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Review
The endophytic continuum
Barbara SCHULZ1 and Christine BOYLE2
1 Institute of Microbiology, Technical University of
Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig,
Germany.2Augustastrasse. 32, D-02826 Gorlitz, Germany.
E-mail : [email protected]
Received 25 February 2004; accepted 15 February 2005.
In spite of the term endophyte being employed for all organisms
that inhabit plants, mycologists have come to usethe term fungal
endophyte for fungi that inhabit plants without causing visible
disease symptoms. The term refersonly to fungi at the moment of
detection without regard for the future status of the interaction.
This paper is a review
of literature on non-balansiaceous fungi involved in
asymptomatic colonisations of plants. These fungal
endophytesrepresent a continuum of fungi with respect to
physiological status, infection modus, colonisation pattern,
secondarymetabolism, life-history strategy, and developmental and
evolutionary stages, but also with respect to the fungal and
host
taxa involved in the symbioses.We hypothesize that there are no
neutral interactions, but rather that endophyte-host interactions
involve a balance of
antagonisms, irrespective of the plant organ infected. There is
always at least a degree of virulence on the part of the
fungus enabling infection, whereas defence of the plant host
limits development of fungal invaders and disease. It is
alsohypothesized that the endophytes, in contrast to known
pathogens, generally have far greater phenotypic plasticity andthus
more options than pathogens: infection, local but also extensive
colonisation, latency, virulence, pathogenity and
(or) saprophytism. This phenotypic plasticity is a motor of
evolution.
INTRODUCTION
Taken literally, the word endophyte means in theplant (endon
Gr., within; phyton, plant). The usage ofthis term is as broad as
its literal denition and spec-trum of potential plant hosts and
inhabitants, includingbacteria (Kobayashi & Palumbo 2000),
fungi (Stone,Bacon & White 2000), algae (Peters 1991), and
insects(Feller 1995). Any organ of the host can be
colonized.Equally variable is the life-history strategy of the
sym-biosis, ranging from facultatively saprobic, to parasitic,to
exploitive, to mutualistic. However, common to allendophytic
interactions is the provision of nutrientsand a buer from external
environmental stresses andmicrobial competition.The endophytic
partners and their relationships
to each other vary. There are pathogenic endophyticalgae
(Bouarab et al. 1999), parasitic endophytic plants(Marler et al.
1999), mutualistic endophytic bacteria(Chanway 1996, Adhikari et
al. 2001, Bai et al. 2002),ectomycorrhizal helper bacteria
(Founoune et al. 2002),as well as endophytic bacteria in pathogenic
and com-mensalistic symbioses (Sturz & Nowak 2000). There
are equally diverse endophytic interactions of fungiwith their
plant hosts : mutualistic mycorrhizal fungiwith the roots of the
host are termed endophytic (Sieber2002), but also those of the
orchid with its fungal endo-phytes, in spite of the host being
myco-heterotrophicand exploiting the fungal endophyte (Gardes
2002).Some dark-septate fungal endophytes that colonize theroots of
many herbaceous plants and trees are mutu-alistic, whereas others
become pathogenic (Jumpponen2001, Sieber 2002). Other fungal
endophytes inhabitsolely above-ground plant organs and these
againmay partake in varied and variable interactions withthe plant
host, ranging from mutualistic (Redmanet al. 2002, Schulz et al.
2002) to cryptic commensal(Deckert, Melville & Petersen 2001)
to latent and viru-lent pathogens (Sinclair & Cerkauskas 1996,
Schulzet al. 1998). In contrast to the broad usage of the
termendophyte , many mycologists have come to employthis term only
for those fungi that colonize a plantwithout causing visible
disease symptoms at anyspecic moment (Petrini 1991, Wilson 1995,
Stone et al.2000). Some even speak of the true endophytes ,
mean-ing those whose colonisation never results in visible
Mycol. Res. 109 (6): 661686 (June 2005). f The British
Mycological Society 661
doi:10.1017/S095375620500273X Printed in the United Kingdom.
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disease symptoms (Mostert, Crous & Petrini 2000).Aware of
the determinative discrepancies, we willnevertheless use the term
fungal endophyte here todescribe those fungi that can be detected
at a particularmoment within the tissues of apparently healthy
planthosts. Fungal endophytes consist of three basic eco-logical
groups: themycorrhizal fungi, the balansiaceousor grass endophytes
, and the non-balansiaceoustaxa. The main emphasis of this review
will be onthe non-balansiaceous endophytes ; however we rstbriey
review these endophytes. Those interested inmycorrhizal fungi are
referred, for example, to thereview by Brundrett (2004), who
distinguished myco-rrhizal from endophytic interactions,
mycorrhizashaving synchronized plant-fungus development andnutrient
transfer at specialized interfaces.
THE BALANSIACEOUS ENDOPHYTES
The endophytes of the Balansiaceae form a uniquegroup of closely
related fungi with ecological require-ments and adaptations
distinct from those of otherendophytes (Petrini 1996). They belong
to the asco-mycetous genera Epichloe and Balansia, and their
ana-morphs Neotyphodium and Ephelis. Genotypes of mostof the
asexual endophytes suggest that these are inter-specic hybrids ;
hybridization presumably havingoccurred after the loss of sexual
expression (Schardl,Leuchtmann & Spiering 2004). Due to their
ecologicaland economic impact, theirs is the best studied of
theabove-ground endophytic interactions. Balansiaceousendophytes
grow systemically, rarely epicuticularly,and intercellularly within
all above-ground plantorgans of grasses, rushes and sedges,
resulting in verti-cal transmission of the endophyte through the
seeds(Bacon & White 2000). Despite the status of
theinteraction, for instance pathogenic Epichloe spp.
ornon-pathogenic Neotyphodium spp., both are calledendophytes
(Schardl et al. 2004) and both developstructures to maximize the
uptake of nutrients intothe mycelium (Christensen, Bennett &
Schmid 2002).The plant tissues incorporated within the stroma
ofEpichloe are modied, their cells may become hyper-trophied
(mesophyll) or soften (epidermal cells) and failto function as
barriers (White, Reddy & Bacon 2000).The balansiaceous
endophytes produce a diverse
array of secondary metabolites. Several well-studiedtoxic
syndromes may develop in mammals feeding onforage grasses colonized
by species of Neotyphodiumor Epichloe (Lane, Christensen &
Miles 2000, Schardlet al. 2004). The toxic alkaloids include the
anti-insectalkaloids peramine and lolines, and the
anti-vertebratealkaloids lolitrem B and ergovaline (Schardl
2001).Whereas most of the metabolites are of fungal origin,there is
evidence of biosynthetic interaction. Shelbyet al. (1997) found
ergopeptide variants that wereapparently modied by plant
metabolism. Justus, Witte& Hartmann (1997) reported traces of
loline even inuninfected plants of Festuca pratensis. Their
chemistry
and activity have been extensively reviewed and sum-marized by
Lane et al. (2000).Since colonisation of the host is primarily
inter-
cellular, the endophytes are dependent on nutrients ofthe
apoplast for growth. As summarized by Bacon &White (2000), only
a few studies deal with translocationand the specic physiology on
nutrient exchange oraccumulation in vivo. For example, Schmid,
Spiering &Christensen (2000) studied distribution and nutrition
ofthe endophytes in planta. And more recently, Pan &Clay (2004)
found that 14C movement from labelledleaves was greater for
infected stolons than for those ofnon-infected plants. Studies on
enzyme activities, con-centrations of amino acids, and ammonium,
indicatethat the interaction signicantly alters at least
nitrogenmetabolism. Nevertheless, there may be an inducedplant
defence reaction; Roberts et al. (1992) found
thatendophyte-infected plants produced greater amountsof chitinase
than uninfected plants.Even though some balansiaceous endophytes
con-
tribute nothing to the tness of their hosts and mayeven be
antagonistic (Sinclair & Cerkauskas 1996,Faeth & Fagan
2002, Sieber 2002, Schardl et al. 2004),their symbiosis with their
hosts is widely accepted asbeing mutualistic (Schardl & Clay
1997). Physiologicalstudies (Schardl 2001) indicate that signals
communi-cating between E. festucae and host plants ensure
adelicately balanced interaction between the partners.The primary
benets for the fungal partner are nutri-tional, but also include
protection from abiotic stress(Bacon & Hill 1996), for example,
desiccation, as wellas from competing epiphytic organisms (White et
al.2000). The main advantage of the interaction forplants is
presumably protection against herbivory bytoxic alkaloids produced
in the symbiotic association.Additionally, the endophyte may
mediate induced re-sistance, i.e. as an extension of the defensive
mutualismhypothesis ; the activation of the host defence
throughconstitutive and induced resistance (Bultman &Murphy
2000). The expectation of induced resistancein infected plants is
strengthened by reports thatenvironmental stresses can stimulate
the productionof mycotoxins in endophyte-infected plants
(Bultman& Murphy 2000).
NON-BALANSIACEOUS ENDOPHYTES
In contrast to the balansiaceous endophytes, the
non-balansiaceous fungal endophytes are diverse,
bothphylogenetically and with respect to life-history strat-egy.
Most of these fungi belong to the Ascomycota(Petrini 1986), and
they have been isolated from everyorgan of almost all sampled
plants (e.g. Petrini 1991,Schulz et al. 1993, Stone et al. 2000).
Colonisation canbe inter- or intracellular, localised or systemic.
Asapplied to this group of fungi, the term endophytegenerally
refers to a fungus capable of cryptic occu-pation of plant tissue
and describes a momentarystatus. Some endophytes have been found to
be
The endophytic continuum 662
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non-aggressive, not causing disease (Freeman & Rodri-guez
1993, Tyler 1993, Sinclair & Cerkauskas 1996),some to be latent
pathogens, others to play mutualisticroles within their hosts
(Carroll 1988, Freeman&Rodri-guez 1993, Stone et al. 1994,
Sinclair & Cerkauskas1996). Important is that the status of the
interactionbetween endophyte and host may be transient.
Thestability or variability of the asymptomatic interactiondepends
on numerous factors. The following sectionsexplore the diverse
aspects of the interactions of thenon-balansiaceous endophytes with
their hosts.
INTERACTIONS
Which fungi are non-balansiaceous endophytes?
It is often extremely dicult to know whether or nota particular
fungus that has been detected in healthyplant tissue has actually
been growing within the hosttissue and thus is more or less adapted
to being an endo-phyte, or has been incidentally isolated, for
examplenormally being found on other substrates. Often little
isknown about the life-history strategies, or the import-ance of
the endophytic phase, of the fungi detected.There are three methods
presently in use for detectingand identifying fungi in plant
tissue: (1) histologicalobservation; (2) surface sterilisation of
the host tissueand isolation of the emerging fungi onto
appropriategrowth media; and (3) detection by specic
chemistry,(e.g. immunological methods or direct amplication
offungal DNA from colonized plant tissues), having rstascertained
that there are no fungal residues on theplant surface. The rst
method is best suited todistinguish to what extent a fungus
actually colonizesthe host.A problem with the histological approach
is dis-
cerning the fungal mycelium in plant tissue, since col-onisation
is often localized and recognition of minutefungal structures in
plant tissue can be equivocal (Stoneet al. 1994, Deckert et al.
2001, Sieber 2002). Lightmicroscopy may be useful for screening
purposes (e.g.ODell & Trappe 1992, Cabral, Stone & Carroll
1993)and SEM and TEM to visualize fungal structureswithin the plant
tissue (Suske & Acker 1989, Sequerraet al. 1995, Christensen et
al. 2002). Tissues for lightmicroscopy may be observed directly,
preferably fol-lowing vital staining to ascertain that the fungus
isliving or after xation, clearing and staining. Addition-ally, if
the visualized hyphae infected the host naturally,it is often
dicult to determine the taxon to whichthey belong. Other
alternatives are direct identication,for example by following
germination of the externalspores, as well as indirect identication
(Cabral et al.1993), that is by comparing the frequency of
isolationwith the prevalence of a certain hyphal morphology.This
last method neither identies fungi that cannot beisolated with
conventional methods, nor dierentiatesmorphologically similar fungi
that could belong todierent species. In vitro inoculations of
axenic hosts,
however, simplify dierentiation between fungal andplant material
for studying infection and colonisation(Schulz et al. 1999a, Boyle
et al. 2001).A more elegant but elaborate method of visualizing
articially inoculated fungi in plants is to insert thegreen
uorescent protein gene (gfp) into the fungalgenome. This enabled
Mikkelsen et al. (2001) toreadily detect colonisation of
Neotyphodium lolii inperennial rye grass (Lolium perenne). Another
sophis-ticated method for detecting specic endophytes,employs
immunoelectron microscopy (Suske & Acker1989).Isolation of
fungi following surface sterilisation
onto appropriate growth media is usually the initialstep for
investigating endophytes. The most commonprocedure rst employs a
surfactant such as ethanoland (or) Tween (Bills 1996), followed by
a sterilisingagent, such as sodium hypochlorite.
Additionally,cyclosporin (Dreyfuss & Chapela 1994) or other
com-pounds may be added to retard the growth of weedyspecies that
otherwise would overgrow the isolationplates (Bills 1996).
Isolation onto media containing leafextracts of the host may also
be useful (Arnold & Herre2003). For more information see Schulz
et al. (1993),Bills (1996), or Sieber (2002).In order to ascertain
that the fungi being isolated
are indeed growing inside the host, every procedureemployed for
surface sterilisation has to be optimizedfor the host with regard
to tissue sensitivity, age, andthe organ being sterilised.
Estimating the eectivenessof common methods of surface
sterilisation by com-paring the fungi isolated as epiphytes with
thoseisolated following surface sterilization is not
optimal,because horizontally transmitted endophytes areinitially
present externally as spores or have a shortepiphytic phase.
Petrini (1984) subjected spores of thefungi isolated to the same
procedure used for foliagesterilisation. If the spores were killed,
he assumed thatthe surface sterilisation procedure was eective.
How-ever, spores of epiphytes can be protected from
aqueoussterilants in situ by structures of the plant surface
tissue(e.g. trichomes, hydrophobic substances). It is farpreferable
to check the eectiveness of surface sterilis-ation by imprinting
treated tissue on a fungal growthmedium. If no colonies develop
from the imprint, thesterilisation can be assumed to have been
eective(Schulz et al. 1998). However, it is also very importantto
assure that the host tissue has not been damaged byan overly
stringent sterilisation procedure.Molecular methods have been used
not only for
fungal taxonomy (e.g. Mitchell, Roberts &Moss 1995),but also
to identify isolates that do not sporulate inculture (Arnold et al.
2000, Guo, Hyde & Liew 2000,Mucciarelli et al. 2002, Guo et al.
2003). Guo et al.(2000, 2003) identied non-sporulating white
morpho-types from Livingstonia chinensis and Pinus tabulae-formis
rst using the relatively non-specic 5.8S geneand subsequently the
more variable ITS1 and 2 regions,in a nested PCR assay. Nested PCR
assays were also
B. Schulz and C. Boyle 663
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found to be eective for identifying the endophytes ofPhragmites
australis (Wirsel et al. 2001).Molecular methods also permit
identication of
fungi that are viable but not culturable from the hosts(Zuccaro,
Schulz & Mitchell 2003). When employingthis approach, it is
important to take into considerationthat surface sterilisation may
not have denaturised theDNA of epiphytes, though sodium
hypochlorite isrelatively eective for this purpose (Anne E.
Arnold,pers. comm.).In order to identify all the fungi actually
colonising
a host, total DNA must be isolated from the environ-mental
sample. It can then be directly amplied withfungal primers;
denaturing gradient gel electrophoresis(DGGE) may then be used to
separate the bands.Subsequent sequencing and phylogenetic
analysistheoretically enables the identication of all the
fungicolonizing a plant (Kowalchuk, Gerards&Woldendorp1997),
provided that the sequences found correspond toknown sequences in
the databanks. Kowalchuk et al.(1997) characterized 13 endophytic
fungal isolates ofmarram grass (Ammophila arenaria) using primers
for18S rDNA. Zuccaro et al. (2003) used DGGE withsubsequent cloning
and sequencing to identify thefungi associated with the macroalga
Fucus serratus.Primers for 28S rRNA were found to be more
specicthan those for 18S rRNA. Conversely, in attemptsto amplify
plant DNA using universal primers forthe ITS and 5.8S regions of
rDNA, Zhang, Wendel& Clark (1997) also amplied fungal DNA from
bam-boos, as did Camacho et al. (1997) from spruce needles,again
emphasizing the importance of using specicprimers.There have been
three recent reports that the diver-
sity of fungi detected with molecular methods dieredfrom that
found using conventional methods of iso-lation. This was the case
for Fucus serratus (Zuccaroet al. 2003), Pinus taeda (Eells et al.
2004, Arnold et al.2005) and Gaultheria shallon (salal ; Berbee et
al. 2004).This raises a key question: Have the many studies
ondiversity of endophytes associated with various hostsidentied
(all the) fungi actually associated with therespective hosts?In
conclusion, in order to detect all the fungi associ-
ated with a photoautotrophic host, it is paramountto: (1) always
optimise surface sterilisation; and (2)not only use conventional
methods of isolation ontoculture media, but also to employ
molecular methods.
Quantication of colonisation
None of the methods for quantifying the degree ofcolonisation of
endophytic fungi within their hosts isoptimal. Colonisation has
frequently been indirectlyquantied by relating the number of
isolates per taxonto the density of colonisation (e.g. Cabral et
al. 1993,Carroll 1995). However, as molecular methods haveshown,
the most frequently isolated fungi are notnecessarily the primary
colonizers. Fungal colonisation
can be quantied using visual methods, for exampleby direct
counts of infections (Stone 1987). Anothermethod is to correlate
biomass with the concentrationof fungal specic ergosterol (Newell,
Arsu & Fallon1988, Weete & Ghandi 1996). This method may
givevariable results because ergosterol concentration varieswith
the age of the mycelium (Olsson et al. 2003).However, Manter,
Kelsey & Stone (2001), usingergosterol analysis to quantify
fungal colonisationwithin Douglas-r needles, found a strong
relationshipbetween ergosterol content and fungal colonisation
byPhaeocryptopus gaeumannii, one of the predominantfoliage
colonists of the host. Phospholipid fatty acidshave been used for
quantication, but their concen-tration varies between fungal genera
(Olsson et al.2003). Fungal biomass can also be measured
usingmonoclonal antibodies. Here the diculty lies indeveloping an
antibody specic enough to accuratelyquantify single endophytic
taxa. Real-time PCR(Schena et al. 2004) is presumably the most
accuratemethod for quantifying fungal colonisation within thehost
and has been successfully employed, for exampleby Winton et al.
(2002), to quantify the density ofcolonisation of Phaeocrytopus
gaeumannii within theneedles of Douglas-r. Hietala et al. (2003)
found astrong correlation quantifying Heterobasidion annosumin
Picea abies with real-time PCR and an ergosterol-based procedure.
Their real-time PCR assay also gavebetter resolution than the
traditional lesion lengthmeasurement assay in screening for
resistant hostclones.
Communities and host adaptation
There have been numerous papers documenting thefungi isolated
from particular hosts (Stone et al. 2000),many correlating the
isolates with ecological para-meters. Thus, we will not concentrate
on that aspect,but rather on diversity and adaptations. The
diversitywithin any particular host may be very high (Carroll1995)
; no two isolates may be identical, even from thesame species or
host (e.g. Lu et al. 2004, Rodrigueset al. 2004). Both diversity
and colonisation densityfrequently increase in the course of the
vegetationperiod, since horizontal transmission
predominates(Carroll 1988, 1995, Petrini 1991, Guske, Boyle
&Schulz 1996, Arnold & Herre 2003).Communities of
endophytes inhabiting a particular
host may be ubiquitous or have what is frequentlyreferred to as
host specicity (e.g. Carroll 1988, Petrini1996, Stone et al. 2000,
Cohen 2004). We concur withCarroll (1999) and Zhou & Hyde
(2001) that the termspecicity should be reserved for organisms that
willonly grow in one host. If this is not the case, it couldbe
termed host preference (Carroll 1999) or host-exclusivity (Zhou
& Hyde 2001).Whether the interac-tion represents specicity,
preference or exclusivity,there has been an adaptation of host and
endophyteto one another. However, some of the fungi occurring
The endophytic continuum 664
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as endophytes, the incidental opportunists, are funginormally
found growing on other substrates, and arenot specically adapted to
their hosts. An examplemight be coprophilous species that are
sometimesdetected as endophytes.Schulz et al. (1993, 1995, 1998)
obtained >6500
endophytic isolates from all organs of more than 500plants from
diverse temperate habitats. The majorityof these isolates belonged
to ubiquitous genera (e.g.Acremonium, Alternaria, Cladosporium,
Coniothyrium,Epicoccum, Fusarium, Geniculosporium,
Phoma,Pleospora), concurring with previous results reviewedby
Petrini (1986), who found that many endophytesbelong to ubiquitous
taxa. Since the assemblages ofendophytes vary with habitat, dierent
ubiquitousgenera are, for example, isolated from tropical thanfrom
temperate climates. Some genera are common inboth tropical and
temperate climates (e.g. Fusarium,Phomopsis, Phoma), while members
of the Xylariaceae,Colletotrichum, Guignardia, Phyllosticta and
Pestalo-tiopsis predominate as endophytes in the tropics(Frohlich
& Hyde 1999, Arnold et al. 2000, Rogers2000, Arnold, Maynard
& Gilbert 2001, Cannon &Simmons 2002, Suryanarayanan,
Venkatesan &Murali2003, Draeger & Schulz, unpubl.). It
would be inter-esting to investigate how the previous occupation of
aninter- or intracellular niche within a plant by one fungalgroup
might aect the subsequent establishment andevolution of other
fungal partnerships.The endophytes from any particular host
usually
include one to several taxa that are adapted to thathost. For
example, Lophodermium spp. are frequentcolonizers of conifers
(Deckert et al. 2001), Disculaumbrinella is primarily found in
Fagus sylvatica (Sieber& Hugentobler 1987), and Physalospora
vaccinii inVaccinium oxycoccus (Schulz et al. 1993).
Followinginoculation of both host endophytes from Phaseolusvulgaris
(bush bean) and those from other hosts (non-host endophytes) onto
the shoots of P. vulgaris, anumber of isolates were found to be
adapted to the host(Schulz 2003). The ratio of reinfection was
considerablyhigher for the host endophytes than for the
non-hostendophytes (Table 1). Additionally, all of the
plantsinoculated with the non-host endophytes developeddisease
symptoms, irrespective of whether or not col-onisation could be
veried by reisolation.Chapela, Petrini & Hagmann (1991)
investigated the
adaptations ofHypoxylon fragiforme to Fagus sylvatica,its
natural host. Extracts of beech bark induced eclo-sion (a
pre-germination response) and germination ina greater proportion of
the fungal ascospores ofH. fragiforme than bark extracts of other
trees (Petrini1996), demonstrating that adaptations can be found
atthe earliest stages of the interaction, i.e. recognitionof the
partner.Some endophytes are primarily isolated from and
adapted to certain organs: Phyllosticta multicorniculatais
adapted to the needles of Abies balsamea (Petrini1996), Cenangium
ferruginosum and Lophodermium
pinastri were primarily isolated from the needle tips ofPinus
mugo ssp. inicata, whereas Cyclaneusma minusoccurred most
frequently in the middle segmentsof the needles (Sieber, Rys &
Holdenrieder 1999).Other endophytes are conned to the bark, such
asMelanconium apiocarpum and a Cryptosporiopsis sp.in Alnus spp.
(Fisher & Petrini 1990). Pestalotiopsiscruenta and Phomopsis
spp were predominantly iso-lated from the twig xylem and bark of
Tripterygiumwilfordii, but not from the roots or leaves (Kumar
&Hyde 2004). The dark-septate endophytes (DSE), in-cluding
Phialocephala fortinii, Chloridium paucisporumand Phialophora spp.
(Jumpponen & Trappe 1998),the basidiomycete Piriformospora
indica (Varma et al.2000), Cryptosporiopsis radicicola (Kowalski
& Bartnik1995), and C. melanigena (Kowalski, Halmschlager&
Schrader 1998), both isolated from Quercus spp.,appear to be specic
to roots.
Adaptative responses in dual culture
In studying interactions of fungi and plant hosts, itis often
advantageous to employ simplied systems.Using dual cultures of
plant calli and endophytes,Peters et al. (1998a) found that in
interactions ofendophytes with their own hosts, metabolites
secretedby the host calli into the growth media resulted inpositive
growth responses of the endophytes (growthtowards the callus or
increased biomass). In dual cul-ture with the callus of a non-host,
this was not the case,suggesting that the endophytes responded to
specicstimuli produced by their respective hosts. Similarly,growth
of an endophyte, Cryptodiaporthe hystrix(Sieber, Sieber-Canavesi
& Dorworth 1990), and ofan EM fungus (Sirrenberg, Salzer &
Hager 1995,Sirrenberg 1996) was greater in dual culture with
callusof the host than it was with that of a non-host. Lu &Clay
(1994) found the same eect with the grassendophyte Aktinsonella,
and suggested that growth offungi correlates positively with host
compatibility.
Table 1. Percentage of colonized segments (measured as rate
of
reisolation) and disease symptoms of the shoots of 34 week
old
decapitated Phaseolus vulgaris plants 21 d after inoculation
with a
fungal spore in water suspension of endophytes from healthy
plants.
Host
endophytes
Non-host
endophytes
no. of isolates 26 19
% isolates that colonized plants 46 16
% colonized segments 69 7
% colonized plants with
symptomsa69 100
% non-colonized plants with
symptomsa45 100
a Symptoms were evaluated in comparison to the controls.
Cultivation at 20 x C, light/dark (16:8) and 100% rh.
Colonisation
was considered to be positive when the strain could be
reisolated
following surface sterilization (Schulz et al. 1993). 40100
segments
(30190 cm2) were evaluated for each interaction.
B. Schulz and C. Boyle 665
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Growth stimulation in the host-interactions studiedby Peters et
al. (1998a) seemed to be due to chemotaxicsignalling with
non-volatile substances (Peters 1998),and not to diusion of specic
nutrients, since the dualcultures grew on a complex medium. The
positivegrowth response of AM and EM fungi to their hosts isalso
chemotaxic, the fungi recognizing various ava-nols, CO2 or vaporous
substances of the host (Gemma& Koske 1988, Becard, Douds &
Pfeer 1992, Koske& Gemma 1992, Martin et al. 2001).The apparent
chemotaxic signalling involved in the
interactions with hosts suggests that these endophyteswere not
mere incidental opportunists in their hostsand that there has been
an evolutionary adaptationbetween some endophytes and their
hosts.
Variability of the interaction
Whether an endophyte grows asymptomatically withinits host or
its colonisation leads to disease depends notonly on adaptations to
a particular host or organ, onthe development stages of the
partners, but also onthe innate but variable virulence of the
endophyte,the host defence response, and on environmental
con-ditions, i.e. the disease triangle. For example, exper-imental
or suboptimal environmental conditions maystress the host and thus
weaken its defence status, re-sulting in disease (Kuldau &
Yates 2000). Under con-ditions of stress, inoculation of endophytic
host isolates(fungi that do not normally cause disease symptomsin a
particular host) onto leaf segments and decapitated(Table 1) and
axenically cultured plants (Schulz et al.1998) resulted in disease
symptoms (necroses, chloroses)and(or) growth inhibition of the host
with most ofthe isolates. The degree of virulence was not
coupledwith particular genera or species of the endophytes.Thus,
being isolated as an endophyte does not excludethe possibility that
a fungus may become pathogenicwhen the host is stressed or
senescent (Kuldau & Yates2000).A fungus occupying asymptomatic
plant tissue may
be a weak pathogen or a virulent strain detected duringlatency,
or possibly just an inhabitant of a niche wait-ing for an
opportunity to propagate. The predisposi-tions of the partners and
the environmental conditionsboth inuence the balance between host
and endo-phyte.
Adaptation to the endophytic life history strategy?
The question now arises : Do the fungi that are isolatedas
endophytes from healthy plant tissue adapt to thatparticular
biotope or niche? The fungi detected at anyone moment in
asymptomatic plant tissue and arbi-trarily termed endophytes
include fungi with dierentlife-history strategies. Some of them
might be patho-gens in a non-host as was suggested by Carroll
(1999),quiescent or latent pathogens, saprophytes hiding,e.g. in a
stomatal cavity and awaiting host senescence
as spores, or virulent pathogens in a latent phase. Eachof these
has a dierent strategy. Or perhaps they aremere incidental
opportunists, fungi normally occurringon other substrates and not
really capable of long-termoccupation of the particular tissue.
That at least someof the fungi frequently reported as endophytes
belongto this group is suggested by the taxa to which manyof these
fungi belong, e.g. taxa otherwise known onlyfrom herbivore dung.
Nevertheless, that certain fungican apparently establish a
long-term occupation oftissues or organs of certain host plants
without causingsymptoms of disease suggests that these fungi
areadapted to an endophytic life strategy with at leastsome
characteristics shared by a diverse group of fungi.Guske et al.
(1996) and Guske, Boyle & Schulz (1999)provided evidence in
support of this by conducting abroad screening (leaf segment tests,
intact plants ingrowth chambers, and eld experiments with
pottedplants) of fungi isolated from healthy (endophytes)
anddiseased Cirsium arvense thistles. Under conditions ofstress for
the host tissue (leaf segment tests, growthchamber experiments),
inoculation with some of theendophytes resulted in disease
symptoms. Under eldconditions, in contrast to the isolates from
diseasedplants, none of the endophytes caused disease (Guskeet al.
1999, Schulz et al. 1998, Guske 2002, Guske,Schulz & Boyle
2004). Thus, these endophytic isolateswere either adapted to the
living plant niche, were in-cidental opportunists, or latent
pathogens that causeddisease under conditions of stress. Comparable
resultswere presented by Photita et al. (2004) with
endophytesisolated from Musa acuminata, and by Mostert et al.(2000)
who isolated Phomopsis viticola and an un-identied Phomopsis sp.
from the shoots and leaves ofVitis vinifera. Only P. viticola, but
not the Phomopsissp., caused disease following inoculation into
healthyhosts. Thus, the Phomopsis sp. seemed to have adaptedto the
endophytic growth strategy. P. viticola, in turn,was considered to
be a latent pathogen. The dierencebetween a pathogen and an
avirulent endophyte maydepend on only one gene. Freeman &
Rodriguez (1993)and Redman, Dunigan & Rodriguez (2001)
demon-strated this by inducing mutations in Colletotrichummagna and
other Colletotrichum spp. They obtainedseveral mutants that were no
longer virulent towardshosts of the former pathogen, and that
diered by onlyone gene each from the wild-type.
SECONDARY METABOLITES
Secondary metabolites, or extrolites (dened bySamson &
Frisvad 2004 as outwardly directed com-pounds produced during
dierentiation of a livingorganism), have mainly been isolated and
characterizedfor industrial purposes (Dreyfuss & Chapela
1994,Tan & Zou 2001, Schulz et al. 2002, Strobel 2003).
Formycologists studying fungal ecology, it is apparentthat
secondary metabolites play a role in vivo andare, for example,
important for numerous metabolic
The endophytic continuum 666
-
interactions between fungi and their plant hosts, suchas
signalling, defence, and regulation of the symbiosis.However, with
the exception of the balansiaceousendophytes, little work has been
done to study therole of secondary metabolites in the
endophyte-hostinteraction.
Role of secondary metabolites within the host
Tan & Zou (2001) reviewed the diversity of metabolitesthat
has been isolated from endophytic fungi, empha-sizing their
potential ecological roles. That plants whoseroots are colonized by
endophytes often grow fasterthan non-infected ones may be due to
the synthesisof phytohormones and other growth-promoting
sub-stances by the fungi (Petrini 1991, Tudzynski 1997,Tudzynski
& Sharon 2002), as had previously beenfound for pathogenic
fungi (Pegg & Ayres 1984).Bargmann & Schoenbeck (1992),
Schulz et al. (1999a, b)and Tan & Zou (2001) also emphasized
that endo-phytic colonisation may improve the hosts
ecologicaladaptability by enhancing tolerance to
environmentalstresses, and by producing antimicrobial
metabolitesagainst phytopathogens (Schulz et al. 1995, 2002)
andpredators (Azevedo et al. 2000, Liu et al. 2001). Astrikingly
high proportion of endophytic fungi (80%)produce biologically
active compounds in tests forantibacterial, fungicidal and
herbicidal activities(Schulz et al. 2002). In spite of plants
colonised byendophytic fungi not exhibiting overt disease
symp-toms, of the fungal isolates from healthy plants, 43%expressed
herbicidal activities, compared to only 27%of the phytopathogenic
isolates, 25% of the epiphytes,18% of the soil isolates, and 13% of
the isolates frommacroalgae (Schulz et al. 1999b, 2002). Are
theseantagonistic substances also synthesized in vivo? Whatrole
could they play in the interaction?To our knowledge, rugulosin is
the only endophytic
secondary metabolite that has been shown to be syn-thesized
within its host, Scots pine, Pinus sylvestris(Miller et al. 2002).
This is presumably due to endo-phytic colonisation by the
non-balansiaceous endo-phytes of the shoots generally being
limited, so thatconcentrations of the metabolites in the tissues
arelow, in contrast to colonisation by Neotyphodium/Epichloe of
grasses, and Fusarium verticillioides ofmaize, where the
concentrations of the alkaloids andfumonisin, respectively, are
measurable, and colonis-ation is extensive (Leuchtmann 1992,
Schardl &Phillips 1997, White et al. 2000, Miller 2001).To
study the role of secondary metabolites in the
endophyte-host interaction, Peters et al. (1998a) andGotz et al.
(2000) confronted endophytes with theirown host calli in dual
culture : Lamium purpureumwithConiothyrium palmarum, Teucrium
scorodoniawithPhomopsis sp., and Phaseolus vulgaris with
Fusariumsp. The calli secreted metabolites that positivelyinuenced
fungal growth (see p. 6656), but becamenecrotic and died before the
fungi had contacted the
calli, suggesting that metabolites toxic to the callihad been
secreted by the endophytes into the medium.This can also be the
case when a host callus is con-fronted with a pathogen (Peipp &
Sonnenbichler 1992).Addition of endophytic culture extract to the
growthmedium resulted in similar necrosis of the callus invarious
interactions (Peters, Dammeyer & Schulz1998b, Hendry, Boddy
& Lonsdale 1993).Not only culture extracts, but also isolated
secondary
metabolites of Coniothyrium palmarum (palmaru-mycins; Krohn et
al. 1994b, 1997a) and Phomopsis sp.(phomopsins and biarylethers;
Krohn et al. 1996) weretoxic to host and non-host seedlings, and to
the algaChlorella fusca (Peters et al. 1998b), indicating thatthe
metabolites produced by the endophytes were nothost-specic. This
was also the case for 94% of thesecondary metabolites isolated from
culture extractsof other endophytes (Krohn et al. 1994b, 1996,
1997a,Schulz et al. 1999b, 2002).Since the secondary metabolites
isolated from
non-balansiaceous endophytic fungi belong to diversestructural
groups (Schulz et al. 2002), the herbicidalactivity is not due to
one or more substances commonto all of these endophytic fungi.
Additionally, as his-tological studies have shown (e.g. Boyle et
al. 2001,Deckert et al. 2001), in many interactions colonisationof
the above-ground organs remains limited, suggest-ing that the
concentrations of metabolites are notnormally adequate to result in
overt disease expression.Nevertheless, it seems probable that these
metabolitesplay a role within the host, and (or) have an
ecologicalsignicance. As hypothesized by Demain (1980) : if afungus
can produce metabolites in vitro, they must alsohave a function in
nature. The multienzyme reactionsequences required for the
synthesis of secondarymetabolites would not be retained by fungi
withoutsome benecial eect for survival. That the diversesecondary
metabolites of endophytes (and other fungi)have an ecological
function is also supported by bothphytopathogenic and soil
inhabiting fungi producingbiologically active secondary metabolites
in vitro andin situ (Demain 1980). Presumably, in an
endophyticinteraction, a nely balanced interaction
hinderspathogenicity. We hypothesize that as long as
fungalvirulence and the host defence reaction are balanced,disease
does not develop (Fig. 1), and that this is thecase regardless of
the life-history strategy of the fungalendophyte.In order to
determine mechanisms of algicidal
and herbicidal inhibitions, Peters & Schulz (unpubl.)tested
the eects of culture extracts of endophyic fungion the oxygen
production of Chlorella fusca. Onlyextracts found to be anti-algal
in preliminary tests in-hibited photosynthesis, as measured by the
productionof oxygen; respiration was not inhibited. Costa Pintoet
al. (2000) observed similar inhibitions of photo-synthesis in two
crop plants when symptomlesslycolonized by endophytes : Fusarium
verticilloides inmaize and Colletotrichum musae in banana.
B. Schulz and C. Boyle 667
-
Pharmaceutical and agrochemical products
Natural products continue to be an important sourceof new
pharmaceutical products (Dreyfuss & Chapela1994, Proudfoot
2002). Considering that six out of 20of the most commonly
prescribed medications areof fungal origin (Gloer 1997), and that
only y5% ofthe worlds fungi have been described (Hawksworth1991,
2001), fungi oer an enormous potential fornew pharmaceuticals. In
optimising this search, it isrelevant to consider that : (1) the
secondary metabolitesa fungus synthesizes may correspond to its
respectivetaxon and ecological niche, e.g. the mycotoxins of
plantpathogens (Dreyfuss & Chapela 1994, Gloer 1997); and(2)
metabolic interactions may enhance the synthesis ofsecondary
metabolites. Thus, endophytic fungi are onesuch source for
intelligent screening. As the biologicalactivities of fungi may
vary with the biotope fromwhich they are isolated (Dreyfuss &
Chapela 1994,Osterhage et al. 2000, Schulz et al. 2002), it is
relevantto consider the habitat from which to isolate, as wellas
recalling that perennial plants growing in tropical orsemitropical
areas are hosts to a greater diversity ofendophytes than those
growing in drier or colderclimates (Bills & Polishook 1994,
Arnold et al. 2000,2001, Strobel 2003).The isolated metabolites of
endophytic fungi belong
to diverse structural groups, including steroids, xan-thones,
phenols, isocoumarines, perylene derivatives,quinones, furandiones,
terpenoids, depsipeptides, andcytochalasines (Krohn et al. 1992a,
b, 1994a, b, 1996,1997a, b, 2002, Konig et al. 1999, Schulz et al.
2002).They are primarily synthesized via the polyketidepathway from
mevalonate-derived C5 units and(or)using the non-ribosomal protein
synthesis. In bothcases clustered genes are involved (Tkacz 2000).
Some
of these metabolites represent novel structural groups,for
example the palmarumycins (Krohn et al. 1997b)and a new
benzopyroanone (Krohn et al. 2002). Thatthe proportion of novel
structures produced by endo-phytes (51%) is considerably higher
than that producedby soil isolates (38%), demonstrates that
endophytesare indeed a good source of novel secondary meta-bolites
(Schulz et al. 2002), and again suggests thatthese metabolites play
a role in the endophytic life-history strategy. The
biotechnological use of thesemetabolites for pharmaceutical or
agrochemical prod-ucts is in the developmental stage. For example,
itis conceivable that rugulosin, produced by a non-sporulating
endophyte of the spruce and active againstthe spruce budworm
(Miller et al. 2002) could beproduced commercially.
COLONISATION
Endophytic colonisation may be intracellular andlimited to
single cells, intercellular and localized,systemic and both inter-
and intracellular (Stone et al.2000), be limited to the roots as is
the case with theDSE (Sieber 2002), be conned to the leaves or
needles(e.g. Lophodermium spp., Deckert et al. 2001; orRhabdocline
parkeri, Stone 1986), be intercellular bothin the roots and shoots
(Fusarium moniliforme, Bacon& Hinton 1996), or adapted to
growth within thebark (e.g. Melanconium apiocarpum in Alnus, Fisher
&Petrini 1990). The fungi may infect with appressoriaand
haustoria (e.g. Discula umbrinella, Stone et al.1994), penetrate
directly through the cell wall (e.g.R. parkeri, Stone 1987), or
enter the host throughthe stomata and substomatal chamber (e.g.
Phaeo-sphaeria juncicola, Cabral et al. 1993).
Fig. 1. Hypothesis : a balance of antagonisms between endophytic
virulence and plant defence response results inasymptomatic
colonisation.
The endophytic continuum 668
-
Foliage and shoots
Although colonisation of the above-ground organsis often
considered to be primarily local (Stone et al.1994, Carroll 1995),
this assumption is based on onlyfew histological studies. For
example, in needles ofPseudotsuga menziesii, Rhabdocline parkeri
occurs asdiscrete intracellular infections, limited to single
epi-dermal cells. It only resumes growth saprophyticallyfollowing
death of the needles (Stone 1987). Cabralet al. (1993) found
infections in Juncus spp. of Clado-sporium cladosporioides to be
restricted to the sub-stomatal chamber, whereas those of
Phaeosphaeriajuncicola developed as limited intercellular
infections.Similarly, growth of Lophodermium spp. in needles
ofPinus strobus is localized, cryptic and intercellular, withhyphae
often growing in coils, presumably to improvenutrient uptake. When
senescence of the host permitsexpansion, pathogenic growth
commences (Deckertet al. 2001); a pattern characteristic of many
endo-phytes. Unlike the balansiaceous endophytes, the
non-balansiaceous ones may cause disease after a period oflatency
(Petrini 1991) and often reproduce only uponand/or after senescence
or death of the host (Sinclair& Cerkauskas 1996).To study the
eects of an intercellular endophytic
colonisation on the constituents of the apoplasticwashing uid
(AWF), Boyle et al. (2001) inoculatedFusarium spp. endophytes onto
the shoots of seedlingsof Hordeum vulgare and Phaseolus vulgaris.
Theseinfected both the leaves (Fig. 2a) of host plants and acallus
of P. vulgaris (Fig. 2b) without penetrating theepidermal cell
walls, only growing intercellularly (aprerequisite for AWF studies)
and without causingdisease symptoms. Even though growth was
inter-cellular, colonisation by the Fusarium endophyteshad no
signicant eects on the constituents of theAWF: glucose, fructose,
sucrose and invertase. Incontrast, colonisation by a pathogen
(Drechslera),which was extensive (histological observation)
andresulted in disease symptoms, led to signicant in-creases in the
concentrations of invertase and glucose(Boyle et al. 2001). We
speculate that limited colon-isation, as indicated both by
histological examinationand ELISA (Boyle et al. 2001), explains why
endo-phytic growth did not aect the constituents of theAWF in
planta.To check for adaptation to intercellular growth,
the isolates were cultivated in various growth mediaincluding
AWF. The dry weights (D.W.)of these endo-phytic Fusarium strains
was signicantly higher inthe AWF than in a complex medium, or in a
mineralmedium with the same carbohydrate concentrations asin the
AWF. Due to the varied sugar concentrationsin the media used, it
was clear that sugar was not alimiting growth factor. Additional
growth factors orinducers only present in sterile ltered AWF
apparentlyenhanced fungal growth, indicating adaptation (Schulzet
al. 2002). These results correlate well with those
of Schmid et al. (2000) who investigated growth ofNeotyphodium
in planta.
Roots
In contrast to colonisation of the shoots, endophyticgrowth
within the roots has frequently been found to beextensive. Root
colonisation can also be both inter- andintracellular, the hyphae
often forming intracellularcoils, e.g. by the DSE (Jumpponen &
Trappe 1998,Stone et al. 2000, Sieber 2002) or by the
basidiomycetePiriformospora indica (Varma et al. 2000). In
conifers,DSE may produce ectomycorrhizal-like structures(Wilcox
& Wang 1987). Many orchid roots are sys-temically colonized by
fungi of the genus Rhizoctonia(Ma, Tan & Wong 2003, Brundrett
2005), and Lepto-dontidium (Bidartondo et al. 2004). The
endophyticcolonisation of maize by an avirulent isolate of
Fusa-rium verticilloides was systemic and intercellular,whereas
pathogenic strains also colonized intracellu-larly (Bacon &
Hinton 1996).For histological studies of endophytic root
colonis-
ation, Schulz et al. (1998, 1999b) inoculated axenicallycultured
roots of Larix decidua seedlings and Hordeumvulgare with
endophytes. Light microscopic examin-ation demonstrated that the
endophytes Cryptosporiop-sis sp. (Fig. 3a) and Phialocephala
fortinii (Fig. 3b)colonized the roots of L. decidua, and Fusarium
sp.those of H. vulgare (Fig. 3c) extensively and both inter-and
intracellularly (Schulz et al. 1999b). Colonisationresulted neither
in growth inhibition nor diseasesymptoms. Similar colonisation
patterns have beenreported in various hosts for DSE other than P.
fortinii,e.g. Phialophora spp., other Phialocephala spp.,
Chlor-idium paucisporum and Leptodontidium orchidicola(Jumpponen
& Trappe 1998, Sieber 2002). The rootendophytic fungus of
cabbage, Heteroconium chaeto-spira, which apparently induced
resistance of thehost to pathogens, grew intra- or intercellularly
inthe root cortical cells (Narisawa, Tokumasu &Hashiba 1998,
Ohki et al. 2002). Cryptosporiopsis sp.occasionally penetrated the
vascular bundles (Fig. 3d;Schulz et al. 1999), which is perhaps not
surprisingsince some Pezicula, and its anamorph Crypto-sporiopsis,
species are latent pathogens (Kehr 1992,Verkley 1999).In
conclusion: endophytic colonisation of the shoot
and root seem to dier. For most of the endophytesthat have been
investigated to date, colonisation ofthe shoot is either
intracellular and then conned toindividual cells or intercellular
but localized. Colon-isation of roots by endophytes, on the other
hand, isusually extensive but may also be inter- or
intracellular.Specialized structures that are presumed to improve
theexchange of metabolites have been observed in bothshoots and
roots. Presently, one can only speculateon the reasons for these
dierent colonisation patterns,since many factors may be involved,
e.g. anatomicaldierences, source-sink relationships, dierences
in
B. Schulz and C. Boyle 669
-
permeability or nutrients supplied by the micropartneror by the
host.
ENDOPHYTES AND HOSTS : FRIENDSOR FOES ?
As discussed above, in dual culture plant calli
secretedmetabolites into the growth medium, resulting in
positive growth responses of the endophyte to thehost. However,
the interaction is more complex : bothendophytes and hosts secreted
metabolites into thegrowth medium that were toxic to the respective
part-ner (Peters et al. 1998a, b).It seems strange that in an
ostensibly asymptomatic
interaction, each of the partners produces metab-olites
potentially toxic to the other (Table 2). In the
A
B
Fig. 2. Intercellular infection and colonisation of Phaseolus
vulgaris leaves with endophytic Fusarium sp., stained withthionine.
(A) infection via the stomata, followed by intercellular growth;
and (B) intercellular growth in bush bean callus.
Bars=20 mm.
The endophytic continuum 670
-
AC D
B
Fig. 3. Inter- and intracellular endophytic colonisation of the
roots of Phaseolus vulgaris and Larix decidua, stained with
thionine. (A) Cryptosporiopsis sp. in the cortex of the rootsof L.
decidua; (B) inter- and intracellular growth of Phialocephala
fortinii in the cortex of the roots of L. decidua; (C) only
intercellular growth of Fusarium sp. in the roots ofP. vulgaris ;
and (D) Cryptosporiopsis sp. penetrates the stele of L. decidua.
Bars=20 mm.
B.SchulzandC.Boyle
671
-
Table 2. Fungal virulence vs plant defence in successful
non-obligately biotrophic fungalhost interactions (root and(or)
shoot).
Result Plant : eector response
Pathogenvirulence factor
Plant defencemechanisms
Endophytevirulence factor
Plant : eector response
Result
infection induction ofpapillae,callose
exoenzymes fordegradation
mechanical barriers:wax, cuticule, cell wall
exoenzymes fordegradation,infection
usually noinductionof barriers
penetration, infection,balanced antagonism
infection,colonisationdisease
degradation,necroses
exoenzymes fordegradation
preformed secondarymetabolites
exoenzymes fordegradation
no degradation infection, colonisation,tolerance,
balancedantagonism
infection,colonisationdisease
degradation,necroses
elicitors,exoenzymes fordegradation
induced defence metabolites,including phytoalexins
elicitors no degradation colonisation, tolerance,balanced
antagonism
colonisation,disease
none elicitors induced fast defencereactions
elicitors none colonisation, tolerance,balanced antagonism
colonisation,disease
none elicitors induced slow defencereactions
elicitors none colonisation, tolerancebalanced antagonism
colonisation,disease
necroses elicitors hypersensitive reaction no elicitation none
colonisation, tolerancebalanced antagonism
colonisation,disease
inhibitions ofphotosynthesisand metabolism
phytotoxicmycotoxins
physiologically active tissue phytotoxicmycotoxins
limited inhibitionof photosynthesis
balanced antagonism
Theendophytic
contin
uum
672
-
interaction of fungal phytopathogen and host, it is wellknown
that the fungus may produce metabolites toxicto the host. The plant
in turn may possess preformeddefence metabolites and, upon
encounter with a fungalinvader, may activate a variety of defence
reactions,including not only mechanical defence, e.g. callose
andpapillae, but also induced defence metabolites (Agrios1997). The
questions are : To what extent is an endo-phytic fungus virulent in
the asymptomatic interactionof endophyte and host? And does the
host respond tothe endophytic colonisation with a defence response
asit does to a pathogen?
Fungal virulence
Only few fungi are actually capable of causing diseasein any one
plant (Heath 1997), since they must rstcross several barriers and
overcome plant defences.Pathogens accomplish this with their
virulence factors,phytotoxic secondary metabolites and
exoenzymes(Agrios 1997). As discussed above, many fungal
endo-phytes produce phytotoxic metabolites in vitro thatare eective
against algal and plant test organisms.In a test for potential
virulence in vivo, inoculation of
most of the screened non-host and host endophytesonto the shoots
of Phaseolus vulgaris caused diseasesymptoms (Table 1). Since some
of these endophytescaused disease symptoms presumably without
eveninfecting the P. vulgaris seedlings (they could not
bereisolated), phytotoxic metabolites and/or exoenzymeshad
apparently been secreted by the fungi epiphytically.All of the
non-host endophytes, i.e. endophytes isolatedfrom dierent plant
species than they were tested on,were known to produce biologically
active secondarymetabolites in culture and caused disease symptoms
inthe P. vulgaris. This suggests that the active metaboliteswere
also being produced following inoculation.Similar results were
presented by Guske et al. (1996)and Schulz et al. (1998) : Half of
the endophytic isolatesfrom apparently healthy Cirsium arvense
thistles causednecroses in leaf segment tests, showing that a
highproportion of the endophytes have the potential tosynthesize
phytotoxic metabolites in contact with thehost.Exoenzymes can also
be virulence factors. As is the
case for pathogenic fungi, in substrate utilisation testsmost
endophytes were able to metabolise in vitro mostsubstrates found on
the surfaces or in the cell wallsof plants, synthesizing proteases,
amylase, phenoloxi-dases, lipases, laccases, polyphenol oxidases,
cellulase,mannase, xylanase and pectin lyase (Sieber et al.
1991,Petrini et al. 1992, Ahlich-Schlegel 1997, Boyle et al.2001,
Lumyong et al. 2002). It is unclear to what extentthe endophytes
use these to decompose organic debrisin the natural environment
(Jumpponen & Trappe1998) and to what extent they are required
for infectionand colonisation. In studying the infection of
beechleaves with the endophyte Discula umbrinella, Viret
&Petrini (1994) obtained microscopic evidence that
these enzymes were synthesized in vivo to infect thehost. All of
the tested endophytic fungi produced exo-enzymes and a high
proportion produced toxins. Butwhy did disease not develop during
colonisation oftheir hosts?
Plant defence reactions
Stone et al. (1994) hypothesized that active host
defencereactions triggered by initial invasion are responsiblefor
restricting endophytic colonisation. Since theninvestigations both
in simplied systems and on intactplants have found active host
responses : induced de-fence metabolites, and induced fast and slow
defencereactions sensu Hahlbrock et al. (1995). A summaryof plant
defence vs fungal virulence is presented inTable 2.
Induced mechanical defence responses
In most interactions no mechanical defence responseswere
observed (e.g. Stone 1986, 1987, Schulz et al.1999b, Bacon &
Hinton 1996, Boyle et al. 2001). How-ever, the formation of
papillae was observed in cellsadjacent to the infection sites of
Stagonospora innu-merosa and Drechslera sp. in Juncus eusus
(Cabralet al. 1993). Narisawa, Usuki & Hashiba (2004) foundcell
wall appositions and thickenings in roots ofChinese cabbage
colonized by an unidentied DSE,whereas Yates, Bacon & Hinton
(1997) found accel-erated lignin deposition in asymptomatic
seedlings ofmaize inoculated with Fusarium verticilloides.
Induced biochemical defences
Peroxidase activity and H2O2 production are diagnosticfor the
fast defence response. Such hypersensitive re-actions were
suggested to explain the accumulationof electron-dense compounds in
the epidermal cells ofbeech colonized by Discula umbrinella (Viret
& Petrini1994). Since then fast defence responses have
beendemonstrated in three endophyte-host interactions.Peters et al.
(1998b) found increased H2O2-productionnot only following
elicitation in suspension cultures ofLamium purpureum with a
pathogen, but also with anendophyte. Boyle et al. (2001) showed
that peroxidaseactivity in the apoplastic washing uid (AWF) ofthe
shoots of Phaseolus vulgaris and Hordeum vulgareincreased bothwhen
hosts were colonized intercellularlyby a pathogen or with an
endophyte. Bishop (2002)found increased peroxidase activity and the
inductionof three novel cationic peroxidase isoenzymes in theAWF
when wheat was infected with an endophyte,but not when colonized by
a pathogen (Bishop 2002).In situ hybridization analysis revealed
that accumu-lation of the related mRNA transcripts coincided
withthe localized areas of F. proliferatum infection. Thus,it seems
that an increase in the fast defence responses
B. Schulz and C. Boyle 673
-
may play a role in limiting growth and virulence ofat least some
endophytes.Phenolic metabolites, generally toxic to microorgan-
isms, are involved in plant defence reactions (Schlosser1997).
They may be preformed or induced, soluble orcell wall bound.
Deposition of phenolics in response toinfection by endophytes was
observed by Stone (1988)and Cabral et al. (1993), who reported the
accumu-lation of phenolics and pigmentation of infected
cellsadjacent to the infection.Phenylalanine ammonium lyase (PAL)
is one of
the marker enzymes for phenolic defence responses(Vidhyasekaran
1997). Peters et al. (1998b) found thatPAL-activity and the
concentrations of soluble pheno-lic metabolites increased following
confrontation indual culture of endophytes with seedlings of
Lamiumpurpureum and in elicited suspension cultures, wherethe cells
must have reacted to structural componentsof the fungal cell walls,
e.g. glycoproteins, glycolipids,or oligosaccharides (Scheel &
Parker 1990).The concentrations of oligomeric proantho-
cyanidins, which function as preformed defence meta-bolites
(Schlosser 1997, Staord 1997) and may serveas a barrier to fungal
penetration (Pankhurst, Craig& Jones 1979), increased during
endophytic colonis-ation of roots of Larix decidua (Schulz et al.
1999b)and Mentha piperita (Mucciarelli et al. 2003).
Theconcentrations of proanthocyanidins also increased ina
mutualistic symbiosis, during the mycorrhization ofL. decidua with
Suillus tridentinus or Boletinus cavipes(Weiss et al. 1997).In
Hordeum vulgare roots the increase of phenyl-
propanoids was greater following infection with anendophyte than
with a pathogen (Schulz et al. 1999b).Bishop et al. (2002) detected
lignin and(or) otherwall-bound aromatic aldehydes at the sites of
plant-fungal contact in the epidermal, mesophyll and vas-cular
bundle sheath cells of wheat when colonized byan endophyte. Lignin
was also rapidly deposited as aresponse to non-pathogenic vs
pathogenic fungal col-onisation of potato (Hammerschmidt 1984),
againdemonstrating the dierent defence responses to endo-phytic and
pathogenic infections and the necessity fordierentiated strategies
of fungal response (Bishopet al. 2002).
Host defence and mutualistic interactions
Host defence reactions have been reported to occur inmutualistic
interactions both with arbuscular (Allen1992) and ectomycorrhizal
fungi. Elicitors of the ecto-mycorrhizal fungus Heboloma
crustuliniforme in con-tact with Picea abies induced signalling
processes thatare regarded as the initial events of a
hypersensitiveresponse (Schwacke & Hager 1992, Salzer et al.
1996).However, the ectomycorrhizal fungi apparently sup-pressed
some of the defence responses (Martin et al.2001). In the AM-host
association digestion or collapseof the arbuscules may have rst
evolved as a defence
against pathogenic fungi (Brundrett 2002). Recently,Hause et al.
(2002) speculated that the elevated levelsof jasmonic acid in
Hordeum vulgare roots infectedwith Glomus intraradices occurring
during myco-rrhization may enhance the defence status of the
host.This might also occur as a response to colonizationby some
endophytes.But nevertheless, the phenolic defence response to
endophytic fungi may dier from that to mycorrhizalfungi. For
example, the sesquiterpenoid cyclohexenonederivative blumenin is
induced during the associationof H. vulgare with the AM-fungus G.
intraradices, butneither with fungal pathogens nor with an
endophyte(Maier et al. 1997). These examples suggest that eachof
these fungi, pathogen, endophyte, AM, and EM,has developed its own
life-history strategy to interactwith its host, ultimately assuring
its own survival andreproduction.
Balanced antagonism
One question has motivated many investigations : Howdoes the
fungal endophyte manage to exist and oftengrow within its host
without causing visible diseasesymptoms? In the following we
propose a workinghypothesis based on observations from the
interactionsthus far studied. A comparison of these
observationswith respect to fungal virulence factors and
hostdefence responses is summarized in Table 2.We hypothesize that
asymptomatic colonisation is a
balance of antagonisms between host and endophyte(Fig. 1).
Endophytes and pathogens both possess manyof the same virulence
factors. The endophytes studiedproduce the exoenzymes necessary to
infect and col-onize the host, even though only some of these
arepresumably latent pathogens. The majority can pro-duce
phytotoxic mycotoxins. The host can react withthe same defence
reactions as to a pathogen, i.e. withpreformed and induced defence
metabolites, mechan-ical defence responses, slow and fast defence
responses.The fact that neither of the partners gains the
upper-hand in the interaction need not be seen as a defector aw of
either of the partners, but rather may be asurvival strategy, for
example (a) of the fungi thatquiescently await host senescence in a
single cell orlocally between cells only subsequently
continuinggrowth as a saprophyte interpreted as true endo-phytes ,
(b) of the DSE that systemically colonize theroots, often as
mutualistic symbionts, or (c) but alsoof the latent weak pathogens,
which slowly producethe critical biomass which enables virulence.
Whereassome endophytes are specically adapted to theirrespective
hosts, others are incidental opportunists.Assemblages of the latter
vary with the respectivelocation, season, and vegetation
surrounding thehost. Nevertheless, their interactions with their
hostsmay also be both balanced and antagonistic, ineect resulting
in a tolerance of the inhabitant. In allof these interactions we
are referring to a momentary
The endophytic continuum 674
-
status , an often fragile balance of antagonisms.Examples of
asymptomatic endophytic colonisationwith varied modes of growth to
which we believe thishypothesis of balanced antagonism may be
appliedinclude:
Foliage and shoots
. Infections are intracellular and limited to single
cells:Stagonospora innumerosa in Juncus eusus (Cabralet al. 1993)
and Rhabdocline parkeri in Douglas r(Stone 1987).
. Colonisation is intercellular, discrete and localized:Fusarium
spp. in shoots of Hordeum vulgare andPhaseolus vulgaris (Boyle et
al. 2001), Lophodermiumsp. in Pinus strobus (Deckert et al. 2001).
Hyphaeare thin and may develop coils to better absorbavailable
nutrients.
. Colonisation is intercellular and systemic within theshoot:
Neotyphodium sp. in grasses (Schardl & Clay1997), Fusarium
moniliforme in maize (Bacon &Hinton 1996).
Roots
. Colonisation is inter- and intracellular and
extensive:Cryptosporiopsis sp. (Schulz et al. 1999b),
Phialo-cephala spp. (Schulz et al. 1999b, Sieber 2002), otherDSE
(Sieber 2002), Piriformospora indica (Varmaet al. 2000), AM-fungi
(Alexopoulos, Mims &Blackwell 1996).
. Colonisation is intercellular and systemic withinthe roots :
Fusarium moniliforme in maize (Bacon &Hinton 1996) and EM fungi
(Alexopoulos et al.1996).
The host-pathogen interaction becomes imbalanced,resulting in
disease (Fig. 1), in contrast to the endo-phyte-host interaction in
which the partners maintaina mutual balance of antagonism.
Nevertheless, it isunclear how this balance is regulated. The
herbicidalmycotoxins may play an important role,
inhibitingphotosynthesis or increasing membrane permeabilityto
improve apoplastic uptake of sugars. Some fungimay be able to
regulate host defence. Perhaps specicrecognition is involved
(Chapela et al. 1991), or thehyphae are able to escape recognition,
e.g. by develop-ing very thin hyphae or altering composition of
thecell wall. Nevertheless, should this balance be disturbedin
favour of the fungus, the endophyte may becomepathogenic, for
example when host defence is weakened(Schulz et al. 1998). In situ
the virulence of weak patho-gens such as Pezicula spp. (Kehr 1992)
is only sucientfor disease development when the host is stressed
orsenescent. The balance of the interaction also dependson the
defence responses of the host. In one host dis-ease develops, in
another it does not (Jumpponen 2001,Sieber 2002). Whether the
interaction is balanced orimbalanced depends on the virulence of
the fungus and
defence of the host, both virulence and defence beingvariable
and inuenced by environmental factors anddevelopmental stages of
the partners.The fragility sometimes characteristic of this
balance is also demonstrated by results of Redman,Rodriguez, and
co-workers, who made mutants ofnormally pathogenic Colletotrichum
spp. A singlemutation transformed a pathogen into an
endophyte,presumably due to the loss of a virulence factor(Freeman
& Rodriguez 1993, Redman, Ranson &Rodriguez 1999), for
example extracellular serineprotease (Redman & Rodriguez 2002).
Additionally, amutant that was virulent in one host was not
necess-arily virulent in another (Redman et al. 2001),
perhapsdemonstrating variability of the plant defence reactionor
lack of recognition.Balanced antagonistic interactions are plastic
in
expression, depending on the momentary status of hostand
endophyte, but also on biotic and abiotic environ-mental factors
and on the tolerance of each of thepartners to these factors. In
particular, many endo-phytes seem to be masters of phenotypic
plasticity: toinfect as a pathogen, to colonize cryptically, and
nallyto sporulate as a pathogen or saprophyte. This ne-cessitates a
balance with the potential for variabilitywhich means that these
endophytic interactions are alsocreative, having the potential for
evolutionary devel-opment ; the symbioses can evolve both in the
directionof more highly specialized mutualisms and in thedirection
of more highly specialized parasitisms andexploitation.
SYMBIOSIS AND MUTUALISM
This may seem to be, but is not the end of the story.The
asymptomatic endophyte-host interaction seemsto involve two
actively antagonistic partners. However,a balanced antagonistic
endophyte-host interactiondoes not exclude the possibility that the
endophyte mayplay a benecial role within its host, for example
byinducing defence metabolites potentially active againstpathogens
(Schulz et al. 1999, Arnold et al. 2003,Mucciarelli et al. 2003),
by secreting phytohormones(Tudzynski & Sharon 2002), by
supplying the host withnutrients from the rhizosphere (Jumpponen,
Mattson& Trappe 1998) and(or) by increasing the
metabolicactivity of the plant host. Colonisation by the
non-balansiaceous endophytes may lead to induced diseaseresistance,
improved growth of the host, and protectionagainst pathogenic
competitors and insect predatorsof the host by the synthesis of
antagonistic second-ary metabolites (Miller et al. 2002, Selosse,
Baudoin &Vandenkoornhuyse 2004).
Mutualisms of endophytes and plant roots
Previously, within plant roots only symbioses ofmycorrhizal
fungi were considered to be mutualistic.
B. Schulz and C. Boyle 675
-
Recently, it has been recognized that many otherfungi can
participate in mutualistic symbioses withthe roots of their hosts
(e.g. Brundrett 2002, Sieber2002).Colonisation of the roots of
Larix decidua seedlings
by endophytes (Phialocephala fortinii, Cryptosporiopsissp.)
signicantly improved lengths (Schulz et al. 2002)and dry weights
(Rommert et al. 2002) of both the rootsand the shoots, as did
application of a mycelial cultureextract of the P. fortinii to the
seedlings (Rommertet al. 2002). Additionally, disease symptoms
decreased(Rommert et al. 2002). Similarly, Varma et al.
(2000)reported that growth of the shoots of maize wasenhanced both
following root-infection with Piriformo-spora indica or treatment
with mycelial culture ltrates.It is possible that the enhancement
of growth of L. de-cidua was in part due to synthesis of indole
acetic acid(IAA) by the endophytes studied, since in vitro
bothsynthesized IAA (Rommert et al. 2002). However,plant hormones
are also produced by pathogens(Tudzynski 1997, Tudzynski &
Sharon 2002), includinga pathogen of L. decidua, Heterobasidion
annosum(Rommert et al. 2002).Interactions of some endophytes with
their hosts are
not only benecial for the host, but provide enoughnutrients for
the endophytes to extensively colonizethe hosts roots (Sieber 2002,
Schulz et al. 2002) andpotentially for growth in the rhizosphere,
which inturn could improve the hosts mineral and nutrientsupply as
mycorrhizal fungi do. Jumpponen (1999)suggested that improvement of
plant growth by theDSE may be due to improved phosphorous
uptake(Jumpponen et al. 1998) or, in a closed system, tothe
increased availability of carbohydrates and/or CO2,both resulting
from fungal metabolism (Jumpponen& Trappe 1998). Whether or not
colonisation byDSE improves growth of the host also depends onthe
host and its metabolic status. For example, in-oculation of the
roots of aseptically grown seedlingsof Carex rma and C. curvula
with DSE led to asignicant increase in production of dry matter
inC. rma but not in C. curvula (Haselwandter & Read1982). Like
AM, DSE may improve phosphoroussupply to the host and even replace
AM and ectomy-corrhizal fungi at sites with extreme
environmentalconditions (Sieber 2002). Similarly, Muller
(2003)found that colonisation of Lolium perenne withendophytes of
the Balansiaceae led to a signicantdecrease of mycorrhizal
colonisation. Phialocephalafortinii has even been found to form a
Hartig netand a thin patchy mantle, considered the
anatomicalhallmarks of ectomycorrhizae, in axenic culture ofSalix
glauca seedlings (Fernando & Currah 1996).DSE also formed good
mantles and Hartig netswith the roots of some of the nursery stocks
of Pinusbanksiana, P. contorta and P. glauca (Danielson &Visser
1990).In addition to DSE, Piriformospora indica (Varma
et al. 2000) and Cryptosporiopsis sp. (Schulz et al. 2002)
are non-mycorrhizal root colonizers that have beenshown to
improve growth of their hosts. Endophyticroot colonisation with
Fusarium spp. and Cladorrhinumfoecundissimum improved growth of
their respectivehosts (Gasoni & Stegman De Gurnkel 1997,
Kuldau& Yates 2000, Sieber 2002), with C. foecundissimumalso
increasing phosphorus uptake. Endophytic colon-isation of the roots
of Hordeum vulgare with Chaeto-mium spp. was found to increase root
fresh weight(Vilich, Dolfen & Sikora 1998); Phoma meti
enhancedroot and shoot biomass, root length, and tiller numbersof
Vulpia ciliata subsp. ambigua (Newsham 1994),and an endophyte of
Mentha piperita both promotedexpansion of the hosts root system and
increasedboth biomass and height (Mucciarelli et al. 2002,2003).
The authors speculate that this may be due tothe synthesis of plant
growth hormones by the fungusor better nutrient supply. Root
colonisation byendophytes may have other mutualistic advantages
forthe partners. The fungus benets by obtaining a
reliablenutritional source. But the hosts may benet from
theinteractions not only with improved growth. Inocu-lations of
various hosts with root endophytes increasedhost tolerance to
stress and induced resistance, asreported by Schoenbeck & Dehne
(1979), Bargmann& Schoenbeck (1992), Hallmann & Sikora
(1994),Redman et al. (2001), and Sieber (2002). This was,
forexample, the case when Fusarium spp. and Acremoniumendophytes
grew systemically and asymptomaticallywithin roots of their hosts
(Raps & Vidal 1996, 1998,Dugassa, Raps & Vidal 1998, Kuldau
& Yates 2000).And colonisation of the roots of the Chinese
cabbage(Brassica campestris) by 16 dierent endophytic fungi,in
particular Heteroconium chaetospira, almost com-pletely suppressed
disease of Plasmodiophora brassicae(Narisawa et al. 1998, Usuki et
al. 2002). Inoculationswith Piriformospora indica improved survival
ratesof tobacco seedlings when planted on polluted sites(Sahay
& Varma 1999). Jallow, Dugassa-Gobena &Vidal (2004) found
that endophytic colonisation ofthe roots was able to reduce larval
ingestion of thefoliage, demonstrating a systemic eect of
coloni-sation. Most interestingly, Redman et al. (2002) foundthat a
novel endophytic Curvularia sp. increased hosttolerance to
temperatures of up to 65 xC (!), evidencefor improved tness by
colonisation by endophytes.One possible mechanism that might help
account forthese diverse eects of endophyte infection is the nd-ing
that root colonisation of Brassica oleracea byAcremonium alternatum
altered the concentrations ofphytosterols in the leaves of the host
Dugassa et al.(1998).Morphologically and physiologically,
endophytic
root colonisations mirror the variability and thusplasticity of
endophytic interactions found at everylevel : of the individual,
the population and the genus,but also of the evolutionary
developmental stages fromendophyte to specialized mycorrhizal
fungus. Equally,they mirror the dierent possible life history
strategies,
The endophytic continuum 676
-
from mutualism to an exploitive strategy, both be-coming more
prevalent with increasing specialization(Brundrett, 2002).
Mutualisms of endophytes and shoots
Most of the reports concerning mutualistic inter-actions of
endophytes with the above-ground organsof their hosts concern
defence against insect herbivory(e.g. Wulf 1990, Vilich et al.
1998, Azevedo et al.2000, Bultman & Murphy 2000, Anke &
Sterner2002, McGee 2002). For example, local colonisationsof
Rhabdocline parkeri protect Douglas r againstattack by Contarinia
larvae (Sherwood-Pike, Stone& Carroll 1986). Even the culture
extracts of endo-phytes were found to be toxic to the spruce
budworm,Choristoneura fumiferana (Johnson & Whitney
1994).Miller et al. (2002) found that an endophyte fromthe needles
of white spruce produces rugulosin, alsotoxic to the spruce
budworm, and the only reportto date in which the metabolite has
been found to besynthesized in vivo. The other relatively few
reportsof mutualistic interactions with non-balansiaceousendophytes
are of those that colonize both the above-ground plant organs and
the roots. For example,colonisation with an avirulent endophytic
strain ofFusarium verticilloides signicantly increased dryweight of
maize seedlings (Yates, Bacon & Hinton1997). Mucciarelli et al.
(2002, 2003) found that colo-nization of Mentha piperita by a
non-sporulatingisolate resulted in taller plants and increased
dryweights of all plant organs. The ratio of leaf dry matterover
leaf area also increased, which according to theauthors, suggests
an improvement of host metabolismand photosynthesis.The advantage
of infection for plants may be that
endophytes are stimulated to increase production ofmycotoxins
after damage to the host has occurred(Bultman & Murphy 2000).
Another basis for mutual-ism could be induced resistance (Dean
& Kuc 1987, vanWyck, Scholtz &Marasas 1988, Wicklow 1988,
Kuldau& Yates 2000), as has also been found for
endophyticcolonisation of the roots (see above). Arnold et
al.(2003) found that inoculation of endophyte-freeleaves of
Theobroma cacao with endophytes resulted ininduced resistance of
the leaves to Phytophthora sp.However, not every endophyte-host
association withthe shoots induces measurable resistance. To
testwhether non-balansiaceous endophytes enhance hoststress
tolerance as Neotyphodium does to the shoots ofgrass (Cheplick
& Clay 1988, Belesky & Malinowski2000), Boyle et al. (2001)
pre-inoculated seedlings ofPhaseolus vulgaris with an endophytic
Fusarium sp. andsubsequently stressed the seedlings with UV, excess
nitro-gen fertilization, or shading, both in growth
chamber(semi-sterile conditions) and eld experiments. In thiscase,
pre-inoculation only slightly reduced diseasesymptoms of plants
subsequently inoculated with afungal pathogen.
Systemic colonisation and mutualism
Mutualistic endophytic associations have been reportedmore
frequently in associations with roots than withthe aboveground
plant organs, perhaps due to the factthat colonisation of the
aboveground organs is fre-quently localized, whereas that of the
roots is moreoften extensive and sometimes systemic (Stone et
al.2000, Sieber 2002). However, it is not possible to gen-eralize
that mutualistic associations of fungi with planthosts generally
involve systemic or extensive root in-fections. For example, there
are mutualistic systemiccolonisations of above-ground organs: (1)
of grasseswith Neotyphodium giving the hosts the same benetsas
systemic root colonisation (Cheplick & Clay 1988,Schardl &
Clay 1997, Belesky & Malinowski 2000,Bultman &Murphy 2000,
Schardl et al. 2004) ; but also(2) limited infections of the shoots
that can result ininduced resistance (see above).The fact that
roots in contrast to the above-ground
organs of the plants are more frequently colonizedsystemically
by microorganisms may be due to the factthat roots are in close
contact with an environmentharbouring many dierent mainly
degradatively activemicro-organisms that can potentially provide
theplants with water and essential minerals. We suggestthat
mutualistic interactions have developed betweenmicroorganisms and
the roots, because the roots as anatural carbon sink of the plants
can supply dual andmulti-organism symbioses with nutrients. In
return thehost can be supplied with minerals and water by
themicroorganisms. Additionally, in contrast to the shoot,infection
and colonisation by microorganisms of theroots are less limited by
xeromorphic tissue structures(epidermal wall, wax, etc.) and water
for spore germi-nation. An endophyte cannot improve the
nutrientstatus of the photosynthetic organs directly. Thus,in
general, a mutualistic systemic interaction withthe roots of a
putative host is more probable thanwith the aboveground organs.
And, recently a mol-ecular basis for mutualistic interactions of
rootswith microorganisms was found (Imaizumi-Anrakuet al.
2005).
Variability of mutualisms
As demonstrated above, the same endophytes thatunder certain
conditions interact mutualistically withtheir hosts may become
pathogenic, for example whenthe host is stressed and the balance of
the antagonismis tilted in favour of the fungus (Kuldau &
Yates2000, Jumpponen 2001, Sieber 2002). The denitionendophyte only
applies to a momentary status. And,just as some plant host species
and cultivars are morelikely to develop mutualistic mycorrhizal
associationsthan others (Francis & Read 1995, Feldmann &
Boyle1998), associations with DSE also vary with their hosts.An
example for AM fungi : of eight hosts inoculatedwith AM fungi, only
one developed a mutualistic
B. Schulz and C. Boyle 677
-
Table 3. Conclusions concerning fungal endophytichost
interactions.
Areas of investigation Conclusions
Diversity of endophytes (1) Surface sterilisation should be
optimized using the imprint method for the respective host/organ
investigated. (2) Bothconventional methods of culture and those of
molecular biology should be used to attempt to identify all fungal
endophytes.(3) The diversity of endophytes is very broad and varies
from incidental opportunists to those with host adaptations.
Colonisation Colonisation of the shoots is primarily local, that
of the roots usually extensive and often systemic within the roots.
In bothorgans inter- and intracellular growth is found.
Variability of the interaction Whether a particular endophytic
isolate causes disease or grows asymptomatically within its host
varies, depends onpre-dispostions of host and endophyte, and on
environmental conditions.
Adaptation to the endophyticlife-history strategy
Whereas some endophytes, e.g. the incidental opportunists, seem
not to be specically adapted to an endophytic life-history
strategy, others are. Evidence for adaptation of the latter : low
virulence, host and organ specicity/adaptations,capability to grow
both endophytically and saprophytically.
Secondary metabolites (1) A high proportion of fungal endophytes
produce biologically active secondary metabolites, residing in a
habitat thathas not been extensively studied with respect to
secondary metabolites. Thus, they are a good source for intelligent
screeningfor novel agrochemical and pharmaceutical products. (2)
The majority of fungal endophytes produce in vitro
non-specicherbicidal secondary metabolites, also toxic to their own
hosts
Fungal virulence Endophytic fungi produce both the exoenzymes
and the herbicidal mycotoxins required to infect and colonize their
plant hosts.
Plant defence The plant defence reaction is induced by
endophytes as it is by pathogens : preformed and induced defence
metabolites,sometimes mechanical defence. With respect to defence
metabolites, the response seems to dier from that to amycorrhizal
fungus.
Balanced antagonism We hypothesize : As long as fungal virulence
and host defence are balanced, colonisation remains
asymptomatic=balancedantagonism. When the fragile balance of powers
is tipped, e.g. by environmental factors or by senescence in favour
of thefungus, disease develops.
Mutualisms Mutualistic interactions with endophytes are more
common when colonisation is extensive and(or) systemic, in
particularin the roots, and may, for example, improve growth of the
host or induce resistance.
Theendophytic
contin
uum
678
-
symbiosis, seven did not (Francis & Read 1995).Similarly,
Fernando & Currah (1996) found that shootbiomass of Potentilla
fruticosa increased followinginoculation with only one of four
strains of Lepto-dontidium orchidicola and decreased following
inocu-lation with two strains. The biomass of Picea glauca,
incontrast, increased when inoculated with the twostrains that had
decreased biomass of P. fructicosa.Thus, whether or not an
interaction is mutualisticdepends on the genotypes of host and
endophyte, aswell as on many factors in the environment,
i.e.presence or absence of stressors, which stretch theplasticity
of phenotype of host and pathogen.
Denition of symbiosis
Every interaction is or may be variable and depends
ondispositions and developmental stages of the partnersas well as
on environmental factors. Thus, symbiosisshould be dened in the
broad sense as used by De Bary(1879) as the living together of
dissimilarly namedorganisms [_des Zusammenlebens
ungleichnamigerOrganismen_ ], i.e. an association between two
ormore organisms of dierent species (Schulz et al. 1998).This
denition avoids anthropocentric prejudgementsabout variable or
unknown aspects of the interaction.In the case of the
endophyte-host interaction, onevariable is the continual ux in the
role of the fungalpartner, which may, for example, at one moment
bethat of an asymptomatically colonizing endophyte andat another
phase in development be that of a pathogen.The other variable is
the frequently unknown physio-logical interactions between two
symbionts, whichprevent specifying the advantages and disadvantages
ofthe interaction for the individual partners. This doesnot exclude
the option of additionally characterizing aninteraction as
mutualistic, commensal or antagonisticwhen enough is known about a
relationship to warrantclassication, because dierent phases or
stages of aninteraction can be dierently characterised.
ENDOPHYTIC CONTINUUM
In consideration of the available results on endophyticfungi
(Table 3), the following conclusions can bedrawn:(1) Which
organisms are endophytes? A survey ofthe literature showed that
this term is employed for allorganisms that inhabit plants :
animals, other plants,eukaryotic and prokaryotic microorganisms,
irrespec-tive of whether or not disease or mutualisms areinvolved.
Even a more limited denition of fungalendophyte to mean fungi that
inhabit plant hosts atsome time in their life, colonizing internal
plant tissueswithout causing apparent harm to their host,
e.g.Petrini (1991), applies to a continuum of organisms,a continuum
of physiological statuses, of life historystrategies, of
developmental and of evolutionary stages.
This is in part because the denition applies to amomentary
status within the host. An examination ofthe fungi that colonize
plants shows that a high diver-sity of taxa are represented. An
examination of theplant taxa that can be colonized shows that
fungiinhabit almost all hosts thus far studied (Stone et al.2000).
A broad spectrum of fungal and of host taxa areinvolved in the
interactions of fungi and plants.(2) The colonisation patterns of
endophytes withintheir hosts spans a continuum and includes fungi
thatare specialized to occupy every niche within the host,those
that grow inter- and those that grow intra-cellularly, those with
organ specicity and those thatcolonize aboveground organs as well
as the roots. Somegrow only endophytically and others endo- and
epi-phytically. And there are those that are adapted tocertain
hosts and others that are ubiquitous withrespect to their hosts.(3)
To explain the apparent macroscopic symptom-lessness of
colonisation, we hypothesize that there is abalanced antagonism
(Fig. 1) between fungal endo-phytes and plant hosts. This is a
conceptual model toexplain the limitations of colonisation and the
preven-tion of the development of disease in roots and shoots.(4)
There is no set life-history strategy of endophyticfungi within
their hosts. The life-history strategies ofthe endophytic fungi
vary depending on developmentalstage of host and fungus, virulence
of the fungus andthe host defence response, but also on
environmentalfactors which inuence the phenotypic plasticity ofboth
partners. There are weak pathogens whosecolonisation remains
asymptomatic until, due to inde-pendent physical and environmental
factors, stress orsenescence, the host defence response weakens.
Othersremain quiescent until the host senesces, only thenbecoming
saprophytic. And then there are the (latent)virulent pathogens that
have not yet caused obviousdisease symptoms. A broad spectrum of
fungi, withdierent life history strategies, many with the
capacityto react exibly, inhabit any one host. It is this
pheno-typic plasticity (sensu West-Eberhard 2003), bothof
endophytes taken as a group, but also of manyindividual endophytes,
that results in a continuum oflife history strategies for
endophytic fungi. Phenotypicplasticity is the intra-individual
variation under thedual inuence of genes and environment
(West-Eberhard 2003). Again, endophytes taken as a group,but also
many individual endophytes, are exible andhave numerous options:
infection, latency, local col-onisation, virulence, and
saprophytism. Some also havethat of systemic colonisation. Their
phenotypic plas-ticity is creative, a motor of evolution.(5)
Determination of the life-history strategies of anendophyte-host
symbiosis : We hypothesize that infungus-plant interactions there
is no neutral interac-tion, but rather, outcomes of these
interactions dependon a balance of antagonisms, that there is
always atleast a degree of virulence on the part of the fungus,if
no more than to enable colonisation and access to
B. Schulz and C. Boyle 679
-
nutrients and shelter, and that there is always host de-fence,
limiting fungal colonisation. This mutual antag-onism may be only
for a limited period of time, e.g.until a chlamydospore develops.
As Smith & Read(1997) concluded, there is always a conict of
interests at all stages of symbioses between fungal and
plantpartners. From an ecological and from an
evolutionarystandpoint (sensu Brundrett 2002), there are
con-tinuums of associations in the development of sapro-phytic
fungi of the rhizosphere to mutualisticmycorrhizal fungus. Examples
of the spectrum of theseassociatio