Epichloe spp. associated with grasses:new insights on life cycles, dissemination and evolution

Mariusz Tadych1

Marshall S. BergenJames F. White Jr.

Department of Plant Biology and Pathology, RutgersUniversity, New Brunswick, New Jersey 08901-8520

Abstract: Epichloe species with their asexual statesare specialized fungi associated with cool-seasongrasses. They grow endophytically in tissues of aerialparts of host plants to form systemic and mostlyasymptomatic associations. Their life cycles mayinvolve vertical transmission through host seedsand/or horizontal transmission from one plant toother plants of the same species through fungalpropagules. Vertical transmission has been wellstudied, but comparatively little research has beendone on horizontal dissemination. The goal of thisreview is to provide new insights on modes ofdissemination of systemic grass endophytes. Thereview addresses recent progress in research on (i)the process of growth of Epichloe endophytes in thehost plant tissues, (ii) the types and development ofreproductive structures of the endophyte, (iii) therole of the reproductive structures in endophytedissemination and host plant infection processes and(iv) some ecological and evolutionary implications oftheir modes of dissemination. Research in theEpichloe grass endophytes has accelerated in the past25 y and has demonstrated the enormous complexityin endophyte-grass symbioses. There still remain largegaps in our understanding of the role and functionsof these fungi in agricultural systems and understand-ing the functions, ecology and evolution of theseendophytes in natural grass populations.

Key words: Clavicipitaceae, conidiation, endo-phytes, epibiotic, horizontal dissemination, hybridiza-tion, vertical dissemination

INTRODUCTION

Epichloe (Fr.) Tul. & C. Tul. species (Clavicipitaceae,Hypocreales, Ascomycota), including their asexualstates, are fungal symbionts of cool-season grasses(Pooideae, Poaceae; TABLE I) (Clay 1990, Glenn et al.1996, Schardl et al. 2004, Spatafora et al. 2007,Leuchtmann et al. 2014). It has been estimated that

up to 30% of all cool-season grass species worldwideare associated with Epichloe spp. (Leuchtmann 1992).These fungi are characterized by their endophytichabit to form systemic and asymptomatic associationsthroughout the aerial parts of the host plant, wherethrough intercellular growth of hyphae they colonizethe apoplastic spaces of culms, leaf sheaths and seeds(Clay 1990; White et al. 1991; Christensen et al. 1997,2002, 2008; Christensen and Voisey 2009). Because oftheir systemic growth, species of Epichloe are fre-quently seed-transmitted (TABLE I) to the next gen-eration of the host plant (i.e. when the seedgerminates, the resulting seedling may contain thefungal symbiont) (Sampson 1933, Siegel et al. 1984,White 1988, Clay 1990, White et al. 1991). However,many Epichloe species, in addition to having primarilyendophytic growth, produce fungal structures thatappear externally on the aboveground plant parts(FIG. 1). The external structures consist of: (i)mycelial networks with conidiogenous cells andconidia, growing mostly epibiotically on leaf blades(White et al. 1996, Christensen et al. 1997, Meijer andLeuchtmann 1999, Moy et al. 2000, Craven et al. 2001,Dugan et al. 2002, Moon et al. 2002, Tadych andWhite 2007, Tadych et al. 2007, Iannone et al. 2009,Oberhofer 2012, Alderman 2013), and (ii) light-colored stromata (i.e. dense masses of hyphae, whichusually envelop inflorescences and upper leaf sheathsof flowering culms and prevent flower and seeddevelopment, a syndrome referred to as ‘‘chokedisease’’ (Bradshaw 1959; White 1988; White et al.1991; Schardl et al. 2004, 2009; Schardl 2010;Christensen et al. 2012).

The relationship of the endophytes to hosts ishighly variable and has been suggested to be anantagonism-mutualism continuum depending onparticular host genotype and endophyte-haplotypecombinations and environmental conditions (Saikko-nen et al. 2004, 2006, 2010; Cheplick and Faeth 2009;Brosi et al. 2012). Epichloe endophytes may modify themetabolic activities of colonized hosts (e.g. they maycause a change in levels of antioxidants and othermetabolites in hosts tissues) (Malinowski et al. 1998;Rasmussen et al. 2008, 2009; White and Torres 2010;Brosi et al. 2012; Hamilton and Bauerle 2012;Hamilton et al. 2012; Koulman et al. 2012; Qawasmehet al. 2012a, b; Torres et al. 2012). The endophytesalso produce a range of secondary metabolites (Bushet al. 1997; Clay and Schardl 2002; Schardl et al. 2012,

Submitted 3 Oct 2013; accepted for publication 10 Oct 2013.1 Corresponding author. E-mail: tadych@aesop.rutgers.edu

Mycologia, 106(2), 2014, pp. 181–201. DOI: 10.3852/106.2.181# 2014 by The Mycological Society of America, Lawrence, KS 66044-8897Issued 22 April 2014

181

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182 MYCOLOGIA

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sen

tn

on

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rid

TADYCH ET AL.: BIOLOGY OF EPICHLOE 183

TA

BL

EI.

Co

nti

nu

ed

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na

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mu

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no

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pre

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th

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han

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an

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d

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spra

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v.]

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en

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serv

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nt

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a(Z

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an

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serv

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184 MYCOLOGIA

TA

BL

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Co

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nu

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Tax

on

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msy

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TADYCH ET AL.: BIOLOGY OF EPICHLOE 185

2013; Takach et al. 2012), including ergot alkaloids,indole-deterpenes, aminopyrrolizidines (lolines)and pyrrolopyrazine (peramine) (Gallagher et al.1984, Lyons et al. 1986, Rowan and Gaynor 1986,Bush et al. 1993). Lolines and peramine exhibitinsecticidal properties, whereas ergovaline (ergotalkaloid) and lolitrem B (indole-diterpene) arepoisonous to grazing livestock. For example, ergova-line and lolitrem B produced by E. coenophiala andE. festucae var. lolii have been established as thecausal agent of fescue toxicosis in cattle (Bacon et al.1977, Schmidt et al. 1982) and ryegrass staggers insheep (Fletcher and Harvey 1981, Latch et al. 1984)respectively. In addition, these metabolites arethought to cause the documented complex effectsof Epichloe spp. in enhancing herbivore deterrence(Belesky et al. 1987; Arechevaleta et al. 1989; Clay1990, Bazely et al. 1997, 2007; Malinowski andBelesky 2000; Panaccione et al. 2001; Ahlholmet al. 2002; Clay and Schardl 2002; Faeth 2002,2009; Faeth and Sullivan 2003; Schardl et al. 2004;Lehtonen et al. 2005; Granath et al. 2007; Olejniczakand Lembicz 2007; Rudgers and Clay 2008; Crawfordet al. 2010; Saikkonen et al. 2010; Schardl 2010;Lembicz et al. 2011; Bultman et al. 2012; Faeth andSarri 2012; Gibert et al. 2012; Gundel et al. 2012,2013; Hamilton et al. 2012; Torres et al. 2012;Czarnoleski et al. 2013). Other classes of fungalmetabolites, siderophores (e.g. epichloenin A, fer-riepichloenin A, epichloenin B, also have beenfound in both endophyte-infected plants and endo-phyte mycelia ( Johnson et al. 2007a, b; Koulmanet al. 2007, 2012; Cao et al. 2008). The function ofsiderophores in the Epichloe-grass associations is notfully understood. They have been proposed to play arole in resistance in endophyte-infected grass plants(Rasmussen et al. 2008). It also has been postulatedthat extracellular siderophore, epichloenin A, mayserve as an important molecular/cellular signal forcontrolling fungal growth and hence the symbioticinteraction of E. festuca with L. perenne ( Johnsonet al. 2013).

Epichloe spp. reproduce by three modes: (i) sexualreproduction resulting in stromata development onflowering culms, (ii) seed colonization from systemicinfections or (iii) a mixed mode where both floweringculms choked by stromata and healthy floweringculms with seeds colonized by endophytic myceliumare present. Epichloe-Pooideae symbioses have longbeen considered static associations. However, thebiology and ecology of Epichloe spp. indicate thatthese fungi may have a more complex life cycle andform more dynamic associations with their hosts.

The subject of this review is to discuss the variationsin life cycle among different species of both theT

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186 MYCOLOGIA

sexual and asexual Epichloe spp. associated withgrasses. This review will concentrate on (i) the processof internal growth of Epichloe endophytes in thevegetative and reproductive tissues of the host plant,

(ii) epibiotic growth and development of sexual and/or asexual reproductive structures by these fungi, (iii)the roles of these fungal structures in endophytedissemination and host plant infection processes and

FIG. 1. Features of Epichloe spp. A. Endophytic hyphae (enh) of Epichloe coenophiala growing in Festuca arundinacealeaf. B. Endophytic hyphae (enh) of E. coenophiala penetrating aleurone layer of F. arundinacea seed. C. Epichloe typhina subsp.poae showing epibiotic hyphae (eph) with conidiogenous cells (cc) and conidia (c). D. Epibiotic colonization of coleoptile of Poasecunda subsp. juncifolia seedling by E. typhina subsp. poae showing aerial proliferation of hyphae (arh) and conidiogenous cells.E. Conidial stromata (cst) of Epichloe festucae on tillers of Festuca rubra. F. Section through conidial stroma of E. typhina subsp.poae developing on P. secunda subsp. juncifolia tiller showing conidiogenous cells (cc) with conidial clusters (ccl) or solitaryconidia (c). G. Sexual stroma (sst) of Epichloe typhina on Dactylis glomerata tiller. H. Longitudial section of perithecium ofEpichloe baconii on Agrostis stolonifera showing centrum and asci (a) surrounded by a thin perithecial wall (w) as well as periphyses(p) bordering the inner wall of the ostiolar neck region. I. Section through maturing asci (a) of E. baconii showing filamentousascospores (as) and ascus tip (at). Bars: A, B, F 5 10 mm. C 5 20 mm. D 5 1 mm. E, G 5 10 mm. H, I 5 100 mm.

TADYCH ET AL.: BIOLOGY OF EPICHLOE 187

(iv) some ecological and evolutionary implications ofthe modes of dissemination.

COLONIZATION OF THE HOST PLANT

Endophytic growth.—The internal systemic associationof Epichloe endophytes with grass hosts is formedduring development of the host plant. The endo-phytes proliferate in intercellular spaces (FIG. 1A)and do not form haustoria. Available data suggest thatthey use nutrients from apoplastic fluids as well asfrom adjacent host cells through production of anumber of hydrolytic enzymes (Lindstrom et al. 1993,Tan et al. 2001, Christensen et al. 2002, Moy et al.2002, Li et al. 2005, Bryant et al. 2007). Theendophyte-host association requires close coordina-tion of life cycles of both partners (Herd et al. 1997).

During the vegetative growth of the host grassbeginning with seed germination, the endophytichyphae asymptomatically colonize the developingshoot apex of the seedling (Clay 1990, Christensenet al. 2002). Further hyphal growth through tissues ofthe shoot apical meristem, leaf primordia, sheathsand blades of the leaves results in systemic coloniza-tion of aerial parts of the seedling (FIG. 1A). Inmeristematic tissues hyphal growth is characterized byhyphal tip growth and branching. In contrast, theintercellular hyphae that reside in the apoplast ofgrowing seedlings form extended longitudinal andusually convoluted strands with little branching. Asthe grass cells expand, mycelium within the culm andleaf tissues extend through intercalary extensionrather than tip growth (Christensen et al. 1997,2000, 2008; Herd et al. 1997; Tan et al. 2001). Thismay be the result of shear force on hyphae due to theelongating plant parenchyma cells. This pattern ofgrowth results in synchronization of hyphal growthand plant cell growth. During the development ofthe host inflorescence, endophytic hyphae proliferatefrom the vegetative apex, penetrate into the inflores-cence primordium and floral apex to colonize theovary and ovules. Following fertilization, hyphae enterthe embryo sac and can be found early on the surfaceof the developing embryo. During maturation of seed,hyphae can be found concentrated throughout theembryo and surrounding tissues including plumuleapex, aleurone layer (FIG. 1B) and scutellum of thegrass seed (Freeman 1904, Philipson and Christey1986, White et al. 1991, May et al. 2008); the presenceof the endophyte in grass seeds is not detrimental (i.e.does not affect seed viability) (Siegel et al. 1984).After the seeds are released from the matureinflorescence and germinate, the resulting seedlingscontain the fungal symbiont (Siegel et al. 1984), thusenabling the endophyte to transmit vertically from

maternal plant to the next host generation (Freeman1904, Clay 1990, Afkhami and Rudgers 2008).

Epibiotic growth.—Growth of most Epichloe endo-phytes, regardless of their ability to reproducesexually or asexually, is usually not limited to internaltissues of the host plant. Some Epichloe species arecapable of growing superficially and producingepibiotic reproductive structures (i.e. mycelial net-works with conidiogenous cells and conidia onaboveground parts, mostly on leaf blades) (FIG. 1C–D).These epibiotic structures have been documented ondiverse grass species, including A. hyemalis, Bro.auleticus, Bro. setifolius, D. glomerata, Ech. ovatus,Festuca arvernensis Auquier, Kerguelen & Markgr.-Dann. (synonym: Festuca ovina var. glauca [P. Beauv.]W.D.J. Koch), F. pratensis, Festuca rubra L. (synonym:Festuca rubra var. commutata Gaudin, Festuca ovina var.rubra [L.] Sm.), Hel. europaeus, H. brevisubulatum,Lolium 3 hybridum Hausskn., L. perenne, P. rigidifolia,P. bonariensis (Lam.) Kunth, P. secunda subsp.juncifolia and Poa sp. (White et al. 1996, Christensenet al. 1997, Moy et al. 2000, Craven et al. 2001, Dugan etal. 2002, Moon et al. 2002, Tadych et al. 2007, Iannoneet al. 2009, Oberhofer 2012, Alderman 2013). Theepibiotic hyphae are usually aligned with the longitu-dinal junctions between epidermal cell walls andoccasionally branching, sometimes interconnectedand overlapping (FIG. 1C). The epibiotic conidia arehyaline, aseptate, reniform to ellipsoid, water-transmit-ted and capable of germinating (Tadych et al. 2007,2012).

Stroma formation.—When a host plant colonized byan Epichloe endophyte undergoes reproductive devel-opment (TABLE I), the fungus in many cases has thepotential to form a compact mycelial structure calleda stroma (FIG. 1E–G). Formation of stromata byendophytes results from unrestricted hyphal growthwithin and outside the developing inflorescence,which envelops an emerging inflorescence and theupper leaf sheath of reproductive tillers (White et al.1991, White 1997). The stromata usually preventflower and seed development on affected culms,leading to partial or total sterility of the host plant(Bradshaw 1959). Stromata of Epichloe spp. arestructures where mating takes place but also whereasexual (FIG. 1E–F) and sexual reproductive propa-gules important for fungal dispersal develop(FIG. 1G–I). As the stroma develops, numerouselongated conidiogenous cells arise on a surface(FIG. 1F), which constitute the anamorph (conidialstroma; FIG. 1E). Some Epichloe endophytes, althoughnot known to complete a sexual cycle, may produceconidial stromata on flowering culms (Sampson 1933,White et al. 1992, Ji et al. 2009, Christensen et al.

188 MYCOLOGIA

2012, Oberhofer and Leuchtmann 2012, Tadych et al.2012). The conidial stroma (FIG. 1E) is light, usuallywhite to gray, covered with a dense layer ofunbranched phialidic conidiogenous cells, wherehyaline, one-celled, typically reniform to ellipsoid,conidia are borne (FIG. 1F). These conidia mayfunction as spermatia (Leyronas and Raynal 2008a)(i.e. gametes in a mating process if the fungusundergoes heterotallic sexual reproduction). Epichloestromata are hermaphroditic; both receptive hyphaeand spermatia are formed on the same stroma (White1997). As a consequence of fertilization, the stromathickens, usually become brightly colored, yellow ororange (FIG. 1G) and superficial perithecia (FIG. 1H)develop, containing asci with ascospores (FIG. 1I)

SEXUAL DEVELOPMENT AND REPRODUCTION

Because Epichloe fungi are heterothallic, successfulcompletion of sexual reproduction relies on crossfertilization between two different fungal matingtypes of the species (White and Bultman 1987). Thisresults in the transfer of gametes from a stroma of onemating type to a stroma of the opposite mating type(Raynal 1991b, White 1997, Bultman et al. 1998,Webster and Weber 2007). Spermatia of Epichloespecies are transferred to ascogonia in perithecialprimordia by specialized flies in the genus Botano-phila Lioy (Diptera, Anthomyiidae), which feed anddevelop on Epichloe stromata (Kohlmeyer and Kohl-meyer 1974; Bultman and White 1988; Bultman et al.1995, 1998). Although this pollination-like associationis probably not highly specific, a study of Epichloe-associated Botanophila flies revealed that in Europeand North America at least six distinct fly taxa areinvolved in gamete transfer (Leuchtmann 2007,Lembicz et al. 2013). Specific volatile compounds,that is sesquiterpenoid alcohol (chokol K), methylester (methyl (Z )-3-methyldodec-2-enoate 5 MME)and perhaps other compounds (e.g. diterpenes,trimethylester 5 methyl 2,4,8-trimethylundecanoate5 MTE) emitted by the fungal stroma attract flies tostroma (Schiestl et al. 2006; Steinebrunner et al.2008a, b, 2008c). During their visit, female flies feedon fungal tissues including conidia that eventuallypass through the digestive system (Bultman andLeuchtmann 2003). Feces that are actively depositedon the stromata by the fly during feeding andoviposition contain intact and viable conidia. Theseconidia function as spermatia and are capable ofcross-fertilization of the fungus. Eventually fly larvaehatch and feed on the stroma tissues, includingperithecia. They then drop and pupate in the soil(Bultman et al. 1998). These fungal stromata are thesole source of nourishment for the Botanophila larvae.

In situations of high egg load and an excessive larvalexploitation of stromata, greater larval mortality hasbeen observed, suggesting the existence of somedegree of mutual dependence among both partners,the fly and the fungus (Bultman et al. 2000).

Some field observations indicate, however, thatthere is no correlation between fly abundance andstroma fertilization (Rao and Baumann 2004; Gor-zynska et al. 2010, 2011; Rao et al. 2012). These studiesnoted that in some grass populations no flies, fly eggsor larvae on stromata were detected but fertilizedstromata were reported universally, with peritheciapresent on 85–100% of examined stromata. However,the absence of eggs or larvae of Botanophila onstromata is not necessarily evidence that the flies orother vectors did not visit the stromata. Becausestromata provide food for the Botanophila females,flies could have visited without laying eggs, anddeposited spermatia during such visits which in turnfertilized the stromata (Parker and Bultman 1991). Forinstance, it has been shown that spermatia may adhereto insect body hairs (Bultman et al. 1995).

It has been suggested that in grass populationswhere no flies were observed, another mechanism(i.e. ascospore fertilization) may be responsible forstroma fertilization (Alderman and Rao 2008, Raoet al. 2012). These authors proposed that at thebeginning of growing season the early emergingstromata of E. typhina developing on D. glomerataplants first are fertilized by flies and later thesestromata produced ascospores. They hypothesizedthat ascospores were disseminated by wind and servedas gametes for subsequent fertilization of developingstromata among neighboring fields. Furthermore,Rao et al. (2010, 2012) reported that the gray gardenslug (Deroceras reticulatum Muller; Agriolimacidae,Gastropoda, Mollusca) and reticulate taildropper(Prophysaon andersoni Cooper; Anadenidae, Gastro-poda, Mollusca) forage on D. glomerata plantsinfected by stroma-producing E. typhina and thatthe fungal spermatia are transmitted through the gutof a slug; the authors suggested that this may result incross fertilization of the fungal stromata. This modeof spermatia transport might be limited; the averagebiomass consumed by grove snails (Cepaea nemoralisL.; Helicidae, Gastropoda, Mollusca) feeding on endo-phyte-infected Pu. distans grass seedlings was less thanthat consumed on uninfected ones (Czarnoleski et al.2010).

Abiotic conditions may also play a role in fertiliza-tion. If a stroma of one mating type is sufficientlyclose to a stroma of the opposite mating type, watersplash may be responsible for transfer of spermatiabetween stromata and their fertilization during rainyand windy conditions (Tadych et al. 2007, 2012; Rao

TADYCH ET AL.: BIOLOGY OF EPICHLOE 189

et al. 2012). Therefore, this mechanism could workefficiently, especially in dense populations of stromataand under moist environmental conditions (Rao andBaumann 2004; Gorzynska et al. 2010, 2011; Rao et al.2012). Further studies are required to determine howgametes in Epichloe spp., in both agricultural andnatural grass populations, are transferred.

FACTORS AFFECTING DEVELOPMENT OF FUNGAL

REPRODUCTIVE STRUCTURES

In natural grass populations, sexual reproduction of ahost is rarely completely eliminated by developmentof stromata, even at high colonization frequencies. Inmany species of Epichloe, the abundance of stromatavaries among infected plant species as well as amongindividual infected plants or their tillers (White andChambless 1991; Bucheli and Leuchtmann 1996;Kover and Clay 1998; Groppe et al. 1999; Meijer andLeuchtmann 1999, 2000; Yanagida et al. 2005;Cheplick and Faeth 2009; Lembicz and Olejniczak2009; Oberhofer and Leuchtmann 2012; Tadych et al.2012) and is regulated by specific host genotype-endophyte co-evolution and environmental factors.

Fungal factors.—Population-structure studies of E.sylvatica associated with Brp. sylvaticum revealed thatstromatal development is controlled by the fungalgenotype (Bucheli and Leuchtmann 1996; Meijer andLeuchtmann 1999, 2001). In E. sylvatica, both stroma-forming strains and non-stroma-forming strains donot belong to a single random mating population butare differentiated genetically into separate subpopu-lations despite their occasional co-occurrence onindividual host plants. Epichloe typhina subsp. poaeas revealed by morphology and phylogenetic analysisis associated with four species of grasses (i.e. P.nemoralis, P. pratensis, P. secunda subsp. juncifolia, P.sylvestris). Nevertheless, this endophyte shows allpossible variations in reproductive modes dependingon the host species (Tadych et al. 2012). In Poapratensis the fungus forms stromata but is not seed-transmitted. In P. nemoralis and P. secunda subsp.juncifolia the fungus produces stromata but it alsoenters seeds. In P. sylvestris, fungal stromata have notbeen reported but the fungus transmits throughseeds. Perithecia with viable ascospores have beenobserved only rarely on stromata of P. nemoralis andP. pratensis (A. Leuchtmann pers comm), whileperithecia with ascospores have not been reportedon P. secunda subsp. juncifolia. The most vigorousfungal endophytes may affect plant growth andreproduction as well as the number of stromataproduced (Groppe et al. 1999). Based on the quantityof Epichloe DNA in vegetative plant tissue, Groppe

et al. (1999) found that plants of Bro. erectus infectedby E. bromicola with the highest concentration of thefungus in the vegetative plant tissues had the highestnumber of stromata (3.4 per plant) with no inflores-cences whereas infected plants with the lowestconcentration of the fungus in the vegetative planttissues had the fewest stromata (0.5 per plant) and themost fertile inflorescences (0.3 per plant). However,this value was much lower than values found foruninfected plants (1.1 inflorescences per plant).

Host factors.—Groppe et al. (1999) found that thenumber of stromata produced by E. bromicola in Bro.erectus plant communities was greater when higherplant species diversity was observed. In another studyon E. bromicola infecting three different Bromus spp.,Brem and Leuchtmann (2003) found evidence thatthe 26 E. bromicola isolates from Bromus hostscollected from natural populations in Switzerlandand France were genetically differentiated accordingof their hosts. Although different host strains wereinterfertile, the strains differed in their reproductivemodes (Schardl and Leuchtmann 2005). The E.bromicola strain naturally infecting Bro. erectus wasalways stroma-forming and failed to transmit in hostseeds, whereas its strains from Bro. benekenii and Bro.ramosus did not form stromata and were seedtransmitted. Furthermore, reciprocal inoculations ofhost plant seedlings showed that asexual strains fromBro. benekenii and Bro. ramosus were incapable ofinfecting Bro. erectus whereas the stroma-formingisolates from Bro. erectus were broadly compatiblewith all three host species. Brem and Leuchtmann(2003) proposed that asexual forms arose from withinsexual populations on Bro. erectus after host shifts toBro. benekenii and Bro. ramosus. In another studyLembicz et al. (2010) found that the sexual form of E.bromicola also occurs on El. repens in Poland,apparently the result of a host shift where thereproductive mode remained constant.

A correlation between plant genotype and stromaexpression also has been found in studies on Brp.sylvaticum infected by E. sylvatica (Meijer andLeuchtmann 2000) and on H. patula infected by E.elymi. (Tintjer et al. 2008). Meijer and Leuchtmann(2000) investigated stroma expression in nine geno-types of Brp. sylvaticum and found that in the firstyear 74.1% of the total variance in stroma productionwas explained by host genotype while this variancecomponent declined to 27.5% in the second year ofobservation. Another grass endophyte, E. festucae,forms stromata on F. rubra (Leuchtmann et al.1994). However, separate field surveys of populationsof this endophyte associated with F. rubra and Festucaovina L. in grasslands of the northern Europe,

190 MYCOLOGIA

European subarctic and the European Mediterraneansemi-desert did not yield stromata with fruiting bodyformation but instead revealed predominantly non-recombining asexual or clonal population structures(Arroyo Garcıa et al. 2002, Bazely et al. 2007, Wali et al.2007). These observations suggest that under condu-cive conditions entire populations of Epichloe spp. canthrive as asymptomatic endophytes perhaps dependingentirely on their asexual reproduction via their hostseeds or on production of epibiotic conidia fordispersal.

In populations of Pu. distans, a perennial Euro-Siberian halophyte grass (Hughes and Hallidays1980), the infection by E. typhina seems to appearonly in plants in anthropogenic habitats (Lembicz1998; Lembicz et al. 2009, 2011). The absence of E.typhina colonization in natural populations of thehost, as suggested by Lembicz et al. (2011), was aresult of widespread distribution of host genotypeswhich are incompatible with E. typhina; on the otherhand, if latently infected, the genotype preventeddevelopment of stromata (Meijer and Leuchtmann2000, Lembicz et al. 2009). In the host genotypeinfected by E. typhina, the number of tillers bearingstromata increased significantly with age of the hostplant (Lembicz et al. 2011). To counteract thenegative influence of choke disease the Pu. distansgrass populations in anthropogenic habitats andinfected by E. typhina produced additional tillerswith stromata in late summer (Lembicz et al. 2009,2011). These tillers did not appear in grass popula-tions of natural saline habitats (Lembicz 1998). Thefungus on these late summer tillers initially developedstromata at a lower frequency, but their numberincreased significantly with the age of the plants(Lembicz et al. 2009, 2011). These late summerstromata were always white and did not produceperithecia with asci and ascospores. The researchersspeculated that the lack of perithecia might be due tothe absence of the vector, the adult Botanophilaflies, in late summer (Bultman et al. 1998, but seeGorzynska et al. 2011). In addition, the authorshypothesized that the production of fungal reproduc-tive structures in late summer might not provide anybenefits to the fungus, E. typhina, or to the plant, Pu.distans (Lembicz et al. 2009, 2011). However, latesummer stromata give rise to abundant conidialstromata and may support local dissemination of thefungus and possibly be responsible for maintenanceof new infections in germinating seeds and develop-ing seedlings of the host grass in late summer or earlyautumn (Tadych et al. 2012). As observed by otherresearchers ( Jackowiak 1984, 1996; Jackowiak et al.1991; Moravcova and Frantık 2002), the new seedlingsof Pu. distans can appear in the field throughout the

region under favorable humid and temperatureconditions in September. High humidity, fluctuatingtemperature of 15/6 C and lower constant tempera-ture 15 C (Moravcova and Frantık 2002) optimal forPu. distans seed germination, are aligned withoptimal infection conditions for Epichloe spp. ( Ju etal. 2006). Clearly the relationship of E. typhina withPu. distans requires further study.

Environmental and geographical factors.—Experimen-tal data suggest that environmental factors may notonly influence the distribution and occurrence ofEpichloe endophytes (Novas et al. 2007, Iannone et al.2013) but also the expression of their reproductivestructures. Groppe et al. (2001) showed that stromaformation increased in E. bromicola infecting Bro. erectusfollowing experimental fragmentation of the hostpopulation. During a 2 y study in natural grasspopulations, the proportion of El. hystrix plants bearingE. elymi stromata was significantly different betweenyears, populations and demonstrated a year by popula-tion interaction, suggesting that stroma expression canvary with environmental conditions (Tintjer et al. 2008).The survey on the distribution of E. typhina in naturalpopulations of C. purpurea in Sweden revealed that thefungal stromata in the field were present only innutrient-rich and wet habitats and totally absent fromnutrient-poor and dry habitats (Wennstrom 1996).However, Sun et al. (1990) reported that the additionof nitrogen fertilizer had a negative effect in theproduction of stromata by E. festucae in infected F.rubra plants. In contrast, observations on Brp. sylvaticumcolonized by E. sylvatica revealed that fertilized plantson average showed slightly higher stroma expressionthan unfertilized plants (Meijer and Leuchtmann2000); less consistent increases in stroma expressionin fertilized plants were reported when the infectedplants were exposed to elevated concentration of CO2.On the other hand, Groppe et al. (1999) found elevatedatmospheric CO2 enhanced the production of fungalstromata in E. bromicola-infected Bro. erectus plants butinhibited inflorescence production in uninfectedplants. Lembicz et al. (2011) proposed that higherconcentrations of CO2 that occurred in anthropogenichabitats occupied by Pu. distans grass colonized by E.typhina might be responsible for higher stromaproduction by the fungus.

Kirby (1961) suggested that other factors thatstimulate plant growth might reduce stroma formationbecause rapidly growing flowering culms can escapefungal infection. In contrast, White and Chambless(1991) and White et al. (1991) found that growth offungal isolates on certain sugars in vitro were highlycorrelated with the number of stromata produced onhost plants. A regulatory mechanism involving sugars

TADYCH ET AL.: BIOLOGY OF EPICHLOE 191

predicts that factors that alter the relative sugarconcentrations in the meristems also might alterstroma expression (White et al. 1991). Otherenvironmental factors also may influence stromaexpression (e.g. the effect of shading). Meijer andLeuchtmann (2000) observed a moderate increase ofstroma production in E. sylvatica-infected Brp.sylvaticum in a 2 y study. The authors proposed thatsuccessful horizontal transmission of stroma-formingstrains in shaded environment had contributed toincreased stroma expression in the shade. Undercontrolled conditions, Alderman (2013) has shownthat stroma formation in E. typhina infected D.glomerata plants could be induced by vernalization ofplants at 8 C and 8 h light for 16 wk, following byplacing the plants in a growth chamber at 15 C and16 h light 2 wk before transferring the plants to agreenhouse, where stromata usually appeared withinseveral weeks.

The distribution of stromata of Epichloe spp. oftendoes not follow the distribution of their mostcommon host grasses. In Fennoscandia, Epichloespecies are widely associated with more than 25 grassspecies (Vare and Itamies 1995, Saikkonen et al. 2000,Bazely et al. 2007, Granath et al. 2007, Wali et al.2007). However, pronounced geographical variationand uneven expression of stromata on a specific grasshost species have been observed. For example, thestroma-producing E. typhina colonizing D. glomeratais common in southern Finland, Norway and Swedenbut rare in north Fennoscandia, although the grasshost occurs in pastures and meadows throughout theregion. A similar pattern was found for Ph. pretenseand several other grass genera widely distributed inFennoscandia, including the northernmost part ofthe region, and colonized by Epichloe spp. (Eriksson1967, Koponen and Makela 1976, Eckblad andTorkelsen 1989, Vare and Itamies 1995). At higherlatitudes of Fennoscandia (65uN and higher) stroma-ta were reported solely on species of Calamagrostisonly, including Calamagrostis lapponica (Wahlenb.)Hartm, C. purpurea and Calamagrostis stricta (Timm)Koeler (Eriksson 1967, Koponen and Makela 1976,Eckblad and Torkelsen 1989). Farther south thestromata were found only on Calamagrostis canescens(G.H. Weber) Roth (60uN) and C. epigeios (64uN) insouthern Finland (Koponen and Makela 1976). Insouthern Norway the white conidial stroma predom-inate in May, June and July but in the north also canbe found as late as mid-August (Eckblad andTorkelsen 1989). The orange perithecia stromatamay appear as early as the end of May and as late asthe beginning of October. Peak formation of peri-thecia is in the first two weeks of July. Thus theasexual and the sexual forms of stromata may occur

simultaneously, even on different culms of the sameplant. However, the teleomorph in the north is rare,perhaps because the development of the fungus isslow as a result of the late spring (Eckblad andTorkelsen 1989). Variations in stroma developmenton grasses in different regions may be a complexinteraction between fungal and plant genetics andenvironmental factors such as soil nutrients, water,temperature and day length.

DISSEMINATION OF FUNGAL PROPAGULES

AND INFECTION OF GRASS HOSTS

The mode of dissemination of Epichloe symbiontsvaries depending on host plant, endophyte popula-tion and endophyte species (White 1988, White andChambless 1991, Brem and Leuchtmann 2003,Leuchtmann 2003, Schardl and Moon 2003, Rodri-guez et al. 2009, Oberhofer and Leuchtmann 2012,Tadych et al. 2012). The vertically transmittedsymbiont is disseminated through the maternal germline, while the horizontally transmitted symbiont istaken up from the environment anew by each hostgeneration (Rodriguez et al. 2009, Bright andBulgheresi 2010). Until recently it was believed thatin asexual lineages of Epichloe endophytes the fungigrow systemically throughout most of the above-ground parts of the host plant, are completelyasymptomatic and do not form any external struc-tures for horizontal dissemination (Sampson 1933,Clay 1988, Schardl et al. 2004, Schardl 2010). It wasthought that dissemination between host generationsis achieved by vegetative growth of mycelium into theseed embryo (Sampson 1933, Siegel et al. 1984, Clay1990, Clay and Schardl 2002, Cheplick and Faeth2009, Schardl 2010). However, some of these asexualendophytes also are able to produce external fungalstructures, such as epibiotic mycelial networks withconidiogenous cells and conidia, growing mostly onleaf blades of the host plants (White et al. 1996,Christensen et al. 1997, Moy et al. 2000, Craven et al.2001, Dugan et al. 2002, Moon et al. 2002, Tadychet al. 2007, Iannone et al. 2009, Oberhofer 2012,Alderman 2013) and conidiogenous cells with conidiadeveloping on partial/full asexual stromata (Whiteet al. 1991, Ji et al. 2009, Christensen et al. 2012,Oberhofer and Leuchtmann 2012, Tadych et al. 2012).The presence of epiphytic fungal structures bearingconidia suggests that some of these endophytes alsomight have the ability for plant-to-plant spread viahorizontal transmission of the conidia; thus theirdependence on the host-mediated vertical transmis-sion may not be absolute. More recently it has beendemonstrated that conidia of E. typhina subsp. poae,frequently formed on the surface of leaves of P.

192 MYCOLOGIA

secunda subsp. juncifolia or on the conidial stromataon this host, are spread by water splash and areresponsible for infection of germinating seeds anddeveloping seedlings. As a consequence, such infec-tions cause systemic colonization of the host plant bythis endophyte (Tadych et al. 2007, 2012).

As referenced earlier, sexual Epichloe species onundeveloped stromata produce conidia but afterfertilization perithecia with viable ascospores develop.Mature ascospores are forcibly ejected from perithe-cia and are transmitted by wind (White 1997).Ascospore dispersal predominantly occurs at nightwith high relative humidity and plant water pressure(Ingold 1948, Raynal 1991a, Welch and Bultman1993, White et al. 1993, White and Camp 1995,Webster and Weber 2007). It has been hypothesizedthat ascospores mediate horizontal transmission ofEpichloe endophytes (Chung and Schardl 1997; Bremand Leuchtmann 1999; Leuchtmann 2003; Leyronasand Raynal 2008a, b; Schardl et al. 2009; Schardl2010), but it has not been confirmed whetherinfection occurs directly from ascospores or byinfective primary or secondary conidia formed afteriterative cycles of conidiation of ascospores (Baconand Hinton 1988, 1991; Leuchtmann 2003). There-fore, the exact mechanisms of contagious infection byascospores are still not fully understood (Leucht-mann 2003, Webster and Weber 2007). Chung andSchardl (1997) proposed that the mechanism ofinfection of grasses by ascospores is invasion of theovule via stigma colonization, a mechanism similar tothat demonstrated for Claviceps purpurea (Fr.) Tul.Western and Cavett (1959) proposed another mech-anism, where ascospores or conidia may infectwounds. In experiments to demonstrate infectionChung and Schardl (1997) and Western and Cavett(1959) found infection rates of 1.3% and 5.4%

respectively. However, inconsistencies in reproducingthe results obtained in these experiments leaves somedoubt about the actual mechanism of infection as wellas site of infection (Brem 2001, Leyronas and Raynal2008b).

In contrast, exposure of D. glomerata seeds tofertilized stroma of E. typhina resulted in an infectionrate of 25% (Leyronas and Raynal 2008b). Theresearchers hypothesized that germinating ascosporesor conidia produced by ascospores might be respon-sible for the infection. In another study, although therate of new infections was relatively low, 5.3% ofyoung seedlings of Brp. sylvaticum, growing in Petridishes and exposed to ascospores-ejecting stromata ofE. sylvatica, became infected (Brem 2001). Similarly,under natural conditions Brem and Leuchtmann(1999), using stroma-producing E. sylvatica coloniz-ing Brp. sylvaticum, demonstrated contagious spread

of the endophytic fungus to mature uninfected Brp.sylvaticum plants growing nearby infected plants. Thenumber of new infections observed in that study waspositively correlated with the amount of stromataobserved within each local population, an infectionrate of 34% at one site and 17% at another site wasreported (Brem and Leuchtmann 1999). Theseresults also were reproduced in experimental plots,in which Brp. sylvaticum plants infected by stroma-producing E. sylvatica served as the inoculum sourceand surrounded by mature uninfected plants; thefrequency of new infection of these previouslyuninfected plants was 16–25% (Brem 2001). Theseauthors concluded that infection of vegetative tillersof the grass host was frequent and hypothesized thatascospores invade the meristematic zone of tillerbuds.

Tadych et al. (2012) revealed that germinatingseeds and seedlings of P. secunda subsp. juncifoliabecome infected when exposed to E. typhina subsp.poae conidia and mycelium produced by theseconidia. In this study 36.4% and 33.7% of plantsharbored the endophyte after exposure to conidiaoriginating respectively from leaves or stromatae.These results suggest that conidia may be responsiblefor horizontal dissemination of Epichloe fungi andthat conidia or mycelium derived from conidia aredirectly responsible for infections of host grass.Ascospores also are responsible for horizontal dis-semination. The water-disseminated conidia may beresponsible for local distribution, whereas the wind-disseminated ascospores may function to vector thefungus longer distances. However, stroma productionalso may enhance horizontal dissemination of conidiaover large distances by facilitating insect vectoring(Bultman et al. 1995, 1998). Flying insect vectorscould transport conidia long distances and initiateinfections via tillers or seedling infections.

In a mixed mode of reproduction that is wide-spread in the northern hemisphere (e.g. TABLE I) theinfected plant forms both stroma-bearing tillers andnormal tillers with fertile inflorescences (White 1988,Schardl 2010); the inflorescences produce fullydeveloped and viable seeds that are asymptomaticallycolonized by fungal hyphae that enable verticaltransmission of the fungal endophyte by the seeds.In this mixed reproduction strategy, the fungus oninfected plants may form either sexual stromata withascospores or partial/full asexual stromata, togetherwith epibiotic mycelial networks bearing conidia; thisstrategy may result in dissemination of the fungus andultimately colonization of new plants. Such complexand diverse modes of reproduction in Epichloe spp.may effectively support wide distribution of distinctEpichloe-grass associations in various ecosystems.

TADYCH ET AL.: BIOLOGY OF EPICHLOE 193

EPIBIOTIC CONIDIA—ROLES IN HYBRIDIZATION

Horizontal dissemination involving epibiotic conidiamay be responsible for widespread associations ofasexual Epichloe endophytes with grasses, especially inthe southern hemisphere and northernmost subarcticregions of the northern hemisphere (e.g. Finland,Norway, Sweden). In these regions asymptomaticEpichloe endophytes showing epibiotic conidial stagesare prevalent, and in the subarctic regions of thenorthern hemisphere stromata stages, with or withoutperithecia, have been found occasionally on someendophytically infected grasses (Koponen and Makela1976; Eckblad and Torkelsen 1989; Bazely et al. 2007;Wali et al. 2007; Saikkonen et al. 2010; Iannone et al.2011, 2012a, b). In South America, studies of field andherbarium grass specimens revealed that almost 40 outof more that 50 grass species in the genera Briza,Bromus, Calamagrostis, Festuca, Hordeum, Melica,Phleum and Poa were infected by Epichloe endophytes(Iannone et al. 2011, 2012a, b, c, 2013; Mc Cargo et al.2014). Even though these grasses inhabit a wide rangeof environments, from tropical through subtropicalrainforests to cold deserts in southern Patagonia, aswell as altitudes of up to 3000 m, none of the infectedgrasses exhibited stroma development (Iannone et al.2012a). Similarly, during intensive field studies inFinland, Norway and Sweden, including the northern-most subarctic regions of the countries, stromata of E.festucae also were never found on F. rubra and F. ovina(Bazely et al. 2007, Wali et al. 2007). Because conidiaare produced epibiotically, sexual stromata may not beessential for horizontal transmission of the fungus to anew host. Host jumps have been proposed recently tooccur in some Epichloe-grass associations (Gentile et al.2005, Oberhofer and Leuchtmann 2012, Tadych et al.2012). Host jumps may be linked with the capabilityfor horizontal dissemination by ascospores (Craven2012). However, in many other cases host jumps maybe due to dissemination and infectivity of conidiarather than ascospores.

Forty-three Epichloe taxa endophytically associatedwith grasses have been described (TABLE I); 14 areknown to develop sexual structures with viableascospores, while in the remaining 29 taxa the sexualstate has not been observed (Leuchtmann et al. 2014).More than half (51.2%) of all Epichloe species arehybrid (Moon et al. 2004, Leuchtmann et al. 2014);they are prevalent in certain grass populations(Leuchtmann 2003, Gentile et al. 2005, Iannone etal. 2012a, Oberhofer and Leuchtmann 2013). Multiplecopies of the translation elongation factor 1-a (tefA), b-tubulin (tubB) or other genes that originated frominterspecific hybridization can be isolated from thesehybrid species. Because the versions of these sequences

reflect the lineage of the progenitor species, phyloge-netic analyses of these sequences into distinct cladeshave been used to infer the parental Epichloe species.Interspecific hybridization has been proposed toexplain the loss of the sexual stage in Epichloe speciesknown to be hybrid (Kuldau et al. 1999, Gentile et al.2005). There is one known exception; E. liyangensis, asexually reproducing species from China, is a hybrid(Kang et al. 2011). Hybridization may be a mechanismto circumvent the lack of recombination in asexualstrains by the acquisition of an additional genome.This also may enable the hybrids to explore a broaderrange of ecological niches compared to non-hybrids(Brasier 2000, Schardl and Craven 2003, Giraud et al.2008, Hamilton et al. 2010, Oberhofer and Leucht-mann 2012). How and where hybridization betweenEpichloe species occurs is not yet known, but parasexualreproduction by somatic vegetative fusion (anastomo-sis) of fungal hyphae from Epichloe species has beenproposed (Schardl et al. 1994, 1997; Tsai et al. 1994;Schardl and Craven 2003; Moon et al. 2004, 2007;Schardl 2010). The origin of hybrids from two or moreEpichloe genomes clearly indicates that host jumps arethe likely basis for hybridization (Moon et al. 2004,Gentile et al. 2005, Oberhofer and Leuchtmann 2012,Tadych et al. 2012).

With the discovery that conidia may infect seedlingsand thus may set up primary infections in grasses, it isclear that biologists must begin to examine theoccurrence and frequency of horizontal transmissionof Epichloe spp. There remains a number of questionsthat need to be addressed including: (i) do ascosporesform hyphae directly or do they produce conidia thatare infective, (ii) what range of distances can conidia orascospores travel, (iii) do insects vector conidia thatmay set up infections, (iv) are multiple vectors involvedin the fertilization of stromata, (v) do environmentalfactors such as day length affect stroma development,(vi) could this account for regional differences instromata development, (vii) are ascospores or conidiainvolved in hybridization and (viii) does hybridizationoccur within plants or outside plants? Not only dovarious aspects of the ecology of fungal endophytes ingrasses need to be systematically examined, but there isenormous complexity that will require elucidation bybiochemical and molecular techniques. Clearly, fur-ther unraveling of the strands of this complexity will bea challenge for all investigators conducting research inEpichloe -Pooideae symbioses.

ACKNOWLEDGMENTS

Our research on grass endophytes was supported in part bythe New Jersey Agricultural Experiment Station and theRutgers Turfgrass Science Center.

194 MYCOLOGIA

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