Epichloë—the obligate mutualist protecting some commercially important temperate grasses from biotic and abiotic stresses

Abstract

The relationship between Epichloë fungal endophytes found in some temperate grasses spans the continuum from antagonistic to mutualistic. The predominant focus here is on those that form mutualistic associations with those temperate grasses of the New World due to their substantial economic impact. This review describes the evolutionary origin, discovery, lifestyle and life cycle of Epichloë as background to understand the close association between plant host and fungal endophyte. Many asexual Epichloë fungal endophytes are unique in being obligate mutualists, lacking a free-living stage in nature, and being maternally inherited through host seed. The compatibility between Epichloë and its natural host plant is so complete and specific that manually transferring an Epichloë endophyte strain to other grasses, even some that are closely related genetically, though possible, often results in endophyte and/or plant death or poorly performing plant genotypes. The stability of the association is facilitated through the biotic and abiotic protection of the host grass through the endophyte promoting endogenous defense responses in the host, or directly through the production of secondary metabolites. Epichloë endophytes are renowned for producing a diverse range of alkaloids that can negatively affect invertebrate and vertebrate herbivores, either through toxicity or feeding deterrence, thereby enhancing host survival. However, Epichloë chemistry can significantly impact the health and welfare of grazing animals resulting in large economic impacts. This includes effects known as ryegrass staggers, heat stress and fescue toxicosis. In some temperate areas of the world, notably New Zealand, Australia and USA, grasses such as perennial ryegrass and tall fescue need to have the Epichloë-derived protection to ensure persistence against invertebrate pests and drought but without the downsides of clinical and subclinical losses for grazing animals. Extensive screening has identified strains of Epichloë which have been successfully commercialised for ryegrasses and fescues that ensure persistence of the grass host without causing serious health and welfare impacts on the grazing animal. The development and deployment of selected Epichloë strains therefore represent a critical intersection of plant protection, animal welfare, and agricultural productivity, a theme explored throughout this review.

1. Background and discovery of Epichloë

Perennial ryegrass (Lolium perenne L.) and tall fescue (Festuca arundinacea Schreb.; Schedonorus arundinaceus) are temperate grass species of substantial economic importance due to their widespread use in grazed pasture systems throughout temperate regions globally (Hannaway et al., 1999; Stewart, 2006; Inda et al., 2014; Filho et al., 2023). Lolium is considered to have originated in the eastern Mediterranean region around 4.1 million years ago, with diversification occurring through autogamous (self-fertilisation) and allogamous (cross-fertilisation) lineages (Ghariani et al., 2020). Tall fescue is an allohexaploid species native to Europe and North Africa, with a probable hybrid origin, involving multiple morphotypes that suggest independent origins from different diploid progenitors (Hunt and Sleper, 1981; Hand et al., 2010; Cuyeu et al., 2013; Ghariani et al., 2020). Both of these grass species, now widely used in the New World (Stewart, 2006), co-evolved with the Epichloë fungus (Clavicipitaceae, Ascomycota) (Schardl et al., 1997; Schardl et al., 2008; Saikkonen et al., 2016; Bastías et al., 2024). Epichloë are biotrophs, requiring living host tissue to survive, grow, and reproduce, rather than simply killing the host upon infection (Florea et al., 2015). This mutualistic association provides Epichloë with a safe environment with adequate nutrients and water, while the grass host gains effective protection from both invertebrate and vertebrate herbivores (Schardl, 1996; Saikkonen et al., 2016; Realini et al., 2024), some foliar diseases (Pérez et al., 2020; Card et al., 2021; Fernando et al., 2022) as well as an enhanced tolerance to some abiotic stresses (Malinowski and Belesky, 2019; Hewitt et al., 2021). It has been described as a “defensive mutualism” in which the fungi defend their hosts against herbivory, thereby defending their own resources (Clay, 1988).

Epichloë was first noted over a century ago in Lolium temulentum, a grass known for its toxicity dating back to Roman times (Freeman, 1903). It is now known to contain E. occultans (Moon et al., 2000). However, the toxicity noted was most probably not due to E. occultans, as this is not known to contain any mammalian toxic alkaloids, but most likely due to the production of other mycotoxins, e.g., ergot in seedheads (Claviceps purpurea). A pathogenic and sexual form, E. typhina, was then noted in Dactylis glomerata with the potential to cause “choke” [i.e., maturation of the inflorescence is halted and no seeds are produced on that tiller (Kirby, 1961)] and identified as systemic, growing intercellularly and distributed by infected seed but also capable of producing fruiting bodies and spores (Sampson, 1933). The maternal inheritance for Epichloë was noted in L. temulentum and L. perenne over six seasons of seed production and the relationship was noted as not being obligate for the host species (Sampson, 1935). The endophyte in L. perenne was noted as being symptomless and for not producing fruiting bodies or “choke” (Sampson, 1937). Also, in the 1930s the fungus in L. perenne was suspected of causing staggers, an animal health neurotoxic disorder causing incoordination, and described in two papers in the early 1940s (Neill, 1940, 1941). However, it was not until the late 1970s and early 1980s that the significance of Epichloë in temperate grasses on animal health disorders, notably with tall fescue (fescue toxicosis; Bacon, 1995; Hoveland, 2009) and perennial ryegrass (ryegrass staggers first noted in 1906; Gilruth, 1906—referenced by Fletcher et al., 1999), was fully documented (Bacon et al., 1977; Fletcher and Harvey, 1981, Gallagher et al., 1981). The discovery in tall fescue in the USA was originally assigned as E. typhina (Bacon et al., 1977) (sexual form of Epichloë) but later confirmed as E. coenophialum (Morgan-Jones and Gams, 1982; Christensen and Latch, 1991) (asexual form of Epichloë). In New Zealand the discovery was made for the asexual form E. festucae var. lolii in perennial ryegrass (Hume et al., 2020; Card et al., 2024).

The aim of this review is to examine the biology and significance of Epichloë fungal endophytes in temperate grasses, and to highlight why these specialised microbes are unique and have been successfully commercialised as an economical and reliable biocontrol technology.

2. Nomenclature of Epichloë

What is now known as Epichloë was originally classified as two genera—Neotyphodium (formerly Acremonium) which included those species with no sexual life cycle (anamorphs) and Epichloë which included all those with a sexual life cycle (teleomorphs). However, nomenclatural rule changes in the International Code of Nomenclature for algae, fungi and plants, adopted at the 18th International Botanical Congress in Melbourne, Australia, in 2011, which required that a single name to be used for each fungal species, meant that Neotyphodium and Epichloë species were required to be brought into the one genus—in this case Epichloë (Leuchtmann et al., 2014).

3. The biology of Epichloë

While research on Epichloë has focused largely on its impact in perennial ryegrass and tall fescue it does occur in many other grass species and genera in the subfamily Pooideae (Clay, 1990; Leuchtmann, 1992; Schardl et al., 2004; Tadych et al., 2012) distributed through Europe, North and South America, Asia and Australasia (Tadych et al., 2014).

Epichloë species, and strains within some species, can vary from being a pathogen to a mutualist (White, 1988; Schardl, 1996; Campbell et al., 2017). Additionally, within a species of Epichloë there can be significant variation in the secondary metabolite profile among strains (Schardl, 2001).

3.1. Evolutionary origin

It has been proposed that “evidence of a significant tendency for co-divergence suggests that the origin of genus Epichloë may have been close in time to the origin of the highly speciose grass subfamily, Pooideae” (Saikkonen et al., 2016). Co-evolution of Epichloë and their grass host (Schardl et al., 2008) might suggest that the center of diversity and perhaps the region of origin of Epichloë was in the Northern Hemisphere (Eurasia or North America) based on ribosomal DNA diversity studies (White et al., 2001). However, other studies have concluded that some Epichloë-host phylogenies are incongruent and do not support codivergence as a characteristic of the association (Jackson, 2004; Ambrose et al., 2014).

The evolutionary origins of Epichloë remain uncertain, but it has been proposed that these fungi arose from plant pathogenic ancestors (Carroll, 1988; White, 1988; Saikkonen et al., 2004a). Repeat-induced point mutations, which are common in pathogenic fungi, have been identified in chromosome-level genome studies of two pathogenic Epichloë sibling species along with bipartite genome organisation (Treindl et al., 2021). This allows high evolutionary flexibility, enabling faster adaptation to new environments or hosts through reassortment of chromosome segments and integration of new genes as seen in other microbes (Sonnenberg and Haugen, 2023). Additionally, there have been examples of where saprophytic or pathogenic fungi have become asymptomatic endophytes due to a single genetic mutation (Freeman and Rodriguez, 1993) or perhaps as a result of phenotypic plasticity (Schulz and Boyle, 2005).

3.2. Lifestyle and life cycle

Epichloë hyphae are predominantly found in the above ground plant, both leaves and flowers, growing in intercellular spaces (Clay and Schardl, 2002; Saikkonen et al., 2016). Adventitious roots can also become infected as they start their development in the true stem below the meristematic zone (Christensen and Voisey, 2007). Growth of the endophyte is synchronised with that of the host at all stages of growth of the host grass (Christensen and Voisey, 2007). Epichloë exist asymptomatically at least for most of their life cycle within plant tissues (White, 1987; Leuchtmann and Clay, 1997). The hyphae are attached to the outside of cells (intercellular) and grow by intercalary extension (expansion of hyphae at locations other than the tips) as the leaf and flowering stem are growing and extending (Christensen et al., 2008; Voisey, 2010). The growth of host plant and endophyte hyphae appear to be synchronised, with hyphae seldom branching and predominantly growing parallel to the leaf axis (Christensen et al., 2002; Liu et al., 2017). While this is a notable feature of Epichloë hyphal growth which is crucial for forming a structured hyphal network within the host plant, contributing to the mutualistic relationship and allowing maternal inheritance, it is not unique and has been documented in other filamentous fungi (Read, 2011). Intercalary growth of hypha is supported by specific genetic and biochemical pathways, such as the cell-wall integrity mitogen-activated protein kinase cascade in Epichloë (Becker et al., 2015), which regulates hyphal network formation (Lowry et al., 2010). In endophytic Epichloë, chitin is absent from hyphal cell walls and replaced by chitosan via the action of chitin deacetylase. Chitin is, however, retained in septa, where it facilitates intercalary hyphal growth and may contribute to evasion of host immune responses (Noorifar et al., 2021). Chitosan is a poor substrate for extracellular plant chitinases (Ride and Barber, 1990; Pusztahelyi, 2018). However, chitin is present when the fungus is grown in axenic culture (Scott et al., 2018) or epiphytically (Noorifar et al., 2021) acting as a protective barrier (Lenardon et al., 2010).

The Epichloë species can be either sexually and/or asexually transmitted (Moon et al., 2004; Tadych et al., 2014; Berry et al., 2022). Sexual reproduction of Epichloë species provides the ability for horizontal transmission of the fungus from one individual host plant to another unrelated plant. Interestingly, where an Epichloë strain can be transmitted both asexually and sexually on an individual plant, it seems likely that this duality of reproductive processes is likely triggered by an epigenetic effect (Saikkonen et al., 2016). A taxonomic revision of Epichloë genus, now over a decade old, determined that of the 43 distinct lineages (i.e., 34 distinct species, 3 subspecies, and 6 varieties), 25 were anamorphic (asexual and reproduce by conidia rather than sexual ascospores) and 18 teleomorphic (produces ascospores through meiosis), with only 3 reproduced only sexually with only horizontal transmission, 28 only asexually and 12 species/subspecies both sexually and asexually (Leuchtmann et al., 2014; Table 1). Since then, several new species of Epichloë have been identified including E. alsodes, E. hybrida, E. novae-zelandiae, E. scottii, E. transsilvanica (Table 1) and unnamed species such as the Epichloë recently identified in Festuca simensis, a grass of highland tropical Uganda, Africa (Card et al., 2025).

TABLE 1.

Characteristics of Epichloë species, including host association, region of origin, interspecific hybrid status, reproductive mode, and transmission pathway to another host.

Epichloë species	Host grass genus	Region of origin	Hybrid status	Sexual and/or asexual reproduction*	Ability to reproduce sexually	Form of transmission
E. alsodes†	Poa	North America	Hybrid	Asexual	Not observed	Vertical
E. amarillans	Ammophila, Agrostis, Hordeum, Sphenopholis	North and South America, Europe, North Africa	Non-hybrid	Sexual	Yes	Vertical
E. aotearoae	Echinopogon	New Zealand	Non-hybrid	Asexual	Not observed	Vertical
E. australiensis	Dichelachne, Echinopogon	New Zealand, Australia	Hybrid	Asexual	Not observed	Vertical
E. baconii	Agrostis	Europe, North Africa	Non-hybrid	Sexual	Yes	Horizontal
E. brachyelytri	Brachyelytrum	Europe, North Africa, North America	Non-hybrid	Sexual	Yes	Vertical
E. bromicola	Agropyron, Brachypodium, Bromus, Elymus, Leymus, Roegneria	Europe, North Africa, Asia, North and South America	Non-hybrid	Sexual	Yes, in some host species	Vertical and horizontal
E. cabralii	Bromus, Phleum	Europe, North Africa, Asia, North America and South America	Hybrid	Asexual	Not observed	Vertical
E. canadensis	Elymus	Europe, North Africa, Asia, North America	Hybrid	Asexual	Not observed	Vertical
E. chisosa	Achnatherum	Asia	Hybrid	Asexual	Not observed	Vertical
E. coenophialum	Festuca	Europe, North Africa, Asia, North and South America	Hybrid	Asexual	Not observed	Vertical
E. danica	Hordelymus	Europe, North Africa	Hybrid	Asexual	Not observed	Vertical
E. disjuncta	Hordelymus	Europe, North Africa	Hybrid	Asexual	Not observed	Vertical
E. elymi	Elymus	Europe, North Africa, Asia, North America	Non-hybrid	Sexual	Yes	Vertical
E. festucae	Fescue	Europe, North Africa, Asia, North and South America	Non-hybrid	Sexual	Yes	Vertical
E. festucae var. lolii	Lolium	Europe, North Africa	Non-hybrid	Sexual	Not observed	Vertical
E. funkii	Achnatherum	Asia	Hybrid	Asexual	Not observed	Vertical
E. gansuensis	Achnatherum	Asia	Non-hybrid	Asexual	Not observed	Vertical
E. gansuensis var. inebrians	Achnatherum	Asia	Non-hybrid	Asexual	Not observed	Vertical
E. glyceriae	Glyceria	Europe, North Africa, North America	Non-hybrid	Sexual	Yes	Horizontal
E. guerinii	Melica	South America, Sub-Saharan Africa	Hybrid	Asexual	Not observed	Vertical
E. hordelymi	Hordelymus	Europe, North Africa	Hybrid	Asexual	Not observed	Vertical
E. hybrida†	Lolium	Europe	Hybrid	Asexual	Not observed	Vertical
E. liyangensis	Poa	Europe, North Africa, Asia, North and South America, New Zealand	Hybrid	Sexual	Yes	Vertical
E. melicicola	Melica	South America, Sub-Saharan, Africa, South America	Hybrid	Asexual	Not observed	Vertical
E. mollis	Holcus	Europe, North Africa	Non-hybrid	Asexual	Yes	Vertical
E. novae-zelandiae†	Poa	New Zealand	Hybrid	Not observed
E. occultans	Lolium	Europe, North Africa	Hybrid	Asexual	Not observed	Vertical
E. pampeana	Bromus	Europe, North Africa, Asia, North and South America	Hybrid	Asexual	Not observed	Vertical
E. schardlii	Cinna	North America	Hybrid	Asexual	Not observed	Vertical
E. scottii var. nov.†	Melica	Europe	Non-hybrid	Sexual	Not observed	Vertical and possibly horizontal
E. sibirica	Achnatherum	Asia	Non-hybrid	Asexual	Not observed	Vertical
E. siegelii	Fescue	Europe, North Africa, Asia, North and South America	Hybrid	Asexual	Not observed	Vertical
E. sinica	Roegneria	Asia	Hybrid	Asexual	Not observed	Vertical
E. sinofestucae	Fescue	Europe, North Africa, Asia, North and South America	Hybrid	Asexual	Not observed	Vertical
E. stromatolonga	Calamagrostis	Asia	Non-hybrid	Asexual	Not observed	Vertical
E. sylvatica	Brachypodium	Europe, North Africa, Asia	Non-hybrid	Sexual	Yes	Vertical
E. sylvatica subsp. polliinensis	Hordelymus	Europe, North Africa	Non-hybrid	Sexual	Yes	Vertical
E. tembladerae	Briza. Bromus, Fescue, Hordeum, Melica, Phleum. Poa	Europe, North Africa, Asia, North and South America, Sub-Saharan Africa, New Zealand	Hybrid	Asexual	Not observed	Vertical
E. typhina	Anthoxanthum, Brachypodium, Bromus, Dactylis, Lolium, Phleum	Europe, North Africa, Asia, North and South America	Non-hybrid	Sexual	Yes, in some host species	Vertical and horizontal
E. typhina var. ammophilae	Ammophila	North America	Non-hybrid	Sexual	Not observed	Not observed
E. typhina sub sp. clarkii	Holcus	Europe, North Africa	Non-hybrid	Sexual	Yes	Horizontal
E. typhina sub sp. poae	Poa	Europe, North Africa, Asia North and South America, New Zealand	Non-hybrid	Sexual	Yes, in some host species	Vertical and horizontal
E. typhina sub sp. poae var. aonikenkana	Bromus	Europe, North Africa, Asia, North and South America	Non-hybrid	Sexual	Not observed	Vertical
E. typhina sub sp. poae var. canariensis	Lolium	Europe, North Africa	Non-hybrid	Sexual	Not observed	Vertical
E. typhina sub sp. poae var. huerfana	Fescue	Europe, North Africa, Asia, North and South America	Non-hybrid	Sexual	Not observed	Vertical
E. uncinata	Fescue	Europe, North Africa, Asia, North and South America	Hybrid	Asexual	Not observed	Vertical

Epichloë species in bold were previously classified as Neotyphodium. Information derived primarily from Leuchtmann et al. (2014), Caradus et al. (2021b) and Newman et al. (2021). Vertical transmission refers to the inheritance of Epichloë endophytes from parent to offspring via host seed, whereas horizontal transmission involves the spread of the fungus between host plants through infectious spores.

* Sexual indicates teleomorphic type and asexual indicates anamorphic type. †Discovered after publication of, or not described in Leuchtmann et al. (2014)—refer either Moon et al. (2007), Leuchtmann et al. (2019), Campbell et al. (2017), Shymanovich et al. (2017), or Thünen et al. (2022).

The rare occurrence of sexually reproducing strains had been previously observed (Meijer and Leuchtmann, 1999). For asexual Epichloë species it has been categorically shown that they are solely maternally inherited (vertical transmission) and do not travel via pollen (Liu et al., 2017; Forester et al., 2025). It is generally accepted that Epichloë species that are interspecific hybrids, which have heteroploid (aneuploid or polyploid) genomes, lack the capacity for sexual reproduction and are vertically transmitted through seed (Clay and Schardl, 2002). In contrast, those that are horizontally (contagiously) transmitted to other hosts through ascospore production are haploids (Schardl, 2001; Moon et al., 2004; Figure 1). In pre-anthesis florets, vertically transmitted Epichloë hyphae grow throughout the ovule nucellus, but not the integument or embryo sac, with a single hypha extending from the ovary placenta into the ovule which when fertilised extends into the developing seed and embryo (Liu et al., 2017).

FIGURE 1.

Asexual and sexual life cycles of Epichloë. A fly from the genus Botanophila is often involved in transferring conidia to the opposite mating type. The fungal stromata are visited by Botanophila flies for feeding and egg laying (Kohlmeyer and Kohlmeyer, 1974; Bultman and Leuchtmann, 2008; Bultman et al., 2022) which are attracted by the volatile compounds—methyl (Z)-3-methyldodec-2-enoate and chokol K (Steinebrunner et al., 2008b).

It has been concluded that “the abundance of hybrid species among grass endophytes, and their prevalence in many host populations suggests a selective advantage of hybridisation for the mutualistic endophytes” (Moon et al., 2004). Hybridisation of Epichloë endophytes has been proposed as a mechanism that may increase host competitive potential in stressful, resource-limited environments (Saari and Faeth, 2012). However, a consideration that it may have been due to their greater genetic variation through combining genomes from different species, which can then lead to increased adaptability and fitness benefits for host plants possibly through producing a wider range of bioactive alkaloids (Tsai et al., 1994; Gentile et al., 2005; Charlton et al., 2012; Schardl et al., 2013b) does not necessarily appear to be the case (Oberhofer et al., 2014; Jia et al., 2016). It is possible that Epichloë strain by host species compatibility may compromise these types of comparisons. It has been convincingly argued, supported by modeling, that Epichloë species that are strictly vertically transmitted through seed can only function as mutualists, as persistence in the environment would be unlikely without conferring some benefit to the host (Newman et al., 2021). That is despite others proposing that vertically transmitted endophytes are “usually parasitic” (Faeth and Sullivan, 2003) or fall along a mutualism-parasitism continuum (Müller and Krauss, 2005). However, it has also been considered that “in mutualistic associations, where one organism is contained within another organism, selection against sexual reproduction of the endobiont frequently occurs” (White, 1988; referring to Law, 1985). There is no doubt that complex and dynamic processes underlie grass-endophyte symbioses (Charlton et al., 2014).

Hybridisation of Epichloë species could potentially occur either during sexual reproduction or through vegetative nuclear fusion between two Epichloë species growing with the same host (Selosse and Schardl, 2007). Both options seem to be problematic. Sexual hybridisation between different Epichloë species was unsuccessful from many attempts with only one generated hybrid which still only produced haploid offspring which had to be rescued from the stroma since ascospores did not eject (Schardl and Leuchtmann, 1999). Most Epichloë species are not interfertile, an exception being E. typhina and E. clarkii (presumably being E. typhina sub clarkii (Craven et al., 2001; Table 1) which can hybridise (Bultman et al., 2011; Treindl and Leuchtmann, 2019). However, even here when ascospores were collected from both species, only 9.3% of fungal fruiting bodies contained spores of interspecific hybrid origin, suggesting that postzygotic isolating mechanisms provide an important means of reproductive isolation for Epichloë species (Bultman et al., 2011). It has also been considered that if selection against hybrids is strong then prezygotic reproductive barriers are likely reinforcing this outcome (Servedio and Noor, 2003). Additionally, sexual reproduction of Epichloë involves stroma formation which prevents host flowering (a disease referred to as choke; Western and Cavett, 1959; Kohlmeyer and Kohlmeyer, 1974; Merlet et al., 2022) whereas asexual reproduction through vertical transmission in host seeds does not affect host fitness (Leuchtmann and Schardl, 1998).

Hybridisation via vegetative nuclear fusion (Schardl and Craven, 2003) is equally challenging. In nature it is most unusual for more than one Epichloë strain to infect a single grass genotype, and generally competitive exclusion will eliminate one quite quickly (Christensen et al., 2000; Wille et al., 2002). However, grove bluegrass (Poa alsodes), a North American woodland grass, can independently host two species of Epichloë that vary in alkaloids produced (Shymanovich et al., 2019). It is therefore hypothesised that “hybridisation events must occur shortly after coinfection” (Selosse and Schardl, 2007). However, Selosse and Schardl (2007) noted that many hybrid Epichloë strains are found in grass species that are quite distantly related to all of their parents’ known hosts (Chung et al., 1997; Moon et al., 2004; Gentile et al., 2005), and that Epichloë strains manually inoculated into a non-host species often cause a defense response leading to spontaneous endophyte extinction after inoculation or plant death (Koga et al., 1993; Christensen, 1995; Christensen et al., 2000).

Mating of sexual Epichloë occurs due to the formation of stroma—a structure that proliferates over the plant host surface. For mating to occur spermatia (conidia) of the opposite mating type within the same mating population can be transferred by a specific fly of the genus Botanophila (Bultman et al., 2000; Bultman and Leuchtmann, 2008; Bultman et al., 2022; Figure 1). However, it has been noted that this interaction with flies is not obligatory, and that fertilisation of the fungus can also occur without the involvement of flies (Rao and Baumann, 2004; Górzyńska et al., 2011). Fertilisation leads to proliferation of thick mycelium over the stroma surface (Schardl and Tsai, 1993; Leuchtmann et al., 1994). The proliferating mycelium appears to be from spermatia or stroma and not the result of heterokaryosis (Chung and Schardl, 1997) where the mycelium would contain two or more genetically distinct nuclei within a single common cytoplasm. However, formation of heterokaryons both within and between Epichloë species is possible when grown on culture media as demonstrated by Chung and Schardl (1997). Despite that it is considered that heterokaryosis occurs so rarely for Epichloë species that it has not been selected against. Similarly, though multiple Epichloë strain infection of individual plants probably does occur (Schardl et al., 1994) they are rare (Meijer and Leuchtmann, 1999) except when in sexually reproducing forms. This may be modulated by the genetic variation of both host and endophyte (Wille et al., 2002).

Recently, the bacterial sexual parasite Wolbachia has been shown to infect 70% of fly larvae emanating from eggs laid by Botanophila flies resulting in larval death which could have far reaching effects on not only the fly host, but also the Epichloë fungi (Pagel et al., 2019). Infection by Wolbachia could therefore limit consumption of Epichloë spores by Botanophila larvae if the bacteria promoted premature larval death. Additionally, the mycoparasite Clonostachys epichloë, also has entomopathogenic activity toward Botanophila flies involved in the sexual cycle of the Epichloë and can colonise the surface of grass seeds infected with the Epichloë endophyte (Górzyńska, 2025).

Though Epichloë are biotrophs, it is possible to culture some species/strains on defined media, which is a core requirement for transferring asexual species/strains to novel hosts (Latch and Christensen, 1985). When isolated from the host plant, Epichloë generally grow slowly on defined media (Chung and Schardl, 1997).

3.3. Host—strain specificity

The genetic compatibility of an Epichloë strain with a host grass can be very specific, such that moving Epichloë strains across taxa can be challenging and often results in poor host plant growth and performance (Song H. et al., 2015; Saikkonen et al., 2016) and also resulting in different levels of alkaloid production than the original host and poorer viability in stored seed (Hennessy et al., 2016; Freitas, 2017). This effect became evident when infecting modern cereals such as rye (Secale cereale) and wheat (Triticum aestivum) with Epichloë strains isolated from wild grass relatives (Simpson et al., 2014) such as Elymus and Hordeum species (Card et al., 2014). It was concluded that “the formation of synthetic symbioses is possible but that issues of compatibility require resolution” with resulting plant phenotypes ranging from heavily stunted (sometimes terminal) through to some that resemble healthy uninfected plants (Simpson et al., 2014). This close strain to host specificity is possibly a result of their co-evolution (Saikkonen et al., 2004b; Easton, 2007; Schardl et al., 2008; Gundel et al., 2010). Genes in Epichloë encoding for secreted proteins have been identified as key determinants of host specialisation of endophyte strains (Schirrmann et al., 2018). Despite the general principle of close strain to host specificity there are examples of where some Epichloë strains are shared between different Elymus species and even across continents (Schirrmann et al., 2015).

3.4. Chemistry

The association of mutual benefit between asexual Epichloë and host grasses is facilitated by the large range of secondary metabolites and specifically alkaloids produced by the endophyte when located in planta (Bush et al., 1997; Johnson et al., 2019; Lee et al., 2021). Secondary metabolites are organic compounds produced by organisms that are not directly involved in their growth, development, or reproduction (Zandavar and Afshari Babazad, 2023). They are associated with defense, signaling, and environmental adaptation, improving ecological fitness but do not directly impact growth (Bharadwaj et al., 2020).

The impact of Epichloë in improving plant defenses is due not only to produced secondary metabolites, but also through these endophytes enhancing plant immunity against chewing insects by promoting endogenous defense responses mediated by the jasmonic acid pathway (Bastías et al., 2017a). Additionally, endophytes have been shown to influence quantitative changes in antioxidant phenolic composition (Qawasmeh et al., 2012a; Qawasmeh et al., 2012b). Some phenolic-like compounds have been shown to be exuded into the rhizosphere resulting in improved phosphorus uptake in low phosphorus environments (Malinowski and Belesky, 2000).

3.4.1. Diversity of secondary metabolites

The secondary metabolite chemistry of Epichloë is only partly understood and includes both alkaloids and non-alkaloids.

3.4.1.1. Alkaloids

An alkaloid is an organic compound containing a nitrogen atom, typically in a heterocyclic ring (Bhambhani et al., 2021). Four major classes of alkaloids are known to be produced by Epichloë: ergot alkaloids, indole diterpenes, pyrrolopyrazines, and aminopyrrolizidines (Siegel and Bush, 1996; Schardl et al., 2012; Schardl et al., 2013a; Schardl et al., 2013b; Panaccione et al., 2014). The chemical structures of compounds representing the different alkaloids expressed by Epichloë are shown in Figure 2. A description of the main categories of known alkaloids associated with Epichloë is summarised below.

FIGURE 2.

Chemical structures of known alkaloids produced by Epichloë in host grasses.

1. Ergot alkaloids: This extensive group of nitrogenous compounds derived from L-tryptophan has four main groups of compounds—clavines [e.g., chanoclavine (1), agroclavine (2)], lysergic acid (3), lysergic acid amides [e.g., ergine (4), ergonovine (5)] and ergopeptines [e.g., ergovaline (6), ergotamine (7), ergocornine (8), ergocristine (9), ergosine (10), ergocryptine (11)] (Schardl et al., 2006; Gerhards et al., 2014; Guerre, 2015). During the evolutionary development of Epichloë this diversity of ergot alkaloids has resulted from gene gains, gene losses, and gene sequence changes (Young et al., 2015). Ergovaline is the most prevalent and toxic ergot alkaloid expressed by Epichloë infected grasses (Lyons et al., 1986; Schardl et al., 2009; Caradus et al., 2020), but other ergot alkaloids produced by Epichloë endophytes can also contribute to animal health and welfare issues (Gadberry et al., 2003; Duringer et al., 2007; Guerre, 2015). The main animal health and welfare concerns associated with ergot alkaloid consumption are summarised in Table 2. Ergovaline has low in-planta mobility and strong regulation of accumulation by the internal plant environment (Spiering et al., 2002) and found principally in the basal tissues of tillers (Spiering et al., 2005). Clavines are the simplest compounds of the ergot-alkaloid class produced by the early steps of the ergot-alkaloid biosynthetic pathway (Young et al., 2015). Chanoclavine, although an intermediate compound in the ergot alkaloid pathway, can accumulate to be at a similar concentration as the ergopeptine alkaloids. However, chanoclavine is not known to cause any animal toxicity issues (Finch et al., 2019) but may have potential insecticidal activity although this is not confirmed (Hudson et al., 2021). Lysergic acid which is metabolised from elymoclavine however had been associated with high levels of African black beetle (Heteronychus arator) deterrence (Hudson et al., 2021), although no apparent effect on fall army worm (Spodoptera frugiperda) (Clay and Cheplick, 1989).

TABLE 2.

Animal health and welfare disorders associated with consuming Epichloë infected grasses.

Animal health disorder	Epichloë species	Potential associated alkaloids	References
Ruminants
Fescue toxicosis in cattle—causes tail loss, decreased feed intake, abortions, lameness due to sloughing off or loss of hooves, rough hair coats, reduction in calving rate, resulting in production losses, e.g., reduced weight gains, reduced milk production in dairy cows	E. coenophiala	Ergovaline and to a lesser extent lysergic acid, primarily acting as a vasoconstrictor	Jacobson et al., 1970; Porter et al., 1979; Hoveland et al., 1983; Hemken et al., 1984; Bacon et al., 1986; Stuedemann and Hoveland, 1988; Porter and Thompson, 1992; Roberts and Andrae, 2004; Klotz et al., 2006, 2008; Strickland et al., 2009; Tor-Agbidye et al., 2001; Guerre, 2015; Hume et al., 2025
Fescue toxicosis in deer—causing abdominal fat accumulation particularly in females	E. coenophiala	Ergovaline	Wolfe et al., 1998
Heat stress in ryegrasses	E. festucae var. lolii	Ergovaline	Fletcher et al., 1994; Fletcher et al., 2017
Ryegrass staggers, resulting in reduced milk production in dairy cows and lower liveweight gain, greater faecal soiling in the breech area (termed “dags”), higher incidence of myiasis (flystrike), compromised reproductive performance and increased mortality	E. festucae var. lolii	Lolitrem B	Fletcher and Harvey, 1981; Gallagher et al., 1981; Gallagher et al., 1982; Gallagher et al., 1984; Mortimer et al., 1984; Smith and Julian, 1998; Tor-Agbidye et al., 2001; Duringer et al., 2021; Philippe, 2016; Fletcher et al., 2017; Hume et al., 2025
	E. festucae var. lolii	Lolitrem A	Munday-Finch et al., 1995
Ryegrass staggers	Epichloë sp. LpTG-3	Epoxyjanthitrems	Babu, 2009; Babu et al., 2018; Finch et al., 2020
Ryegrass staggers (mild)	E. festucae var. lolii	Paxilline and terpendole C	Gallagher and Hawkes, 1986; Vassiliadis et al., 2023
Perennial ryegrass toxicosis—noted in Australia	E. festucae var. lolii	Ergovaline and lolitrem B	van Heeswijck and McDonald, 1992; Reed, 2005; Reed et al., 2016
“Huecu” toxicosis, a staggers-like syndrome	E. tembladerae	Indole-diterpenoid tremorgens other than lolitrem B; and glycoproteins	Miles et al., 1995; Cabral et al., 1999
Drunken horse grass poisoning	E. gansuense	Ergonovine and ergonovinine	Zhang et al., 2014
Pseudoruminants
Ryegrass staggers, brain oedema, degenerative renal and hepatic lesions in camels	E. festucae var. lolii	Lolitrem B (and possible ergovaline)—in ryegrass hay and straw	Alabdouli et al., 2014
Staggers in alpacas, along with subclinical effects—heat sensitivity and ill-thrift and reduced fertility	E. festucae var. lolii	Lolitrem B and ergovaline	Sampaio et al., 2008
Non-ruminants
Abortions in pregnant horses—increased gestation lengths, agalactia, foal and mare mortality, tough and thickened placentas, weak and dysmature foals	E. festucae	Ergovaline	Cross, 1997
Ryegrass staggers in a range of non-ruminants	E. festucae var. lolii	Lolitrem B	Hume et al., 2016; Zebeli et al., 2026
Horse oedema	E. coenophiala in Mediterranean-type tall fescue	Speculated to be loline, but essentially unknown	Bourke et al., 2009; Finch et al., 2017
White rhinoceros (Ceratotherium simum)	E. festucae var. lolii	Lolitrem B	Bluett et al., 2004

More detailed impacts of Epichloë alkaloids on animal heath symptoms are summarised by Caradus and Johnson (2020).

2. Indole diterpenoids: Indole diterpenoids are a structurally diverse group of compounds with a common core structure of a cyclic diterpene skeleton (derived from geranylgeranyl diphosphate), and an indole structure derived from tryptophan (Saikia et al., 2006). They include the compounds paspaline (12), paxilline (13), terpendoles (14–17), lolitriol (18), lolitrems (19, 20), and epoxyjanthitrems (21–25) (Weedon and Mantle, 1987; Munday-Finch et al., 1997; Munday-Finch et al., 1998; Gatenby et al., 1999; Tapper and Lane, 2004; Finch et al., 2020). Some lolitrem compounds are neurotoxins and cause tremorgens or staggers in livestock (Gallagher et al., 1984; Philippe, 2016). However, not all lolitrem compounds are tremorgenic—for example lolitrem E (19) the precursor of lolitrem B (20) is noted as not being tremorgenic when fed to mice (Miles et al., 1994). Lolitrem B, which is tremorgenic, has highest concentration in seed, the base of tillers and then in older leaf sheaths and dead leaves (Ball et al., 1997b; Spiering et al., 2005).

3. Pyrrolopyrazines: Peramine (26) is a pyrrolopyrazine-1-one metabolite (Rowan et al., 1986) found widely across Epichloë species but not outside of the epichloae (Siegel et al., 1990; Panaccione et al., 2014; Tanaka et al., 2005). Peramine is the product of a single multifunctional enzyme (PerA) as opposed to a complex pathway (Tanaka et al., 2005). Natural mutations and truncations of the PerA enzyme have been shown to produce a range of pyrrolopyrazine-1,4-dione and pyrrolopyrazine-1-one derivatives (Berry et al., 2019). Peramine is water-soluble and is distributed throughout the host plant, including tissues remote from fungal hyphae (Ball et al., 1997a; Spiering et al., 2002, 2005). Its detection in cut leaf fluid and guttation fluid suggests that peramine is transported within the plant, potentially via the xylem (Koulman et al., 2007). Peramine has no known negative effects on vertebrates (Rowan, 1993), although it has been speculated to be associated with high fecal moisture with lambs, and consequent fecal soiling (Pownall et al., 1993).

4. Lolines (1-aminopyrrolizidines): Three main loline derivatives can be found in Epichloë infected grasses—N-acetylloline (NAL 27), N-acetylnorloline (NANL 28) and N-formylloline (NFL 29) (Schardl et al., 2012). Trace amounts of loline (30), norloline (31) and N-methylloline (32) have also been observed in endophyte infected meadow fescue (Festuca pratensis) (Justus et al., 1997; Pan et al., 2014). Lolines are found in high concentrations in tall fescue and even higher levels in meadow fescue, also in some other Epichloë infected grasses (Schardl et al., 2007; Schardl et al., 2012), but not in perennial ryegrass infected with E. festucae var. lolii (Siegel et al., 1990; Ball and Tapper, 1999). In meadow and tall fescue, evidence from insect bioassays indicates that loline alkaloids are translocated in, or readily accessible from, the phloem (Popay et al., 2023) and translocated to the root system (Patchett et al., 2008b; Patchett et al., 2011b). Lolines have also been observed at detectable levels in guttation fluid (Koulman et al., 2007). Lolines appear to have no negative effects on vertebrates (Schardl et al., 2007; Finch et al., 2016a).

3.4.1.2. Non-alkaloids

Non-alkaloid secondary metabolites associated with Epichloë include flavonoids such as tricin (33) and isoorientin (34) (Ju et al., 1998), sesquiterpenoids (e.g., chokols (35–38), Hiroyuki et al., 1989), fatty acids (Koshino et al., 1987), a dipeptide-polyketide (Song et al., 2017) and cyclic octa/nona-peptides (Johnson et al., 2015; Figure 3). Some of these have been associated with anti-fungal activity (refer section 3.2.2) while the function of others remains unknown. The recently identified dipeptide-polyketide dahurelmusin A (39) has been indicated to provide resistance to aphids (Song et al., 2017). The epichloëcyclins [e.g., epichloëcyclin A (40)] are octa- and nonapeptides produced by non-ribosomally synthesised peptide (NRPS) that is cyclised via a ribosomally synthesised and post-translationally modified peptide system (RiPPs). Though produced by one of the most highly expressed genes in planta, the function of the epichloëcyclins is still unknown.

FIGURE 3.

Chemical structures of known non-alkaloid secondary metabolites produced by Epichloë in host grasses.

Additionally, fungal reactive oxygen species (i.e., highly reactive, unstable oxygen-containing molecules that are crucial for cellular communication and metabolism) production is critical in maintaining a mutualistic fungus–plant interaction (Tanaka et al., 2006).

3.5. Vulnerability of endophytes

The viability of endophyte in seed is primarily determined by the length of storage, temperature, and relative humidity which directly affects seed moisture levels (Rolston and Agee, 2007; Hume et al., 2013; Bradford et al., 2016). For valuable commercial seed lots, it is recommended that they are packed into moisture proof packaging and stored at a temperature of 5°C or less, relative humidity of 30% or less, and seed moisture of 8% or less (Hume et al., 2013). Transmission of endophyte to the developing seed is crucial for the life cycle of Epichloë, which at least for ryegrass, is heavily influenced by host genetics (Gagic et al., 2018).

4. The role of Epichloë in grazed temperate grasslands

The secondary metabolite chemistry produced in planta by Epichloë is primarily directed toward deterring herbivores from consuming the grass host. This ensures enduring plant persistence which is evident with Kentucky 31 tall fescue in United States (Bouton et al., 1993; Satognon, 2024) and ryegrasses in New Zealand and Australia with “wild type” standard endophyte (van Heeswijck and McDonald, 1992; Hume et al., 2020; Caradus, 2024; Hewitt et al., 2025). In both cases these endophytes produce ergovaline and/or lolitrem B that directly affect the health and wellbeing of the grazing ruminant in addition to deterring grazing (Ball et al., 1991; Hoveland, 1993; Bouton et al., 2001; Ball et al., 2019). Secondary metabolites from Epichloë also protect the host grass from insect pests (Breen, 1994; Caradus and Johnson, 2020) and pathogens (Card et al., 2021). It has been argued with reference to Epichloë that “plant secondary chemicals and endophytic alkaloids often fail to protect plants because: (1) specialist herbivores evolve to detoxify and use defensive chemicals for growth and survival; and (2) natural enemies of herbivores may be more negatively affected by alkaloids than are herbivores” (Faeth and Saari, 2012). However, over many decades in New Zealand where insect pressure can be severe, this has not been observed with both “wild type” (also called “common toxic” or “standard endophyte”) and commercial (novel selected strains) Epichloë strains used in perennial ryegrass. Improved drought tolerance due to Epichloë has been observed in tall fescue, although the biochemistry and/or mechanism causing this effect are largely unknown (Elmi and West, 1995; Elbersen and West, 1996; Nagabhyru et al., 2013; Xu et al., 2017).

4.1. Impact on grazing vertebrates

Epichloë produce alkaloids that can cause a variety of health and welfare concerns, and productivity losses, for both grazing ruminants and non-ruminants (Table 2). In United States, the cost of fescue toxicosis was estimated to be US$600 million per year over 20 years ago (Fribourg and Waller, 2004). In New Zealand, the estimated cost of ryegrass staggers was NZ$100 million per year 20 years ago (NZ Veterinary Association, 2007). Similarly, in Australia the estimated cost for perennial ryegrass toxicosis in sheep production systems was A$100 million per year (MLA, 2012). These negative effects of Epichloë endophytes on animals led to the proposition that endophyte-free grasses would be more beneficial for livestock production (Stuedemann and Hoveland, 1988; Bouton et al., 1993). However, it was soon realised that the endophyte was crucial to persistence of the grass host (Bacon and Siegel, 1988; Bacon, 1995; Easton, 1999; Easton et al., 2001) and resulted in programmes to deliver novel less mammalian toxic strains, which have been successfully commercialised for over 25 years (Bouton et al., 2002; Parish et al., 2003; Franzluebbers et al., 2009; Hopkins et al., 2010; Caldwell et al., 2013; Johnson and Caradus, 2019; Caradus and Johnson, 2020).

In addition to impacts of animal health and welfare disorders indirectly reducing levels of production (Table 2) some Epichloë chemistry (e.g., ergovaline) can also directly cause reduced levels of production through feeding deterrence (Stuedemann et al., 1989; Seman et al., 1990) resulting in reduce animal liveweight gains (Layton et al., 2004). However, the feeding deterrence due largely to ergovaline has been used as a benefit in turf grasses to deter birds from feeding on them when used in airports and playgrounds (Pennell et al., 2010; Pennell and Rolston, 2013; Pennell et al., 2016; Pennell et al., 2017b; Pennell et al., 2017a). Similar Epichloë chemistry, such as ergovaline, that maybe toxic to mammals and deter insect pests, aid in deterring insectivorous and predatory birds from those areas as well (Popay and Wyatt, 1995; Ball et al., 1997c; Popay and Ball, 1998).

The movement of Epichloë endophyte alkaloids to animal meat and milk has been examined and raise no food safety concerns. It has been shown that concentrations of epoxyjanthitrems (20–24) and lolitrem B (19) in sheep and dairy cow fat can increase when concentrations in pasture increase but then decrease when animals were removed from pastures containing these compounds (Finch et al., 2012; Finch et al., 2013; Duringer et al., 2021). There is no convincing evidence that ergot alkaloids accumulate in edible tissues of animals fed ergot alkaloids (Anon, 2005; Schumann et al., 2009; Zbib et al., 2015).

4.2. Impact on growth and persistence of pasture grasses

Epichloë endophytes particularly in New Zealand, Australia and United States have been shown to improve both the persistence and yield of important temperate grasses (Hume and Sewell, 2014; Thom et al., 2014; Caradus, 2024). Interestingly, with many of these originating in Europe (particularly for perennial ryegrass and tall fescue) the selective advantage of Epichloë endophyte is not immediately obvious but has been suggested as an option for sustainable agriculture (Kauppinen et al., 2016; von Cräutlein et al., 2021). However, where Epichloë is important for yield and persistence it is due primarily to providing resistance and/or tolerance to biotic and abiotic stress (Lee et al., 2021). It has been stated that “over 40 insect pests and diseases have been documented as responding to endophyte infection” (Stewart et al., 2022).

4.2.1. Insect pest resistance

In some temperate grassland environments (e.g., New Zealand; Caradus, 2024; Card et al., 2024) Epichloë is crucial for the persistence of grasses against a wide range of invertebrate pests. The known impact of various alkaloids produced by Epichloë on insect pests are primarily either due to toxicity or deterring feeding and are summarised in Table 3. There are however several Epichloë strains that effectively control the impact of insect pests but for which the chemistry responsible is still unknown. For example, the control of root aphid (Aploneura lentisci) by strains AR5 and AR37 (Popay et al., 2021); reduced numbers of African black beetle and reduced feeding by Argentine stem weevil (Listronotus bonariensis) by strain AR37 (Popay et al., 2008); reduced numbers of Philobota spp.—pasture tunnel moths and Pseudococcidae—mealybugs by strain AR37 (Moate et al., 2012); and reduced fall army (Spodoptera frugiperda) larval growth and feeding with Epichloë strains in meadow fescue and selected strains in tall fescue (Lee et al., 2025); and strains AR47 and AR48 have been shown to have a deterrent and/or toxic effects on wheat sheath miner (Cerodontha australis) (Jensen et al., 2022); an undocumented Epichloë strain that provided resistance to sod webworm (Crambus spp.) (Funk et al., 1983); and naturally Epichloë-infected native Iranian tall fescue providing biological control of Elm-grass root aphid (Tetraneura ulmi) and barley aphid (Sipha maydis) (Mohammadi et al., 2024).

TABLE 3.

Epichloë alkaloids associated with providing protection of grass host against invertebrate pests.

Alkaloid compound	Invertebrate pest	Modes of protection	References
Compounds not known to cause animal health and welfare concerns
Lolines	Aphids—Rhopalosiphum padi and Schizaphis graminum	Toxicity	Johnson et al., 1985; Latch et al., 1985; Siegel et al., 1990; Eichenseer et al., 1991; Wilkinson et al., 2000; Bultman et al., 2006; Bastías et al., 2017b
	Grass grub—Costelytra giveni	Feeding deterrent	Popay et al., 2003a; Patchett et al., 2008b; Patchett et al., 2011a
	Root aphid—Aploneura lentisci	Reduced root aphid numbers per plant	Jensen and Popay, 2007; Schmidt, 1993
	Red-headed cockchafer—Adoryphorus coulonii	Feeding deterrent	Bryant et al., 2010
	African black beetle—Heteronychus arator	Deterrent, antifeeding effect on larval and adult stages	Bryant et al., 2010; Barker et al., 2015a
	Argentine stem weevil—Listronotus bonariensis	Feeding deterrent and death of larvae	Patchett et al., 2008a; Jensen et al., 2009; Popay et al., 2009; Barker et al., 2015c
	Porina—Wiseana cervinata	Reduce feeding and weight gain	Popay and Lane, 2000
	Black field cricket—Teleogryllus commodus	Deterrent	Barker et al., 2015b
	Fall armyworm larvae—Spodoptera frugiperda	Deterrent—reduced worm survival and liveweight gains	Riedell et al., 1991
	Greenbugs—Schizaphis graminum	Toxicity	Riedell et al., 1991
	Japanese beetle larvae—Popillia japonica	Toxicity and deterrence	Patterson et al., 1991; Bush et al., 1993
	Rice leaf bug—Trigonotylus caelestialium	Toxicity and deterrence	Shiba and Sugawara, 2009
N-formyl loline	Cat flea larvae—Ctenocephalides felis	Contact toxicity	Bush et al., 1993
	Grass grub—Costelytra giveni	Toxicity	Popay and Lane, 2000; Popay and Tapper, 2007
	Large milkweed bug—Oncopeltus fasciatus	Feeding deterrent and toxic	Johnson et al., 1985; Yates et al., 1989
	American cockroach—Periplaneta americana	Contact toxicity	Bush et al., 1993
	Lesion nematode—Pratylenchus scribneri	Toxicity and deterrence	Kimmons et al., 1990; Bacetty et al., 2009
N-acetylnorloline	Argentine stem weevil—Listronotus bonariensis	Deterrent	Jensen et al., 2009
N-acetyl loline	European corn borer larvae—Ostrinia nubilalis	Toxic effects and reduced larval weight gain	Riedell et al., 1991
	Grass grub—Costelytra giveni	Deterrent	Popay and Tapper, 2007
Possibly loline (Epichloë strain was in cultivar Kentucky 31 in tall fescue)	Soil nematodes—Pratylenchus scribneri and Tylenchorhynchus acutus	Reduced infection of roots of tall fescue	West et al., 1988
Peramine	Argentine stem weevil—Listronotus bonariensis (larva), pasture mealy bug—Balanococcus poae	Feeding deterrent for both adults and larvae; reduced oviposition	Prestidge et al., 1982; Gaynor and Rowan, 1985; Rowan and Gaynor, 1986; Rowan et al., 1990; Pennell et al., 2005; Chacón-Fuentes et al., 2025
	Cutworm—Graphania mutans	Not a deterrent, but disrupted development	Dymock et al., 1989b
	Aphid—Schizaphis graminum	Feeding deterrent and toxic	Siegel et al., 1990
Compounds known to cause some animal health and welfare concerns
Ergovaline	Argentine stem weevil—Listronotus bonariensis	Feeding deterrent	Dymock et al., 1989b; Popay et al., 1990
	African black beetle—Heteronychus arator	Ergopeptine alkaloids—ergotamine, ergovaline, ergocryptine	Ball and Prestidge, 1992, 1993; Popay and Ball, 1998
Ergovaline, ergotamine	Lesion nematode—Pratylenchus scribneri	Attractant and causes death	Bacetty et al., 2009
Ergovaline and/or ergine Ergonovine Ergocryptine, ergonovine	Black cutworm—Agrostis ipsilon	Deterrence and toxicity	Baldauf et al., 2011
	Aphids—Rhopalosiphum padi	Toxicity	Shymanovich et al., 2015
	Lesion nematode—Pratylenchus scribneri	Deterrent	(Bacetty et al., 2009)
	Large milkweed bug—Oncopeltus fasciatus	Toxic	Yates et al., 1989
Ergotamine, ergonovine, ergocryptine	Fall army worm—Spodoptera frugiperda	Reduced worm survival and liveweight gains	Clay and Cheplick, 1989
Chanoclavine	African Black beetle—Heteronychus arator	Mild deterrent	Hudson et al., 2021
Lysergic acid	African black beetle—Heteronychus arator	Deterrent	Hudson et al., 2021
Lolitrem B	Porina—Wiseana spp.	Reduce feeding and weight gain	Kaufononga, 2019
	Argentine stem weevil—Listronotus bonariensis	Toxic/feeding deterrent of larvae	Prestidge and Gallagher, 1985; Dymock et al., 1989a; Rowan, 1993; Popay et al., 2003b
	Mollusc—Deroceras reticulatum	Reduced feeding	Barker, 2008
Paxilline	Porina—Wiseana cervinata	Reduce feeding and weight gain	Babu, 2009; Kaufononga, 2019
	Argentine stem weevil—Listronotus bonariensis	Feeding deterrent	Rowan, 1993
Epoxyjanthitrems	Porina—Wiseana spp.	Reduced feeding and toxicity	Popay et al., 2012; Hennessy et al., 2016; Finch et al., 2020

While the focus for the impact of Epichloë on insect pests has been through alkaloid production there is evidence that some insect resistance may come from genes transferred horizontally into Epichloë from bacterial donors (Ambrose et al., 2014).

4.2.2. Pathogen resistance

Early Epichloë studies referred to production of antifungal substance(s) in in vitro assays with fungal pathogens such as Rhizoctonia cerealis (White and Cole, 1986). Other reports of Epichloë suppressing other fungal diseases of their host grass include dollar spot disease (Sclerotinia homoeocarpa) in fine fescues (Festuca rubra subsp. rubra) (Clarke et al., 2006), Rhizoctonia zeae in tall fescue (Gwinn and Gavin, 1992), and barley yellow dwarf virus in meadow fescue due to reduced aphid numbers (Lehtonen et al., 2006). Epichloë can protect the grass host plant against pathogens both directly through the production of fungicidal compounds, competition for a common resource, or the induction of plant defenses, and indirectly through endophyte-generated changes in the abiotic or the biotic environment (Pérez et al., 2020). A meta-analysis of 22 publications, covering a total of 18 host grass species, 11 Epichloë endophyte species, and 22 fungal pathogen species, demonstrated a negative overall effect of Epichloë on infection by pathogens (Pérez et al., 2020). Interestingly, but perhaps not surprisingly, in that analysis the protective impact of the Epichloë was greater in laboratory and glasshouse trials than in field trials. An extensive summary of both in vitro and in planta bioactivity exhibited by species of Epichloë toward fungal saprophytes or phytopathogens is provide by Card et al. (2021).

Using in vitro assays, it has been confirmed that the chemistry responsible for the antifungal activity observed by Epichloë endophytes is not due to the known Epichloë-derived antimammalian and insecticidal alkaloids and their intermediates [peramine (26), N-formylloline (29), N-acetylloline (27), lolitrem B (20), epoxyjanthitrem I (22), paxilline (13), terpendole E (14), terpendole C (16), ergovaline (6)] (Siegel and Latch, 1991; Christensen, 1996; Fernando et al., 2021). Chemistry associated with providing antifungal activity is described in Table 4. Some Epichloë species do not necessarily produce some of these compound, e.g., E. festucae and E. bromicola did not produce chokol K (38) (Steinebrunner et al., 2008a). Additionally, the inhibition of fungal pathogens can be endophyte strain specific within a species of Epichloë (Niones and Takemoto, 2014; Fernando et al., 2021; Li et al., 2023).

TABLE 4.

Chemistry associated with antifungal activity provide by Epichloë.

Chemical	Epichloë species	Pathogens active against	Effect and testing procedure	References
Sesquiterpinoid—Chokol K	E. sylvatica E. clarkii	Stagonospora nodorum Mycosphaerella graminicola	Reduced the spore germination of all tested fungi—tested in culture	Steinebrunner et al., 2008a
Sesquiterpinoids—Chokols A, B, C	E. typhina		Known to be fungitoxic	Yoshihara et al., 1985
Sesquiterpenoids—Chokols A, B, C, D, E, F and G	E. typhina		Known to be fungitoxic	Hiroyuki et al., 1989
Protein—designated Efe-AfpA	E. festucae	Sclerotinia homoeocarpa	In vitro petri dish assay with extracted compound	Tian et al., 2017
Indole derivatives—indole-3-acetic acid and indole-3-ethanol	E. festucae	Cryphonectria parasitica	Isolated from organic extracts from liquid fermentation cultures	Yue et al., 2000
Fatty acids—C-18 hydroxy unsaturated fatty acids	E. typhina		Noted as fungitoxic	Koshino et al., 1987
Peptide—ε-poly-L-lysine	E. festucae	Drechslera erythrospila, Botrytis cinerea, Phytophthora infestans	Reduced the spore germination of all tested fungi and hyphal growth in vitro	Purev et al., 2020
Gamahonolides—Gamahonolides A and B, gamahorin, and 5-hydroxyl-4-phenyl-2(5H)-furanone	E. typhina		Noted as fungitoxic	Koshino et al., 1992
Phenolics—1,3-O-di-trans-p-coumaroylglycerol, 1,2-O-di-trans-p-coumaroylglycerol, chokorin	E. typhina		Noted as fungitoxic	Koshino et al., 1988
Cyclic peptide—epichlicin	E. typhina	Cladosporium phlei	Reduced the spore germination	Seto et al., 2007
Cyclic peptide—Cyclosporin T	E. bromicola	Alternaria alternata, Fusarium avenaceum, Bipolaris sorokiniana, Curvularia lunata		Song Q. Y. et al., 2015
Protease and endoglucanase activity possibly related to vibA gene which was associated with anti-fungal activity	E. festucae	Drechslera erythrospila, D. siccans, D. dictyoides, Colletotrichum graminicola, Bipolaris sorokiniana	Inhibited hyphal growth conidial germination	Niones and Takemoto, 2014; Niones and Takemoto, 2015

The antagonism of Epichloë toward multiple species of saprophytic and pathogenic microbes associated with some temperate grasses has been reviewed by Card et al. (2021). In addition to the secondary metabolites list in Table 4, Epichloë-mediated induction of the plant’s own defenses has been proposed as another means of providing resistance to phytopathogens (Xia et al., 2018; Pérez et al., 2020).

4.2.3. Abiotic stress tolerance

In addition to “protecting” the host plant from numerous biotic challenges, Epichloë also provide some benefits that reduce abiotic stress impacts (Malinowski et al., 2005; Malinowski and Belesky, 2019; Lee et al., 2021). The most notable is improved tolerance to drought or low soil moisture environments (Arachevaleta et al., 1989; Eerens et al., 1998; West et al., 1993; Malinowski and Belesky, 2000; Kannadan and Rudgers, 2008; Li et al., 2008; Kane, 2011; Decunta et al., 2021). However, there are some studies reporting that endophyte status does not affect plant responses to drought (White et al., 1992; West, 1994; Cheplick et al., 2000; Rudgers and Swafford, 2009; He et al., 2017), although these were glasshouse pot trials. Where Epichloë has shown improved drought tolerance, the effect in fescue has been a reduction in stomatal conductance and improved osmotic adjustment, particularly in the meristematic and growing zone of the plant resulting in improved recovery from drought (Bacon, 1993; Elmi and West, 1995; Elbersen and West, 1996). The imposition of water deficit has also been shown to increase free glucose, fructose, trehalose, sugar alcohols, proline and glutamic acid in shoots and roots, along with the fungal metabolites, mannitol, loline alkaloids (Nagabhyru et al., 2013) and in ryegrass ergovaline and lolitrem B (Hahn et al., 2008). For perennial ryegrass, some Epichloë strains such as AR37 have been shown to increase root system growth (Hume, 2018) which may provide the shoot with a better supply of nutrients and water in periods of stress. Changes in antioxidant enzyme activity due to Epichloë presence has also been linked to improved recovery from drought (Xu et al., 2017). The positive effects of Epichloë on ryegrass persistence during drought become even more important if coinciding with insect attack, confirming their critical importance in many intensively managed pastoral systems (Hewitt et al., 2021).

Improvements have also been shown for tolerance to salinity (Li et al., 2008; Sabzalian and Mirlohi, 2010; Yin et al., 2014; Wang et al., 2020), tolerance to high aluminum (Malinowski and Belesky, 1999; Zaurov et al., 2001) or high nickel (Mirzahosseini et al., 2014) soils, and improved nutrient uptake in low nutrient (e.g., phosphorus) soils (Malinowski and Belesky, 2000) due to Epichloë infection in grasses. However, the mechanisms for these improvements are not necessarily well understood. Some may be due to improved resilience with the host plants protected from some insect pests and pathogens by the Epichloë endophyte (Lee et al., 2021).

5. The development of commercially viable Epichloë strains

Following the discovery of Epichloë endophytes in temperate grasses, such as ryegrass and fescue, and their links to both animal health and welfare issues, as well as enhanced host-plant persistence, efforts were initiated to identify Epichloë strains that retained agronomic benefits while mitigating negative impacts on livestock. This search focused on endophyte strains capable of sustaining host persistence without compromising animal health and welfare (Easton, 1999; Easton et al., 2001; Gunter and Beck, 2004; Phillips and Aiken, 2009; Johnson et al., 2013; Johnson and Caradus, 2019; Caradus and Johnson, 2020; Caradus et al., 2021b; Eady, 2021; Caradus, 2024; Hewitt et al., 2025). This led to the identification for what has been termed “novel” or “selected” endophytes.

5.1. Ryegrasses

In New Zealand, and to a lesser extent in Australia, the focus on identifying novel Epichloë for ryegrasses has been to reduce the potential for ryegrass staggers and heat stress, and as a result cause less dags/flystrike in sheep and remove the hand brake on milk production and live weight gain (Hume et al., 2025). To ensure success, plant breeders needed to ensure that the genetic compatibility between the selected Epichloë strain and host plant was effective, so that transmission through seed and establishment in resulting seedlings was effective (Christensen et al., 1997; Gagic et al., 2018; Zhang et al., 2022). The first strain successfully commercially released was AR1 in 2001—aimed at providing resistance to Argentine stem weevil with very low levels of ryegrass staggers and no heat stress (Fletcher, 1999; Popay et al., 1999). Ryegrass cultivars with AR1 endophyte strain were rapidly adopted by farmers (Caradus et al., 2013). Evaluation under dairying indicated that AR1-infected ryegrass while producing similar pasture yields as standard endophyte-infected ryegrass, it also provided small (6.7%) improvements in milk yield with no incidence of ryegrass staggers in grazing animals (Bluett et al., 2005). This has been followed by numerous other novel strains with different profiles in terms of insect pest resistance while keeping animal health and welfare concerns to a minimum (Caradus et al., 2021b; Table 5). The most notable outcome of this work has been the development of the Epichloë strain AR37. This endophyte has been shown to substantially increase perennial ryegrass production, with national gains of approximately 12% and increases of up to 37% in northern New Zealand compared with standard Epichloë strains over a 4-year period (Hume et al., 2007). More recent evaluations report production increases of up to 33% and 55% relative to AR1 and endophyte-free cultivars, respectively (Ferguson et al., 2025). In addition, AR37 confers resistance against five of the six major insect pests affecting ryegrass (Young et al., 2013), extending pasture persistence such that average renewal intervals may increase from 5 to 8 years (Thom et al., 2013; PGWS, 2026). Importantly, it also reduces the severity of ryegrass staggers compared with standard endophyte strains (Fletcher et al., 2017; Caradus and Johnson, 2020; Caradus et al., 2021b; Eady, 2021). Consequently, AR37, 1924 has remained the dominant Epichloë endophyte incorporated into proprietary ryegrass cultivars in New Zealand through to the mid-2020s (Caradus et al., 2025). The success of Epichloë endophytes has resulted in > 90% of all New Zealand bred or marketed proprietary forage tall fescue, perennial and hybrid ryegrass cultivars containing a novel Epichloë endophyte (Milne, 2007; Chynoweth et al., 2015).

TABLE 5.

Insect pests that commercialized strains of Epichloë for use in pasture grasses have been documented to protect their host plant.

Insect pest	Epichloë Brands (or strains)		References
	High level of protection	Moderate to low level of protection
Epichloë festucae var. lolii in ryegrasses
Argentine stem weevil—Listronotus bonariensis	Standard, AR1, NEA2, NEA4, AR371, NEA121, AR128, Endo5, RGT18, CM142	(Edge)	Popay and Hume, 2011; Thom et al., 2014; Popay et al., 2017; Caradus et al., 2025; PBRA, 2026a
Pasture mealy bug—Balanococcus poae	Standard, AR1, NEA2, NEA42, AR371, Endo5, NEA, (Happe4)	(Edge)	Pennell et al., 2005; Popay and Hume, 2011; Popay et al., 2021; PBRA, 2026a; Ferguson, personal communication April 2026
African black beetle—Heteronychus arator	Standard, NEA2, NEA42, AR371, NEA121, AR128, Endo5, NEA, RGT18, CM142, (Happe4)	AR1	Popay and Hume, 2011; Thom et al., 2014; Caradus et al., 2025; PBRA, 2026a; Ferguson, personal communication April 2026
Root aphid—Aploneura lentisci	AR371, (AR63) NEA121, AR128, RGT18, CM142, (Happe4)	Standard, NEA2, NEA4	Popay and Gerard, 2007; Pennell et al., 2005; Popay and Hume, 2011; Thom et al., 2014; Popay and Cox, 2016; Popay et al., 2021; Hewitt et al., 2024a; Caradus et al., 2025; PBRA, 2026a; Ferguson, personal communication April 2026
Porina—Wiseana cervinata	AR371, NEA121, AR128, RGT18, CM142, (Happe4)	Standard	Jensen and Popay, 2004; Popay and Hume, 2011; Caradus et al., 2025; PBRA, 2026a; Ferguson, personal communication April, 2026
Grass grub—Costelytra giveni		AR37, (Happe4)	Popay and Thom, 2009; Hewitt et al., 2024b; PBRA, 2026a; Ferguson, personal communication April, 2026
Epichloë coenophialum in fescues
Argentine stem weevil—Listronotus bonariensis	AR542 (MaxQ)		Popay et al., 2005
African black beetle—Heteronychus arator	AR542 (MaxQ), AR584—MaxQII,		Popay et al., 2005; PBRA, 2026a
Root aphid—Aploneura lentisci	AR542 (MaxQ), AR584—MaxQII		Popay and Jensen, 2005; PBRA, 2026a
Grass grub—Costelytra giveni	AR10175 MaxR	AR542 (MaxQ), AR584—MaxQII,	Popay and Tapper, 2007; Hewitt et al., 2024b; PBRA, 2026a
Black field cricket—Teleogryllus commodus	AR584—MaxQII		PBRA, 2026a
Epichloë uncinata in festulolium
Argentine stem weevil—Listronotus bonariensis	U2		Patchett et al., 2008a; Barker et al., 2015c; Popay et al., 2017; PBRA, 2026a
Pasture mealy bug—Balanococcus poae	U2		PBRA, 2026a
African black beetle—Heteronychus arator	U2		Barker et al., 2015a; PBRA, 2026a
Root aphid—Aploneura lentisci	U2		PBRA, 2026a
Porina—Wiseana cervinata	U2		PBRA, 2026a
Grass grub—Costelytra giveni		U2	Patchett et al., 2011a; PBRA, 2026a
Black field cricket—Teleogryllus commodus	U2		Barker et al., 2015b; PBRA, 2026a

Information from Caradus and Johnson (2020), Caradus et al. (2021a,b), Caradus (2024) and PBRA (2026a) has been integrated into this table. * Brackets indicate the strain is no longer commercially available.

¹Epichloë sp. LpTG-3.

²Epichloë sp. LpTG-2.

³ Epichloë hybrida.

⁴ Epichloë siegelii.

⁵E. uncinata.

5.2. Fescues

The priority for a novel Epichloë endophyte for fescues was to eliminate the occurrence of fescue toxicosis. This was effectively achieved through the release of tall fescue endophyte strain AR542 (branded MaxQ in USA and MaxP in New Zealand/ Australia) in 2000 and then AR584 (branded MaxQII in USA and MaxP in New Zealand/ Australia) (Bouton, 2009; Phillips and Aiken, 2009; Young et al., 2014; Johnson and Caradus, 2019; Card et al., 2024). To date there have been relatively few other tall fescue novel Epichloë strains successfully commercialised, with only ArkShield, Protek, and E34, joining MaxQII on the market (Smith and Phillips, 2016; Caradus and Johnson, 2020; Allen et al., 2021). In New Zealand, a novel strain for meadow fescue (strain AR1017, branded MaxR) has also been commercialised (Stewart et al., 2025).

5.3. Other pasture grasses

While grasses other than ryegrass or fescue do form mutualistic associations with Epichloë, none have been commercialised successfully.

5.4. Turf grasses

The Epichloë–grass mutualism, and resulting impacts on insect pests and mammals, makes it an ideal technology for use in areas where grazing animals are absent and grass cover is required, such as in parks, sports playing surfaces and airports (Funk et al., 1993; Meyer et al., 2013; Stewart et al., 2022; Reid et al., 2025). It has been shown that some of the mammalian toxic alkaloids expressed by Epichloë act as deterrents to not just insects, but also birds, mice and rabbits (Pennell et al., 2010; Finch et al., 2016b; Pennell et al., 2016). As a result, an Epichloë endophyte product has been released for specific use at airports and in parks and playing areas and branded Avanex™, using endophyte strains AR95 for ryegrasses and AR601 for tall fescue (Pennell and Rolston, 2013; Pennell et al., 2016; Pennell et al., 2017b; Pennell et al., 2017a).

5.5. Commercialisation code of practice

Proprietary forage seed companies in New Zealand (PBRA, 2026b) and USA (Alliance for Grassland Renewal, 2026) have agreed that for a tall fescue or ryegrass seed line to be labeled as “endophytic” then at least 70% of seed must contain viable Epichloë endophyte. For New Zealand the agreed wording includes “its members, adopt the position that for grade endophytic ryegrass and fescue seed to be sold, that there is a current seed test to confirm that at least 70% of seed will be infected with live endophyte at the time of sale to the retailer.” In Australia, a unified seed company position is still to be confirmed but individually some companies agree that for an endophyte to be effective on farm, at least 70% of the seed needs to contain a live endophyte (DLF Seeds Australia, 2026).

6. Concluding comments

Asexual Epichloë endophytes in New World pastures (e.g., New Zealand, Australia, and USA) provide considerable benefit to the persistence of temperate grass pastures (Bouton, 2009; Thom et al., 2012; Figure 4). They are highly efficacious, making them one of the most successful biocontrol products. However, balancing that benefit while ensuring reduced or nil animal health and welfare concerns has been a major driver in delivering Epichloë strains of commercial value. Clearly in these pastures the absence of Epichloë endophyte results in poor persistence and the need for ongoing pasture renewal, which is both expensive and has considerable environmental downside through soil disturbance. Over the last 25 years several strains of Epichloë have been successful commercialised in New Zealand, Australia and USA. Being maternally inherited they are part of the seed and move into the seedling grass plant upon germination. Pastoral farmers can use Epichloë endophytic grasses without it being disruptive to their traditional on-farm management systems. To ensure viable endophyte is maintained in seed during storage, seed is recommended to be kept at 5°C or less and relative humidity of 30% or less. While Epichloë has to date been used only in temperate grasses it may have application in some cereals which while not having Epichloë are genetically related to some wild grasses that do harbor Epichloë (Card et al., 2014; Simpson et al., 2014; Wood et al., 2024).

FIGURE 4.

Effect of Epichloë endophyte strain AR37 (right) compared with nil endophyte (left) on perennial ryegrass persistence and growth in New Zealand.

The Epichloë–grass association represents a stable, co-evolved symbiosis in which microbial function is embedded within plant life history, supporting consistent performance across seasons and variable stress regimes. Its success challenges conventional distinctions between genetics, management, and environment by demonstrating that adaptive capacity can be distributed across plant–microbe assemblages rather than residing solely within the plant genome. In New Zealand, the implications extend beyond individual pastures; by underpinning the persistence and productivity of temperate grasslands, Epichloë endophytes have helped stabilise the pastoral foundation of the national agricultural economy. Their widespread adoption illustrates how biologically embedded microbial symbioses can enhance resilience, reduce system vulnerability, and support long-term agricultural viability at national scale. There is little doubt that Epichloë typifies the stunning role that microbial symbioses can play in improving resilience and yield in plants of economic importance.

Funding Statement

The author(s) declared that financial support was not received for this work and/or its publication.

Author contributions

JC: Conceptualization, Writing – original draft, Writing – review & editing. KH: Writing – review & editing. DH: Writing – review & editing. WM: Writing – review & editing.

Conflict of interest

JC was employed by the Grasslanz Technology Ltd, a company that funds research and development of Epichloë strains for commercial use.

The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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The author(s) declared that Generative AI was not used in the creation of this manuscript.

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