Endophytic Continuum

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.

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

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

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


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.


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


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.


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.


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


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.


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


preformed secondarymetabolites


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


none elicitors induced slow defencereactions

elicitors none colonisation, tolerancebalanced antagonism


necroses elicitors hypersensitive reaction no elicitation none colonisation, tolerancebalanced antagonism


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


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


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.


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,


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


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

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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


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

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Endophytic Continuum