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ENDOPHYTIC FUNGI
DIVERSITY, CHARACTERIZATION
AND BIOCONTROL
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ENDOPHYTIC FUNGI
DIVERSITY, CHARACTERIZATION
AND BIOCONTROL
EVELYN HUGHES
EDITOR
New York
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Preface vii
Chapter 1 Endophytic Fungi: Occurrence, Classification,
Function and Natural Products 1
Afra Khiralla, Rosella Spina, Sakina Yagi,
Ietidal Mohamed and Dominique Laurain-Mattar
Chapter 2 Endophytic Fungi Are Multifunctional
Biosynthesizers: Ecological Role
and Chemical Diversity 39
Khaled A. Selim, Mohamed M. S. Nagia
and Dina E. El. Ghwas
Chapter 3 Endophytic Fungi Isolated from Vochysia divergens
in the Pantanal, Mato Grosso Do Sul: Diversity,
Phylogeny and Biocontrol of Phyllosticta citricarpa 93
Y. M. Hokama, D. C. Savi, B. Assad, R. Aluizio,
J. A. Gomes-Figueiredo, D. M. Adamoski,
Y. M. Possiede and C. Glienke
Chapter 4 Dark Septate Endophytes (DSE) in Polluted Areas 125
Elena Fernández-Miranda Cagigal
Index 147
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.
Chapter 1
ABSTRACT
Introduction: Researches on endophytic fungi have proven they are a
promising source of biocontrol agents. These organisms are present in the
internal healthy plant tissues during a part or/all of their life cycle without
causing apparent harm to their hosts. They influence greatly the
physiological activities of their host plants. Fungal endophytes enhance
their host resistance against abiotic stress, disease, insects and
mammalian herbivores by producing a broad range of fungal metabolites.
Indeed several interesting metabolites isolated from endophytic fungi
belong to diverse chemical classes, including: alkaloids, steroids,
flavonoids, terpenoids, quinones and phenols. Since the isolation of
paclitaxel in 1993 from an endophytic fungus of Pacific Yew, fungal
Corresponding author E-mail address: dominique.mattar@univ-lorraine.fr.
1. INTRODUCTION
The term “endophyte” is derived from the Greek, endon = within and
phyte = plant. It was first introduced in 1866 by de Bary. It was used broadly
to refer to any organism found within tissues of living plants; including
everything from virulent foliar pathogens to mycorrhizal root sombionts;
subsequent re-definitions led to confusion regarding the meaning of the term.
Modern mycologists generally agree that endophytes are organisms that
colonize internal plant tissues without causing apparent harm to their host.
Different groups of organisms such as fungi, bacteria, actinomycetes and
mycoplasma are reported as endophytes of plants (Arnold, 2007).
Collectively, more than 100 years of research suggest that most, if not all,
plants in natural ecosystems are symbiotic with mycorrhizal fungi and/or
fungal endophytes (Petrini, 1986). Unlike mycorrhizal fungi that colonize
plant roots and grow into the rhizosphere, endophytes reside entirely within
plant tissues and may grow within roots, stems and/or leaves, emerging to
often occur sparsely as hypha in the intercellular fluids and wall spaces of their
plant hosts, sporulate at plant or host-tissue senescence (Bacon and White,
2000). Studies of endophytic fungi were initiated nearly 200 years ago, when
Person in 1772 described the species Sphaeria typhena, now known as
Epichloe typhina (Pers.) Tul. (Khan, 2007). Fossils, in a 400-million-year-old,
indicated that plants have been associated with endophytes. Krings et al.,
(2007) studied petrographic thin sections of the Rhynie chert plant Nothia
aphylla, they found that three fungal endophytes occur in prostrata axes of this
plant.
Endophytic fungi 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).
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).
Insects deterrence
Reduction of nematodes
Increase of biomass
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).
6.2.3. Steroids
Many steroids are produced by endophytes, but most of the isolated
compounds showed moderate antimicrobial activities. Along with ergosterol,
3β,5α,6β-trihydroxyergosta-7,22-diene; 3β-hydroxyergosta-5-ene; 3-
oxoergosta-4,6,8,22-tetraene; 3β-hydroxy-5α,8α-epidioxyergosta-6,22-diene;
3β-hydroxy-5α,8α-epidioxyergosta-6,9,22-triene and 3-oxoergosta-4-ene, two
new steroids, 3β,5α-dihydroxy-6β-acetoxyergosta-7,22-diene and 3β,5α-
dihydroxy-6β-phenyl- acetoxyergosta-7,22-diene were characterized from the
liquid culture of an fungal endophyte Colletotrichum sp. of Artemisia annua.
Some of these metabolites were shown to be antifungal against some crop
pathogens Gaeumannomyces graminis var. tritici, Rhizoctonia cerealis,
Helminthosporium sativum and Phytophthora capisici (Lu et al., 2000; Yu et
al., 2010).
6.2.4. Terpenoids
Sesquiterpenes, diterpenoids and triterpenoids are the major terpenoids
isolated from endophytes (Yu et al., 2010). In period of 2006-2010, sixty five
sesquiterpenes, fourty five diterpenes, five monoterpenes and twelve other
terpenes, amounting to 127 terpenoids were isolated from endophytic fungi
and all have biological activity such as anti-microbial, anti-cancer and anti-
protozoa (Souza et al., 2011).
Three novel eremophilane-type sesquiterpenes were isolated from the
endophyte Xylaria sp. associated with Licuala spinosa. The three compounds,
eremophilanolide 1, 2 and 3 exhibited moderate cytotoxic activity with IC50
values of 3.8–21 µM against cancer cell lines KB, MCF-7, and NCI-H187
(Isaka et al., 2010). Two ent-eudesmane sesquiterpenes, ent-4(15)-eudesmen-
11-ol-1-one and ent-4(15)-eudesmen-1R, 11-diol were isolated from the
endophytic fungus Eutypella sp. BCC 13199 from the plant Etlingera littoralis
(Earth ginger) (Isaka et al., 2009). Four cytotoxic sesquiterpene compounds, 8-
deoxytrichothecin, trichothecolone, 7α-hydroxytrichodermol and 7α-
hydroxyscirpene, were isolated from fungal isolate KLAR 5, a mitosporic
Hypocreales found in a healthy twig of the Thai medicinal plant Knema
laurina. Also, tauranin, merulin A and C, sesquiterpene compounds, were
isolated from endophytic fungi with cytotoxic activity (Kharwar et al., 2011).
Two insect toxins of a pimarane diterpene framework were isolated from
the broth of an unidentified endophyte from a needle of the balsam fir Abies
balsamea (Tan and Zou, 2001). Subglutinol A and B, immunosuppressive but
noncytotoxic, were produced by Fusarium subglutinans, an endophytic fungus
from the perennial twining vine Tripterygium wilfordii (Lee et al., 1995).
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
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
6.2.8. Acids
Li et al., (2016) stated that an endophytic fungus from Salvia miltiorrhiza
produces salvianolic acid C as its host plant. This fungal endophyte identified
as Phoma glomerata D14. Khiralla (2015) isolated a new acid 3,7,11,15-
Tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethyl- hexacosa-
4E,8E,12E,16,18-pentaenoic acid; Khair acid from the solid culture of the
endophyte Curvularia papendorfii isolated from Vernonia amygdalina. This
acid revealed an average antibacterial effect against methicillin-resistant
Staphylococcus aureus with MIC value of 62.5 μg mL-1. Zhao et al., (2012)
reported three endophytic fungi Fusarium solani, F. oxysporum and F.
proliferatum from pigeon pea Cajanus cajan producing cajaninstilbene acid
(CSA). This acid is one of the major stilbenes found in pigeon pea. However,
cajaninstilbene acid revealed hypotriglycerimic, hypoglycemic, anti-
inflammatory, analgesic and antioxidant activities. Cytonic acids A and B have
been isolated from the solid-state fermentation of the endophytic fungi
Cytonaema sp. Cytonic acids A and B showed potential anti-viral effect
against human cytomegalovirus (hCMV) protease with values of IC50 = 43µM
and IC50 =11µM respectively (Guo et al., 2000). Two new 10-oxo-10H-
phenaleno[1,2,3-de]chromene-2-carboxylic acids, xanalteric acids I and II
were purified from extracts of the endophytic fungus Alternaria sp., isolated
from the mangrove plant Sonneratia alba collected in China. The two acids
exhibited weak antibiotic activity with MIC values of 125 and 250 μg mL-1
respectively against multidrug-resistant Staphylococcus aureus (Kjer et al.,
2009). The endophytic fungus Cryptosporiopsis cf. quercina produces
cryptocin in culture. This unique tetramic acid displays powerful antimycotic
activity against several plant pathogenic fungal strains including Pythium
ultimum, Pyricularia oryzae, with MIC values 0.39 and 0.78 μg mL-1
respectively (Li et al., 2000). Two new acids were isolated from Mangrove
endophytic fungus (No. ZZF13) (Xia et al., 2008).
REFERENCES
Abdalla, M. A.; Matasyoh, J. C. Endophytes as producers of peptides: an
overview about the recently discovered peptides from endophytic
microbes. Nat. Prod. Bioprospect. 2014, 4(5), 257–270.
Alves, R. R. N.; Rosa, I. L. Biodiversity, traditional medicine and public
health: where do they meet?. J. Ethnobiol. Ethnomed. 2007, 3(14), 1–9.
Aly, A. H.; Edrada-Ebel, R.; Wray, V.; et al., Bioactive metabolites from the
endophytic fungus Ampelomyces sp. isolated from the medicinal plant
Urospermum picroides. Phytochemistry. 2008, 69(8), 1716–1725.
Amna, T.; Puri, S. C.; Verma, V.; et al., Bioreactor studies on the endophytic
fungus Entrophospora infrequens for the production of an anticancer
alkaloid camptothecin. Can. J. Microbiol. 2006, 52, 189–196.
Arechavaleta, M.; Bacon, C. W.; Hoveland, C. S.; et al., Effect of the tall
fescue endophyte on plant response to environmental stress. Agron. J.
1989, 81, 83–90.
Arnold, A. E. In Endophytic Fungi: Hidden Components of Tropical
Community Ecology; Schnitzer, S.; Carson, W., Ed.; Tropical Forest
Community Ecology; Blackwell Scientific, Inc: Malden, MA, 2008.
Arnold, A. E. Understanding the diversity of foliar endophytic fungi: progress,
challenges, and frontiers. Fungal. Biol. 2007, 21(2–3), 51–66.
Arnold, A. E.; Engelbrecht, B. M. J. Fungal endophytes double minimum leaf
conductance in seedlings of a tropical tree. J. Trop. Ecol. 2007, 23, 369–
372.
Arnold, A. E.; Lutzoni, F. Diversity and host range of foliar fungal
endophytes: are tropical leaves biodiversity hotspots?. Ecology. 2007, 88,
541–549.
Arnold, A. E.; Maynard, Z.; Gilbert, G.; et al., Are tropical endophytic fungi
hyper diverse. Ecol. Lett. 2000, 3, 267–274.
Bacon, C. W.; Porter, J. K.; Robbins, J. D.; et al., Epichloe typhina from toxic
tall fescue grasses. Appl. Environ. Microbiol. 1977, 34, 576–581.
Bacon, C. W.; White, J. F. J. In Physiological Adaptations in the Evolution of
Endophytism in the Clavicipitaceae. Bacon, C. W.; White, J. F. J.; Ed.;
Microbial Endophytes; Marcel Dekker Inc: New York, NY, 2000.
Bagchi, B.; Banerjee, D. Diversity of fungal endophytes in Bauhinia vahlii (a
lianas) from different regions of Paschim medinipur district of west
Bengal. Int. J. Sci. Environ. Technol. 2013, 2(4), 748–756.
Fröhlich, J.; Hyde, K. D. Biodiversity of palm fungi in the tropics: are global
fungal diversity estimates realistic?. Biodiver. Conserv. 1999, 8, 977–
1004.
Gamboa, M. A.; Bayman, P. Communities of endophytic fungi in leaves of a
tropical timber tree (Guarea guidonia: Meliaceae). Biotropica. 2001, 33,
352–360.
Gentile, A. M.; Rossi, S.; Cabral, D.; et al., Origin divergence and phylogeny
of Epichloë endophytes of native argentine grasses. Mol. Phylogenet.
Evol. 1999, 35, 196–208.
Gonzalez, C. M.; Gonzalez, M. J. M.; Anayab, A. L.; et al., Acremoxanthone
E, a novel member of heterodimeric polyketides with a
bicyclo[3.2.2]nonene ring, produced by Acremonium camptosporum W.
Gams (Clavicipitaceae) endophytic fungus. Chem. Biodiver. 2015, 12,
133–147.
Gu, W.; Ge, H. M.; Song, Y. C.; et al., Cytotoxic benzo[j]fluoranthene
metabolites from Hypoxylon truncatum IFB-18, an endophyte of
Artemisia annua. J. Nat. Prod. 2007, 70, 114–117.
Guerin, D. Sur la presence d’un champignon dans l’lvraie. J. Bot. 1898, 12,
230–238. [About a fungus in the rye-grass. J. Bot. 1898, 12, 230–238.]
Guo, B.; Dai, J. R.; Ng, S.; et al., Cytonic Acids A and B: novel tridepside
inhibitors of hCMV protease from the endophytic fungus Cytonaema
Species. J. Nat. Prod. 2000, 63, 602–604.
Guo, B.; Li, H.; Zhang, L. Isolation of the fungus producing vinblastine. J.
Yunnan. Univ., (Natural Science Edition). 1998, 20, 214–215.
Harper, J. K.; Arif, A. M.; Ford, E. J.; et al., Pestacin: a 1,3-dihydro
isobenzofuran from Pestalotiopsis microspora possessing antioxidant and
antimycotic activities. Tetrahedron. 2003, 59, 2471–2476.
Hatakeyama, T.; Koseki, T.; Murayama, T.; et al., Eremophilane
sesquiterpenes from the endophyte Microdiplodia sp. KS 75-1 and
revision of the stereochemistries of phomadecalins C and D. Phytochem.
Lett. 2010, 3, 148–151.
Hiroyuki, K.; Teruhiko, Y.; Michi, O.; et al., Gamahonolides A, B, and
gamahorin, novel antifungal compounds from stromata of Epichloe
typhina on Phleum pratense. Biosci. Biotechnol. Biochem. 1992, 56,
1096–1099.
Holliday, P. A. Dictionary of Plant Pathology; Cambridge University Press:
Cambridge, 1998.
Li, E.; Tian, R.; Liu, S.; et al., Pestalotheols A−D, Bioactive metabolites from
the plant endophytic fungus Pestalotiopsis theae. J. Nat. Prod. 2008b,
71(4), 664–8.
Li, H. J.; Lan, W. J.; Yao, J. H.; et al., High performance liquid
chromatography-electrospray ionization tandemmass spectrometry
confirms the sequences of new cyclic peptides produced by marine
microorganism. Chin. J. Instrument. Anal. 2005, 24, 46–49.
Li, H. J.; Lin, Y. C.; Yao, J. H.; et al., Two new metabolites from the
mangrove endophytic fungus No. 2524. J. Asian Nat. Prod. Res. 2004, 6,
185–191.
Li, H.; Xiao, J.; Gao, Y.; et al., Chaetoglobosins from Chaetomium globosum,
an endophytic fungus in Ginkgo biloba, and their phytotoxic and cytotoxic
activities. J. Agric. Food Chem. 2014, 62(17), 3734–3741.
Li, J. Y.; Strobel, G.; Harper, J.; et al., Cryptocin, a potent tetramic acid
antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina.
Org. Lett. 2000, 2(6), 767–770.
Li, X.; Zhai, X.; Shu, Z.; et al., Phoma glomerata D14: an endophytic fungus
from Salvia miltiorrhiza that produces salvianolic acid C. Curr. Microbiol.
2016, 73, 31–37.
Lim, W.; Park, J.; Lee, Y. H.; et al., Subglutinol A, an immunosuppressive α-
pyrone diterpenoid from Fusarium subglutinans, acts as an estrogen
receptor antagonist. Biochem. Biophys. Res. Commun. 2015, 461(3), 507–
512.
Liu, L.; Liu, S.; Niu, S.; et al., Isoprenylated chromone derivatives from the
plant endophytic fungus Pestalotiopsis fici. J. Nat. Prod. 2009, 72, 1482–
1486.
Lodge, D. J.; Fisher, P. J.; Sutton, B. C. Endophytic fungi of Manilkara
bidentata leaves in Puerto Rico. Mycologia. 1996, 88, 733–738.
Lösgen, S.; Magull, J.; Schulz, B.; et al., Isofusidienols: novel chromone-3-
oxepines produced by the endophytic fungus Chalara sp. Eur. J. Org.
Chem. 2008, 4, 698–703.
Lu, H.; Zou, W. X.; Meng, J. C.; et al., New bioactive metabolites produced by
Colletotrichum sp., an endophytic fungus in Artemisia annua. Plant
Science. 2000, 151, 67–73.
Malinowski, D. P.; Belesky, D. P. Adaptations of endophtye-infected cool-
season grasses to environmental stresses: mechanisms of drought and
mineral stress tolerance. Crop. Sci. 2000, 40, 923–940.
BIOGRAPHICAL SKETCH
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Communications:
Chapter 2
ABSTRACT
Symbiosis is a widespread phenomenon in nature. Endophytes are
defined as all microorganisms that colonize asymptomatically within
living healthy tissues. In general, endophytes are considered as
commensalistic symbionts, where they receive nutrients and shelter from
the host. In return, they are thought to provide the host with chemical
*
Corresponding Author address: Email: khaled.selim@uni-tuebingen.de.
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
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:
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).
(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.
5. CLASSIFICATION OF ENDOPHYTES
Endophytic fungi have a great effect on the plant fitness, ecology and
evolution. Various groups of these microorganisms are able to produce
number of bioactive agents (Brundrett et al., 2006). In the past, endophytic
fungi were classified into two groups: - Clavicipitaceous and non-
Clavicipitaceous according to their taxonomy, evolution, ecological function
and host specificity. After that, Rodriguez et al., 2009 illustrated that, there are
four special groups depending on six properties which are tissue(s) colonized,
plant colonization pattern, host range, plant biodiversity levels, ecological
functions and mechanism of transmission between host generations. Class (1)
is Clavicipitaceous endophytes while; non-Clavicipitaceous endophytes are
divided into three special groups class (2), class (3) and class (4) (Rodriguez et
al., 2009). We will summarize each class briefly:
European investigators in 19th century were the first who noted the class
(1) (Clavicipitaceous endophytes) was found in the seeds of Lolium linicolum,
Lolium arvense, Lolium remotum and Lolium temulentum (Vogl, 1898). After
that, Clay, 1988 demonstrated that they are defensive mutualists of host
grasses. From this time, investigations on endophyte natural history, ecology,
physiology and evolution has been followed (Schardl and Moon, 2003; Rao
et al., 2005 and Koulman et al., 2007). Usually these endophytes found within
plant shoots and make systemic intercellular infections. Colonized plants
always harbor one controlling fungal isolate/ genotype. According to Clay and
Schardl, 2002 there are three types of Clavicipitaceous endophytes extended
from pathogenic and symptomatic species (Type I) to mixed combination and
asymptomatic endophytes (Types II and III) respectively. The class (1)
endophytes are primarily transmitted vertically and horizontally on offspring
via seed infections (Saikkonen et al., 2002). Class (1) endophytes confer
drought tolerance, increase plant biomass and produced chemicals that toxic to
herbivory and animals (Clay, 1988). However, the importance of these fungi is
due to the host genotype, environmental conditions and host species (Faeth
and Sullivan, 2003).
On the other hand, class (2) (non-Clavicipitaceous endophytes) are highly
various, symbolizing a polyphyletic aggregation of primarily ascomycete's
fungi with diverse and always bad unknown ecological role. Non-
Clavicipitaceous endophytes have been isolated from everywhere of land plant
and from all terrestrial ecosystems, containing both biomes and ecosystems
ranging from the tundra to the tropics (Arnold and Lutzoni, 2007). The
capacity of lot of fungi to switch between free-living lifestyles and endophytic,
the prudence they supply into the evolution of different ecological modes in
fungi, their ecological roles, their potential applications and the scale of their
diversity are attractive to ecologists, mycologists, applied scientists and
physiologists (Vasiliauskas et al., 2007and Selosse et al., 2008). Non-
Clavicipitaceous endophytes can be distinguish into three classes depend on
mechanism of transmission between host generations, host colonization
patterns, ecological function and planta biodiversity levels as shown in Table
(2).
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
The search for other sources of energy is becoming important because the
sources of liquid fuel are decreasing. The microorganisms in unique ecological
niches are the most effective ways to search for unusually metabolically active
secondary metabolites. The endophytic fungi have been recently discovered to
produce fuel related compound. These compounds are compatible with the
presence motive infrastructure and renewable which, depend on drop fuel
technology. Beside, these microorganisms have the abilities of growing and
releasing gaseous products on cellulosic wastes and agricultural instead of
agriculture products that may act as feed and food sources. These endophytic
fungi produce bioactive volatile compounds and a lot of them are fuel related.
Reviews on the endophytic microorganisms that producing these volatile
compounds have been appeared recently (Strobel, 2006, 2011 and 2012).
This encouraged researchers to directly plan their researches for the
discovery of new endophytic fungi that could produce hydrocarbons like
Muscodor albus (Worapong et al., 2001). Till now, all members of genus
Muscodor albus release volatile compound that kill and/or inhibit pathogenic
bacteria and fungi and some are detrimental to insects too (Strobel, 2011 and
2012). Due to the effective nature and the broad range of its volatile organic
compounds (VOCs), this fungal genus has been studies and promoted for use
as biological control (Strobel, 2011 and 2012). M. vitigenus, M. crispans, M.
sutura, and M. roseus all are identified as new species of this fungus, which
were isolated from different parts of the earth such as China, India and
Thailand (Strobel, 2012).
Furthermore, screening the chemistry of the volatile compounds released
by these endophytes, detected the presence of alkenes, benzene derivatives,
alkylesters, alkanes, polyaromatic hydrocarbons, terpenoids and other
compounds that either similar to or are linked to the chemical compound
families of diesel fuel. Therefore VOCs were called mycodiesel (Strobel et al.,
2008). Some samples acquired from stems of Eucryphia cordifolia in
rainforest of Chile. These patterns contain an organism that grow in the
appearance of the VOCs of M. albus and also release VOCs with antimicrobial
activities (Stinson et al., 2003a). This organism was identified morphologically
as Gliocladium roseum, however through taxonomic studies the organism was
strange deficient stage of the ascomycetous fungus-Ascocoryne sarcoides
(Strobel et al., 2010). This fungus is important due to its ability to create a
wide range of the acetate esters of straight chained alkanes containing heptyl
and sec-octyl alcohols (Strobel et al., 2008). After that, the scientist knew that
the alkyl alcohol side chains of these acetate esters act the basic free alkanes of
diesel as they specially have been detected in every diesel sample that had
been analyzed. Moreover, breakages of the ester bond go after by a reduction
of the alcohol to a methyl group and resulting in alkane which similar to that
found in diesel.
Moreover, further work has indicated the presence of decyl esters and
nonyl in the components of fungal VOCs (Griffin et al., 2010). However,
diesel fuels have series of straight chained hydrocarbons; also there they have
cyclic alkanes, branched alkanes and benzene derivatives along with a lot of
polyaromatic hydrocarbons (Griffin et al., 2010 and Mallette et al., 2014). In
addition, the analysis of VOCs of this fungus is resulted in an array of ketones,
alcohols, esters and different other hydrocarbons such as benzenes derivatives,
cycloalkenes and cycloalkanes (Mallette et al., 2014).
In the past few decades, scientists have begun to realize that almost all
higher plants may serve as a reservoir of indescribable numbers of endophytic
microbes (Bacon and White 2000). These endophytic microbes are fungi and
bacteria, and live in the intercellular spaces of their hosts and this also true for
marine plants including algae or seagrasses and also for invertebrate animals
including soft corals and sponges. Almost all endophytes release bioactive
secondary metabolites which may be implicated in a host-endophyte
relationship (Selim et al., 2012). As a direct consequence of the natural role of
endophytic secondary metabolites, they may ultimately to have applicability in
medical field for improving the drug discovery (Selim et al., 2011 and 2012).
A worldwide scientific effort is to isolate endophytes and to study their natural
products is now under way (Strobel, 2003).
Overall, the rational responses for studying endophytes as promising
sources of new therapeutic agents is the fact that this field of the science is an
unexplored area of biochemical diversity. Additionally, the focus on
endophytes as a potential source for pharmaceutical industry is due to the
reality that the endophytes contribute to the host by providing protection to
them with a variety of secondary metabolites (volatiles, insecticidal,
antimicrobial, hormones-like compounds and …. etc.). Some of endophytic
metabolites may be of interest clinically since they possess antibacterial,
antifungal, antimalarial and other interesting biological activities (Verma et al.,
2009 and Selim et al., 2012). Finally, one of major concern to the
pharmaceutical companies is the toxicity of any potential probable drug to
human tissues. Apparently, plants as a pool of endophytic microbes are a
eukaryotic system in which the endophyte exists. The metabolites synthesized
by the endophytes may have less cell toxicity; otherwise, they could destroy
the target tissues of the host. Thus, the host itself has naturally served as a
selection system for microbes producing bioactive molecules with reduced
toxicity toward eukaryotes. Therefore, the endophytes consider being an
important source of novel natural compounds and a notable growing trend is
the characterization of endophytic secondary metabolites in drug discovery
(Strobel, 2003; Verma et al., 2009 and Selim et al., 2012).
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.
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).
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.
10.1. Alkaloids
Figure 3. (Continued).
Figure 3. Chemical structures (1-17) show the diversity of alkaloid metabolites isolated
from endophytic fungi.
10.3. Glycosides
10.4. Peptides
system contains six α-amino acid residues connected to a β-amino acid from
the metabolites of Talaromyces wortmannii an Aloe vera derived endophyt
(Bara et al., 2013). Very recently this year this method was used to identify the
absolute configuration of a novel cyclic hepta-peptide unguisin F (41) obtained
from the Mucor irregularis that was associated with the medicinal plant
Moringa stenopetala found in Cameroon (Akone et al., 2016).
By means of spectectral, chiral HPLC and X-ray crystallographic data two
new cyclic peptides were identified from the broth of mangrove endophytic
fungus isolated from the leaves of Kandelia candel growing in Hong Kong.
Their structures were assigned as cyclo-(D-Leu-Gly-L-Tyr-L-Val-Gly-S-
OLeu) and cyclo-(D-Leu-Gly-L-Phe-L-Val-Gly-S-O-Leu) (Huang et al.,
2007). Cycloaspeptide A (42) is a cyclic peptide isolated from the extracts of
the fermentation broth of Penicillium janczewskii isolated from the phloem of
the Chileangymnosperm Prumnopitys andina (Schmeda-Hirschmann et al.,
2008). The bioassay guided fractionation of a plant associated fungus
identified as Aspergillus tubingensis isolated from Brucea javanica lead to the
isolation of a cyclic penta-peptide namely malformin A1 (43) which showed a
high anti-Tobacco mosaic virus (TMV) activities (Tan et al., 2015). Figure (6)
summarize the diversity of peptides metabolites isolated from endophytic
fungi (structures from 35-43).
10.5. Steroids
10.6. Terpenoids
isolated for the first time from Penicillium sp. SXH-65 in 2014 together with
five analouges compounds (Sun et al., 2014). Based on the interpretation of
extensive 1D and 2D NMR, mass spectroscopic and x-ray crystallographic
data, four novel merterpenoids compounds (62-65) were isolated from the
mangrove plant Acanthus ilicifolius associated-endophyte Aspergillus flavipes
(Bai et al., 2015).
Figure 7. (Continued).
Figure 8. (Continued).
Figure 9. Chemical structures (85-97) show the diversity of new classes of endophytic
metabolites.
REFERENCES
Adhikari, T. G., Joseph, C. M., Yang, G., Philips, D. A., Nelson, L. M. (2001).
Evaluation of bacteria isolated fromrice for plant growth promotion and
biological control of seedling disease of rice. Can. J. Microbiol. 47, 916–
924.
Akone, S. H., Daletos, G., Lin W., Proksch, P. (2016). Unguisin F, a new
cyclic peptide from the endophytic fungus Mucor irregularis. Z.
Naturforsch., C: J. Biosci. 71(1-2), 15-19.
Aly, A. H., Debbab, A., Proksch, P. (2011). Fungal endophytes: unique plant
inhabitants with great promises. Appl. Microbiol. Biotechnol. 90(6),
1829–1845.
Amatangelo, K. L., Vitousek, P. M. (2008). Stoichiometry of ferns in Hawaii:
implications for nutrient cycling. Oecologia 157(4), 619–627.
Arnold, A. E., Lutzoni, F. (2007). Diversity and host range of foliar fungal
endophytes: are tropical leaves biodiversity hotspots? Ecology 88, 541–
549.
Arnold, A. E., Maynard, Z., Gilbert, G. S., Coley, P. D., Kursar, T. A. (2000).
Are tropical fungal endophytes hyperdiverse? Ecology Letters 3, 267–274.
Arnold, A. E., Mejía, L. C., Kyllo, D., Rojas, E. I., Maynard, Z., Robbins, N.,
Herre, E. A. (2003). Fungal endophytes limit pathogen damage in a
tropical tree. PNAS 100, 15649–15654.
Azevedo, J. L., Maccheroni, W. Jr., Pereira, J. O., Araujo, W. L. (2000).
Endophytic microorganisms: a review on insect control and recent
advances on tropical plants. Elect J Biotechnol 3, 40–65.
Bacon, C. W., Porter, J. K., Robbins, J. D. (1975). Toxicity and occurrence of
Balansia on grasses from toxic fescue pastures. Appl. Environ. Microbiol.
29, 553–556.
Bacon, C. W., White, J. F. (2000). Microbial Endophytes. Marcel Deker Inc.,
New York.
Bai, Y., Aoust, F., Smith, D., Driscoll, B. (2002). Isolation of plant-growth-
promoting Bacillus strains from soybean root nodules. Can. J. Microbiol.
48, 230–238.
Bai, Z.-Q., Lin, X., Wang, J., Zhou, X., Liu, J., Yang, B., Yang, X., Liao, S.,
Wang L., Liu Y. (2015). New meroterpenoids from the endophytic fungus
Aspergillus flavipes AIL8 derived from the mangrove plant Acanthus
ilicifolius. Mar. Drugs 13(1), 237-248, 212.
Bara, R., Aly, A. H., Wray, V., Lin, W. H., Proksch, P., Debbab, A. (2013).
Talaromins A and B, new cyclic peptides from the endophytic fungus
Talaromyces wortmannii. Tetrahedron Lett. 54(13), 1686-1689.
Blunt, J. W., Copp, B. R., Keyzers, R. A., Munro, M. H., Prinsep, M. R.
(2016). Marine natural products. Nat Prod Rep. 33(3), 382-431.
Bouarab, K., Potin, P., Correa, J., Kloareg, B. (1999). Sulfated
oligosaccharides mediate the interaction between a marine red alga and its
green algal pathogenic endophyte. Plant Cell. 11, 1635–1650.
Brader, G., Compant, S., Mitter, B., Trognitz, F., Sessitsch, A. (2014).
Metabolic potential of endophytic bacteria. Curr. Opin. Biotechnol. Sci.
27, 30–37.
Brown, K. B., Hyde, K. D., Guest, D. I. (1998). Preliminary studies on
endophytic fungal communities of Musa acuminata species complex in
Hong Kong and Australia. Fungal Diversity 1, 27–51.
Brundrett, M. C. (2006). Understanding the roles of multifunctional
mycorrhizal and endophytic fungi. In: Schulz BJE, Boyle CJC, Sieber TN,
eds. Microbial root endophytes. Berlin, Germany: Springer-Verlag, 281–
293.
Bugni, T. S., Ireland, C. M. (2004). Marine-derived fungi: a chemically and
biologically diverse group of microorganisms. Natural Product Reports
21(1), 143-163.
Iliopoulou, D., Mihopoulos, N., Vagias, C., Papazafiri, P., Roussis, V. (2003).
Novel Cytotoxic Brominated Diterpenes from the Red Alga Laurencia
obtusa. J. Org. Chem. 68, 7667-7674.
Iqbal, J., Nelson, J. A., McCulley, R. L. (2013). Fungal endophyte presence
and genotype affect plant diversity and soil-to-atmosphere trace gas
fluxes. Plant Soil 364, 15–27.
Jinu, M. V., Gini C. K., Jayabaskaran, C. (2015). In vitro antioxidant activity
of cholestanol glucoside from an endophytic fungus Lasiodiplodia
theobromae isolated from Saraca asoca. J. Chem. Pharm. Res. 7(1), 952-
962.
Jumpponen, A. (2001). Dark septate endophytes are they mycorrhizal?
Mycorrhiza 11, 207–211.
Kharwar, R. N., Verma, V. C., Strobel, G., Ezra, D. (2008).The endophytic
fungal complex of Catharanthus roseus (L.) G. Don. Curr. Sci. 95, 228-
233.
Kiers, E. T., Denison, R. F. (2008). Sanctions, cooperation and the stability of
plant-rhizosphere mutualisms. Annual Review of Ecology, Evolution and
Systematics 39, 215-236.
Kobayashi, D. Y., Palumbo, J. D. (2000). Bacterial endophytes and their
effects on plants and uses in agriculture. In: Bacon CW, White JF (eds)
Microbial endophytes. Dekker, New York, pp 199–236.
Koulman, A., Lane, G. A., Christensen, M. J., Fraser, K., Tapper, B. A.
(2007). Peramine and other fungal alkaloids are exuded in the guttation
fluid of endophyte-infected grasses. Phytochemistry 68, 355–360.
Kowalski, T., Kehr, R. D. (1992). Endophytic fungal colonization of branch
bases in several forest tree species. Sydowia 44, 137–168.
Krohn, K., Bahramsari, R., Florke, U., Ludewig, K., Kliche-Spory, C., Michel,
A., Aust, H. J., Draeger, S., Schulz, B., Antus, S. (1997).
Dihydroisocoumarins from fungi: Isolation, structure elucidation, circular
dichroism and biological activity. Phytochemistry 45(2), 313-320.
Krohn, K., I. Kock, B. Elisaesser, U. Floerke, B. Schulz, S. Draeger, G.
Pescitelli, S. Antus and T. Kurtan (2007). Bioactive natural products from
the endophytic fungus Ascochyta sp. from Meliotus dentatus -
configurational assignment by solid-state CD and TDDFT calculations.
Eur. J. Org. Chem. (7), 1123-1129.
Kuldau, G., Bacon, C. (2008). Clavicipitaceous endophytes: Their ability to
enhance grass resistance to multiple stresses. Biological Control 46, 57-
71.
Mallette, N., Pankratz, E., Busse, S., Strobel, G. A., Carlson, R., Peyton, B.
(2014) Evaluation of cellulose as a substrate for hydrocarbon-fuel
production by Ascocoryne sarcoides (NRRL 50072). J Sustain Bioener
Syst 4, 33-49.
Merlin. E. (1922). On the mycorrhizas of Pinus sylvestris L. and Picea
abiesKarst. A preliminary note. Journal of Ecology 9, 254–257.
Ming, Ge. H., Yun Zhang, W., Ding, G., Saparpakorn, P., Chun Song, Y.,
Hannongbua, S., Xiang Tan, R. (2008). Chaetoglobins A and B, two
unusual alkaloids from endophytic Chaetomium globosum culture.
Chemical Communications. (45), 5978-5980.
Mitter, B., Petric, A., Shin, M. W., Chain, P. S., Hauberg-Lotte, L.,
ReinholdHurek, B. (2013). Comparative genome analysis of Burkholderia
phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based
on interaction strategies with host plants. Front. Plant Sci. 4, 120.
Mohali, S., Burgess, T. I.; Wingfield, M. J. (2005). Diversity and host
association of the tropical tree endophyte Lasiodiplodia the obromae
revealed using simple sequence repeat markers. Forest Pathol. 35, 385-
396.
Moon, C. D., Schardl, C. L., Christensen, M. J. (2000). The evolutionary
origins of Epichloë endophytes from annual ryegrass. Mycologia 92,
1103–1118.
Mostert, L., Crous, P. W., Petrini, O. (2000). Endophytic fungi associated with
shoots and leaves of Vitis vinifera, with specific reference to the
Phomopsis viticola complex. Sydowia 52, 46–58.
Murali, T. S., Suryanarayanan, T. S., Venkatesan, G. (2007). Fungal
endophyte communities in two tropical forests of southern India: diversity
and host affiliation. Mycological Progress. 6, 191–199.
Nagia, M. M., El-Metwally, M. M., Shaaban, M., El-Zalabani, S. M., Hanna,
A. G. (2012). Four butyrolactones and diverse bioactive secondary
metabolites from terrestrial Aspergillus flavipes MM2: isolation and
structure determination. Organic and medicinal chemistry letters 2(1), 1.
Nishikawa, H. (1993). Biochemistry of filamentous fungi II and III. A
metabolic product of Aspergillus melleus. Bull. Agric. Chem. Soc. Jpn, 9,
107-109 and148-151.
Ohtani, M., Narisada, M. (1991). Sulfur-sulfur bond formation reaction using
bis(1-methyl-1H-tetrazol-5-yl) disulfide. The Journal of Organic
Chemistry 56(18), 5475-5478.
Oliveira, C. M., Regasini, L. O., Silva, G. H., Pfenning, L. H., Young, M. C.
M., Berlinck, R. G. S., Bolzani, V. S., Araujo, A. R. (2011).
Qiao, M. F., Ji, N.-Y., Liu, X.-H., Li, F., Xue, Q.-Z. (2010). Asporyergosterol,
a new steroid from an algicolous isolate of Aspergillus oryzae. Nat. Prod.
Commun. 5(10), 1575-1578.
Qiao, M.-F., N.-Y. Ji, F.-P. Miao and X.-L. Yin (2011). Steroids and an
oxylipin from an algicolous isolate of Aspergillus flavus. Magn. Reson.
Chem. 49(6), 366-369.
Qin, J. C., Gao, J. M., Zhang, Y. M., Yang, S. X., Bai, M. S., Ma, Y.-T.,
Laatsch, H. (2009). Polyhydroxylated steroids from an endophytic fungus
Chaetomium globosum ZY-22 isolated from Ginkgo biloba. Steroids
74(9), 786-790.
Rao, S., Alderman, S. C., Takeyasu, J., Matson, B. (2005). The Botanophila-
Epichloë association in cultivated Festuca in Oregon: evidence of simple
fungi vory. Entomologia Experimentalis et Applicata 115, 427–433.
Redell, P. and Gordon, V. (2000). Lessons from nature: can ecology provide
new leads in the search for novel bioactive chemicals from rainforests? In:
S. K. Wrigley, M. A. Hayes R., Thomas, E. J., T., Chrystal T., Nicholson
N. (ed.), Biodiversity: new leads for pharmaceutical and agrochemical
industries. The Royal Society of Chemistry. Cambridge, United Kingdom
p. 205–212.
Redman, R. S., Sheehan, K. B., Stout, R. G., Rodriguez, R. J., Henson, J. M.
(2002). Thermo tolerance conferred to plant host and fungal endophyte
during mutualistic symbiosis. Science. 298, 1581.
Rice-Evans, C. A., Diplock, A. T. (1993). Current status of antioxidant
therapy. Free Radical Biology and Medicine 15(1), 77-96.
Rodrigues, K. F. (1994). The foliar fungal endophytes of the Amazonian palm
Euterpe oleracea. Mycology, 86, 376–385.
Rodrigues, K. F., Samuels, G. J. (1992). Idriella species endophytic in palms.
Mycotaxon 43, 271-276.
Rodriguez, R. J., Henson, J., Volkenburgh, Van. E., Hoy, M., Wright, L.,
Beckwith, F., Kim, Y., Redman, R. S. (2008). Stress tolerance in plants
via habitat-adapted symbiosis. International Society of Microbial Ecology
2, 404–416.
Rodriguez, R. J., White, J. F. Jr., Arnold, A. E., Redman, R. S. (2009). Fungal
endophytes: diversity and functional roles. New Phytol. 182, 314–330.
Romao, A. S., Sposito, M. B., Andreote, F. D., Azevedo, J. L., Araujo, W. L.
(2011) Enzymatic differences between the endophyte Guignardia
mangiferae (Botryosphaeriaceae) and the citrus pathogen G. citricarpa.
Genet Mol Res 10, 243–252.
Rubini, M. R., Silva-Ribeiro, R., Pomella, A. W. V., Maki, C., Araújo, W. L.,
Santos, D. R., Azevedo, J. L. (2005) Diversity of endophytic fungal
community of cacao ( Theobromacacao) L. and biological control of
Crinipellisperniciosa causal agent of Witches’ broom disease Int J Biol
Sci 1, 24–33.
Saikkonen, K., Faeth, S. H., Helander, M., Sullivan, T. J. (1998). Fungal
endophytes: a continuum of interactions with host plants. Annu. Rev. Ecol.
Syst. 29, 319–343.
Saikkonen, K., Ion, D., Gyllenberg, M. (2002). The persistence of vertically
transmitted fungi in grass meta populations. Proceedings of the Royal
SocietyB: Biological Sciences 269, 1397–1403.
Schardl, C. L., Moon, C. D. (2003). Processes of species evolution in
Epichloë/ Neotyphodium endophytes of grasses. In: White JFJ, Bacon
CW, Hywel-Jones NL, Spatafora JW, eds. Clavicipitalean fungi:
evolutionarybiology, chemistry, biocontrol and cultural impacts. New
York, NY, USA: Marcel-Dekker, 273–310.
Schmeda-Hirschmann, G., Hormazabal, E., Rodriguez, J. A., Theoduloz, C.
(2008). Cycloaspeptide A and pseurotin A from the endophytic fungus
Penicillium janczewskii. Z. Naturforsch., C: J. Biosci. 63(5/6), 383-388.
Schulz, B., Boyle, C. (2005). The endophytic continuum. Mycol. Res. 109,
661–687
Schulz, B., Boyle, C., Sieber, T. N. (2006). Microbial root endophytes.
Springer, Berlin Heidelberg New York.
Schulz, B., Sucker, J., Aust, H. J., Krohn, K., Ludewig, K., Jones, P. G.
(1995). Biologically active secondary metabolites of endophytic Pezicula
species. Mycological Research. 99, 1007-1015.
Selim, K. A., El-Beih, A. A., AbdEl-Rahman, T. M., El-Diwany, A. I. (2012).
Biology of Endophytic Fungi. Current Research in Environmental and
Applied Mycology 2(1), 31-82.
Selim, K. A., El-Beih, A. A., AbdEl-Rahman, T. M., El-Diwany, A. I. (2011).
Biodiversity and antimicrobial activity of endophytes associated with
Egyptian medicinal plants. Mycosphere 2(6), 669-678.
Selim, K. A., El-Beih, A. A., AbdEl-Rahman, T. M., El-Diwany, A. I. (2014).
Biological evaluation of endophytic fungus Chaetomium globosum
JN711454 as potential candidate for improving drug discovery. Cell
Biochem Biophys. 68(1), 67-82.
Selim, K. A., El-Beih, A. A., AbdEl-Rahman, T. M., El-Diwany, A. I. (2016).
High expression level of antioxidants and pharmaceutical bioactivities of
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), 951-
953.
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
ABSTRACT
Endophytic fungi are important biotechnological tools because they
produce many secondary metabolites. However, to access this important
source of bioactive molecules, it is essential to explore the diversity of
endophytic fungi and catalog their species richness in different
ecosystems. Tropical regions are recognized as areas of high diversity,
although many areas remain unexplored, such as the Pantanal of Mato
*
Corresponding author: Dr. Chirlei Glienke, ch.glienke@gmail.com, Phone: (55) 4133611562.
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.
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
To extract DNA, the isolates were cultured in PDA for 3 days at 25°C and
then subjected to a MOBIO Ultraclean Microbial DNA Isolation Kit (MO
Bio, Carlsbad, US), following the manufacturer’s instructions.
Figure 1. Map showing the Pantanal colored in gray and the collection site is in the
magnification box, with the points at Santa Emilia Farm illustrating the nine sampled
trees. The collection points were identified by letters and numbers:
1(19.501944ºS/55.601389ºW), 2(19.500833ºS/55.600556ºW),
3(19.500556ºS/55.600278ºW), 4(19.501111ºS/55.600556ºW),
A(19.508548ºS/55.628775ºW), B(19.506286°S/55.610172ºW),
C(19.509930ºS/55.627039ºW), D(19.509038ºS/55.626354ºW) and
E(19.507243ºS/55.615815ºW).
Extract Production
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.
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.
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.
Sordariomycetes
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
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.
Nigrospora
In the phylogenetic analysis (Figure 6A and 6B), isolates LGMF1121,
LGMF1133, LGMF1138, and LGMF1156 were found to be closely related to
the species Nigrospora oryzae, whereas isolate LGMF1125 was most closely
related to strain Nigrospora sphaerica (Figure 6 A–B). Strains LGMF1121
and LGMF1133 did not cluster with any type species in this genus; however,
there are few sequences available for this genus (Figure 6). In contrast,
Nigrospora is a genus that has been studied widely because of its secondary
metabolites (Kim et al. 2001; Zhang et al. 2009), and strains in this genus have
been isolated as endophytes from a large number of tropical plant species,
especially medicinal plants (Martinez-Luis et al. 2011).
Colletotrichum
Based on ITS sequence analysis, strains LGMF1135, LGMF1137, and
LGMF1173 were classified as species belonging to the Colletotrichum
gloeosporioides complex. However, as expected, ITS sequences are not
enough to identify the species in this complex (Figure 7). Species of the genus
Colletotrichum are pathogenic (Huang et al. 2009) as well as endophytic in
several plant species, mainly from tropical regions (Refaei et al. 2011; Vega et
al. 2010).
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.
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.
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
and LGMF141 are clustered with strains named as Diaporthe sp. 1 from Brazil
(Gomes et al. 2013), showing 100% of similarity. However, strains
LGMF1144 and LGMF1157 formed a clade with long branch suggesting these
isolates belong to a new species of Diaporthe. To more precisely assign strains
LGMF1144 and LGMF1157 to a taxon, multilocus sequencing is suggested, as
used previously for endophytic strains from Brazilian medicinal plants (Gomes
et al. 2013). Diaporthe species are frequently isolated endophytes found in
several plants (Hakizimana et al. 2011; Singh et al. 2011; Gomes et al. 2013).
Lanceispora
Isolates LGMF1142, LGMF1143, LGMF151, and LGMF1152 showed
high sequence similarity with the species Lanceispora amphibia (family
Amphisphaeriaceae; GenBank accession number LC146743). Even though the
genus Lanceispora was described by Nakagiri et al. in 1997 and was found to
comprise two species, L. amphibia and L. phyllophila, there are no L.
phyllophila sequences available in public databases, thus complicating the
identification of our isolates at the species level. Both L. amphibia and L.
phyllophila were identified as endophytes, first isolated from Bruguiera
gymnorrhiza in mangrove forests in the Southwest Islands of Japan and from
unknown dicotyledonous leaves in Singapore (Nakagiri et al. 1997; Sarma et
al. 2001). It is interesting that we isolated strains of this genus from medicinal
plants in Brazil, showing the high level of biodiversity in Pantanal of Mato
Grosso do Sul.
Dothideomycetes
Dothideomycetes order are commonly isolated as endophytes from a wide
number of plants (Crous et al. 2006). In our study, based on the phylogenetic
trees (Figures 9 to 12) the endophytic strains belonging to Dothideomycetes
were attributed to six genera: Phyllosticta (n = 38), Pseudofusicoccum (n = 2),
Neofusicoccum (n = 2), Cladosporium (n = 1), Annellosympodiella (n = 1) and
Phaeosphaeria (n = 1).
Phyllosticta
Among the isolates belonging to the genus Phyllosticta genus (Table 1,
Figure 9), strains LGMF1172 and LGMF1193 are most closely related to the
species Phyllosticta podocarpi, P. pseudotsudae, P. owaniana, and P.
bifrenariae, however with high genetic distance when compared with others
Phyllosticta species in the phylogenetic tree (Figure 9). The remaining
Phyllosticta isolates were identified as Phyllosticta capitalensis, supported by
a 100% bootstrap value and low intraspecific diversity (Figure 9). Phyllosticta
capitalensis is a common endophyte found in a large number of plants
(presently described in >70 plant families) distributed worldwide (Glienke et
al. 2011).
Figure 10. Bayesian phylogenetic tree based on ITS sequence, showing the
relationships between the species of Neofusicoccum and Pseudofusicoccum genera.
The tree is rooted with Botryosphaeria dothidea sequence obtained from GenBank.
Scale bar shows 2 changes and Bayesian posterior probability values are indicated at
the nodes.
Botryosphaeriaceae
In the phylogenetic analysis strains LGMF1122 and LGMF1118 grouped
with Pseudofusicoccum stromaticum, whereas the isolate LGMF1116
clustered with Neofusicoccum brasiliense type strain (Figure 10). Both
Pseudofusicoccum and Neofusicoccum belong to Botryosphaeriaceae family,
and they were recently separated from the genus Fusicoccum based on
morphological differences in their conidia and molecular sequences (Crous
et al. 2006). Species in these genera are normally found as endophytes and
pathogens of woody plant species such as Eucalyptus and Acacia (Mohali et
al. 2007; Pérez et al. 2010), and they have caused serious economic losses in
Pinus cultures in South America (Burgess et al. 2007).
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.
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.
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).
REFERENCES
Altschul, S. F. BLAST algorithm. Encyclopedia of Life Sciences, 2005, 1–4.
Arieira, J. Nunes da Cunha, C. Fitossociologia de uma floresta inundável
monodominante de Vochysia divergens Pohl (Vochysiaceae), no Pantanal
Norte, MT, Brasil. Acta Botanica Brasilica, 2006, 20, 569–580.
Arruda, W. S., Oldeland, J., Filho, A. C. P., Pott, A., Cunha, N. L., Ishii, I. H.,
Damasceno-Junior, G. A. Inundation and fire shape the structure of
riparian forests in the Pantanal, Brazil. PloS One, 2016, 11, 1–18.
Bensch, K., Groenewald, J. Z., Dijksterhuis, J., Starink-wilemse, M.,
Andersen, B., Summerell, B. A., Shin, H. D., Dugan, F. M., Schoroers, H.
J., Braun, U., Crous, P. W. Species and ecological diversity within the
Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales).
Studies in Mycology, 2010, 67, 1–94.
Burgess, T. I., Barber, P. A., Sufaati, S., Xu, D., Hardy, G. E. S., Dell, B.
Mycosphaerella spp. on Eucalyptus in Asia: new species, new hosts and
new records. Fungal Diversity, 2007, 24, 135–157.
Buchanan, M., Hashimoto, T., Asakawa, Y. Five 10-phenyl-[11]-
cytochalasans from a Daldinia fungal species. Phytochemistry, 1995, 40,
135–140.
Chowdhary, L. K. Kaushik, N. Fungal endophyte diversity and bioactivity in
the Indian medicinal plant Ocimum sanctum Linn. PLoS One, 2015, 10, 1–
25.
Crous, P. W., Slippers, B., Wingfield, M. J., Rheeder, J., Marasas, W. F. O.,
Philips, A. J. L., Alves, A., Burgess, T., Barber, P., Groenewald, J. Z.
Phylogenetic lineages in the Botryosphaeriaceae. Studies in Mycology,
2006, 55, 235–53.
Crous, P. W., Shivas, R. G., Quaedylieg, W., van der Bank, M., Zhang, Y.,
Summerell, B. A., Guarro, J., Wingfield, M. J., Wood, A. R., Alfenas, A.
C., Braun, U., Cano-Lira, J. F., García, D., Marin-Felix, Y., Alvarado, P.,
Andrade, J. P., Armengol, J., Assefa, A., den Breeven, A., Camele, I.,
Cheewangkoon, R., de Souza, J. T., Duong, T. A., Esteve-Raventós, F.,
Fournier, J., Frisullo, S., García-Jiménez, J., Gardiennet, A., Gené, J.,
Hernández-Restrepo, M., Hirooka, Y., Hospenthal, D. R., King, A.,
Lechat, C., Lombard, L., Mang, S. M., Marbach, P. A., Marincowitz, S.,
Marin-Felix, Y., Montaño-Mata, N. J., Moreno, G., Perez, C. A., Peréz, S.
A. M., Robertson, J. L., Roux, J., Rubio, E., Schumacher, R. K., Stchigel,
A. M., Sutton, D. A., Tan, Y. P., Thompson, E. H., van der Linde, E.
Walker, A. K., Walker, D. M., Wickes, B. L., Wong, P. T., Groenewald, J.
Nakagiri, A., Okane, I., Ito, T., Katumoto, K. Lanceispora amphibia gen. et
sp. nov., a new amphisphaeriaceous ascomycete inhabiting senescent and
fallen leaves of mangrove. Mycoscience, 1997, 38, 207–213.
Niu, J. Z., Sanders, H. H., Zhang, Y. X., Lin, J. Z., Dou, W., Wang, J. J.
Biological control of arthropod pests in citrus orchands in China.
Biological Control, 2014, 68, 15–22.
Petrini, O., Sieber, T. N., Toti, L., Viret, O. Ecology, metabolite production,
and substrate utilization in endophytic fungi. Natural Toxins, 1993, 1,
185–196.
Pérez, C. A., Wingfield, M. J., Slippers, B., Altier, N. A., Blanchette, R. A.
Endophytic and canker-associated Botryosphaeriaceae occurring on non-
native Eucalyptus and native Myrtaceae trees in Uruguay. Fungal
Diversity, 2010, 41, 53–69.
Perryman, A. M., Clark, S. J., West, J. S. Splash dispersal of Phyllosticta
citricarpa conidia from infected citrus fruit. Scientific Reports, 2014, 4, 1–
8.
Pongcharoen, W., Rukachaisirikul, V., Phongpaichit, S., Kuhn, T., Pelzing,
M., Sakayaroj, J., Taylor, W. C. Metabolites from the endophytic fungus
Xylaria sp. PSU-D14. Phytochemistry, 2008, 69, 1900–1902.
Pott, A. Pott, V. J. Plantas do Pantanal. 1 Ed. Corumbá, Embrapa - CPAP.
1994.
Rambaut, A. 2012. Tree Figure Drawing Tool Version 1.4.0. Institute of
Evolutionary Biology, University of Edinburgh, 2006-2012. [http://tree.
bio.ed.ac.uk/software/figtree/)].
Refaei, J., Jones, E., Sakayaroj, J., Santhanam, J. Endophytic fungi from
Rafflesia cantleyi: species diversity and antimicrobial activity.
Mycosphere, 2011, 2, 429–447.
Rivera-Orduña, F. N., Suarez-Sanchez, R. A., Flores-Bustamante, Z. R.,
Gracida-Rodriguez, J. N., Flores-Cotera, L. B. Diversity of endophytic
fungi of Taxus globosa (Mexican yew). Fungal Diversity, 2011, 47, 65–
74.
Ronquist, F., Teslenko, M., Van Der Mark, P., Ayres, D. L., Darling, A.
MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice
across a large model space. Systematic Biology, 2011, 61, 539–42.
Rosa, L. H., Vaz, A. B. M., Caligiorne, R. B., Campolina, S., Rosa, C. A.
Endophytic fungi associated with the Antarctic grass Deschampsia
antarctica Desv. (Poaceae). Polar Biology, 2009, 32, 161-167.
Rungjindamai, N., Pinruan, U., Choeyklin, R., Hattori, T., Jones, E. B. G.
Molecular characterization of Basidiomycetous endophytes isolated from
leaves, rachis and petioles of the oil palm, Elaeis guineensis in Thailand.
Fungal Divers, 2008, 33, 139–161.
Sarma, V. V., Hyde, K. D. Lanceispora phyllophila sp. nov. on petioles of
unknown dicotyledonous leaves in Singapore. Mycoscience, 2001, 42, 97–
99.
Schreiber, A. Bailey. L. Rapid Quantitation and Identification of Carbendazim
in Orange Juice Using the New AB SCIEX QTRAP® 4500 LCMS/MS
System Fast method development in response to contaminated orange
juice imports to the U.S. Food and Environmental, 2012, 1, 1–4.
Sinegani, A., Emtiazi, G., Hajrasuliha, S., Shariatmadari, H. Biodegradation of
some agricultural residues by fungi in agitated submerged cultures.
African Journal of Biotechnology, 2011, 4, 1058–1061.
Singh, S. K., Strobel, G. A., Knighton, B., Geary, B., Sears, J., Ezra, D. An
endophytic Phomopsis sp. possessing bioactivity and fuel potential with
its volatile organic compounds. Microbial Ecology, 2011, 61, 729–39.
Somaiah, M. N., Sunayana, N., Prakash, H. S. Endophytic fungal diversity in
medicinal plants of Western Ghats, India. International Journal of
Biodiversity, 2014, 1–9.
Strobel, G., Daisy, B., Castillo, U., Harper, J. Natural products from
endophytic microorganisms. Journal of Natural Products, 2004, 67, 257–
268.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S.
MEGA5: Molecular evolutionary genetics analysis using maximum
likelihood, evolutionary distance, and maximum parsimony methods.
Molecular Biology and Evolution, 2013, 28, 2731–2739.
Tavčar, M., Svobodová, K., Kuplenk, J., Novotný, C., Pavko, A.
Biodegradation of azo dye RO16 in different reactors by immobilized
Irpex lacteus. Acta Chimica Slovenica, 2006, 53, 338–343.
Thompson, J. D., Higgins, D. G., Gibson, T. J. Clustal w: Improving the
sensitivity of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Research, 1994, 22, 4673–4680.
Vega, F. E., Simpkins, A., Aime, M. C., Posada, F., Peterson, S. W., Rehner,
S. A., Infante, F., Castilho, A., Arnold, A. E. Fungal endophyte diversity
in coffee plants from Colombia, Hawaii, Mexico and Puerto Rico. Fungal
Ecology, 2010, 3, 122–138.
Wang, Y., Guo, L., Hyde, K. D. Taxonomic placement of sterile morphotypes
of endophytic fungi from Pinus tabulaeformis (Pinaceae) in northeast
China based on rDNA sequences. Fungal Diversity, 2005, 20, 235–260.
Chapter 4
ABSTRACT
Dark septate endophytes (DSE) constitute a very heterogeneous
group of Ascomycetes characterized by a septate and melanized
mycelium. Inside, tissues show intra- and intercellular development and
are able not only to generate mantle and Hartig net but also to produce
typical intracellular structures (microsclerotia), all without causing
apparent damage to the plant. DSE were previously thought to be
restricted to infertile boreal or alpine habitats, where arbuscular
mycorrhizal fungi cannot persist. However, in recent years DSE have
been found extensively distributed in polluted areas around the world,
supporting a growing body of evidence that points to a prominent
ecological role, even when these organisms have not been studied from
the physiological role of a host-fungi perspective. It has been
hypothesized that DSE dominance as root endophytes might relate to
their melanised cell walls, known to play an important function in heavy
metal immobilization by sequestration. In addition to the improved
nutritional performance associated with mycorrhizal fungi, this capacity
*
Corresponding Author: Email: cagigal_11@hotmail.com, Tel.: +34985104835, Fax:
+34985104867.
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.
Given the stable nature of these compounds, heavy metals are a group of
very persistent pollutants in the soil. Of the 118 elements of the periodic table,
49 are considered as heavy metals. Not all these high-density metals are
particularly toxic in normal concentrations; for instance, iron (Fe),
molybdenum (Mo), Zn and manganese (Mn) are essential micronutrients.
However, others are toxic at low concentrations and have no known function,
such as silver (Ag), arsenic (As), Hg, Cd, Pb and antimony (Sb) (Niess, 1999).
The risk of heavy metals resides in the fact that they can be chemically or
biologically degraded (Kabata-Pendias, 2000), and in addition, they tend to
bioaccumulate provoking diverse toxic effects. High concentrations of these
metals (essential or not) in the soil can produce symptoms of toxicity in plants,
since they displace the essential elements and disable multifaceted activity
causing, for example, inhibition of growth (Van Assche & Clijster, 1990).
Among its negative effects on plant biology, it can result in growth inhibition
or in oxidative stress-related damage, due to the formation of reactive oxygen
species (ROS) (Mudipalli, 2008). Moreover, these metals can also bind to
organic molecules, such as pigments or enzymes, replacing some essential
metals, altering their specific function (Malayeri et al., 2008). In this regard,
heavy metals show a remarkably high affinity for the sulfhydryl, amino,
phosphate, carboxyl and hydroxyl groups. For example in the carbonic
anhydrase enzyme, the Zn atom present in its active center can be replaced by
a heavy metal, thereby reducing Calvin cycle efficiency (Navarro-Aviñó et al.,
2007).
A wealth of physical (cancer, hurts in the kidney, autoimmunity, etc) and
psychical problems (anxiety, passiveness, etc) have been described for
humans. Hence, their persistence, progressive accumulation and the
transference likelihood to other systems pose a threat both to human health and
ecosystems (Becerril et al., 2007). For these reasons, the adoption of necessary
measures to enable the restoration of soil becomes essential.
SOIL (BIO)REMEDIATION
Soil remediation represents a technological challenge both for industries
and governmental institutions. Contaminated soil can be remediated by
chemical, physical or biological techniques (Mc Eldeowney et al., 1993). In
turn, technologies can be classified in two categories: (a) ex-situ, which
requires removal of the polluted soil to treat it (inside/outside) and (b) in situ,
in which remediation is performed without removing the contaminated soil.
This latter technique is more convenient given its lower cost and impact on the
ecosystem. In contrast, ex situ techniques such as excavation, washing and soil
storage together with the removal of chemical or physical contaminants causes
a physicochemical alteration leading to loss of soil functionality due to the
elimination of its biological activity (including nitrogen-fixing bacteria,
mycorrhizal fungi and fauna) (Ghosh & Singh, 2005).
For all the above reasons, it is necessary to develop new techniques, more
safe and secure and with less associated costs. Among the most widely
accepted in situ biological techniques of recent years for decontamination of
polluted soils stand phytoremediation. This technique is based on the use of
plants that accumulate high concentrations of heavy metals in their tissues
(Miransari, 2011) or at least tolerate it, allowing for reforestation of these
polluted areas.
Considering the plants tolerance/intolerance to growth on contaminated
soil heavy metal, we can classify them as metallophyte or intolerant.
Metallophytes are those plants, which have developed physiological
mechanisms to resist, tolerate and survive in soils with high levels of metals
(Becerril et al., 2007). And inside of the metallophytes we can distinguish
between “strict metallophytes,” restricted entirely to metal-rich soils and
“facultative metallophytes,” with populations on both metalliferous and
nonmetalliferous soils. Among them, some support the presence of metals but
do not allow the entry into the root and therefore the translocation to other
parts of the plant. Conversely, others may accumulate metals in their aerial
tissues. This accumulation depends on type of metal and plant species. These
plants are called hyperaccumulators and are used in phytoextraction.
Not all heavy metals are considered harmful to plant development.
Toxicity varies depending on concentration, persistence and origin. Therefore,
phytoavailability of metals can be defined as the ratio of metals found in the
soil that can be absorbed by a given plant genotype (Prasad, 2004).
There are several biochemical and genetic mechanisms that allow plants
develop in habitats a priori harmful to their development. These mechanisms
can be summarized as follows (Navarro-Aviñó et al., 2007):
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).
FUNGAL ASSOCIATIONS
The dependency of trees from mycorrhizal fungi has been long
established, especially where soil characteristics are adverse. Numerous papers
have demonstrated that if degraded areas are reforested using mycorrhized
trees, the survival and development rates achieved are superior to those
achieved with trees without mycorrhizal fungi (Duñabeitia et al., 2004;
Meharg & Cairney, 2000).
The two most widespread types of mycorrhizal association are arbuscular
mycorrhizal (AM) and ectomycorrhizal (ECM). It is known that AM establish
symbiosis with nearly 75% of all families of vascular plants from natural
ecosystems, (Brundrett, 2009) as well as in degraded and contaminated areas
(Jeffries et al., 2003). These associations improve nutrition, tolerance and
accumulation of heavy metals in the plants (Tonin et al., 2001). In case of
ECM, estimated point towards only 4.5% of plants with this type of
association (Brundrett, 2009). Despite its great importance in plants with
agroforestry interest (Marx & Cordell, 1989). ECM fungi are able to mobilize
soil nutrients by secreting low molecular weight organic compounds (oxalates,
siderophores or citrates), and are involved in several biogeochemical cycles
such as the carbon (C) and nitrogen (N).
Nonetheless, other fungi that can be found abundantly and with worldwide
distribution in these stressful environmental conditions (Cevnik et al., 2000;
Vrålstad et al., 2002a; Wilberforce et al., 2003; Li et al., 2012; Fernández-
Miranda, 2014) are dark septate endophytes (DSE). Jumpponen (2001) defined
them as conidial or sterile ascomycetes fungi mostly belonging to the order
Helotiales with darkly pigmented and septate hyphae that colonise living plant
roots without causing apparent negative effect. Colonization by DSE produces
typical intracellular structures called microsclerotia (Figure 1) inside root cells.
The role of these fungi on host plant is controversial because while some
authors observe his influence as a promoter of plant hormones (Schulz, 2006)
and positive effects, as would a mycorrhizal fungus (Jumpponen, 2001;
Newsham, 2011) others observe negative effects (Wilcox & Wang, 1987;
Tellenbach et al., 2011). The predominance of DSE as endophytes in heavy
metal contaminated areas can relate to their melanised cell walls, which can
sequester heavy metals and therefore confer protective effects to the plant.
However, despite this evidence, a limited number of studies have focused on
DSE function.
DISTRIBUTION
DSE fungi were previously thought to be restricted to infertile boreal or
cold-stressed habitats (Jumpponen & Trappe, 1998), where AM fungi, the
typical mutualists of herbaceous plant roots at lower latitudes and altitudes,
cannot persist or occur only sporadically (Upson et al., 2007; Newsham et al.,
2011). However, in recent years there have been reports of more than 600
plant species spanning 100 plant families worldwide (Barrow & Aaltonen,
2001; Addy et al., 2005). They appear in diverse habitats such as arid, arctic,
boreal, alpine, temperate forest, or tropical ecosystems (Jumpponen & Trappe,
1998; Mandyam & Jumpponen, 2005). Moreover, latest studies have shown
that DSE are the dominant fungi in metal contaminated sites (Cevnik et al.,
2000; Vrålstad et al., 2002a; Wilberforce et al., 2003; Li et al., 2012;
Fernández-Miranda, 2014). They colonize both herbaceous and ligneous
plants, and particularly roots of metal hyperaccumulators, halophytes, orchids,
or marine macrophytes (Mandyam & Jumpponen, 2005). And recent studies
demonstrated that DSE reported that DSE are the dominant fungi from healthy
fine roots of Erica herbacea (Cevnik et al., 2000) Betula pubescens, Populus
tremula, Picea abies, Pinus sylvestris, Calluna vulgaris, Vaccinium myrtillus,
V. vitis-idaea, Deschampsia flexuosa (Vrålstad et al., 2002a) Arabis hirsuta,
Acacia decurrens, Symplocos paniculata, Rabbosia eriocalyx, Arenaria
serpyllifolia, Rosa longicuspis (Li et al., 2012) B. celtiberica and Salix
atrocinerea (Fernández-Miranda, 2014) in polluted soils in around the world.
With all these data, Mandyam & Jumpponen (2005) analyzed the reports
of present of DSE in alpine, arctic, antarctic, boreal, temperate and tropical
habitats and conclude that DSE may be as abundant as mycorrhizal fungi, are
ubiquitous in occurrence, co-occur with different types of mycorrhizae c) are
most prevalent in stressed environments. So what is the function of these fungi
in a natural ecosystem?
TAXONOMY OF DSE
These fungi belong to a wide range of ascomycete taxa, but are often
members of the order Helotiales, such as Phialocephala fortinii,
Leptodontidium orchidicola or Meliniomyces bicolor (Jumpponen & Trappe,
1998; Addy et al., 2005; Vrålstad et al., 2002a). Belonging to this order we can
find an ecologically diverse and complex group of pathogenic fungi,
saprophytes, DSE, parasites of others fungi and ectomycorrhizal and ericoid
mycorrhizal fungi (Vrålstad et al., 2002a; Tedersoo et al., 2009).
Teleomorphic species of the Helotiales are characterised by inoperculate asci
and discoid, turbinate or clavate ascocarps ranging in size from the hardly
visible members of the Hyaloscyphaceae to more prominent members of the
Geoglossaceae and Sclerotiniaceae. The Helotiales was erected by Nannfeldt
(1932), and was replaced with Leotiales (Carpenter, 1988). However, the
Helotiales sensu str. and Leotiales sensu str. are currently recognised as two
separate orders, the latter only comprising the Leotiaceae sensu str. (Korf &
Lizon, 2001).
As shown in Figure 2 (Fernández-Miranda, 2014), the position of many
species within the order Helotiales is very complex, and there are species with
an uncertain position as seen in the last reorganization of the
Hyaloscypaheceae family (Han et al., 2014).
Based on rDNA ITS sequence analysis, Tedersoo et al., (2009)
differentiated 6 complexes. Several Helotiales subgroups such as the
Phialocephala–Acephala and Rhizoscyphus–Meliniomyces complexes and
Lachnum spp.
Figure 2. Maximum likelihood (ML) consensus tree derived from the ITS data set. On
each branch, the percentages (%) of 10,000 bootstrapping replicates supported by ML
are shown and the Bayesian PPs. The sequence of our isolation is represented in bold.
The tree was rooted using a sequence from Bulgaria inquinans. The scale shows the
expected number of changes per nucleotide. Only Bayesian posterior probability
values above 50% are indicated.
The term mycorrhiza was coined by the German botanist Albert Berhhard
Frank in 1885, who defined it as a symbiotic association between the roots
(rhizos) of a plant and certain groups of soil fungi (mycos), where both
members of the association benefit and actively involved in the transport and
absorption of nutrients (Mykorhizen). Brundrett (2004) redefined the term
mycorrhizas as symbiotic associations between a fungus (specialised for life in
soils and plants) and a root (or other substrate-contacting organ) of a living
plant that is primarily responsible for nutrient transfer, and that could be
essential for one or both partners. Mycorrhizas take place in a specialised plant
organ where intimate contact results from synchronised plant-fungus
development. This last definition is based on developmental and functional
characteristics that can summarized on 5 criteria (Brundrett, 2009):
It is clear that DSE establish an intimate contact with the host plant and
could generate modified root systems, but the crucial point of nutrient and
metabolite exchange is until unclear. For example, Peterson et al., (2008)
described that both DSE’s hyphae and microsclerotia in root cells lack a host-
derived perifungal membrane and interfacial matrix material, and hence cannot
be regarded as specialized interfaces for nutrient transfer between plant and
fungus. On the other hand, however, it is been widely reported that
Rhizoscyphus ericae (Current Name Pezoloma ericae), a member of the order
Helotiales and the typical mycorrhizal associate of ericaceous plant species,
enhances the uptake of organic N from the acidic heathland soils that its hosts
inhabit (Smith & Read, 2008). DSE’s effect on host plants is controversial,
with plant responses to experimental inoculation ranging from negative
Several hypotheses have been put forward to explain the observed positive
responses to root endophyte colonization, being the two most prominent: 1)
modulation of plant growth via nutrient mineralization (as in mycorrhizae)
(Jumpponen, 2001; Mandyam & Jumpponen 2005; Upson et al., 2009;
Newsham, 2011) and 2) production of plant growth promoting phytohormones
(Mucciarelli et al., 2002; Schulz & Boyle, 2005; Schulz, 2006).
In view of the above evidence, a meta-analysis performed by Mayerhofer
et al., (2013) showed that identity of the inoculated endophyte affects plant
response, as was the case for plants inoculated with Phialocephala, which tend
to have smaller biomass than controls (Tellenbach et al., 2011; Reininger et
al., 2012). Not to mention differences in experimental conditions, which
undoubtedly contribute to the high levels of variability in plant response seen
in the literature. In conclusion, it appears that the effects depend on the host-
symbiont combination, and therefore more studies are necessary to clarify
which species inside this genus could be considered mycorrhizal fungi and
which not.
most abundant cellular thiol-rich heavy metal binding peptide in fungi (Singh
et al., 1997) and some works tend to consider the soluble tripeptide as the first
line of defence against heavy metal cytotoxicity (Viarengo & Nott, 1993).
Superoxide dismutase and catalase are crucial for cellular detoxification,
controlling the levels of superoxide anion radical and hydrogen peroxide
(Pócsi et al., 2004; Bai et al., 2003). Zhan et al., (2011) found that superoxide
dismutase and catalase activities in the hyphae of Exophiala pisciphila had
positive correlations with Pb(II) and Cd(II) concentrations, and Ban et al.,
(2012) confirmed these results observing that these enzymes play an effective
role in protecting G. cylindrosporus against oxidative stress induced by Pb(II).
Moreover superoxide dismutase appears to act as the primary defence against
acute Pb(II) stress.
The presence of melanins and antioxidant enzymes are not the only
mechanisms that these fungi possess. It has been demonstrated, the
relationship between toxic metal mineral solubilisation and metal in Cu-
tolerant isolates of P. ericae demonstrated a much higher ability to solubilize
Cd, Cu and Zn phosphates than isolates from non-polluted areas (Fomina &
Gadd, 2007). Bartholdy et al., (2001) revealed that P. fortinii excreted
hydroxamate siderophore for Fe(III) mobilization (ferricrocin, ferrirubin and
ferrichrome C). All these results, further and more in-depth knowledge relating
to these metal tolerance mechanisms in fungal endophytes is necessary and
their potential positive effects on the survival of their plant hosts in metal-
enriched soils will ultimately lead to powerful applications in bioremediation.
CONCLUSION
Based on the studies available, we can conclude that DSE fungi are
prevalent in various habitats and colonise a high rage of plant species. This
group of fungi cannot be overlooked while assessing the fungal communities
of any ecosystem. Even in the absence of clear consensus about positive
impacts on host fitness, growth or performance, DSE can be said to be likely
to perform functions similar to those attributed to mycorrhizal fungi. Although
experimental evidence is limited and experimental results may conflict, DSE
are seems to be involved in host nutrient uptake. And finally, the presence of
melanins may indicate a function of altering environmental tolerances.
Further and more in-depth studies on the metal tolerance mechanisms in
DSE and their effects on host plant survival in metal-enriched soils, but the
possibility of pre-inoculation of seedling with DSE as a strategy for achieving
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.
REFERENCES
Addy, H. D., Piercey, M. M., & Currah, R. S. (2005) Microfungal endophytes
in roots. Canadian Journal of Botany, 83, 1–13.
Bai, Z. H., Harvey, L. M., & McNeil, B. (2003). Physiological responses of
chemostat cultures of Aspergillus niger (B1-D) to simulated and actual
oxidative stress. Biotechnology Bioengineering, 82, 691–701.
Ban, Y., Tang, M., Chen, H., Xu, Z., Zhang, H., & Yang, Y. (2012). The
response of dark septate endophytes (DSE) to heavy metals in pure
culture. PloS one, 7(10), e47968.
Barrow, J. R., & Aaltonen, R. E. (2001). Evaluation of the internal
colonization of Atriplex canescens (Pursh) Nutt. Roots bay dark septate
fungi and the influence of host physiological activity. Mycorrhiza, 13,
239-247.
Bartholdy, B. A., Berreck, M., & Haselwandter, K. (2001). Hydroxamate
siderophore synthesis by Phialocephala fortinii, a typical dark septate
fungal root endophyte. Biometals, 14(1), 33-42.
Becerril, J. M., Barrutia, O., Plazaola, J. G., Hernández, A., Olano, J. M., &
Garbisu, C. (2007). Especies nativas de suelos contaminados por metales:
aspectos ecofisiológicos y su uso en fitorremediación. Revista
Ecosistemas, 16(2).
Bell, A. A, & Wheeler, M. H. (1986). Biosynthesis and functions of fungal
melanins. Annual Review in Phytopathology, 24, 4111–451.
Brundrett, M. C. (2004). Diversity and classification of mycorrhizal
associations. Biological reviews of the Cambridge Philosophical Society,
79, 473-495.
Jeffries, P., Gianinazzi, S., Perotto, S., Katarzyna, T. & Barea, J. M. (2003).
The contribution of arbuscular mycorrhizal fungi in sustanaible
maintenance of plant health and soil fertility. Biology and Fertility of
Soils, 37, 1-16.
Han, J. G., Hosoya, T., Sung, G. H., & Shin, H. D. (2014). Phylogenetic
reassessment of Hyaloscyphaceae sensu lato (Helotiales, Leotiomycetes)
based on multigene analyses. Fungal Biology, 118, 150-167.
Harms, H., Schlosser, D., & Wick, L. Y. (2011). Untapped potential:
exploiting fungi in bioremediation of hazardous chemicals. Nature
Reviews Microbiology, 9, 177-192.
Hrynkiewicz, K., Baum, C., & Leinweber, P. (2009). Mycorrhizal community
structure, microbial biomass P and phosphatase activities under Salix
polaris as influenced by nutrient availability. European Journal of Soil
Biology, 45, 168-175.
Jumpponen, A., & Trappe, J. M. (1998). Dark septate endophytes: a review of
facultative biotrophic root-colonizing fungi. New Phytologist, 140, 295–
310.
Jumpponen, A. (2001). Dark septate endophytes-are they mycorrhizal?.
Mycorrhiza, 11, 207-211.
Kabata-Pendias, A. (2000). The Anthroposphere. In: Trace elements in Soils
and Plants. 3rd Edition. CRC Press. Boca Raton, Florida 3-26.
Kalač, P. (2010). Trace element contents in European species of wild growing
edible mushrooms: A review for the period 2000-2009. Food Chemistry,
122, 2-15.
Kapoor, A., Viraraghavan, T., Cullimore, R., & Cullimore D. (1999). Removal
of heavy metals using the fungus Aspergillus niger. Bioresource
Technology, 70, 95-104.
Korf, R. P., & Lizon, P. (2001). The status of the ordinal name Leotiales.
Czech Mycology, 52.
Kuo, M. J., & Alexander, M. (1967). Inhibition of the lysis of fungi by
melanins. Journal of Bacteriology, 94, 624–629.
Lado, L. R., Hengl, T. & Reuter, H. I. (2008). Heavy metals in European soils:
A geostatistical analysis of the FOREGS Geochemical database.
Geoderma, 148, 189-199.
Li, H. Y., Li, D. W., He, C. M., Zhou, Z. P., Mei, T. & Xu, H. M. (2012).
Diversity and heavy metal tolerance of endophytic fungi from six
dominant plant species in a Pb–Zn mine wasteland in China. Fungal
Ecology, 5, 309-315.
Mandyam, K., & Jumpponen, A. (2005). Seeking the elusive function of the
root-colonising dark septate endophytic fungi. Studies in Mycology, 53,
173-189.
Malayeri, B. E., Chehregani, A., Yousefi, N. & Lorestani, B. (2008).
Identification of the hyperaccumulator plants in copper and iron mine in
Iran. Pakistan journal of biological sciences: PJBS, 11, 490-492.
Martino, E., Turnau, K., Girlanda, M., Bonfante, P., & Perotto, S. (2000).
Ericoid mycorrhizal fungi from heavy metal polluted soils: their
identification and growth in the presence of zinc ions. Mycological
research, 104, 338-344.
Marx, D. H., & Cordell, C. E. (1989). The use of specific ectomycorrhizas to
improve artificial forestation practise. En: Wippa, J. M. & Lumsdem, R.
D. (eds.). Biotecnology of fungi for improving plant growth. Cambridge
Univ. Press. Cambridge, pp. 1-25.
Mayerhofer, M. S., Kernaghan, G., & Harper, K. A. (2013). The effects of
fungal root endophytes on plant growth: a meta-analysis. Mycorrhiza, 23,
119-128.
McEldeowney, S., Hardman, D. J., & Waite, S. (1993). Treatment
Technologies. En: Pollution Ecology and Biotreatment Technologies.
McEldeowney, S., Hardman, J. & Waite, S. (Eds). Longman Singapore
Publishers, Singapore.
Meharg, A. A., & Cairney, J. W. G. (2000). Ectomycorrhizas: extending the
capabilities of rhizosphere remediation?. Soil Biology and Biochemistry,
32, 1475-1484.
Michelot, D., Sibobud, E., Doré, J-C., Viel, C., & Poirier, F. (1998). Update on
metal content profiles in mushrooms-Toxicological implications and
tentative approach to the mechanisms of bioaccumulation. Toxicon, 36,
1997-2012.
Miransari, M. (2011). Hyperaccumulators, arbuscular mycorrhizal fungi and
stress of heavy metals. Biotechnology Advances, 29, 645-653.
Mucciarelli, M., Scannerini, S., Bertea, C. M., & Maffei, M. (2002). An
ascomycetous endophyte isolated from Mentha piperita L.: biological
features and molecular studies. Mycologia, 94, 28–39.
Mudipalli, A. (2008). Metals (micro nutrients or toxicants) and global health.
The Indian journal of medical research, 128, 331.
Nannfeldt, J. A. (1932). Studien uber die Morphologie und Systematik der
nicht-lichenisierten inoperculaten Discomyceten.
Basidiomycetes, 7
A Bauhinia vahlii, 4, 23
biofuel, viii, 40
A. nidulans var. echinulatus, 12
Botryodiplodia, 15
Abies balsamea, 11
Botrytis, 13, 15, 56
Acremonium camptosporum, 13, 26
Botrytis cinerea, 13, 56
Acremonium coenophialum, 6
Bursera simaruba, 14
Acremonium sp., 12, 19, 32
Acremoxanthones A, B, C, 13
alkaloids, vii, 1, 5, 6, 10, 30, 45, 50, 57, 58, C
59, 81, 82, 83, 84, 89
Alternaria, 12, 14, 15, 17, 18, 20, 25, 27, cajaninstilbene acid, 14, 21, 35
28, 62, 79 Cajanus cajan, 14, 21
Alternaria sp., 14, 15, 17, 18, 25, 27, 28 Camptothecin, 10, 15, 18, 19, 23, 30, 31
altersolanol A, 12 Candida albicans, 13, 56
Annona muricata, 13 Catharanthus roseus, 10, 15, 19, 35, 81
Aphanomyces cochlioides, 14 Catunaregam tomentosa, 13, 20
Artemia salina, 13 chaetoglobosin U, 10, 19, 25
Artemisia annua, 11, 18, 26, 29 chaetoglobosins A and C, 13
Nonclavicipitaceous, 5, 7
I Nothapodytes, 10, 16, 19, 30, 31
Nothapodytes foetida, 10, 19, 30, 31
Imperata cylindrical, 10
Nothia aphylla, 2
isocoumarins, 27, 33, 61, 62, 63, 78, 91
O
J
Oncovin®, 10
Juniperus cedre, 10, 25
opium, 15
Ozonium, 15
K
P
Khair acid, 14, 17
Knema laurina, 11
paclitaxel, vii, 1, 15, 19
Papulaspora, 15
L Penicillium, 12, 16, 17, 59, 62, 64, 66, 68,
69, 70, 78, 79, 80, 82, 84, 86, 87, 88, 91
L. arvense, 6 Penicillium lilacum, 12
L. linicolum, 6 peptides, 12, 23, 29, 58, 65, 66, 67, 77, 79,
L. remotum, 6 80
Larix laricina, 12, 25 peramine, 6, 81
leucinostatin A, 12, 19 Periconia, 13, 15, 19, 34, 61, 89
Licuala spinosa, 11 Periconia sp., 13, 34
loline, 6, 31 peronosporomycete, 14
lolitrem, 6 pestalachloride A and B, 10
Lolium temulentum, 6, 48 pestalachloride C and D, 10
Pestalotia, 15, 17
Pestalotiopsis, 12, 15, 18, 19, 20, 21, 22, 26,
M 28, 29, 33, 61, 73, 82, 90, 91
Pestalotiopsis microspora, 12, 19, 21, 26,
merulin A and C, 18 28
Metarhizium, 15 Pezicula sp., 12
methicillin-resistant, 12, 14 Phialocephala, 16, 133, 136, 139, 141, 145
Monilia,, 16 Phoma glomerata, 14, 29
Monochaetia, 15 Phoma multirostrata, 12, 34
Mucor, 13, 15, 66, 76 phylloplane, 8
Mucor miehei, 13 Phyllosticta, v, viii, 15, 18, 34, 93, 94, 95,
96, 97, 99, 100, 109, 110, 114, 115, 116,
N 117, 119, 121, 123
Phytophthora capisici, 11
Neothyphodium coenophialum, 7 Pinus sylvestris, 9, 12, 30, 83, 132
Neotyphodium coenophialum, 6 Pithomyces, 15
Neurospora, 16, 18, 31, 102, 106 Plasmodium falciparum, 13
Nodulisporium sp., 10, 20, 25, 80 Poa ampla, 10, 33
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