Fungal endophytes from higher plants

Prévia do material em texto

REVIEW
Fungal endophytes from higher plants: a prolific source
of phytochemicals and other bioactive natural products
Amal H. Aly & Abdessamad Debbab & Julia Kjer &
Peter Proksch
Received: 24 November 2009 /Accepted: 1 February 2010 /Published online: 13 March 2010
# Kevin D. Hyde 2010
Abstract Bioactive natural products from endophytic fungi,
isolated from higher plants, are attracting considerable
attention from natural product chemists and biologists alike
as indicated by the steady increase of publications devoted to
this topic during recent years (113 research articles on
secondary metabolites from endophytic fungi in the period of
2008–2009, 69 in 2006–2007, 36 in 2004–2005, 14 in 2002–
2003, and 18 in 2000–2001). This overview will highlight
the chemical potential of endophytic fungi with focus on the
detection of pharmaceutically valuable plant constituents,
e.g. paclitaxel, camptothecin and podophyllotoxin, as prod-
ucts of fungal biosynthesis. In addition, it will cover new
bioactive metabolites reported in recent years (2008–2009)
from fungal endophytes of terrestrial and mangrove plants.
The presented compounds are selected based on their
antimicrobial, antiparasitic, cytotoxic as well as neuropro-
tective activities. Furthermore, possible factors influencing
natural product production in endophytes cultivated in vitro
and hence the success of bioprospecting from endophytes are
likewise discussed in this review.
Keywords Endophytes . Fungi . Host plants .
Bioactive metabolites
Introduction
Endophytic fungi, a polyphyletic group of highly diverse,
primarily ascomycetous fungi that are defined functionally
by their occurrence within tissues of plants without causing
any immediate overt effects (Bacon and White 2000; Hyde
and Soytong 2008), are found in liverworts, hornworts,
mosses, lycophytes, equisetopsids, ferns, and seed plants
from the arctic tundra to the tropics (Strobel 2006b; Arnold
2007; Huang et al. 2008, 2009; Hyde and Soytong 2008;
Oses et al. 2008; Raghukumar 2008). Once inside their host
plant, endophytes usually assume a quiescent (latent) state
either for the whole lifetime of the infected plant tissue or
for an extended period of time, i.e. until environmental
conditions are favorable for the fungus or the ontogenetic
state of the host changes to the advantage of the fungus
which may then turn pathogenic (Sieber 2007; Rodriguez
and Redman 2008). It is worth mentioning that, of the
nearly 300,000 species of higher plants existing on earth,
each plant contains a diversity of endophytes (Strobel and
Daisy 2003). Analysis of the fungal diversity within host
plants is, however, often biased towards fast-growing,
ubiquitous species, whereas rare species with minor compet-
itive strength and specialized requirements may remain
undiscovered upon isolation and cultivation attempts (Duong
et al. 2006; Unterseher and Schnittler 2009). Recently, a
more precise identification and phylogenetic accommodation
of sterile morphotypes and unculturable fungi was achieved
by highly discriminant DNA-based techniques which
improve our knowledge and appreciation of fungal endo-
phytic biodiversity (Guo et al. 2003; Wang et al. 2005;
Arnold and Lutzoni 2007; Arnold et al. 2007; Tao et al.
2008a; Tejesvi et al. 2009).
Colonization of host plants by endophytic fungi is believed
to contribute to host plant adaptation to biotic and abiotic stress
factors (Redman et al. 2002; Arnold et al. 2003; Waller et al.
2005; Zhang et al. 2006; Akello et al. 2007; Bae et al. 2009;
Giordano et al. 2009). It is of special interest that in many
cases host plant tolerance to biotic stress has been correlated
A. H. Aly :A. Debbab : J. Kjer : P. Proksch (*)
Heinrich-Heine-Universität Düsseldorf, Institut für
Pharmazeutische Biologie und Biotechnologie,
Universitätsstr. 1, Geb. 26.23,
40225 Düsseldorf, Germany
e-mail: proksch@uni-duesseldorf.de
Fungal Diversity (2010) 41:1–16
DOI 10.1007/s13225-010-0034-4
with fungal natural products (Saikkonen et al. 1998; Tan and
Zou 2001; Strobel et al. 2004; Zhang et al. 2006). The
majority of natural products occurring in endophytic micro-
organisms have been shown to have antimicrobial activity,
and in many cases these have been implicated in protecting
the host plant against phytopathogenic microorganisms
(Gunatilaka 2006) even though evidence for the production
of these compounds in planta is up till now mostly lacking
(for a noteworthy exception see Aly et al. 2008a).
Although the first discovery of endophytes already dates
back to 1904, this group of microorganisms did at first not
receive much attention in the decades to follow. This
changed dramatically after the detection of paclitaxel
(taxol®) (1) in the endophytic fungus Taxomyces andreanae
that had been isolated from Taxus brevifolia, the latter being
the original source of this important anti-cancer drug
(Stierle et al. 1993, 1995). This spectacular discovery
which raised questions with regard to horizontal gene
transfer between host and endophyte (or vice versa) that are
still unresolved came unexpected at that time and was even
at first questioned (Stierle et al. 1995). In the years to
follow, paclitaxel production could be detected in numerous
other endophytic fungi that had been isolated from a wide
range of host plants that are not known to produce
paclitaxel thereby indicating that the biogenetic capacity
for the production of this important drug is far more wide
spread in fungi than it is in plants. Recently it was
demonstrated that the production of known herbal com-
pounds in endophytes is by no means restricted to
paclitaxel but extends also to further pharmacologically
important natural products such as camptothecin (2) (Puri et
al. 2005; Amna et al. 2006), podophyllotoxin (3) (Puri et al.
2006; Eyberger et al. 2006; Kour et al. 2008) and others. In
addition to being alternative sources for secondary metab-
olites known from plants, endophytes accumulate a wealth
of other biologically active and structurally diverse natural
products that are unprecedented in nature (Tan and Zou
2001; Strobel and Daisy 2003; Strobel et al. 2004;
Gunatilaka 2006; Zhang et al. 2006; Verma et al. 2009)
and are of importance for drug discovery or as lead
compounds for agriculture (Strobel 2006a, b; Mitchell et
al. 2008). It is hence now generally accepted that
endophytes represent an important and largely untapped
reservoir of unique chemical structures that have been
modified through evolution and are believed to be involved
in host plant protection and communication (Gunatilaka
2006). Meanwhile, understanding the ecology, evolution,
and importance of fungal endophytes has grown to be a
formidable prospect, due to the huge number of fungi that
are able of forming endophytic associations and their
uniqueness relative to other plant-associated microbes
(Arnold 2007; Hyde and Soytong 2008; Sánchez Márquez
et al. 2008).
In the following, pharmaceutically valuable plant sec-
ondary metabolites found to be produced by fungal
endophytes are reported. Moreover, selected examples of
secondary metabolites from endophytic fungi, obtained
from terrestrial and mangrove plants, published in the
period 2008–2009 (88 compounds including 67 new natural
products) are presented, with special emphasis on bioactive
products, source organisms and country of origin. The
compounds are selected and grouped according to their
antimicrobial, antiparasitic, cytotoxic as well as neuro-
protective properties. The structures are shown only for
new compounds, or for previously reported compounds
with newly reported biological activities.
Endophytic fungi as sources of plant secondary
metabolites
Recently, several studies have led to the discovery of
important plant secondary metabolites from endophytic
fungi thus raising the prospect of using such organisms as
alternative sources of these metabolites (Priti et al. 2009).
The discovery of the paclitaxel (taxol®) (1) producing
endophytic fungus Taxomyces andreanae from the yew
plant Taxusbrevifolia (Stierle et al. 1993, 1995) set the
stage for a more comprehensive examination of other Taxus
species and other plants for the presence of paclitaxel
producing endophytes, so as to apply it to the industrial
production of this pharmacologically important drug.
Paclitaxel, the multi-billion dollar anti-cancer compound
produced by the yew plant, has activity against a broad
band of tumor types, including breast, ovarian, lung, head
and neck cancers, as well as advanced forms of Kaposi’s
sarcoma. It was found to bind to polymerized tubulin
promoting microtubule formation and microtubule stabili-
zation against disassembly and hence inhibiting mitosis and
therefore cancer growth. Many other endophytic fungi,
such as Seimatoantlerium tepuiense, S. nepalense (Bashyal
et al. 1999), and Tubercularia sp. strain TF5 (Wang et al.
2000), have meanwhile been reported to produce paclitaxel.
In a recent study, investigating the endophytic fungal
diversity of Taxus chinensis, thirteen species belonging to
different genera were verified for producing paclitaxel in vitro.
Among the paclitaxel-producing fungi, the yield of the drug
produced by Metarhizium anisopliae was 846.1!g/L (Liu et
2 Fungal Diversity (2010) 41:1–16
al. 2009). Furthermore, Pestalotiopsis microspora (Strobel et
al. 1996), Periconia sp. (Li et al. 1998), Bartalinia robillar-
doides and Colletotrichum gloeosporioides (Gangadevi and
Muthumary 2008a, b), residing in plants other than Taxus
species were also found to produce paclitaxel. HPLC
quantification showed that the amount of paclitaxel produced
by the latter two fungi was 187.6 and 163.4!g/L, respectively
(Gangadevi and Muthumary 2008a, b). However, up to now
paclitaxel contents from fungal isolates are too low for
industrial production as alternative processes exist which
include semi-synthesis of the anti-cancer drug starting from
the naturally occurring precursor desacetylbaccatin III which
can be isolated in sufficient amounts from needles of other
Taxus species such as T. baccata.
OMe
OMeMeO
OH
O
O
O
OH
H
3
O O
O
O
O
O
O
O
MeO
OMe
OH
H
H
HO
H
HO
H
S
5b
O O
O
H
HO
O
H
H O
O
O
O
OMe
OMe
OH
H
H
5a
O
O
O
HO
N
N
2
O OH
O
O
HO
O
O
OH
HN
Ph
OAc
H
AcO
1
O
Ph
O
Ph
N
H
N
OH
N
N
H
H
HO
OAcMeO
H
4
O
MeO
O OMe
O
Another mitotic inhibitor, which is clinically used in
chemotherapy treatment for certain types of cancer includ-
ing leukemia, lymphoma, breast and lung cancer for many
years, is the dimeric indole alkaloid vincristine (4). Unlike
paclitaxel, vincristine arrests mitosis by binding to tubulin
dimers thus inhibiting their assembly to microtubule
structures. Preliminary evidence was reported for the
production of vincristine, originally obtained from Cathar-
anthus roseus, by endophytic Fusarium oxysporum isolated
from the same plant (Lingqi et al. 2000).
Fungal Diversity (2010) 41:1–16 3
The lignan podophyllotoxin (3), synthesized by Podo-
phyllum species, is highly valued as the precursor to
clinically used anti-cancer drugs such as etoposide (5a)
and teniposide (5b). The mechanism of action of these
drugs is confined to inhibition of topoisomerase II, thus
blocking the ligation step of the cell cycle, harming the
integrity of the genome, and subsequently leading to
apoptosis and cell death. Topoisomerases are a class of
enzymes that catalyze and guide the unknotting of DNA
during DNA transcription. The endophytic fungal strains
Trametes hirsuta and Phialocephala fortinii, isolated from
Podophyllum hexandrum and P. peltatum, respectively,
were reported to produce podophyllotoxin (Puri et al.
2006; Eyberger et al. 2006) at a yield ranging from 0.5 to
189!g/L (Eyberger et al. 2006). Recently, podophyllotoxin
was also reported from Fusarium oxysporum which is an
endophyte of the medicinal plant Juniperus recurva that
accumulates podophyllotoxin. The highest yield of podo-
phyllotoxin produced by these endophytes amounted to
28!g/g of dry mass (Kour et al. 2008). Similarly, the
anticancer pro-drug desoxypodophyllotoxin (6) was
reported from Aspergillus fumigatus which is an endophyte
of J. communis. The maximum yield of fungal desoxy-
podophyllotoxin was in this case in the range of 4±2!g/
100 g dry weight of mycelia and 3±2!g/L of spent broth
(Kusari et al. 2009).
 
 
 
 
OMe
OMeMeO
O
O
O
OH
H
6
N
N
O
O
O
ON
O
OH
N
7a
N
N
O
O
O
HO
OH
N
7b
OHOOH
HO
OHOOH
HO
8
HN
N
H
H
NO
N
O N
O
HOH
O
9
N
O
N
O
10
N
OH
OHHOH
11
O
O
OH
OR
HO
12 R = Me
14 R = H
O
OH13
4 Fungal Diversity (2010) 41:1–16
Examples of naturally occuring topoisomerase I inhibitors
include the cytotoxic plant alkaloid, camptothecin (2) as well
as its semi-synthetic water soluble derivatives irinotecan
(7a) and topotecan (7b). Camptothecin was originally
obtained from Camptotheca acuminata (Nyssaceae), but
occurs also in systematically unrelated plant families such as
Icacinaceae (Nothapodytes foetida, Pyrenacantha klaineana,
Merrilliodendron megacrapum), Apocynaceae (Ervatamia
heyneana), Rubiaceae (Ophiorrhiza pumila, O. mungos), or
Gelsemiaceae (Mostuea brunonis) (Wink 2008). Recently,
camptothecin was identified in cultures of the endophyte
Entrophospora infrequens isolated from Nothapodytes foetida
with a maximum yield of 0.575±0.031 and 4.96±0.73
mg/100 g of dry cell mass in shake flasks and in a bioreactor,
respectively (Puri et al. 2005; Amna et al. 2006). It is
speculated by some authors that the patchy distribution of this
alkaloid was originally caused by endophytes through
infection of the respective plants or gene transfer (Wink
2008). Studies on fungal biogenetic gene clusters responsible
for production of this alkaloid are, however, not available.
Further examples of plant secondary metabolites detected in
endophytic fungi include naphthodianthrones, such as hyper-
icin (8), which are well known constituents of St. John’s wort
(Hypericum perforatum). Hypericum species have been used
for centuries against mild forms of depression and anxiety. An
endophytic fungus from H. perforatum was found to produce
hypericin in culture (Kusari et al. 2008).
The occurrence of ergot alkaloids (9) in different fungal
families such as Clavicipitaceae and Eurotiaceae on one hand
and in the dicotyledonous plant families Convolvulaceae and
Polygalaceae, on the other hand, seemed to be a puzzling
coincidence. Recently it was shown that plant-associated seed-
transmitted epibiotic clavicipitalean fungi that colonize the
adaxial leaf surfaces of certain Convolvulaceae plant species
such as Ipomoea asarifolia, are equipped with genetic material
responsible for ergoline alkaloid biosynthesis (Ahimsa-
Müller et al. 2007; Markert et al. 2008). Interestingly, the
alkaloids were not detectable in the mycelium of leaf
associated fungi, which indicated that a transport system for
translocation of the alkaloids from the epibiotic fungus into
the plant may exist (Markert et al. 2008).
Endophyte-infected grasses are known for their ability to
produce loline alkaloids (10) which exhibit deterrent and
toxic effects towards invertebrate and vertebrate herbivores
and are thus possibly involved in protection of endophyte
infected grasses against herbivores (Schardl et al. 2007).
Loline alkaloids, which resemble simplified pyrrolizidine
alkaloids with potent broad-spectrum insecticidal activity,
were detected in the grass, Festuca pratensis, and in the
root hemiparasitic plant Rhinanthus serotinus (Lehtonen et
al. 2005). It was demonstrated that Neotyphodium uncina-
tum, a common endophyte of F. pratensis, was fully able to
synthesize some of the most common loline alkaloids
(Blankenship et al. 2001), and provided these defense
compounds to the grass and its hemiparasite resulting in an
increased resistance of the host plant against insect
herbivores (Lehtonen et al. 2005). Likewise, the legume
genera Astragalus and Oxytropis are notable for the
production of toxic indolizidine alkaloids, such as swainsonine(11), causing locoweed poisoning in livestock. These
alkaloids are apparently produced by endophytic Embellisia
spp. (Ralphs et al. 2008). A similar example was reported for
the mangrove plant Hibiscus tiliaceus. Chemical examination
of the plant as well as fermentation broth of its endophyte
Phomopsis sp. revealed the presence of oleanane-type
triterpenes, suggesting a possible transfer of the biosynthetic
machinery of the oleanane skeleton during evolution (Li et al.
2006, 2008b).
As shown above many endophytes are apparently able to
synthesize the same natural products that also occur in
plants. It is nevertheless assumed that production of these
respective compounds in planta does not proceed exclu-
sively by endophytes but is rather the consequence of
concomitant plant and fungal biosynthesis (Kusari et al.
2008). It remains an open question whether a horizontal
gene transfer occurred at some time during co-evolution of
plants and endophytes that enables the receiving partner to
perform the same biosynthetic reactions as present in the
donor. An answer to this intriguing question can only be
given after biogenetic gene clusters from host plants and
endophytes have been elucidated.
Detection of known plant constituents in cultivated
endophytic fungi is by no means as rare as initially thought,
but evidence for the presence of known fungal metabolites
in higher plants also exists. For example, the fungal
metabolite alternariol monomethyl ether (also known as
djalonensone) (12), reported from several Alternaria species,
was even described for the first time from a plant source,
Anthocleista djalonensis (Onocha et al. 1995). Further-
more, aureonitol (13), a fungal metabolite known from
Chaetomium species, was isolated from an extract of
Helichrysum aureo-nitens (Bohlmann and Ziesche 1979).
Fungal Diversity (2010) 41:1–16 5
Fungal metabolites including alternariol (14), alternariol
monomethyl ether (12), altenusin (15), macrosporin (16)
and methylalaternin (17) that had been isolated in our
laboratory from endophytic strains of Alternaria and
Ampelomyces , were traced in fractions of their
corresponding host plants Polygonum senegalense and
Urospermum picroides by means of LC/MS (Aly et al.
2008a, b). This analytical technique provides high sensi-
tivity and specificity of detection even for very complex
extracts or fractions. As a future direction of work it will
be of great interest to determine the contribution of fungal
biosynthesis to the secondary metabolite profiles of plants
as this would offer an additional explanation for the patchy
distribution of natural products, including certain alkaloids,
cardiac glycosides and anthraquinones in the plant king-
dom as stated by Wink (2008).
 
 
 
O
OH
OHOMe
18
O
OH
19
HO
MeO
O
OH
20
O OH
HO
21
OHOH
O
OH
OH
O
OH
25
O
OOH
OH
O
24
O
OH
OH
O
OH
26
O
OH
R1
 R1 R2 R3
27 OH Cl H
28 OH H H
29 H H OH
R2
R3
O
O
O
O O
OH
30
O
O
OH
ROH
O
16 R = H
17 R = OH
HO
O
OH
OMe
HO
HO
15
O
OH
OOH
22
O
OH
23
O
OH
OH
Newly reported examples of bioactive compounds
from endophytic fungi
In this part of our review we present an overview on natural
products reported from endophytic fungi during the years 2008
and 2009 that were selected based on their bioactivities in the
following important fields of indication: microbial and
parasitic infections, cancer and neuronal injury or degenera-
tion. These compounds do not match known plant metabolites
but are of interest due to their unique structural features and
biological activities. This overview is intended to be a
continuation of previous reviews covering this field (Tan and
Zou 2001; Strobel and Daisy 2003; Strobel et al. 2004;
Gunatilaka 2006; Zhang et al. 2006; Verma et al. 2009). The
mounting interest in endophytic fungi as sources of drug
leads is clearly reflected by the increasing number of
6 Fungal Diversity (2010) 41:1–16
publications on this subject over the past 10 years (113 CAS
referenced research articles on secondary metabolites from
endophytic fungi in the period of 2008–2009, 69 in 2006–
2007, 36 in 2004–2005, 14 in 2002–2003, and 18 in 2000–
20011). In the last 2 years (2008–2009), research on fungal
endophytes has led to the discovery of more than 100 new
natural products, whereas almost the same number of new
compounds was reported for the period 2000–2007.
Antimicrobial secondary metabolites
Chemical analysis of the culture extract of the endo-
phyte Nodulisporium sp. (Xylariaceae), isolated from the
plant Erica arborea (Ericaceae) from the island of
Gomera (Canary islands), yielded six novel metabolites
including nodulisporins D–F (18–20), (3S,4S,5R)-2,4,
6-trimethyloct-6-ene-3,5-diol (21), 5-hydroxy-2-hydroxy-
methyl-4H-chromen-4-one (22) and 3-(2,3-dihydroxyphe-
noxy)-butanoic acid (23) which were isolated together
with seven known compounds. The antibacterial, fungi-
cidal, and algicidal properties of the six novel substances
were tested in an agar diffusion assay in comparison to
standard antibiotics. All substances showed antifungal
and antialgal activities and 18–20 were also antibacterial.
The strongest zones of inhibition were caused by 20 (Dai
et al. 2009).
O
O
MeO
37
OH
OH
OH
OH
OH
O
OOH
HO
OH
R1
 R1 R2 
31 OH H 
32 Cl Cl
R2 O
O
OH
OMe
R1O
R2
 R1 R2 
34 SO3-Na+ H 
35 SO3-Na+ OH
36 H OHOH
O
MeO
33
H
H
OH
OH
OH
OH
OH
O
O
O OH
R
OMeO
38 R = H
39 R = OH O
O
O OH
R
OMeO
40 R = H
41 R = OH
O
OOH
OH
OH
43
OH
O
O
OOH
OH
42
OH
OH
O
O
O
O
R
OH
OH
44 R = OH
45 R = H
HO
O
OH
46
O
O
O
O
OH
47
1 Inserted inquiry in SciFinder: endophytic+fungi+metabolites.
Fungal Diversity (2010) 41:1–16 7
The endophytic fungus Phomopsis sp. (Valsaceae),
isolated from leaves of Laurus azorica (Lauraceae),
growing on Gomera island, yielded six new metabolites
(24–29). Among the isolated compounds, the new
metabolites cycloepoxytriol B (26) and cycloepoxylac-
tone (24) showed good antibacterial and antifungal
activities against Bacillus megaterium and Microbotryum
violaceum with a radius of zone of inhibition of 5 and
6 mm for 26, 5 and 10 mm for 24, respectively (Hussain
et al. 2009).
Cultures of the fungal endophyte Ampelomyces sp.
(Leptosphaeriaceae), isolated from the medicinal plant
Urospermum picroides (Asteraceae), collected in Egypt,
yielded six new natural products (30–35). The known
compounds 6-O-methylalaternin (36) and altersolanol A
(37), which were also isolated from this endophyte,
displayed antimicrobial activity against the gram positive
pathogens, Staphylococcus aureus, S. epidermidis and
Enterococcus faecalis at minimal inhibitory concentra-
tions (MIC) of 41.7 and 37.2–74.4!M, respectively
(tetracycline: 0.9!M against S. epidermidis; gentamycin:
52.4!M against E. faecalis). Interestingly, 6-O-methyl-
alaternin (36) and the known compound macrosporin were
also identified as constituents of an extract derived from
the host plant U. picroides thereby indicating that the
production of bioactive natural products by the endophyte
proceeds also under in situ conditions within the host (Aly
et al. 2008b).
The endophytic fungus Chalara sp. (strain 6661, order
Helotiales2) was isolated from Artemisia vulgaris (Aster-
aceae) growing along the coast of the Baltic Sea. Chemical
analysis revealed four novel metabolites, named isofusi-
dienol A–D (38–41), besides other known fungal metab-
olites. Compounds 38 and 39 exhibited strong
antibacterial activity against B. subtilis. An inhibition
zone of 23 and 22 mm was caused by 15!g of 38 and 39
on 6-mm filter disks, respectively. Under the same
conditions, 15!g of penicillin G caused an inhibition zone
with a 50 mm diameter. The minimal amount of 38
causing inhibitory effects against this bacterium was
determined to be 0.625!g (on 6-mm filter disks) (Lösgen
et al. 2008).
The fungus Alternariasp. (Pleosporaceae), isolated from
fresh healthy leaves of the Chinese Mangrove plant
Sonneratia alba (Sonneratiaceae), yielded two new com-
pounds named xanalteric acids I (42) and II (43) beside
eleven known metabolites. The two new compounds 42 and
43 exhibited weak antibiotic activity against multidrug-
resistant Staphylococcus aureus with MIC values of
343.40–686.81µM (Kjer et al. 2009).
Recently, chemical investigation of the endophytic
fungus Pestalotiopsis theae (Amphisphaeriaceae), which
was isolated from branches of an unidentified tree near
Jianfeng Mountain, Hainan Province, China, yielded four
new metabolites named pestalotheols A–D (44–47). Among
the new compounds, pestalotheol C (46) displayed inhib-
2 No rank available in NCBI taxonomy browser.
8 Fungal Diversity (2010) 41:1–16
itory effects toward HIV-1LAI replication in C8166 cells
with an EC50 value of 16.1µM (the positive control
indinavir sulfate showed an EC50 value of 8.18µM) (Li et
al. 2008a).
O
O
O
O
OHMeO
OH
OH
MeO
OH
OH
OH
OH
48/49
OH
OOH
MeO
OH
OH
OH
50
H
H
O
OHOH
MeO
OH
OH
OH
51
H
H
52
O O
OMe
OH
O
O
OH
OR3
R1
R2O
 R1 R2 R3
53 H H SO3H
54 H SO3H Me
55 OH H Me
HO
O
OH
OH
HO
HO
56
OH
OH
57
O
HO
OH
O
O
HO
OH
OOH
MeO
58
OH
OMe
59
O
O
HO
HO
Cytotoxic secondary metabolites
From stem tissues of the Moroccan medicinal plant Mentha
pulegium (Lamiacae), the endophytic fungus Stemphylium
globuliferum (Pleosporaceae) was isolated. Extracts of the
fungus, which was grown on solid rice medium, exhibited
considerable cytotoxicity when tested in vitro against L5178Y
lymphoma cells. Chemical investigation yielded five new
secondary metabolites (48–52). An inseparable mixture of the
new atropisomers alterporriol G and H (48/49) exhibited
considerable cytotoxicity against L5178Y lymphoma cells
with an EC50 value of 3.7µM (kahalalide F: 4.3µM, positive
control), whereas the other isolated compounds showed only
moderate activity. All compounds were also tested for protein
kinase inhibitory activity in an assay involving 24 different
human protein kinases. The known compounds 6-O-methyl-
Fungal Diversity (2010) 41:1–16 9
alaternin (36), macrosporin, altersolanol A (37), and the
atropisomers 48 and 49 were the most potent inhibitors,
displaying EC50 values between 1.9–4.0µM toward individual
kinases. Among the 24 different enzymes tested, kinases
Aurora-B and CDK4/CycD1 proved most susceptible toward
the tested compounds (Debbab et al. 2009).
Chromatographic separation of an extract of the endophytic
fungus Alternaria sp. (Pleosporaceae), isolated from the
Egyptian medicinal plant Polygonum senegalense (Polygala-
ceae), yielded 15 natural products, including seven new
compounds (53–59) as well as the known compounds 12,
14 and 15. Compounds 12, 14, 15, 53 and 56 showed
cytotoxic activity against L5178Y lymphoma cells with EC50
values ranging from 6.6–28.7µM. When analyzed in vitro for
their inhibitory potential against 24 different protein kinases,
compounds 12, 14, 15, 53, 55 and 56 inhibited several of
these enzymes (IC50 values 0.6–36.4µM). Interestingly,
compounds 12, 14 and 15 were also identified as constituents
of an extract derived from healthy leaves of the host plant P.
senegalense, thereby indicating that the production of natural
products by the endophyte proceeds also under in situ
conditions within the plant host (Aly et al. 2008a).
O
O
OH
H
HO
O
OH
H
OH
60 61
O
O
OH
H
OH
R2
R1
 R1 R2
62 OH, H H 
63 H2 OH 
64 O H 
65 H2 H
!- "- 
NH
O
OH
OH
66
O
H
H
OH
H
HN
OH
O
O
O
H
67
O
N
O
OH
OH
OH
OH
N
O
O
MeO
OMe
68
Wijeratne et al. (2008) reported five new metabolites
including hydroxymethylcyclozonarone (60), phyllospinar-
one (61), 3"-hydroxytauranin (62), 12-hydroxytauranin
(63), and 3-ketotauranin (64) together with the known
compound tauranin (65) from the endophyte Phyllosticta
spinarum (Botryosphaeriaceae), isolated from Platycladus
orientalis (Cupressaceae) collected in Arizona, USA. The
isolated compounds were evaluated for inhibition of cell
proliferation using a panel of five cancer cell lines. Only 65
showed activity with EC50 values of 4.3, 1.5, 1.8, 3.5 and
2.8µM against NCI-H460 (non small cell lung cancer),
MCF-7 (breast cancer), SF-268 (CNS cancer (glioma)), PC-
3 M (metastatic prostate cancer), and MIA Pa Ca-2
(pancreatic carcinoma), respectively (doxorubicin as posi-
tive control 0.01, 0.07, 0.04 and 1.11µM, respectively). In
order to determine the mechanism(s) responsible for the
10 Fungal Diversity (2010) 41:1–16
antiproliferative activity, tauranin was tested against drug-
sensitive PC-3 M and NIH 3 T3 (mouse fibroblast) cells
where it induced apoptosis in both cell lines.
A new natural product, named phomopsin A (66), together
with the known compound cytochalasin H (67), was isolated
from the endophytic fungus Phomopsis sp. (Valsaceae)
obtained from the bark of an unidentified Mangrove plant
collected in China. Antitumor activities of the isolated
compounds against KB cells (human nasopharyngeal epi-
dermal carcinoma) and multidrug resistant KBv200 cells
were determined. Compound 66 showed moderate cytotox-
icity toward KB and KBv200 cells with IC50 values of 110.7
and 66.4µM, respectively, while compound 67 exhibited
strong cytotoxicity toward KB cells and KBv200 cells with
an IC50 value of less than 2.5µM (Tao et al. 2008b).
Chemical investigation of the culture broth of the
endophytic fungus Eupenicillium sp. (Trichocomaceae),
isolated from the rainforest tree Glochidion ferdinandi
(Euphorbiaceae) collected in Australia, afforded the new
modified dipeptide trichodermamide C (68). Trichoderma-
mide C was shown to display cytotoxicity towards human
colorectal carcinoma cell line HCT116 and human lung
carcinoma cell line A549 with IC50 values of 1.5 and 9.3µM,
respectively (Davis et al. 2008).
Recently, we obtained an endophytic Pestalotiopsis sp.
(Amphisphaeriaceae) from fresh healthy leaf material of
Rhizophora mucronata (Rhizophoraceae) collected in Dong
Zhai Gang-Mangrove Garden on Hainan Island, China.
Chemical investigation of this endophyte yielded five new
cytosporones J–N (69–73), new coumarins, pestalasins A–E
(74–78), a new alkaloid named pestalotiopsoid A (79) (Xu et
al. 2009a), and six new chromones, pestalotiopsones A–F
(80–85) (Xu et al. 2009b). Among the isolated compounds
only pestalotiopsone F (85) exhibited moderate cytotoxicity,
with an EC50 value of 26.89!M, when tested against the
murine cancer cell line L5178Y (Xu et al. 2009b).
O
OH
OH
O
R
OH
69 R =
70 R =
71 R =
O
O
O
O
OH
OHR1
OH
72 R1 =
73 R1 =
OR2O
O
R2 = Me
R2 = Me
O
R3R1
R2
OH
O
74 R1 = OMe R2 = OMe R3 =
75 R1 = OMe R2 = OMe
76 R1 = OMe R2 = OMe
77 R1 = OMe R2 = OH
78 R1 = OH R2 = OMe
 R3 =
 R3 =
 R3 =
 R3 = CH2OH
OH
OH
OH
OH
OH
OH
N OO
HO
OO
79
Antiparasitic secondary metabolites
A study aimed at the discovery of novel antiparasitic agents
yielded two new natural products 86 and 87 from an isolate
of Edenia sp. (Pleosporaceae), obtained from mature leaves
of Petrea volubilis (Verbenaceae) collected in Coiba
National Park, Panama, besides other known compounds
including preussomerin EG1, palmarumycin CP2 and CJ-
Fungal Diversity (2010) 41:1–16 11
12,371 (Martìnez-Luis et al. 2008). The new metabolites
palmarumycin CP17 (86) and palmarumycin CP18 (87)
caused significant inhibition of the growth of Leishmania
donovani in the amastigote form, with IC50 values of 1.34
and 0.62µM, respectively (amphotericin B: IC50 0.09!M,
positive control). These compounds were inactive, how-
ever, when tested against Plasmodium falciparum or
Trypanasoma cruzi at a concentration of 10µg/mL, indicat-
ing that they have selective activity against Leishmaniaparasites. The two new metabolites showed also weak
cytotoxicity toward Vero cells (African green monkey kidney
epithelial cells), with IC50 values of 174 and 152µM,
respectively. The therapeutic window of these compounds,
however, is quite significant since their antileishmanial
activity is 130 or 245 times stronger than their cytotoxic
properties (Martìnez-Luis et al. 2008).
Altenusin (15), a biphenyl fungal metabolite, was
isolated from Alternaria sp. (Pleosporaceae) endophytic
in Trixis vauthieri (Asteraceae) collected in Brazil, a plant
known to contain trypanocidal compounds. The com-
pound showed trypanothione reductase (TR) inhibitory
activity with an IC50 value of 4.3 ± 0.3!M. This
compound is the first one in its class of metabolites for
which TR inhibitory activity is demonstrated, thereby
opening up new perspectives for the design of more
effective derivatives that could serve as drug leads for new
chemotherapeutic agents to treat trypanosomiasis and
leishmaniasis (Cota et al. 2008).
O
O
80 R1 = R2 =
81 R1 = R2 =
82 R1 = R2 =
83 R1 = R2 =
84 R1 = R2 =
O
O
OH
O
O
O
O R1
OR2
HO
85 R1 = R2 =
OH
OH
O
O
O
O
OO
OH
OH
O
86
OO
O
O
OH
87
HN NNH
O
O
88
12 Fungal Diversity (2010) 41:1–16
Neuroprotective secondary metabolites
A study designed to discover novel neuroprotective agents
yielded chrysogenamide A (88), a new member of the
macfortine group of alkaloids, from Penicillium chrysoge-
num (Trichocomaceae), an endophytic fungus associated
with Cistanche deserticola (Orobanchaceae) collected from
Inner Mongolia, in northwest China. The new compound
exhibited neurocyte protection against oxidative stress-
induced cell death in SH-SY5Y cells. Chrysogenamide A
inhibited cell death induced by hydrogen peroxide by
improving cell viability by 59.6% at a concentration of 1!
10!4µM (Lin et al. 2008).
Possible factors influencing natural product production
by cultured endophytes
Recent gene sequencing studies of biogenetic gene clusters
involved in fungal secondary metabolism suggest that the
potential number of expected natural products exceeds by
far the one known today (Szewczyk et al. 2008; Pettit 2009;
Schroeckh et al. 2009). This discrepancy is not so much
due to insensitive analytical methods that fail to pick up
minor metabolites but more to the fact that certain
biogenetic fungal gene clusters are apparently not expressed
under the usual laboratory culture conditions (Szewczyk et
al. 2008). For example, the gene cluster for lolitrem
biosynthesis in Neotyphodium lolii, a mutualistic endophyte
of perennial ryegrass, was found to be highly expressed in
planta but expression was very low or undetectable in
culture-grown fungal mycelia, strongly suggesting that
plant signaling is required to induce expression (Young et
al. 2006). In this context it is of interest that homoserine
and asparagine, abundant amino acids in pea seedlings, act
as host signals to activate expression of a pathogenesis-
related gene in virulent strains of Nectria haematococca
which is only expressed in planta (Yang et al. 2005). Lack
of such host stimuli in culture media may explain why
production of natural products by a nascent endophyte
isolate is often severely attenuated through subculturing (Li
et al. 1998).
Production of secondary constituents by endophytes may
furthermore be influenced by developmental stages of the
fungal culture. Thus, a number of developmental pathways
mediated by G-protein signaling, including vegetative
growth and sexual/asexual development, were found to
regulate also secondary metabolite cluster expression (Tag
et al. 2000; Shwab and Keller 2008). For example,
signaling by the " subunit of a heterotrimeric G-protein,
FadA, was found to promote vegetative growth and repress
sexual/asexual development, sterigmatocystin production in
Aspergillus nidulans, as well as aflatoxin production in A.
flavus and A. parasiticus (Calvo et al. 2002). Oxylipins,
hormone-like signaling molecules mediating the balance of
sexual to asexual spore production in Aspergilli, were
reported to contribute to regulation of secondary metabolite
biosynthesis (Tsitsigiannis and Keller 2007). Small
hormone-like molecules, such as conidiogenone which
induces conidiation, may also induce secondary metabolism
(Roncal et al. 2002). A more general model of secondary
metabolite regulation was recently identified when the
nuclear protein, LaeA, that is required for the transcription
of several secondary metabolite gene clusters in Aspergillus
nidulans, was discovered. Deletion of LaeA was found to
block the expression of sterigmatocystin, penicillin, and
lovastatin gene clusters. On the other hand, overexpression
of LaeA triggered increased penicillin and lovastatin
production (Bok and Keller 2004). In addition to
pathway-specific stimulators and regulators, fungal second-
ary metabolite production is also influenced by general
environmental factors, such as carbon and nitrogen sources,
temperature, light, and pH. It is likely that fungi are able to
regulate the energetically costly process of secondary
metabolite production according to environmental condi-
tions and specific needs (e.g. competitive forces, host plant
interaction and communication). In this context it should be
pointed out that most studies on natural products from
endophytes have so far been conducted using axenically
growing cultures whereas in planta different fungal strains
always coexist e.g. in leaves of a given host plant.
Competition among different fungi or among endophytic
fungi and endophytic bacteria is likely to be another major
factor triggering natural product accumulation (Cueto et al.
2001; Ho et al. 2003; Oh et al. 2007; Glauser et al. 2009;
Pettit 2009; Schroeckh et al. 2009). Future attempts to
optimize culture conditions should take this aspect into
consideration e.g. by performing co-cultivation experiments
or by adding cell lysates (from other fungi or from bacteria)
as elicitors of secondary metabolism to cultivation media.
Conclusion
The significance of fungi as unconventional sources of
bioactive compounds was first realized by the discovery of
penicillin from Penicillium notatum, almost 80 years ago
(1928), by Alexander Fleming. In the continuous search for
novel drug sources, endophytic fungi have proven to be a
promising, largely untapped reservoir of natural products,
with great chemical diversity. These compounds have been
optimized by evolutionary, ecological and environmental
factors yielding effective bioactive agents. Despite intensive
research, different aspects of the relationship between
endophytes and their hosts are yet unclear. In some cases
endophytes were found to produce medicinally important
Fungal Diversity (2010) 41:1–16 13
natural products originally known exclusively from their
host plants, thus raising the prospect of using such
organisms as alternative and sustainable sources of these
substances. However, the feasibility of industrial production
of such substances by endophytic fungal sources has still to
be proven.
Literature surveys showed that the fungal genus Pesta-
lotiopsis is of the most productive (30% of the 67 new
compounds reported in this review were isolated from
Pestalotiopsis). The genus is characterized by its extensive
distribution and the wide genetic and biological diversity of
its members. Pestalotiopsis species occur on a wide range
of substrata, and many are saprobes, while others are either
pathogenic or endophytic. It has been suggested that this
vast variability may have arisen by mutation, genetic
crossing, or by other yet uncorroborated mechanisms, such
as genetic exchange with host plants, making this genus a
“microbial factory” of bioactive natural products with
potential use in agriculture and medicine (Strobel 2002).
For this reason, Pestalotiopsis species have been subject of
investigation in our research program on secondary
metabolism of endophytic fungi from plants.
Recent advancements in molecular biologyof fungal
secondary metabolism offer a better insight into how
biogenetic gene clusters are regulated and whether their
expression is affected by environmental changes and
culture conditions. A deeper understanding of the host-
endophyte relationship at the molecular and genetic levels
may help to induce and optimize secondary metabolite
production under laboratory conditions to yield bioactive
natural products with significant medicinal and agricultural
potential.
Acknowledgement P.P. wishes to thank BMBF for support.
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