Endophytic fungi-a source of novel biologically active secondary metabolites

Prévia do material em texto

996
Barbara SCHULZ1†, Christine BOYLE1, Siegfried DRAEGER1, Anne-Katrin RO> MMERT1
and Karsten KROHN2
" Institute of Microbiology, Technical University of Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
#Fachbereich Chemie, UniversitaX t Paderborn, Warburger Strasse 100, D-33095 Paderborn, Germany.
E-mail : b.schulz!tu-bs.de
Received 6 October 2001; accepted 28 June 2002.
In the continual search by both pharmaceutical and agricultural industries for new products, natural selection has been
found to be superior to combinatorial chemistry for discovering novel substances that have the potential to be
developed into new industrial products. Since natural products are adapted to a specific function in nature, the search
for novel secondary metabolites should concentrate on organisms that inhabit novel biotopes. Endophytic fungi inhabit
such a biotope. In the course of the last 12 years, we have isolatedC 6500 endophytic fungi from herbaceous plants
and trees, screened them for biological activities, and have isolated and determined the structures of the biologically
active compounds. Correlations were found between biological activity and biotope, e.g. a higher proportion of the
fungal endophytes, in contrast to the soil isolates, inhibited at least one of the test organisms for antialgal and
herbicidal activities. The substances isolated originated from different biosynthetic pathways: isoprenoid, polyketide,
amino acid derivatives, and belonged to diverse structural groups: terpenoids, steroids, xanthones, chinones, phenols,
isocumarines, benzopyranones, tetralones, cytochalasines, and enniatines. The potential role of the endophyte and its
biologically active metabolites in its association with its host has been investigated. The fungal endophytes possess the
exoenzymes necessary to colonize their hosts and they grow well in the apoplastic washing fluid of the host. When the
roots of larch are colonized, the association with the host may be mutualistic, improving growth of the host and
supplying the mycobiont with enough nourishment to extensively colonize the host’s roots. The concentrations of some
plant defence metabolites are lower than in the control when the host is infected with a pathogen than with an
endophyte. We hypothesize that the interaction fungal endophyte–plant host is characterized by a finely tuned
equilibrium between fungal virulence and plant defence. If this balance is disturbed by either a decrease in plant
defence or an increase in fungal virulence, disease develops. Not only must the endophyte synthesize metabolites to
compete first with epiphytes and then with pathogens in order to colonize the host, but presumably also to regulate
metabolism of the host in their delicately balanced association. The utilization of a biotope such as that of the fungal
endophyte is one aspect of intelligent screening, another very important one is the taxonomy of the fungus in order to
avoid redundant structural isolations. It is not a random walk through a random forest. Many groups of fungi in
different biotopes are waiting to be exploited.
INTRODUCTION
The search for new products for the pharmaceutical
and agrochemical industries is an on-going process that
requires continual optimization (Dreyfuss & Chapela
1994). Previously, the screening of 10000 natural
products resulted in one commercial product. In the
* Paper presented at the British Mycological Society symposium
on Fungal Bioactive Compounds, held at the University of Wales
Swansea on 22–27 April 2001.
† Corresponding author.
advent of combinatorial chemistry, this relationship
changed. Presently, the screening of 100000 structures
day−" from combinatorial chemistry together with the
natural products screened yields less than one com-
mercial product year−" (F. Hansske, pers. comm.). Its
development takes approximately 12 yr and costs
C $350 M (Gloer 1997). Considering that 6 out of 20
of the most commonly prescribed medications are of
fungal origin (Gloer 1997) and onlyC 5% of the fungi
have been described (Hawksworth 1991, 2001), fungi
offer an enormous potential for new products.
Mycol. Res. 106 (9) : 996–1004 (September 2002). # The British Mycological Society
DOI: 10.1017}S0953756202006342 Printed in the United Kingdom.
Review
Endophytic fungi : a source of novel biologically active
secondary metabolites*
B. Schulz and others 997
Until recently, the search for new fungal metabolites
concentrated mainly on the random screening of
isolates. In optimizing the search for new bioactive
secondary metabolites, it is relevant to consider that :
(1) the secondary metabolites a fungus synthesizes may
correspond with its respective ecological niche, e.g. the
mycotoxins of plant pathogens (Gloer 1997) ; and (2)
that metabolic interactions may enhance the synthesis
of secondary metabolites. Thus, the fungi screened
should originate from biotopes from which fungi have
not been previously isolated for biochemical purposes
and they should have metabolic interactions with their
environment. This is an example of intelligent screening
and is a strategy for exploiting the untapped potential
for secondary metabolites that fungi offer.
Endophytic fungi are one source for intelligent
screening and fulfill both criteria. They growwithin their
plant hosts without causing apparent disease symptoms
(Petrini 1991, Wilson 1995) and growth in this habitat
involves continual metabolic interaction between fun-
gus and host. Additionally, in comparison to fungal
plant pathogens and fungal soil isolates, relatively few
secondary metabolites have been isolated from endo-
phytic fungi (Tan & Zou 2001). Consequently, we iso-
lated C 6500 endophytic fungi from herbaceous plants
and trees as well as from algal thalli and screened them
for their biological activities and chemical profiles.
MATERIAL AND METHODS
Organisms
Fungal endophytes were isolated from all organs of
numerous asymptomatic herbaceous plants, trees, and
marine algal thalli (Fucus vesiculosus, F. serratus, F.
spiralis., Laminaria sp., Ceramium sp., Ascophyllum
nodosum, Halarachnion ligulatum, Plumaria elegans,
Enteromorpha sp.) following optimization of surface
sterilization with 70% ethanol and sodium hypo-
chlorite. The plants were primarily from Lower Saxony,
Germany (5900 isolates) but some originated from
Costa Rica (180 isolates) and Mallorca (120 isolates).
The marine algae were from the North and Baltic Seas
(C 300 isolates). Surface sterilization was optimized
according to Schulz et al. (1998). The isolates were
determined and the taxon compared with those listed in
data banks (Turner 1971, Turner & Aldridge 1983,
Chapman & Hall 2000) for known secondary metabo-
lites to avoid redundant isolation and determination of
structures, since fungi of a given taxon often produce the
same metabolites (Frisvad, Bridge & Arora 1998). The
C 2800 fungal soil isolates originated from numerous
countries (Schulz et al. 1999).
The endophytes tested for the synthesis of exoen-
zymes were Fusarium spp. 1 & 2 from Hordeum vulgare
(barley), Fusarium sp. 3 and Alternaria sp. from
Phaseolus vulgaris (bean), Phialophora sp. and Crypto-
sporiopsis sp. from Larix decidua (larch), Coniothyrium
palmarum and Phomopsis sp. from Lamium purpureum,
and Geniculosporium sp. from Teucrium scorodonia.
Modes of penetration and colonization were studied
in bean and barley using the three Fusarium isolates and
the pathogen Drechslera sp. (Boyle et al. 2001).
Screening and chemical profiling
The isolates were tested for fungicidal, antibacterial,
antialgal and herbicidal activities as described pre-
viously (Schulz et al. 1995). Thin-layer chromatography
and bioautogramms were conducted to obtain meta-
bolic profiles of the culture extracts and used to
optimize the conditions of culture (Schulz et al. 1995).
Media
Media used for the scale-up culturing of the isolates
were semi-solid biomaltand malt extract soya media
(Ho$ ller et al. 2000). Growth of selected Fusarium
isolates in the apoplastic washing fluid (AWF) was
compared to that in the synthetic media SNA and
SNA5 and biomalt (Boyle et al. 2001). Concentrations
of sugars in SNA are comparable to those in AWF and
those in SNA5 to those in the apoplastic fluid.
Influence of endophytic colonization of the roots on the
growth of larch
Sterilized larch seedlings were cultivated axenically in a
liquid medium in Lecaton2 expanded clay and their
roots inoculated at the age of 3 months with either an
endophyte, Cryptosporiopsis sp. or Phialophora sp., or
with a pathogen, Heterobasidion annosum, according
to Schulz et al. (1999). Growth was evaluated as a
subjective estimation in cm of the increase or decrease
in height from the last period of evaluation.
Isolation and structural analyses of secondary
metabolites
Isolation of the bioactive metabolites and their struc-
tural analyses were according to Krohn et al. (e.g.
1992a, 1994b, 1995).
Isolation of apoplastic washing fluid
Apoplastic washing fluid (AWF) was extracted ac-
cording to a modified version of the method employed
by Mu$ hling & Sattelmacher (1995) from barley and
bean plants cultivated in growth chambers (Boyle et al.
2001).
Synthesis of exoenzymes
Selected endophytic isolates were tested for their
capabilities to produce the exoenzymes necessary for
penetrating and colonizing their hosts. These included
protease and amylase (Boyle et al. 2001), phenol oxidase
(Bavendamm 1928), lipase and cellulase (Carroll &
Petrini 1983), xylanase according to Carroll & Petrini
Secondary metabolites from endophytic fungi 998
(1983) but with xylan (Sigma) as a substrate, and pectin
lyase (Dingle, Reid & Solomons 1953, Obi 1981).
RESULTS AND DISCUSSION
Secondary metabolites
A higher proportion of the endophytic fungi exhibited
biological activity than the soil isolates did. Whereas
83% of the algal isolates and 80% of the endophytic
fungi from plants inhibited at least one of the test
plant endophytes algal endophytes soil isolates
all inhibitions
algicidal (herbicidal)
fungicidal
antibacterial
0
20
40
60
80
100
%
 b
io
lo
gi
ca
lly
 a
ct
iv
e 
st
ra
in
s
Fig. 1. Proportion of biologically active isolates from different sources (healthy plants, algae, soils) tested for antibacterial,
fungicidal and algicidal (herbicidal) activities. ‘All inhibitions ’ designates the proportion of isolates active in at least one
test for biological activity.
all inhibitions algicidalfungicidal antibacterial
0
20
40
60
80
100
%
 b
io
lo
gi
ca
lly
 a
ct
iv
e 
st
ra
in
s
Lower Saxony Costa Rica Mallorca
Fig. 2. Proportion of biologically active isolates from different sources (endophytes from healthy plants from Lower
Saxony, Costa Rica, and Mallorca) tested for antibacterial, fungicidal and algicidal (herbicidal) activities. ‘All inhibitions ’
designates the proportion of isolates active in at least one test for biological activity.
organisms for antibacterial, fungicidal, algicidal or
herbicidal activities, only 64% of those from soils did
(Fig. 1). The differences in the proportions of activities
from the various biotopes, e.g. plant vs. soil, is striking.
Of the endophytes isolated from plants, 43% inhibited
the alga Chlorella fusca, and the phanerogam test org-
anisms, Lemna minor and Lepidium sativum, whereas
only 27% of the phytopathogenic isolates, 25% of the
epiphytes, 13% of the algal and 18% of the soil isolates
did (Schulz et al. 1999). The highest proportion of anti-
B. Schulz and others 999
perylene derivatives palmarumycins dimeric anthrone phenols benzopyroanone
mycorrhizin steroids isocumarines xanthones quinones
furandiones preussomarins terpenoids enniatines cytochalasines
Fig. 3. Structural diversity of metabolites isolated from endophytic fungi.
bacterial activity was found among the isolates from
algae, 55% inhibiting one or both of the test organisms.
An analysis of the proportions of biologically active
endophytic isolates according to geographical biotope
shows that 87% of the isolates from Lower Saxony were
inhibitory, in contrast to 52% of those from Costa Rica
(Fig. 2). These results suggest that the metabolites a
fungus produces may vary with the biotope in which it
grows and to which it is adapted. Similar results have
been obtained from fungi isolated from other biotopes
(Gloer 1997). For example, Dreyfuss & Chapela (1994)
found that the production of cyclosporin A, enchino-
candin B, papulacandins and verrucarins varied with
both habitat and substrate and Gloer (1997) demon-
strated that various dung isolates all produced mixtures
of antifungal peptides.
Tan & Zou (2001) recently reviewed the diversity of
metabolites that have been isolated from endophytic
fungi emphasizing their potential ecological role. These
secondary metabolites of endophytes are synthesized
via various metabolic pathways (Tkacz 2000, Tan &
Zou 2001), e.g. polyketide, isoprenoid, amino acid deri-
vation. Those isolated in our cooperations belong to
diverse structural groups (Fig. 3), i.e. steroids, xan-
thones, phenols, isocumarines, perylene derivatives,
quinones, furandiones, terpenoids, depsipeptides and
cytochalasines (Krohn et al. 1992a, b, 1994a–c, 1996,
1997a–c, 1999a, b, 2001a, b, Ko$ nig et al. 1999, John
et al. 1999). Some of them represent novel structural
groups, for example the palmarumycins (Krohn et al.
1997a, b) and a new benzopyroanone (Krohn et al.
2002).
The industrial partners of scientists screening for
novel biologically active secondary metabolites are both
interested in previously unknown activities for known
metabolites (Anke & Erkel 2002), and in attaining a
high proportion of novel structures from the culture
extracts. A comparison of 135 isolated metabolites
whose structures were determined shows that the pro-
portion of novel structures produced by endophytes
(51%) is considerably higher (Fig. 4) than that produced
by soil isolates (38%). Since structural determination is
very time consuming, finding a source of organisms in
which the proportion of novel structures determined
is high means that this is an interesting group of fungi
for further investigations. But why are endophytes so
creative with respect to novel metabolites? On the one
hand, this group of organisms has not been as exten-
sively studied as, for example, the plant pathogenic
fungi have, meaning that the metabolites isolated are
less likely to be known structures. This was to be
expected according to Dreyfuss & Chapela (1994) and
Gloer (1997). On the other hand, the metabolic
interactions of the endophyte with its host may favour
the synthesis of biologically active secondary metabo-
lites.
Secondary metabolites from endophytic fungi 1000
38% new structures
62% known structures
51% new structures
49% known structures
endophytes
soil isolates
Fig. 4. Proportion of novel metabolite structures from soil
isolates and endophytes.
In the search for groups of microorganisms that are
good producers of biologically active metabolites, it
can be concluded:
(1) Endophytic fungi are a good source of novel
secondary metabolites.
(2) Screening is not a random walk through a
ramdom forest. The biological activities and thus the
metabolites produced are associated with the respective
biotope and}or host.
Table 1. Exoenzyme synthesis of endophytic fungi in vitro.
Isolate Protease Amylase
Phenol
oxidase Lipase Cellulase Xylanase
Pectin
lyase
Fusarium sp. 1 ­ ­ ® ­ ­ ­ ­
Fusarium sp. 2 ® ­ ­ ­ ­ ­ ®
Fusarium sp. 3 ® ­ ­ ­ ­ ­ ®
Alternaria sp. ® ­ ­ ­ ­ ­ ®
Cryptosporiopsis sp. ­ ­ ­ nt ® ® ®
Phialophora sp. ® ­ ­ ­ nt nt ®
Coniothyrium palmarum ­ ­ ­ ­ ­ ­ ®
Geniculosporium sp. ­ ® ® ­ ­ ® ®
Phomopsis sp. ­ ® ® ­ ­ ­ ­
nt, not tested; ­, present ; ®, absent.
(3) Thus, if you are looking for water go to the
stream, and consequently, if you are lookingfor novel
secondary metabolites, isolate the fungi from a biotope
that requires the desired metabolic activity.
Interaction of endophyte and host
The following questions arise : Why do such a high
proportion of endophytes produce biologically active
secondary metabolites and what role do these secondary
metabolites play in the plant–fungal interaction? How
do fungal endophytes differ from pathogenic fungi?
Why does endophytic colonization not lead to disease?
Although the enzymes produced varied from isolate
to isolate, the endophytic fungi tested all synthesized in
vitro the enzymes necessary for penetrating and
colonizing their plant hosts (Table 1; cfr Agrios 1997).
Penetration, colonization and growth were monitored
microscopically, revealing differences between endo-
phytes and pathogens. Endophytic penetration of bean
and barley was via the stomata and along the anticlinal
epidermal cells ; the pathogen, in contrast, was also able
to penetrate directly through the cell wall. Colonization
of the shoots of bean and barley was limited, localized
and intercellular, that of the pathogen also intracellular
(Boyle et al. 2001). Growth of endophytes and patho-
gens within the roots of larch and barley was extensive,
systemic and both inter- and intracellular (Schulz et al.
1999). These histological results are in agreement with
most of those reported for other endophyte–host inter-
actions. Whereas colonization of the above-ground
organs by the non-clavicipitaceous endophytes is con-
sidered to be primarily local and intercellular (Stone
et al. 1994, Carroll 1995), that of the root endophytes
is usually extensive and systemic and may be inter- and}
or intracellular, e.g. endo- and ectomycorrhizal fungi
(Allen 1992), Penicillium sp. (Capellano et al. 1987),
dark-septate endophytes (Jumpponen & Trappe 1998),
Piriformospora indica (Varma et al. 2000), Fusarium
spp. (Kuldau & Yates 2000).
If fungal endophytes colonize the shoot intercellu-
larly, then they should be able to use the apoplastic
fluid as a nutrient source. An endophytic isolate,
Fusarium sp. 1, was cultured in apoplastic washing fluid
(AWF) of bean, the synthetic medium SNA with sugar
B. Schulz and others 1001
8 16 32 48
Days post inoculation
0
0.5
1
1.5
gr
ow
th
(e
va
lu
at
io
n 
po
in
ts
)
control
Cryptosporiopsis
Phialophora
Heterobasidion
Fig. 5. Growth of larch seedlings cultivated for three months axenically in a synthetic medium in expanded clay and
inoculated at day 0 with either an endophyte, Cryptosporiopsis sp. or Phialophora sp., or with the pathogen Heterobasidion
annosum. Growth was evaluated as a subjective estimation in cm of the increase or decrease in height from the last period
of evaluation. n¯ 29–55; *¯P% 0±05, significant in comparison to the control according to Kruskal–Wallis ANOVA on
ranks.
concentrations as in the AWF, the synthetic medium
with sugars concentrated as in the apoplastic fluid
(SNA5) and in biomalt medium. Growth of the
endophyte was significantly better in AWF than in any
of the other media, reaching a dry weight of 16±5 mg in
AWF in contrast to 2±2 mg in biomalt, 3 mg in SNA
and 8±4 mg in SNA5 (Boyle et al. 2001), suggesting an
adaptation of the endophyte to growth in the apoplast.
Not only the grass endophytes of the Balansiaceae
have been reported to interact mutualistically with their
hosts (e.g. Carroll 1988). The ‘non-grass ’ endophytes
may also convey to the host disease protection (Villich,
Dolfen & Sikora 1998, Redman, Ranson & Rodriguez
1999, Redman, Dunigan & Rodriguez 2001), produce
secondary metabolites antagonistic against pathogenic
competitors of the host (Noble et al. 1991, Calhoun et
al. 1992, Schulz et al. 1995, Liu et al. 2001), be involved
in the death of insect predators (Azevedo et al. 2000;
Anke & Sterner 2002) or improve growth of the host,
by e.g. dark-septate endophytes (Jumpponen 2001),
Piriformospora indica (Varma et al. 2000), or mycor-
rhizal fungi (Allen 1992). In our experiments, we found
that colonization of the roots of axenically cultured
larch with either the endophyte Cryptosporiopsis sp. or
Phialophora sp. resulted in a significant increase in
plant growth, both in comparison to the control and to
seedlings infected with the pathogen Heterobasidion
annosum (Fig. 5). The interaction was not only beneficial
for the host, but provided enough nourishment for the
endophyte to extensively colonize the host’s roots and
potentially for growth in the rhizosphere, which in turn
could improve the host’s mineral supply.
No improvement of growth was achieved when the
above-ground organs of plants were inoculated, pre-
sumably because these infections were localized in
contrast to the systemic infections of the roots (Boyle et
al. 2001).
A pathogen surmounts the plant defence system, e.g.
by reducing the concentrations of phenolic defence
metabolites (Agrios 1997). In contrast, when the roots
were colonized by an endophyte, the concentrations of
these metabolites were as in the control or even higher
(Schulz et al. 1999). For example, when the roots of
larch were inoculated with Cryptosporiopsis sp., the
concentrations of soluble proanthocyanidins increased
in comparison to the control ; when infected with the
pathogen Heterobasidium annosum they decreased. In
barley roots, the increase in the concentrations of N-4-
coumaroylputrescine, N-4-coumaroylagmatine and 4-
coumaric acid was less when infected by the pathogen
Drechslera sp. than in the controls or when colonized
endophytically by Fusarium sp. (Schulz et al. 1999).
Not only the plant can exhibit a defence reaction
towards the fungus, fungi can also be aggressive towards
their hosts. There are several facets of fungal virulence
(Agrios 1997). One is the secretion of exoenzymes to
colonize a plant host, and another is mycotoxins. Costa
Pinto et al. (2000) found that colonization of the shoots
of maize and banana with the endophytes Fusarium
moniliforme and Colletotrichum musae, respectively,
inhibited the photosynthetic capacity of the host plants.
These crop plants remained asymptomatic. The authors
suggest that the inhibition is due to toxins produced by
the endophytes. We observed a similar effect. In vitro,
the endophytes produced a high proportion of herbi-
cidal and algicidal secondary metabolites (Peters,
Dammeyer & Schulz 1998, Schulz et al. 1999). Culture
extracts of a number of these endophytes inhibit
photosynthesis of the alga, Chlorella fusca (Peters &
Schulz, unpubl.).
Summarizing the nature of the interaction of fungal
endophytes with plant hosts studied in these investiga-
tions, it can be said that :
(1) Fungal endophytes produced the enzymes necess-
ary to penetrate and colonize their hosts, colonizing the
above-ground organs locally and intercellularly, the
roots in contrast extensively, systemically, and inter-
and intracellularly.
(2) They grew well using only the apoplastic washing
fluid as a growth medium.
Secondary metabolites from endophytic fungi 1002
Fungal
virulence
Plant
defence
Endophytic interaction
Balanced antagonism
Pathogenic interaction
Fig. 6. Balanced antagonism between fungal virulence and plant defence.
(3) The association of fungal endophyte and plant
host may be mutualistic.
(4) Pathogenic infection resulted in lower concen-
trations of phenolic defence metabolites than endo-
phytic colonization did.
(5) Some biologically active fungal metabolites inhibit
photosynthesis of the host plant.
The status of the ‘endophyte ’ must be regulated by
several well-evolved steps at different physiological
levels. We therefore hypothesize that the fungal endo-
phyte–plant host interaction is characterized by a finely
tuned equilibrium between fungal virulence and plant
defence (Fig. 6). If this balance is disturbed by either a
decrease in plant defence or an increase in fungal viru-
lence, disease develops. Not only must the endophytesynthesize metabolites to compete first with epiphytes
and then with pathogens in order to colonize the host,
but presumably also to regulate metabolism of the host
in their delicately balanced association.
The utilization of a biotope such as that of the fungal
endophyte is one aspect of intelligent screening, another
very important one being the taxonomy of the fungus in
order to avoid redundant structural isolations. It is not
a random walk through a random forest. Many groups
of fungi in different biotopes are waiting to be exploited.
ACKNOWLEDGEMENTS
The expert technical assistance of Qunxiu Hu, Gudrun Schmid and
Inga Hilbrich is greatfully acknowledged. We thank Alga Zuccaro for
providing us with fungal strains from algae, BASF and the Bundes-
ministerium fu$ r Bildung, Wissenschaft, Forschung und Technologie,
AnalytiCon Discovery, and the Deutsche Forschungsgemeinschaft
for financial support.
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Corresponding Editors: T. M. Butt & D. L. Hawksworth