refaei et al. 2011; Vega et al. 2010).

Figure 7. Bayesian phylogenetic tree based on ITS sequence, showing the relationships 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.


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Figure 8. Bayesian phylogenetic tree based on ITS sequence, showing the relationships 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.



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Figure 9. Bayesian phylogenetic tree based on ITS sequence, showing the relationships 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


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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



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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


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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).

Figure 11. Bayesian phylogenetic tree based on ITS sequence, showing the 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.



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Figure 12. Bayesian phylogenetic tree based on ITS sequence, showing the 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).


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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 Phyllosticta citricarpa 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



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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).


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


49,5 53,9 15,0 40,1 37,7 65,3 0 mg/mL);


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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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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: [email protected], Tel.: +34985104835, Fax: +34985104867.


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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.


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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.


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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



  



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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).


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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



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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; FernándezMiranda, 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


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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?



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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


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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.



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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 hostderived 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


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(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) CrossSection 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 hostsymbiont combination, and therefore more studies are necessary to clarify which species inside this genus could be considered mycorrhizal fungi and which not.



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ROLE IN POLLUTED AREAS Despite apparent toxicity, DSE survive, grow and flourish in metalpolluted 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 fungusplant 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


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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 Cutolerant 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 metalenriched 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



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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 # 10-oxo-10H-phenaleno[1,2,3-de]chromene2-carboxylic acids, 14 3,7,11,15-Tetrahydroxy-18-hydroxymethyl14,16,20,22,24-pentamethyl- hexacosa4E,8E,12E,16,18-pentaenoic acid, 14 7α-hydroxyscirpene, 11 7α-hydroxytrichodermol, 11 8,1’,5’-trihydroxy-3’,4’dihydro-1’H[2,4’]binaphthalenyl-1,4,2’-trione, 12

A A. nidulans var. echinulatus, 12 Abies balsamea, 11 Acremonium camptosporum, 13, 26 Acremonium coenophialum, 6 Acremonium sp., 12, 19, 32 Acremoxanthones A, B, C, 13 alkaloids, vii, 1, 5, 6, 10, 30, 45, 50, 57, 58, 59, 81, 82, 83, 84, 89 Alternaria, 12, 14, 15, 17, 18, 20, 25, 27, 28, 62, 79 Alternaria sp., 14, 15, 17, 18, 25, 27, 28 altersolanol A, 12 Annona muricata, 13 Aphanomyces cochlioides, 14 Artemia salina, 13 Artemisia annua, 11, 18, 26, 29

Ascomycetes, ix, 7, 80, 114, 125, 131, 145, 146 Ascomycota, 5, 49, 96, 97, 101, 103 Aspergillus, 12, 15, 17, 19, 22, 34, 56, 64, 66, 69, 72, 73, 77, 78, 80, 83, 85, 89, 91, 139, 141, 142 Aspergillus rugulosus, 12 aspirin, 15 Avicennia marina, 13, 19, 64

B Basidiomycetes, 7 Bauhinia vahlii, 4, 23 biofuel, viii, 40 Botryodiplodia, 15 Botrytis, 13, 15, 56 Botrytis cinerea, 13, 56 Bursera simaruba, 14

C cajaninstilbene acid, 14, 21, 35 Cajanus cajan, 14, 21 Camptothecin, 10, 15, 18, 19, 23, 30, 31 Candida albicans, 13, 56 Catharanthus roseus, 10, 15, 19, 35, 81 Catunaregam tomentosa, 13, 20 chaetoglobosin U, 10, 19, 25 chaetoglobosins A and C, 13


148

Index

chaetoglobosins A, G, V, Vb, and C, 10 Chaetomium globosum, 3, 10, 13, 17, 18, 19, 21, 24, 25, 29, 30, 33, 34, 58, 59, 68, 82, 83, 85, 86, 87 Chaetomugilin D, 13 citric acid, 9 Cladosporium, viii, 15, 33, 94, 97, 110, 112, 113, 114, 118 Clavicipitaceous, 5, 6, 7, 30, 47, 48, 49, 50, 81 Coccidioides posadasii, 3 Codinaeopsin, 13, 22, 28 Colletotrichum sp., 11, 21, 29, 33, 96 Colletotrichum spp., 8 Coniothyrium sp., 12 Coumarins, 61, 62, 63, 90 Cryptocandin, 13, 20, 32, 56, 57, 88 Cryptosporiopsis cf. quercina, 13, 14, 20, 29, 32, 55, 88 Cryptosporiopsis sp., 12 Curvularia geniculata, 13, 20, 24 Curvularia papendorfii, 14, 17, 37 Curvularia protuberate, 8 curvularides A–E, 20 Cytonaema sp., 10, 14, 22 Cytonic acids A and B, 14, 22

D Daphnopsis americana, 12, 17 digitalin, 15

E Ectostroma, 15 Edenia gomzpompae, 12 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, 42, 44, 45, 46, 47, 49, 51, 52, 55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 72, 73, 77, 80, 83, 84, 87, 89, 90, 93, 94, 101, 103, 120, 121, 122, 142, 143 ent-4(15)-eudesmen-11-ol-1-one, 11

Enterococcus faecium, 12 ent-eudesmane sesquiterpenes, 11, 27 Entrophosphora infrequens, 10 Epichloë festucae, 7 Epichloe typhina, 2, 21, 23, 26, 65, 87 epicoccolides A and B, 19 Epicoccum sp., 14, 19, 33 epicolactone, 14, 19 epiphytes, 8, 31 eremophilanolide 1, 2 and 3, 11 ergosterol, 11, 17, 68 ergot, 6 etopophose phosphate, 16 etoposide, 16

F F. oxysporum, 14, 21 F. proliferatum, 14, 21 Fagus sylvatica, 12, 20 Festuca arundinacea, 6 Fusarium, 8, 10, 11, 14, 15, 18, 19, 21, 22, 25, 28, 29, 33, 34, 56, 58, 59, 82, 89, 91 Fusarium culmorum, 8 Fusarium oxysporum, 10, 19, 33, 34 Fusarium solani, 14, 21 Fusarium subglutinans, 11, 22, 28, 29, 56, 82

G Gaeumannomyces graminis var. tritici, 11 ginkgo biloba, 10, 13, 17, 21, 29, 30, 34, 68, 72, 85 glycosides, 10, 58, 64, 65 guanacastepene, 12, 17, 32

H Helminthosporium sativum, 11 Histoplasma capsulatum, 13 Hormonema dematioides, 9, 12 hypocreales, 5, 11


149

Index

I Imperata cylindrical, 10 isocoumarins, 27, 33, 61, 62, 63, 78, 91

Nonclavicipitaceous, 5, 7 Nothapodytes, 10, 16, 19, 30, 31 Nothapodytes foetida, 10, 19, 30, 31 Nothia aphylla, 2

O

J Juniperus cedre, 10, 25

Oncovin®, 10 opium, 15 Ozonium, 15

K P

Khair acid, 14, 17 Knema laurina, 11

L L. arvense, 6 L. linicolum, 6 L. remotum, 6 Larix laricina, 12, 25 leucinostatin A, 12, 19 Licuala spinosa, 11 loline, 6, 31 lolitrem, 6 Lolium temulentum, 6, 48

M merulin A and C, 18 Metarhizium, 15 methicillin-resistant, 12, 14 Monilia,, 16 Monochaetia, 15 Mucor, 13, 15, 66, 76 Mucor miehei, 13

N Neothyphodium coenophialum, 7 Neotyphodium coenophialum, 6 Neurospora, 16, 18, 31, 102, 106 Nodulisporium sp., 10, 20, 25, 80

paclitaxel, vii, 1, 15, 19 Papulaspora, 15 Penicillium, 12, 16, 17, 59, 62, 64, 66, 68, 69, 70, 78, 79, 80, 82, 84, 86, 87, 88, 91 Penicillium lilacum, 12 peptides, 12, 23, 29, 58, 65, 66, 67, 77, 79, 80 peramine, 6, 81 Periconia, 13, 15, 19, 34, 61, 89 Periconia sp., 13, 34 peronosporomycete, 14 pestalachloride A and B, 10 pestalachloride C and D, 10 Pestalotia, 15, 17 Pestalotiopsis, 12, 15, 18, 19, 20, 21, 22, 26, 28, 29, 33, 61, 73, 82, 90, 91 Pestalotiopsis microspora, 12, 19, 21, 26, 28 Pezicula sp., 12 Phialocephala, 16, 133, 136, 139, 141, 145 Phoma glomerata, 14, 29 Phoma multirostrata, 12, 34 phylloplane, 8 Phyllosticta, v, viii, 15, 18, 34, 93, 94, 95, 96, 97, 99, 100, 109, 110, 114, 115, 116, 117, 119, 121, 123 Phytophthora capisici, 11 Pinus sylvestris, 9, 12, 30, 83, 132 Pithomyces, 15 Plasmodium falciparum, 13 Poa ampla, 10, 33


150

Index

podophyllotoxin, 16, 18, 30 Preussomerin N1, 12 Pyricularia oryzae, 14 Pythium ultimum, 14

Q Quercus sp, 10, 22 quinine, 15

R Rhizoctonia cerealis, 11 Rhizoctonia solani, 14 Rhodotorula minuta, 9 Rhyncholacis penicillat, 16, 20 riboflavin, 9

S Salvia miltiorrhiza, 14, 29 Sclerotinia sclerotiorum, 13, 56 secondary metabolites, vii, viii, 2, 8, 9, 10, 31, 34, 35, 40, 41, 45, 46, 50, 52, 54, 82, 83, 86, 93, 106, 115 Serratia marcescens, 16, 20, 32 Sonneratia alba, 14, 17, 28 Sphaeria typhena, 2 spiroketals, 12 Staphylococcus aureus, 12, 14, 59 steroids, vii, 1, 9, 11, 45, 50, 66, 68, 78, 79, 85, 90, 91 symbiosis, vii, 4, 25, 27, 30, 31, 39, 42, 44, 45, 78, 85, 131, 137, 144, 145

T T. brevifolia, 12 tauranin, 11, 18 Taxomyces, 12, 15, 19, 32, 55, 73, 87, 88 Taxomyces andreanae, 12, 15, 19, 32, 55, 73, 87, 88 Taxus baccata, 12, 19, 32 Taxus brevifolia, 12, 15, 19, 55, 73 teniposide, 16 terpenoids, vii, 1, 9, 11, 32, 50, 52, 68, 72, 90 Theobroma cacao, 14, 19, 33 Torreya taxifolia, 12, 19 torreyanic acid, 12, 19, 28 Trametes, 16, 18, 30 Trichophyton mentagrophytes, 13 Trichophyton rubrum, 13 tricin, 10 Tripterygium wilfordii, 11, 19, 20, 22 Tubercularia, 15

V vancomycin-resistant, 12 Vernonia amygdalina, 14, 17, 38 vincristine, 10, 19 Vochysia guatemalensis, 13, 22

X xanalteric acids I and II, 28 Xylaria sp., 11, 20, 27, 31, 62, 65, 82, 84, 121
