the endophyte-host interaction: a balanced antagonism?

1275

BARBARA SCHULZ1, ANNE-KATRIN RO$ MMERT1, ULRIKE DAMMANN1, HANS-JU$ RGEN AUST1

AND DIETER STRACK2

" Institut fuX r Mikrobiologie, Technische UniversitaX t Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany

# Institut fuX r Pflanzenbiochemie, Weinberg 3, D-06109 Halle (Saale), Germany

Since secondary metabolites are involved in fungal-host interactions, those of endophytes and their hosts were studied to try to

understand why endophytic infections remain symptomless. A screening of fungal isolates for biologically active secondary

metabolites (antibacterial, antifungal, herbicidal) showed that the proportion of endophytic isolates that produced herbicidally active

substances was three times that of the soil isolates and twice that of the phytopathogenic fungi. As markers for the plant defence

reaction, the concentrations of certain phenolic metabolites were chosen. Those that differed in concentration were higher in the

roots of plants infected with an endophyte than in those infected with a pathogen. The results presented here were regarded

together with previous studies on other aspects of the plant defence response using dual cultures of plant host calli and endophytes,

and of cell suspension cultures following endophytic as compared to pathogenic elicitation. The following hypothesis was

developed : both the pathogen-host and the endophyte-host interactions involve constant mutual antagonisms at least in part based

on the secondary metabolites the partners produce. Whereas the pathogen-host interaction is imbalanced and results in disease, that

of the endophyte and its host is a balanced antagonism.

Fungal endophytes infect their hosts without causing visible

disease symptoms and have been isolated from almost every

host thus far studied (Petrini, 1991 ; Schulz et al., 1993 ; Schulz

et al., 1998). In our investigations we have concentrated on the

non-clavicipitaceous endophytes. These endophytes primarily

belong to the Ascomycetes and the related mitosporic fungi

(Bills, 1996). A limited amount of cytological work has been

done showing that the infections of these endophytes in host

plants may be inter- or intracellular and are often localized in

single cells (Stone, 1988 ; Suske & Acker, 1989 ; Cabral, Stone

& Carroll, 1993). Additionally, there have been several reports

that endophytic fungi sporulate after death of the host tissue,

i.e. essentially as saprotrophs (e.g. Stone, 1987), but there is

also some evidence that they may be weak pathogens (Kehr,

1992 ; Kowalski & Kehr, 1992). Carroll (1988) postulated that

certain endophytes enter into mutualistic relationships with

their hosts. Supporting this hypothesis, Wulf (1990) found

that the endophyte Diplodina acerina was apparently involved

in death of the gall wasp in leaves of Acer pseudoplatanus. Thus,

the status ‘ endophyte ’ describes an asymptomatic infection at

one moment without specifying the role of the fungus in the

host, nor its development at a later period (Petrini, 1991 ;

Schulz et al., 1998).

Although it has long been known that fungal secondary

metabolites are crucial to the pathogenicity of many fungi

(Harborne, 1993 ; Agrios, 1997), only little experimental work

has been done to study the role of secondary metabolites in

the endophyte-host interaction. Investigations have primarily

concentrated on isolating secondary metabolites and charac-

terizing their biological activity and the potential significance

of this activity for the host. Whereas, in the interactions of the

clavicipitaceous endophytes with their hosts, the mutualistic

role of alkaloids in conferring resistance to herbivory has been

well studied (Leuchtmann, 1992), there have been no reports

on the role these or other metabolites may play in the fungal-

plant interaction. Not only in the endophyte-host symbioses

of clavicipitaceous fungi, but also in those of the non-

clavicipitaceous endophytes with their hosts, secondary

metabolites may be a contribution of the endophytic partner

to a mutualistic relationship. For example, endophytic fungi

from conifer needles were found to produce metabolites toxic

to the spruce budworm (Calhoun et al., 1992). Endophytic

isolates of Pezicula were shown to produce fungicidally active

metabolites that are toxic to pathogens of their hosts (Schulz

et al., 1995).

Additionally, there have been only few investigations on

the physiological mechanisms of the endophyte-host inter-

action. A knowledge of these mechanisms is, however, a

prerequisite to understanding how the fungal endophyte is

able to grow within the plant host without causing

macroscopically visible disease symptoms. Cabral et al. (1993)

showed that there is indeed a host defence response to

Mycol. Res. 103 (10) : 1275–1283 (1999) Printed in the United Kingdom

The endophyte-host interaction: a balanced antagonism?

Endophyte-host interaction : a balanced antagonism? 1276

endophytic infection. They observed callose formation in

individual cells following infection of Juncus spp. with the

endophytes Stagnospora innumerosa and Drechslera sp. Sieber et

al. (1991) found that endophytic Melanconium spp. synthesize

highly specialized enzymes which may be relevant for the

penetration of the cuticular layers of the host. Several research

groups have investigated the growth responses of host calli

and endophytes in dual culture (Sieber, Sieber-Canavesi &

Dorworth, 1990 ; Hendry, Boddy & Lonsdale, 1993 ; Peters et

al., 1998a). As yet, however, there are only few reports on the

metabolic interactions involved (Peters, Dammeyer & Schulz,

1998b ; Schulz et al., 1998).

Our goal is to understand the nature of the endophyte-host

interactions of the non-clavicipitaceous fungal endophytes as

compared to those of other plant-fungal interactions, e.g.

mutualistic and antagonistic symbioses of mycorrhiza and

pathogens. In this paper we report our initial results on

contributions of fungal endophytes and pathogens and their

plant hosts to these interactions, concentrating on the

secondary metabolites the partners produce. These include : (i)

a screening for biologically active secondary metabolites of

more than 4000 fungal isolates from both soils and plants of

very diverse habitats ; (ii) comparative analyses of the

concentrations of phenolic metabolites of axenically grown

seedlings whose roots were infected with an endophyte with

those produced following infection with a pathogen. The two

plant-fungal systems studied used the larch (Larix decidua

Mill.) which was infected with either the endophyte

Cryptosporiopsis sp. or the pathogen Heterobasidion annosum

(Fr.) Bref. [Syn. Fomes annosus (Fr.) Cooke] and barley (Hordeum

vulgare L.), infected with the endophyte Fusarium sp. or the

pathogen Drechslera sp.

MATERIAL AND METHODS

Isolation and screening of fungal isolates. Approximately

1250 endophytic strains were obtained from a wide range of

deciduous and coniferous trees, shrubs and herbaceous plants

(details from authors) growing in Lower Saxony, Germany.

Surface sterilization of leaf, stem and root segments was done

with ethanol and sodium hypochlorite, the concentrations of

the latter and the times of sterilization being varied according

to the consistency of the tissue to be sterilized. The

effectiveness of the surface sterilization was checked by

making an imprint of the sterilized tissue on an antibacterial

malt-peptone-yeast extract agar (MPYA) medium (Schulz et

al., 1993). When the surface sterilization was successful, no

fungal colonies developed on the medium used for the

imprint. The sterilized segments were then cultivated on

antibacterial agar and the mycelia isolated as they emerged

from the sterilized tissue (Schulz et al., 1993). Some isolates of

ubiquitous genera (e.g. Penicillium, Cladosporium, Trichoderma,

Aspergillus) were not maintained in culture.

For a comparison, we also screened approx. 2100 soil

isolates, 150 epiphytes and 500 plant pathogenic strains, 250

of these from macerated tissues. The soils were from temperate

climates and from extreme habitats (Germany, Canada,

Greenland, U.S.A., Turkey, Tenerife, Egypt, Israel, Ethiopia,

Kenya, Mali, Philippines, Borneo, Thailand, China, Japan, and

Antarctica). Soil was suspended in sterile tap water with one

drop of Tween 80, agitated and then plated onto antibiotic

MPYA. Isolates were separately cultivated as they appeared

on MPY agar medium. Again, most isolates of ubiquitous

genera were not maintained in culture. Plant pathogenic

strains were isolated following surface sterilization as above

for the endophytic strains, but from diseased plant tissue of

Brassica napus L., Cirsium arvense (L.) Scop., Hordeum vulgare L.,

Petroselinum crispum (Mill.) Nym., Phaseolus vulgaris L., Solanum

tuberosum L., Triticum aestivum L. and Zea mays L. Epiphytes

were isolated before surface sterilization onto antibacterial

MPYA from some of the same plants from which endophytes

had been obtained.

A screening for fungicidal, antibacterial and herbicidal

metabolites was conducted as described previously (Schulz et

al., 1995) with all of the isolates using four fungal (Eurotium

repens Corda, Fusarium oxysporum Schltdl., Mycotypha micro-

spora Fenner, Ustilago violacea (Pers.) Roussel), two bacterial

(Escherichia coli (Mig.) Castell. & Chambers and Bacillus

megaterium de Bary), one algal (Chlorella fusca Shih Krauss) and

two phanerogam (Lepidium sativum L. and Lemna minor L.) test

organisms. Evaluation had shown that the results from

screening with the alga and phanerogams could be grouped

together as ‘herbicidal ’ activity (Schulz, unpublished).

Fungal isolates of larch and barley. The endophyte of larch

used in these studies (Cryptosporiopsis sp.) was isolated

following surface sterilization from the fine roots of a 27 yr

old tree growing in a mixed forest of predominantly beech,

larch and fir near Evessen, Lower Saxony, Germany in May,

1994. We chose an isolate of this genus because Crypto-

sporiopsis, along with its teleomorph Pezicula sp., are the

most commonly isolated endophytic genera of the European

larch (Kowalski, 1982 ; Kowalski & Kehr, 1992). Heterobasidion

annosum (Deutsche Stammsammlung fu$ r Mikroorganismen,

Braunschweig, 2728) was chosen as the pathogen since it is

the most problematic root parasite of conifers in Germany

(Butin, 1989).

Endophytic fungi were isolated from healthy roots of

barley following surface sterilization. The barley plants (spring

barley, cv. Salome) had been cultivated for 6–8 wk as

described by Maier et al. (1995). The Fusarium sp. and

Drechslera sp. were chosen from these isolates following tests

for pathogenicity (Schulz et al., 1998).

Axenic culture and inoculation of host seedlings. Larch seeds

were pre-soaked (500 seeds in 500 ml sterile tap water,

changed daily) for 4 d on a rotary shaker at 120 rpm and 4 °C.

On the fourth day of pre-soaking the antibiotic ClaforanTM

(Ch.-B. L588, Hoechst, Germany) was added to a final

concentration of 60 mg l−". Following surface sterilization, the

seeds were plated for germination on 5% (w}v) biomalt agar

(Vitaborn, Hameln, Germany) and cultured at 20° with a

photoperiod of 16 h, light (PAR: 200 µE m−# s−", Philips TLD

32W}84HF, Netherlands). After approx. 2 wk, sterile plants

that had developed needles and roots were transplanted into

sterilized plastic growth pots (height 108 mm; PhytaconTM,

Sigma P-4928, Deisenhofen, Germany) containing 100 ml

Seramis (Effem GmbH, Verden, Aller, Germany) as an

Barbara Schulz and others 1277

(a)80

70

60

50

40

30

20

10

0

Inhi

biti

ng s

trai

ns (

%)

Soil

Plant

All inhibitions Fungicide Herbicide

Soil

Plant

(b)80

70

60

50

40

30

20

10

0

Inhi

biti

ng s

trai

ns (

%)

Soil Endophytic Pathogenic Epithytic

Isolates with herbicidal activity

Fig. 1. Antifungal, herbidical and antibacterial activities of approx.

1900 fungal plant and 2100 fungal soil isolates, grown on solid media

and sprayed with a test organism (see text). (a) All inhibitions of

bacterial, fungal and plant test organisms ; fungicidal¯ inhibition of

the fungal test organisms ; herbicidal¯ inhibition of the plant test

organisms ; (b) algal}herbicidal activity of soil and plant isolates, the

latter according to source (approx. 1250 endophytic, 500 phyto-

pathogenic and 150 epiphytic fungal isolates).

expanded clay substrate and 40 ml modified Melin-Norkrans

(MMN) medium (Kottke et al., 1987). The seedlings were

then cultivated in a growth chamber (Weiss, Reiskirchen,

Germany) for 3 mo at 20}16° (day}night) with a photoperiod

of 16 h and light intensity of 100 µE m−# s−". At an age of

3 mo the larch roots were inoculated with Cryptosporiopsis sp.

or H. annosum by axenically placing a 4¬4 mm segment of

mycelial culture (30 d, grown on 2% w}v biomalt agar

medium) 1 cm deep into the expanded clay substrate

immediately adjacent to the root. Five pots of four plants were

inoculated for each time of harvest (0, 8, 16 and 32 dpi) and

each fungal isolate. The same number of uninoculated plants

served as controls, resulting in a total of 240 plants.

The barley seeds were surface sterilized (Schulz et al., 1998)

and transplanted into Phytacon growth pots at the age of

5–7 d. Inoculation of the plant roots occurred parallel to

transplantation (Schulz et al., 1998) with the Fusarium sp. or

Drechslera sp. For each of the five times of harvest (0, 7, 14, 21

and 28 dpi), five pots of four plants were inoculated for

pathogen and endophyte, five uninoculated pots of four plants

serving as controls and resulting in a total of 300 plants.

Harvest, extraction of sterile plants and determination of

phenolic components. At 2, 4, 8, 16 and 32 days growth and

colouring of the plants were evaluated and at each time of

harvest the success of the infections was ascertained by

reisolation following surface sterilization of a 1 cm segment of

each of the roots included in a sample (four or five plants).

Microscopic examination of the roots served as additional

verification of infection. Longitudinal and cross sections

(20 µm) of the roots were made using a cryo-microtome

(Reichert-Jung, Nußloch, Germany) and stained in trypan

blue-lactoglycerin for 15 h (Brundett, 1984) or for 30 s in

0±1% aniline blue in lactoglycerin (lactate, glycerin, aqua dest.,

v}v}v, 1 :1 :1) and viewed in lactoglycerin (Zeiss Axioscope,

Go$ ttingen, Germany). The remains of the roots were frozen in

liquid nitrogen and temporarily stored at ®20° pending

lyophilization (1–2 h at ®70°, two days in a Christ Alpha

1-4, LDC-1, Osterode, Germany) and then stored at room

temperature in closed vesicles. As controls, mycelial cultures

of the fungi (larch grown on MMN and barley on MS

(Murashige & Skooge, 1962)) were frozen and lyophilized

according to the same procedure to assure that the metabolites

that were to be determined with HPLC were not of fungal

origin. The assays for soluble phenolics, cell wall-bound

products and proanthocyanidins as well as the HPLC analyses

were as previously described (Maier et al., 1995 ; Weiss et al.,

1997).

RESULTS

Fungal secondary metabolites. In our screening for antifungal,

antibacterial and algicidal}herbicidal metabolites, we tested

approx. 4000 isolates from plants and soils. About the same

proportion of soil and plant isolates inhibited at least one of

the test organisms (Fig. 1), 70% and 78%, respectively.

Regarding the herbicidal}algicidal and antifungal activities

separately, we again found that approx. the same proportion

of the plant and soil isolates, nearly 50% of all fungal isolates,

inhibited one of the fungal test organisms (Fig. 1). Only 18%

of the fungal isolates from soils showed herbicidal activity, but

52% of those from plants did.

In order to ascertain whether all plant isolates or only those

with a particular ecological niche produce a high proportion of

herbicidal metabolites, we examined the isolates from plants

according to their sources. The endophytes, with 57% of the

strains inhibiting at least one of the test organisms for

algal}herbicidal activity, were responsible for the high

proportion of activity among the plant isolates. In contrast,

only 27% of the phytopathogens and 25% of the epiphytes

produced herbicidally active secondary metabolites (Fig. 1).

Endophytic and pathogenic infections. Of the inoculated

roots, almost 100% were infected by the endophytes or

pathogens, as shown by incubation following surface

sterilization of the inoculated roots. Noninfected roots were

excluded from further experiments. The endophytic infections

of the roots of larch and barley resulted in neither growth

inhibition nor disease symptoms, whereas infections with the

pathogens led to both disease symptoms and diminished

growth of the seedlings (Figs 2, 3). This discrepancy in


(a)45

40

35

30

25

20

15

10

0

Sho

ot le

ngth

(cm

)

(b)

80

70

60

50

40

30

20

10

0

Rel

ativ

e di

seas

e se

veri

ty

Days post inoculation

ControlFusarium sp. (endophyte)Drechslera sp. (pathogen)

5

90

0 7 14 21 28

Fig. 2. Growth and disease symptoms of barley following inoculation

of the roots of axenically grown seedlings (1 wk) with Fusarium or

Drechslera. Evaluation of the relative disease symptoms including

necroses and chloroses in % of total leaf area. n¯ 20.

development of symptoms between endophytic and patho-

genic infections occurred in spite of the fact that both

endophytes and pathogens extensively colonized the roots of

the two hosts, both inter- and intracellularly (Fig. 4).

Plant secondary metabolites. Phenolic metabolites are

considered to be involved in the plant defense reaction. To

investigate whether their concentrations differ as a result of

endophytic and pathogenic infections, we measured the

concentrations of soluble and cell wall bound phenolic

metabolites of the roots following infection.

In the roots of larch the phenolic metabolites (phenyl-

propanoids) that could be identified were catechin, epicatechin,

maltol glucoside, 4-hydroxybenzoate, 4-hydroxybenzoyl-

glucose, vanillin, ferulate, tetrahydroxystilbene and a quercitin

glycoside. None of these metabolites were found in the fungal

controls. Whereas there were no significant differences in the

concentrations of most metabolites in the axenically grown

control plants and in those infected with one of the two fungal

isolates, the concentrations of oligomeric proanthocyanidins

increased 440% in the roots infected with Cryptosporiopsis sp.

in comparison to the controls (Fig. 5). In contrast, in those

infected with H. annosum the concentration decreased to

almost zero 16 d following infection.

The phenylpropanoids detected in roots of barley were

ferulic acid, 4-coumaric acid, N-4-coumaroylputrescine, N-4-

(a)1·5

0·0

Incr

ease

of

shoo

t len

gth

(cm

)

(b)

3

2

1

0

Rel

ativ

e di

seas

e se

veri

ty


ControlCryptosporiopsis sp. (endophyte)H. annosum (pathogen)

4

0 2 4 8 16

1·0

0·5

32

Fig. 3. Growth and disease symptoms of larch following inoculation

of the roots of axenically grown seedlings (3 mo) with Crypto-

sporiopsis sp. or Heterobasidion annosum. Relative disease severity,

based on the degree of chlorosis : 0¯ no chlorosis, 4¯ brown

needles. No necrosis developed on needles of the seedlings. n¯ 20.

coumaroylagmatine, N-feruloylputrescine and N-feruloyl-

agmatine. None of these metabolites was found in the fungal

controls. The soluble metabolites N-4-coumaroylputrescine

and N-4-coumaroylagmatine could not be detected in barley

roots at the time of inoculation (Fig. 6). Commencing at the

time of inoculation (0 d), however, during the subsequent

28 d the concentrations of agmatine increased in the roots of

the control plants and in those infected with the endophytic

Fusarium, to approx. 0±7 µmol g−" .. and 0±65 µmol g−"

.., respectively, those of putrescine increasing to 7±7 and

7±0, respectively. In contrast, during the same period these

substances were not detectable in roots of the seedlings

infected with Drechslera sp. It is important to note that

between 0 and 7 d after inoculation, the roots infected with

Drechslera sp. had not yet developed disease symptoms. The

concentrations of the cell wall-bound metabolites 4-coumaric

acid and ferulic acid increased less in barley plants infected

with the Drechslera during the 28 d following infection than in

the controls, or in those roots infected with the endophyte.

Whereas the concentration of 4-coumaric acid increased from

zero to 6±8 µmol g−" .. in roots infected with the endophyte

and in the controls, the concentration of this cell wall-bound

metabolite only increased to 2±1 µmol g−" .. in the plants

infected with the pathogen (Fig. 7). The concentration of

ferulic acid increased from approx. 12 to 21 µmol g−" root


Fig. 4. Inter- and intracellular colonization (arrows) of larch with Cryptosporiopsis sp. (a) and H. annosum (b), and of barley with Fusarium

sp. (c) and Drechslera sp. (d ). Scale bars¯ 20 µm.

15

10

5

0


ControlCryptosporiopsis sp. (endophyte)H. annosum (pathogen)

0 8 16 32

Sol

uble

pro

anth

ocya

nidi

ns(µ

mol

cya

nidi

n ch

lori

de e

quiv

alen

ts g

–1

..)

Fig. 5. Concentration of soluble proanthocyanidins in the roots of

larch seedlings grown axenically for 3 mo before inoculation with

endophyte or pathogen and cultured in expanded clay in MMN-

medium at 20° with a photoperiod of 16 h (PAR: 100 µE m−# s−").

n¯ 4 samples of two plants each.

.. in the controls and in the plants infected with the

endophyte, the increase occurring more rapidly in roots

infected with Fusarium sp. than in the controls (Fig. 7). In the

roots colonized by the pathogen, the concentration of ferulic

acid only increased from 11±5 to 16 µmol g−" .. during the

28 d following infection.

DISCUSSION

Summarizing our results concerning the metabolic interactions

of endophyte and host, we have found that :

(1) In our screening for biologically active secondary

metabolites, a significantly higher proportion of endophytic

isolates produced metabolites that are herbicidally active than

those isolated from other sources.

2. In the cases in which the concentrations of phenolic

metabolites differed between roots infected with an endophyte

and those infected with a pathogen, the concentrations were

higher in the roots, infected with an endophyte.

Fungal metabolites. Previously, it was thought that fungal

secondary metabolites might only be waste products of

primary metabolism (Za$ hner, 1977). In recent years it has

become more and more obvious that, as is the case for plant

secondary metabolites (Harborne, 1993), these metabolites

may play an ecological role, e.g. the host selective toxins of

phytopathogenic fungi, antifungal metabolites from myco-

parasitic fungi, mycotoxins that protect fungal reproductive


(a)12

6

N-4

-cou

mar

oylp

utre

scin

e (µ

mol

g–1

.

.)

(b)0·8

0·2



4

0 7 14

10

8

2

0

0·6

0·4

0·0

21 28

N-4

-cou

mar

oyla

gmat

ine

(µm

ol g

–1

..)

Fig. 6. Concentration of N-4-coumaroylagmatine and N-4-

coumaroylputrescine in the roots of barley seedlings grown axenically

for 1 wk before inoculation with endophyte or pathogen and

cultured in expanded clay in MS-medium at 20° with a photoperiod

of 16 h (PAR: 100 µE m−# s−"). n¯ 3 samples of five plants each.

structures from herbivory and those involved in competitive

or antagonistic interactions (Gloer, 1997). Nevertheless,

finding that fungal endophytes, in comparison to phyto-

pathogens and isolates from soils, have the highest

proportion of herbicidally active isolates was quite unexpected,

since by definition endophytic fungi cause no visible disease

symptoms in their hosts (Petrini, 1991). It had seemed more

plausible that phytopathogens would produce a high pro-

portion of herbicidally active metabolites.

It is not the case that the herbicidal activity of these strains

is due to one or more substances common to all of these

endophytic fungi. The structures of some of these secondary

metabolites have been determined and belong to very diverse

structural groups including : steroids (Krohn et al., 1992a),

imides (Krohn et al., 1992b), diterpenes (Ko$ nig et al., in press),

depsipeptides (K. Krohn, R. Bahramsari, H.-J. Aust, S. Draeger

& B. Schulz, unpublished), cytochalasines (Ko$ nig et al., in

press), biarylethers (Krohn et al., 1996), epoxydones and

xanthenes (K. Krohn, K. Beckmann, H. J. Aust, S. Draeger &

B. Schulz, unpublished), isocoumarines (Schulz et al., 1995 ;

Krohn et al., 1997b) and furofuranones (Krohn et al., 1994a).

There is also a previously unknown structural group, the

palmarumycins (Krohn et al., 1994b, c, 1997a, c). Not only the

culture extracts but also the pure substances are herbicidally

active : 31 of 33 of these metabolites inhibited at least one of

the test organisms for herbicidal activity (data not shown).

Finding that such a high proportion of the metabolites isolated

(a)

6

4-co

umar

ic a

cid

(µm

ol g

–1

..)

(b)25

10



0 7 14

4

2

0

20

15

021 28

Feru

lic

acid

(µ

mol

g–1

.

.)

Fig. 7. Concentrations of 4-coumaric and ferulic acids in the roots of

barley seedlings grown axenically for 1 wk before inoculation with

endophyte or pathogen in expanded clay in MS-medium at 20° with

a photoperiod of 16 h (PAR: 100 µE m−# s−"). n¯ 3 samples of five

plants each.

from endophytes are herbicidally active not only suggests an

ecological role for these endophytic secondary metabolites,

but also supports Demain’s hypothesis that these substances

have a natural function. The multienzyme reaction sequences

of secondary metabolites would not be retained by fungi

without some beneficial effect for survival. Additionally, it has

been shown that both phytopathogenic and soil inhabiting

fungi produce biologically active secondary metabolites in

nature (Demain, 1980). Thus, it seems plausible that these

substances are synthesized at some stage in the endophytic

life cycle in vivo. Nevertheless, the question remains : why do

endophytic fungi synthesize such diverse metabolites which

are potentially toxic for their hosts ?

In order to understand the role of these metabolites in the

endophyte-host interaction, Peters et al. (1998a) investigated

a simplified system using dual cultures of plant host calli and

endophytes. It was found that not only in monoculture, but

also in dual culture, the endophytes excreted nonspecific

herbicidal metabolites causing necroses, growth inhibition and

death of the host calli. The host calli in turn excreted non-

specific antifungal metabolites. Thus, both the endophytes and

the plant host calli excreted metabolites toxic to their partners,

suggesting a mutual antagonism of host and endophytic

fungus.


Plant metabolites and defence reaction. The great extent

to which the endophytes Cryptosporiopsis sp. and Fusarium

sp. colonized the roots of their hosts was unexpected, since

infections of plant hosts by non-clavicipitaceous endophytes

have often been assumed to be localized (e.g. Stone et al.,

1994) and neither disease symptoms nor growth inhibition

had been observed. It should be borne in mind, however, that

only a limited amount of cytological work has been done on

these endophytic infections (Suske & Acker, 1989 ; Cabral et

al., 1993 ; Viret, Scheidegger & Petrini, 1993 ; Stone et al.,

1994), so that extended colonization may not be infrequent.

Additionally, the plants used in these experiments had been

cultured in a growth chamber under conditions which

undoubtedly influenced the predisposition of the plant hosts.

The phenolic metabolites found in the roots of larch had

been previously identified by Weiss et al. (1996) in mycorrhizal

larch roots. Mu$ nzenberger, Kottke & Oberwinkler (1995) had

found catechin, epicatechin, 4-hydroxybenzoyl-glucose, 4-

hydroxybenzoate and additionally picein in mycorrhizal fine

roots of larch. The phenylpropanoids found in the roots of

barley were those that had been determined by Peipp et al.

(1997). In uninoculated barley plants of the same age as those

investigated in these experiments, the authors also found an

increase in the concentrations of the hydroxycinnamic acid

amides N-4-coumaroylputrescine and N-4-coumaroylagmatine

during mycorrhization. At a later stage than measured in these

experiments, the authors found a decrease in the concentration

of these metabolites.

Since phenolic metabolites are generally toxic to micro-

organisms (Schlo$ sser, 1997), they presumably play this role in

the systems we studied. The proanthocyanidins have been

shown to function as preformed defence metabolites in their

hosts (Stafford, 1997 ; Schlo$ sser, 1997). For example, Jersch et

al. (1989) found that as long as proanthocyanidins are present

at a sufficiently high concentration in strawberries, Botrytis

cinerea remains in a quiescent state. When the strawberries

ripen and the concentration of proanthocyanidins decrease, B.

cinerea becomes pathogenic. Weiss et al. (1996), investigating

the interaction of larch with the ectomycorrhizal fungi

Boletinus cavipes and Suillus tridentinus, showed that

mycorrhization of larch roots resulted in elevated levels of

phenylpropanoids, including cell wall-bound ferulate. The

secondary metabolites accumulated in the central part of the

subapical cortex tissue and the endodermis. The authors

suggested that such metabolites may provide a barrier to

growth of the mycorrhizal fungi into the apical meristem and

stele, a situation which may also exist in the roots of the larch

we investigated that were infected with the endophyte

Cryptosporiopsis sp. Since proanthocyanidins are located in the

epidermis of Lotus pedunculatus (Pankhurst, Craig & Jones,

1979), they may also serve as a barrier to fungal penetration

in this host. The hydroxycinnamic acid amides studied in

barley may be precursors of antifungal substances, since the

agmatines are precursors of the hordatines, antifungal

metabolites of barley (Stoessl, 1966a, b).

The concentrations of most of the phenolic metabolites

determined in these studies did not differ between control

plants and those infected with endophyte or pathogen. In

those cases in which the concentrations of these metabolites

differed, however, they were higher in the endophyte infected

roots. The increase in phenolic metabolites found in

comparison to the controls following endophytic infection

(proanthocyanidins in larch, ferulic acid in barley) is at first

glance unexpected since the infections remained symptomless.

The higher concentrations of these phenolic defence meta-

bolites may have been due to continual elicitation by the

presence of the fungal endophytes, which the latter – if they

came into contact with them – apparently could tolerate.

When infected with a pathogen, in contrast to the controls,

the concentrations of these phenylpropanoids in the roots

either increased less (ferulic and coumaric acid in barley),

remained undectable as at the time of inoculation (hydroxy-

cinnamic acid amides in barley) or decreased (proantho-

cyanidins in larch) during the course of the experiment.

This is presumably related to the pathogen’s ability to

overcome the host’s defence reaction and cause disease, e.g.

by suppression of synthesis or degradation of the metabolites

so that the concentrations did not attain those they had in the

controls.

In other experiments conducted under the same conditions

of culture the results also suggest that proanthocyanidins play

a role in the plant defence reaction of the larch. Under

nitrogen stress (two and four times the normal concentration

in the growth medium), the concentration of proantho-

cyanidins decreased concomittant with an increased sus-

ceptibility to fungal infection (Ro$ mmert et al., unpublished).

Although these metabolites are primarily contained in the

vacuoles, they are also found in the apoplast (Stafford, 1997)

and may there contribute to limiting potential pathogenicity

of the endophyte.

Previously, not only the concentrations of the phenyl-

propanoids, but also other parameters of the plant defence

response were found to be stronger towards endophytic than

towards pathogenic infection. The activity of PAL was greater

in the axenically grown host Lamium purpureum grown in dual

culture with the endophyte Coniothyrium palmarum than with a

pathogenic Alternaria sp. Elicitation of cell suspension cultures

of L. purpureum with C. palmarum resulted in greater PAL-

activity, a greater release of H#O

#and a higher concentration

of total phenols than when elicited with the Alternaria (Peters

et al., 1998b). Induction of PAL was also higher following

elicitation of suspension cultures of the larch with an

endophyte than with a pathogen (A. Fengler & B. Schulz,

unpublished).

Hypothesis. How does an endophyte manage to exist and

grow within its host without causing visible disease

symptoms? Fig. 8 illustrates a working hypothesis comparing

the endophyte-plant host interaction with that of pathogen

and plant host. Whereas the pathogen overcomes the defence

reaction to the extent that disease develops, the endophyte

only overcomes defence to the extent that it is able to infect

and colonize the plant host. The endophytic infection may

then remain localized as is the case for Rhabdocline parkeri in

Douglas fir (Stone, 1987) or it may become an extensive intra-

and intercellular colonization as we have observed following

inoculation of barley and larch with the endophytes Fusarium

sp. or Cryptosporiopsis sp. Even though disease symptoms do


Balancedantagonism

Suppression ofplant

defence reaction

Plant

Fungicidally active metabolitesEndophyte PathogenD

efence

Def

ence

Tolerance Disease

Her

bici

dally

act

ive

met

abol

ites

Herbicidally active

metabolites

Fig. 8. Hypothesis : balanced antagonism between fungal endophyte and plant host.

not develop, not only pathogen and host, but also endophyte

and host, seem to be actively antagonistic against each other,

as suggested by :

– the high proportion of herbicidal metabolites synthesized

by endophytic isolates ;

– the production of toxic metabolites to the respective

partners by both endophyte and callus in dual culture ;

– the higher concentrations of some phenolic metabolites

during endophytic as compared to pathogenic infection ;

– the greater plant defence reaction to endophytic in contrast

to pathogenic elicitation of suspension cell cultures.

Additionally, the antifungal secondary metabolites that both

endophyte and pathogen can synthesize may, if produced in

vivo, be directed against each other to decrease competition.

The plant defence reactions are active against both fungal

invaders, and both endophyte and pathogen may produce

metabolites toxic to their hosts, but, the pathogen, and not the

endophyte, suppresses the plant defence reaction. Although

this defence may limit the development of disease, the

endophyte apparently can tolerate the plant defence reaction.

The pathogen-host interaction is imbalanced resulting in

disease (Fig. 8), whereas in the endophyte-host interaction the

partners maintain a balanced antagonism without the

development of disease. Should this balance be disturbed in

favour of the fungus, then the endophyte could become a

pathogen. This tolerated endophytic infection does not

exclude the possibility that the endophyte may play a

mutualistic role within its host, for example by increasing the

concentration of defence metabolites potentially active against

pathogens, by excreting phytohormones and}or by increasing

the general metabolic activity of the plant host.

We would like to thank BASF, the Bundesministerium fu$ rBildung und Forschung, the Deutsche Forschungsgemeinschaft

and the Fonds der Chemischen Industrie for financial aid. We

are grateful for the excellent and reliable technical assistance

of Qunxiu Hu and to Dr Siegfried Draeger for help identifying

the fungi. We thank Dr W. Maier for patient assistance with

the HPLC analyses and Dr Christine Boyle for astute criticism

of the manuscript and fruitful discussions of the results that led

to development of the hypothesis.

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the endophyte-host interaction: a balanced antagonism?

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