Bioactive alkaloids in vertically transmitted fungal endophytes



  1. Plants form mutualistic symbioses with a variety of microorganisms including endophytic fungi that live inside the plant and cause no overt symptoms of infection. Some endophytic fungi form defensive mutualisms based on the production of bioactive metabolites that protect the plant from herbivores in exchange for a protected niche and nutrition from the host plant. Key elements of these symbioses are vertical transmission of the fungus through seed of the host plant, a narrow host range, and production of bioactive metabolites by the fungus.
  2. Grasses frequently form symbioses with endophytic fungi belonging to the family Clavicipitaceae. These symbioses have been studied extensively because of their significant impacts on insect and mammalian herbivores. Many of the impacts are likely due to the production of four classes of bioactive alkaloids – ergot alkaloids, lolines, indole-diterpenes and peramine – that are distributed in different combinations among endophyte taxa.
  3. Several legumes, including locoweeds, are associated with a toxic syndrome called locoism as a result of their accumulation of swainsonine. Species in two genera were recently found to contain previously undescribed endophytic fungi (Undifilum spp., family Pleosporaceae) that are the source of that toxin. The fungi are strictly vertically transmitted and have narrow host ranges.
  4. Some plant species in the morning glory family (Convolvulaceae) also form symbioses with endophytic fungi of the Clavicipitaceae that produce ergot alkaloids and, perhaps in at least one case, lolines. Other species in this plant family form symbioses with undescribed fungi that produce swainsonine. The swainsonine-producing endophytes associated with the Convolvulaceae are distinct from the Undifilum spp. associated with locoweeds and the Clavicipitaceous fungi associated with Convolvulaceae.
  5. In the establishment of vertically transmitted symbioses, fungi must have entered the symbiosis with traits that were immediately useful to the plant. Bioactive metabolites are likely candidates for such pre-adapted traits which were likely useful to the free-living fungi as well. With future research, vertically transmitted fungi from diverse clades with narrow host ranges and that produce bioactive compounds are likely to be found as important mutualists in additional plants.


Several classes of mutualisms have been recognized based upon the benefits exchanged between the partners including energetic (e.g. photosynthetic Chlorella in Anthozoans), nutritional (e.g. mycorrhizae), transport (e.g. pollination) and defensive (Boucher, James & Keeler 1982; Janzen 1985; Douglas 1994). In defensive mutualisms, one partner provides protection from or resistance to one or more of its partner's natural enemies. A classic example is that of the Ant-Acacia system in which voracious ants protect Acacia trees from megaherbivores (e.g. elephants) in exchange for shelter in plant structures called domatia (Janzen 1966; Palmer et al. 2008). Defensive mutualisms involving microbial symbionts that produce protective chemistry have repeatedly evolved in diverse taxa and can have resounding effects on host success as well as community structure and dynamics (White & Torres 2009). For example, marine bryozoans harbour bacterial symbionts that produce polyketide bryostatins, without which hosts are vulnerable to fish predation (Lopanik, Lindquist & Targett 2004; Lopanik, this issue), leaf cutter ants utilize an actinomycete that produces antibiotics to ward off yeasts that would otherwise degrade the ants' fungal garden (Currie, Mueller & Malloch 1999), and aphids host facultative bacterial symbionts that can provide resistance to both abiotic (e.g. heat) and biotic (e.g. parasitoids) stressors (Oliver et al. 2010; Oliver, this issue). Defensive mutualism was proposed by Clay (1988) to describe the relationship between certain fungi and their grass hosts, in which the fungi are afforded a habitat and carbohydrates by their host plant and provide their host with protection from biotic stress (e.g. herbivory).

Fungi that participate in defensive mutualisms typically are referred to as endophytes, even though some produce epiphytic structures. The term ‘endophyte’, however, also is used more broadly for any fungus, bacterium or other microorganism that colonizes living plants without causing overt detrimental symptoms and typically has no obvious external signs of infection. Rodriguez et al. (2009) proposed four classes of endophytic fungi that were grouped according to criteria such as host range, tissues colonized, in planta colonization, in planta biodiversity, mode of transmission and the nature of the benefits afforded to the host. Two criteria we feel are diagnostic in defining fungal endophytes that participate in defensive mutualisms are the capacity for vertical transmission of the endophyte via seed and a narrow host range, both of which are characteristic of class 1 endophytes as described by Rodriguez et al. (2009), although those authors limited class 1 endophytes to species in the fungal family Clavicipitaceae.

Hereditary symbionts that are strictly vertically transmitted are completely dependent on host reproduction for their own propagation and reproductive fitness. When a vertically transmitted symbiont benefits the host, it has an indirect positive effect on its own fitness. Any symbiont that is strictly vertically transmitted and puts its host at a disadvantage would go extinct because its host would be outcompeted by non-infected conspecifics (Ewald 1987). Vertically transmitted symbionts are essentially a trait of the host and if one is ‘in the least degree injurious’, first principles would suggest it would be ‘rigidly destroyed’ by natural selection (Darwin 1859). Horizontally transmitted symbionts, on the other hand, exploit the host's ability to survive and contact non-infected individuals and are often more virulent as the symbiont's reproduction is not completely dependent upon the host's reproductive success (Ferdy & Godelle 2005).

The natural host range of a symbiont (as opposed to the range of species it could infect in a laboratory setting) is another defining characteristic of endophytic fungi associated with defensive symbioses. Clavicipitaceous endophytes capable of vertical transmission are associated with select taxa of the Poaceae and Convolvulaceae and have narrow host ranges (Clay & Schardl 2002; Steiner et al. 2011). In most instances, a given fungal taxon is associated with a specific plant host species (Schardl 2010). In contrast, many horizontally transmitted endophytes represent fungal genera that are ubiquitous in the environment and are often associated with multiple host taxa (Rodriguez et al. 2009).

The defensive mutualism hypothesis does not exclude the possibility of negative effects to the host under certain environmental conditions, especially since the host must incur some due to endophyte infection. For example, Cheplick, Clay & Marks (1989) observed reduced growth in endophyte-infected tall fescue under low nutrient conditions. Turf varieties of tall fescue infected with a particular Neotyphodium endophyte were more susceptible to root disease caused by Pythium graminicola (Rodriguez et al. 2009). Wäli et al. (2006) showed that red fescue (Festuca rubra) infected with Epichloë festucae in subarctic regions suffered more damage from the snow mould, Typhula ishikariensis than did non-infected red fescue growing in these same areas. Despite the occasional example that indicates a detriment of endophyte infection to the host, complete dependence on the host plant for transmission of the endophyte provides a means for selecting fungi that are beneficial to their host plants. Fungal endophytes that are strictly vertically transmitted must be beneficial in order for the host plants to retain them (Ewald 1987). In cases where negative effects have been seen, it does not preclude the existence of some positive benefit that has not been measured that results in maintenance of the mutualism, such as reduced survival but increased regeneration resulting in net positive population growth (Rudgers et al. 2012).

In addition to vertical transmission and a narrow host range, a third and striking feature of many plant-endophytic fungus associations – particularly those that fit well with the definition of defensive mutualism – is the production of bioactive secondary metabolites by the fungal symbionts. Whereas fungi in general produce a wide array of secondary metabolites, we propose that bioactive metabolites are particularly important to vertically transmitted fungi, which in the establishment of their fungus–plant symbioses must have provided some immediate benefit to the host plant. We describe here selected examples of such vertically transmitted endophytic fungi, the chemicals they produce and the ways their chemicals contribute to the symbioses. We also include examples of vertically transmitted symbiotic fungi that produce noteworthy bioactive chemicals in their symbiotic state but for which roles of the chemicals in the symbioses have not been experimentally established. In each case, the metabolites observed originally appeared to be components of the plant or would have appeared as such if the symbiosis had not already been discovered.

Clavicipitaceous endophytes of grasses

Fungi of the genera Epichloë and Neotyphodium grow symbiotically with many cool season grasses. Neotyphodium species are exclusively asexual and grow within the intercellular spaces of their grass hosts (Fig. 1a). Epichloë species represent the teleomorphic states of several Neotyphodium species. In addition to the symbiotic phase typical of Neotyphodium species, Epichloë species are capable of exiting their plant hosts via the formation of sexual reproductive stroma on plant inflorescences. Those sexually reproducing Epichloë species are thus capable of vertical transmission through their asexual stage and occasional horizontal transmission when they reproduce sexually. Schardl (2010) referred to representatives of the two genera collectively as epichloae.

Figure 1.

Class 1 endophytic fungi. (a) Aniline blue-stained hypha of Neotyphodium coenophialum growing intercellularly in a peeled leaf sheath of tall fescue (Lolium arundinaceum); (b) Hyphae of GFP-expressing Undifilum oxytropis in locoweed vascular tissue; (c) Numerous, small colonies of Periglandula ipomoeae growing epiphytically on the adaxial leaf surface of Ipomoea asarifolia; (d) Fungicide-treated I. asarifolia leaf lacking fungal colonies; (e) Aniline blue-stained colonies of P. ipomoeae from the adaxial leaf surface of I. asarifolia. (Photos: the authors)

In addition to direct effects on herbivores, epichloid endophytes of grasses can significantly affect ecological communities. Neotyphodium coenophialum-infected tall fescue (Lolium arundinaceum) suppresses both plant (Clay & Holah 1999) and arthropod diversity (Finkes et al. 2006), affects the outcome of competitive interactions (Clay, Marks & Cheplick 1993), alters plant soil feedbacks (Matthews & Clay 2001), slows succession to forest communities (Rudgers et al. 2007), and disrupts relationships between diversity and ecosystem properties such as productivity (Rudgers, Koslow & Clay 2004).

The epichloae collectively produce four classes of bioactive metabolites in their symbiotic associations with plants: ergot alkaloids, indole-diterpenes, loline alkaloids and peramine. Although each of these four classes of alkaloids is derived in some way from amino acid precursors, the four pathways are completely independent of one another. No individual fungal isolate is known to produce representatives of all four classes; most epichloae produce metabolites belonging to one to three of the chemical classes (Schardl et al. 2011).

Ergot alkaloids

Ergot alkaloids are a diverse family of secondary metabolites produced by certain epichloae, ergot fungi (Claviceps spp.) and related species of Balansia and Periglandula (discussed later), and in the opportunistic human pathogen Aspergillus fumigatus. The biosynthetic pathway has been studied extensively and recently reviewed (Lorenz et al. 2009; Panaccione 2010; Wallwey & Li 2011). The diverse metabolites in the ergot alkaloid family can be grouped as clavines, simple amides of lysergic acid, or ergopeptines based on their complexity and relative position in the pathway (Fig. 2). Various ergot alkaloids interact as agonists or antagonists at receptors for the monoamine neurotransmitters serotonin, dopamine, adrenaline and noradrenaline. Resulting activities include vasoconstriction, uncontrolled muscle contraction and disturbance in the central nervous system and reproductive systems (reviewed in Lorenz et al. 2009; Panaccione 2010; Wallwey & Li 2011). Numerous feeding studies with a variety of mammals indicate that ergot alkaloids, at concentrations at which they are found in endophyte-infected grasses, have significant detrimental effects on mammalian health and reproduction (e.g. Hill et al. 1994; Filipov et al. 1998; Gadberry et al. 2003; Parish et al. 2003a,b). Ergot alkaloids also affect insects and nematodes, which contain homologous neurotransmitters. Activities in insects include feeding deterrence, delayed development and increased mortality (Clay & Cheplick 1989; Ball, Miles & Prestidge 1997; Potter et al. 2008).

Figure 2.

Diversification of ergot alkaloids associated with endophyte–plant symbioses. Double arrows indicate one or more omitted intermediates. Dashed arrows indicate uncharacterized steps. Relevant enzymes associated with catalysis at branch points are indicated. At the first branch point, alternative forms of EasA form festuclavine (not pictured) and agroclavine (Coyle et al. 2010); additional alternative forms are hypothesized to produce cycloclavine and lysergol in Periglandula-infected Ipomoea spp. At the second branch point, combinations of peptide synthetases Lps1, Lps2 and Lps3 are required to produce ergopeptines or simple amides of lysergic acid (Lorenz et al. 2009; Ortel & Keller 2009). Lysergic acid is bracketed to indicate that it is not typically considered a clavine.

The ergot alkaloid pathway is notable for its accumulation of intermediates and spur products to concentrations that approach or exceed the amounts of the pathway end product (Panaccione et al. 2003; Panaccione & Coyle 2005). Panaccione (2005) hypothesized that this inefficiency in turning over intermediates has been selected for because those accumulating intermediates or spur products provide some benefit to the producing fungi (or its grass host, in the case of endophytes) that differs from the benefit(s) provided by the pathway end product. Differences in activities of clavine intermediates and spur products compared to ergopeptines or the simple amides are apparent from direct exposure of bacteria and nematodes to these alkaloids in vitro (Panaccione 2005; Panaccione et al. 2006a; Bacetty et al. 2009a,b). In a more natural setting, studies with perennial ryegrass (Lolium perenne) and gene knockout mutants of the endophyte Epichloë typhina × Neotyphodium lolii isolate Lp1 (hereafter simply Lp1) provide more support for this hypothesis. A knockout mutant that accumulated certain clavines but not ergopeptines or simple amides of lysergic acid deterred rabbit feeding on infected grasses as well as or better than the wild type of the fungus (Panaccione et al. 2003, 2006b). In contrast, perennial ryegrass containing the same knockout endophyte had reduced insecticidal and insect feeding deterrent properties compared to wild-type endophyte, indicating a role for ergopeptines and simple amides of lysergic acid in these anti-insect traits (Potter et al. 2008). Notably, the knockout strain accumulated the same molar quantity of ergot alkaloids as the wild type, but the alkaloids were restricted to earlier pathway intermediates and spur products. Thus, the accumulation of both intermediates and end products is beneficial to the fungus and its grass host in resisting vertebrate and invertebrate herbivore pressures.

The general significance of ergot alkaloids to Lp1-infected perennial ryegrass was apparent from the observation that perennial ryegrass containing a different knockout mutant, which was completely devoid of ergot alkaloids but still colonized by the fungus (Wang et al. 2004), was strongly preferred by rabbits, even over the endophyte-free perennial ryegrass (Panaccione et al. 2006b). Thus, without any ergot alkaloids this grass would be subject to increased herbivory and likely at competitive disadvantage compared to grasses containing ergot alkaloid-producing endophytes.


The indole-diterpenes represent another important class of diverse alkaloids produced by some epichloae as well as by certain Claviceps spp. and some members of the Trichocomaceae (e.g. Aspergillus and Penicillium spp.) (Saikia et al. 2008). Indole-diterpenes have been studied intensively because certain members of this class of metabolites have strong tremorgenic activity in mammals. For example, the lolitrems produced by Neotyphodium lolii in perennial ryegrass cause ryegrass staggers (Gallagher, White & Mortimer 1981; Gallagher et al. 1982, 1984), which can result in significant economic losses.

Similar to the ergot alkaloids, the indole-diterpenes of endophytes are very diverse. A simplistic view of the diversification of indole-diterpenes can be based on the oxidation and prenylation of intermediate terpendole I and its subsequent metabolites independently, resulting in different end products including terpendoles, lolitrems and janthitrems (Fig. 3).

Figure 3.

Diversification of indole-diterpenes associated with endophyte-plant symbioses. Double arrows indicate one or more omitted intermediates. Dashed arrows indicate uncharacterized steps. LtmE/LtmJ and LtmF/LtmK represent separate prenyl transferase/monooxygenase (respectively) combinations that work on opposite ends of members of the indole-diterpene family (Young et al. 2006, 2009). Each combination can act on multiple substrates.

Much of the early analysis of grass endophytes for indole-diterpenes focused intensively on lolitrem B. Evidence for lolitrem B as the key tremorgenic toxin in N. lolii-infected perennial ryegrass has come from animal feeding studies (e.g. Gallagher, White & Mortimer 1981; Gallagher et al. 1982) as well as from comparisons of naturally occurring isolates that vary in indole-diterpene profiles (e.g. Bluett et al. 2005a,b). More recent genetic screening, facilitated by a more thorough understanding of indole-diterpene biosynthesis, indicated that some epichloid endophytes that do not produce lolitrem B still produce less complicated indole-diterpenes such as terpendoles (Gatenby et al. 1999; Young et al. 2009; Schardl et al. 2011). Interestingly, fungal endophytes producing terpendoles but lacking lolitrem B have been successfully marketed in forage varieties of perennial ryegrass in New Zealand as less toxic alternatives to traditional perennial ryegrass varieties (Bluett et al. 2005a,b). Lolitrem B-deficient varieties may still induce minor tremoring in mammals, presumably due to the presence of janthitrems or other indole-diterpenes, but the effects are minimal (Bluett et al. 2005a,b).

The observation that some non-tremorgenic epichloae retain the ability to produce intermediates in the indole-diterpene pathway is interesting, considering their negligible anti-mammalian activity compared to the lolitrems. Young et al. (2009) speculated that the less tremorgenic indole-diterpenes could be beneficial to their host by acting against insects, as has been demonstrated for the biogenically related yet structurally distinct compound nodulisporic acid. Nodulosporic acid is produced in culture by Nodulisporium sp. (an anamorphic fungus in the Xylariaceae that was isolated from an unidentified woody plant) and has good insecticidal activity against a range of insects (Byrne, Smith & Ondeyka 2002). The less commonly encountered but biogenically related janthitrems also may be associated with insecticidal activity. Janthitrems accumulate in plants with N. lolii isolate AR37, an endophyte strain that is included in some commercial varieties of perennial ryegrass because of its low tremorgenic activity. AR37-infected perennial ryegrass varieties are notably resistant to the insect pest Wiseana cervinata (porina) (Jensen & Popay 2004); however, a direct linkage of the anti-insect activities of AR37 with the janthitrems has not been established. Several other indole-diterpenes have been isolated from the sclerotia of various Aspergillus spp. (Trichocomaceae, Eurotiales), and these indole-diterpenes have been demonstrated to have anti-insect activities through feeding and topical assays (Gloer 1995).

The production of indole-diterpenes and ergot alkaloids by certain representatives of two phylogenetically disjunct families, the Clavicipitaceae and the Trichocomaceae (and very rarely by fungi outside these families), is remarkable. Whereas the known alkaloid-producing Clavicipitaceae (order Hypocreales) all are associated with living plants, the alkaloid-producing Trichocomaceae (order Eurotiales) are primarily saprotrophs on plant matter. Although ergot alkaloids and indole-diterpenes are assembled from some common precursors, the biosynthetic pathways for these alkaloids are completely independent. The polyphyletic distribution of the two independent pathways among such diverse fungi cannot be explained at present.


The loline alkaloids are a family of aminopyrrolozidine alkaloids, derived from homoserine and proline joined in a non-peptidic manner. Lolines have been most intensively studied in endophytic Neotyphodium spp. but also have been reported in the plants Adenocarpus decorticans (Fabaceae) (reviewed by Schardl et al. 2007) and Argyreia mollis (Convolvulaceae) (Tofern et al. 1999). Whether the lolines in the Adenocarpus and Argyreia species are of plant origin or derived from endophytic fungi has not yet been determined. The biosynthesis and activities of lolines has been reviewed in detail by Schardl et al. (2007). Lolines occur in exceptionally high concentration in Neotyphodium coenophialum-infected tall fescue (Lolium arundinaceum) and also are found abundantly and in variable forms in several other epichloae-infected grasses (Schardl et al. 2007, 2011). Variability among lolines is mainly generated by the presence or absence of methyl, formyl or acetyl groups on the homoserine-derived amine group (Fig. 4a).

Figure 4.

Structures of (a) lolines and (b) peramine. Variation among lolines derives from substituents on the indicated nitrogen (Schardl et al. 2007).

The insecticidal and insect feeding deterrent activities of lolines have been shown in a series of feeding experiments with either endophyte-infected plants (e.g. Yates, Fenster & Bartelt 1989; Siegel et al. 1990; Jensen, Popay & Tapper 2009) or with purified lolines (e.g. Riedell et al. 1991). Riedell et al. (1991) also applied lolines topically to aphids and noted that toxicity of lolines was comparable to that of nicotine. A convincing demonstration of the significance of lolines to insect resistance in an endophyte-infected grass came from an elegant genetic study conducted by Wilkinson et al. (2000) who observed co-segregation of activity against two different aphid species and loline production in a genetic cross among Epichloë festucae isolates in meadow fescue (Lolium pratense). Moreover, aphid mortality increased with increasing concentrations of lolines in plants containing loline-positive progeny.

In addition to the well-documented effect on insects, lolines also are nematicidal (Bacetty et al. 2009a). The effects of lolines appear to be restricted to invertebrates. Schardl et al. (2007) carefully reviewed studies on vertebrate toxicity of lolines and concluded that anti-vertebrate effects of lolines were likely to be negligible, because studies indicating such effects were either confounded by the presence of ergot alkaloids in the same plant tissues or conducted with exceptionally high concentrations of lolines.


Peramine is the most widely distributed of the four classes of epichloae-derived secondary metabolites (Schardl et al. 2011), but its production is not known outside of the epichloae (Clay & Schardl 2002; Tanaka et al. 2005). Its origin as a fungal metabolite was shown by its production by isolated fungi in vitro (Rowan 1993), and more convincingly by its disappearance from grass–endophyte symbiota upon mutation of the relevant gene in the fungus (Tanaka et al. 2005). Peramine is unique among the four major classes of epichloae-produced alkaloids, in that it is a single chemical as opposed to a family of chemicals, and it appears to be the product of a single multifunctional enzyme as opposed to a complex pathway (Tanaka et al. 2005). Peramine is derived from a dipeptide possibly made up of arginine and a precursor to proline (Fig. 4b).

Peramine is a strong feeding deterrent for Argentine stem weevil, an important pest of perennial ryegrass in New Zealand, and several other insects (Clay, Hardy & Hammond 1985; Johnson et al. 1985; Rowan, Hunt & Gaynor 1986; Rowan, Dymock & Brimble 1990; Rowan 1993). The anti-feeding effects of peramine are not universal, however, as the aphid Rhopalosiphum padi appears not to be deterred by its presence (Johnson et al. 1985; Gaynor & Rowan 1986). The significance of peramine to defending plant material against herbivory by Argentine stem weevil was convincingly demonstrated in a study by Tanaka et al. (2005) in which the gene encoding the multifunctional enzyme responsible for peramine biosynthesis was inactivated by knockout. Resulting peramine-deficient mutants were as susceptible to feeding by the Argentine stem weevil as endophyte-free plants of the same variety.

Unlike the ergot alkaloids and indole-diterpenes, which are found mainly in tissues that are colonized by the endophyte (pseudostem or seeds), peramine is water soluble and dispersed throughout the plant (Ball et al. 1997a,b; Spiering et al. 2002, 2005; Koulman et al. 2007). Peramine is found in fluids exuded from cut leaves of all tested endophyte-infected varieties of perennial ryegrass, tall fescue, and Elymus sp. and in the guttation fluid of endophyte-infected perennial ryegrass and Elymus sp. (Koulman et al. 2007). This localization pattern would allow peramine to protect tissues remote from the fungus, and its presence in guttation fluid would conceivably allow it to deter feeding by sensitive insects without the insects breaching the cuticle. The activity of peramine against many phloem feeders and its presence in roots (a tissue not well colonized by peramine-producing fungi) indicates the presence of peramine in phloem.

Distribution of bioactive alkaloid classes among epichloae

The capacity to produce the four classes of bioactive alkaloids varies among epichloae taxa. Two classes of epichloae-produced alkaloids – the ergot alkaloids and the indole-diterpenes – have anti-vertebrate activities, whereas three classes – the ergot alkaloids, lolines and peramine – have anti-invertebrate properties (with the anti-insect activities of epichloae-derived indole-diterpenes still uncertain). In the list of grass–epichloae symbiota compiled by Schardl et al. (2011), there are 29 symbiota for which the presence of all four classes of endophyte alkaloids has been tested. Among these 29 symbiota, 86% produce at least one of the three established anti-insect classes of alkaloids, and 48% have at least two classes of anti-insect alkaloids (Table 1). The common toxic endophyte isolate of N. coenophialum is the only endophyte known to produce all three classes of anti-insect alkaloids. The anti-vertebrate alkaloids are less common than the anti-insect compounds among this same set of 29 symbiota for which data are available. Approximately one-half (15 of 29) of the symbiota contain at least one class of anti-vertebrate compound, and only four of those 15 produce both ergot alkaloids and indole-diterpenes (Table 1). The data show that anti-insect alkaloid classes are more likely to be present in plants containing epichloae endophytes than are anti-vertebrate alkaloids.

Table 1. Distribution of anti-vertebrate and anti-insect alkaloids among epichloae–grass symbiota in which all four classes of bioactive alkaloids have been assayeda
FungusHost plantAnti-vertebrate alkaloidsbAnti-insect alkaloidsc
  1. a

    Refer to Schardl et al. (2011) for details on symbiota.

  2. b

    ERG, ergot alkaloids; IDT, indole-diterpenes.

  3. c

    ERG, ergot alkaloids; LOL, loines; PER, permine.

Epichloë elymi Elymus canadensis ERGERG, PER
Epichloë festucae Festuca longifolia ERG, IDTERG, PER
E. festucae Festuca ovina ERGERG, PER
E. festucae Festuca rubra subsp. commutataERGERG, PER
E. festucae F. rubra subsp. commutataPER
E. festucae F. rubra subsp. rubraIDT
E. festucae F. rubra subs. rubraERGERG
E. festucae Lolium giganteum ERGERG, LOL
Epichloë typhina Lolium perenne PER
Neotyphodium aotearoae Echinopogon ovatus LOL
Neotyphodium coenophialum Lolium arundinaceum ERGERG, LOL, PER
N. coenophialum L. arundinaceum LOL, PER
Neotyphodium huerfanum Festuca arizonica PER
Neotyphodium gansuense Achnatherum inebrians ERGERG
Neotyphodium lolii Lolium perenne ERG, IDTERG, PER
N. lolii L. perenne IDTPER
N. lolii L. perenne IDT
N. lolii × E. typhina isolate Lp1 L. perenne ERG, IDTERG, PER
Neotyphodium siegelii Lolium pratense LOL, PER
Neotyphodium starrii Bromus anomalus ERGERG, PER
Neotyphodium sp. E55 Poa autumnalis LOL, PER
Neotyphodium sp. E4074Lolium sp.LOL, PER
Neotyphodium sp. E4078Lolium sp.ERG, IDTERG, PER
Neotyphodium sp. Festuca paradoxa PER
Neotyphodium sp. Festuca subverticillata
Neotyphodium sp. Hordelymus europaeus
Neotyphodium tembladerae F. arizonica PER
Neotyphodium typhinum Poa ampla PER
Neotyphodium uncinatum L. pratense LOL

The distribution of lolines, ergopeptines (but not other ergot alkaloids) and peramine among sexual stroma-producing Epichloë spp. (those capable of horizontal transmission) compared to strictly asexual, vertically transmitted Neotyphodium spp. was investigated by Leuchtmann, Schmidt & Bush (2000) who observed greater production of lolines and ergopeptines in the vertically transmitted endophytes. The reduced level of anti-insect alkaloids in grasses hosting sexually reproducing epichloae is consistent with the dependence of the sexual Epichloë spp. on insects for spermatization, or transfer of gametes, among fungi of opposite mating types.

Clavicipitaceous endophytes of Convolvulaceae

The genus Periglandula consists of clavicipitaceous epibiotic fungal symbionts of the Convolvulaceae (morning glories) that produce ergot alkaloids in the seeds and, in some cases, the foliage of infected plants (Markert et al. 2008; Steiner et al. 2011). The two species described of Periglandula are fungi that form systemic infections in the above-ground parts of their host plants and are vertically transmitted (Steiner et al. 2006). Epiphytic mycelia are visible to the naked eye on young leaves (Fig. 1c). Steiner et al. (2011) have stated that there is no evidence that the fungi ever penetrate the host plant; however, the seed transmissibility of the fungi indicates that there must be some internal growth of the fungus. Workers demonstrated that this fungus was responsible for ergot alkaloid production when they observed that treatment of Ipomoea asarifolia with fungicides resulted in the loss of epiphytic mycelia concomitantly with loss of detectable ergot alkaloids in the foliage (Kucht et al. 2004). Unlike other occurrences of plant-associated Clavicipitaceae, Periglandula species are the only clavicipitaceous fungi known to associate with a dicotyledonous host (Steiner et al. 2006). These ‘endophytes’ also appear to have a narrow host range: the two described Periglandula species are chemically dissimilar and occur on different host plants (Steiner et al. 2011). A third, undescribed, Clavicipitaceae from Ipomoea tricolor, which does not form epiphytic mycelia, was detectable via PCR and groups with described Periglandula species, yet it is also phylogenetically distinguishable (Ahimsa-Muller et al. 2007).

Prior to the discovery of Periglandula species, the occurrence of ergot alkaloids in the Convolvulaceae was thought to be a case of convergent evolution or horizontal gene transfer (Steiner, Hellwig & Leistner 2008). Although published evidence for Periglandula species colonization has been provided for only three species of Convolvulaceae, ergot alkaloids are known to occur in many more species in this diverse plant family (Eich 2008), each of which likely harbours a species of Periglandula. Whereas ergot alkaloids were discovered in grasses due to their influence on agriculture (Lyons, Plattner & Bacon 1986), their discovery in the Convolvulaceae followed from the work of ethnobotanist Richard Schultes in Central America in the late 1930s (Schultes 1941; Schultes 1969). He reported that the seeds of Turbina corymbosa (host to P. turbinae), called ‘ololiuqui’ by the Aztecs, and the seeds of I. tricolor, called ‘badoh negro’ by the Zapotecs, were consumed ritualistically for divination. Two decades later, Albert Hofmann isolated the lysergic acid amides ergine and lysergic acid α-hydroxyethylamide from T. corymbosa (Hofmann & Tscherter 1960; Hofmann 1961), which were likely responsible for the hallucinogenic effects of these plants.

The discovery of ergot alkaloids in a dicotyledonous plant spurred several studies on the occurrence of ergot alkaloids in the Convolvulaceae; Eich (2008) has critically reviewed this work. In the genus Ipomoea (ca. 500 spp.), 79 species have been screened for ergot alkaloids and 23 (29%) are unambiguously positive (Eich 2008). The genera Argyreia, Stictocardia and Turbina also have ergot alkaloid-positive representatives (Eich 2008). Moreover, the three major types of ergot alkaloids (clavines, lysergic acid amides and ergopeptines) have each been reported from the Convolvulaceae (Eich 2008), including the ergopeptine ergobalansine (Jenett-Siems, Kaloga & Eich 1994), originally discovered in the clavicipitaceous fungi Balansia obtecta which form epiphytic infections of Cenchrus echinatus (Sandbur Grass) (Cyperaceae) (Powell et al. 1990). In addition to ergot alkaloids known from other Clavicipitaceae, unique ergot alkaloids have been discovered in the Convolvulaceae, notably cycloclavine from Ipomoea hildebrandtii, an African shrub (Stauffacher et al. 1969). All reports of ergot alkaloids from the Convolvulaceae have come from the speciose tribe Ipomoeeae (ca. 900 species) and show no clear phylogenetic pattern (Eich 2008); however, the phylogeny of this large family is still not clearly resolved. Both major clades within the Ipomoeeae (Stefanovic, Krueger & Olmstead 2002) have ergot alkaloid-positive representatives, and there is variation within sections with respect to the presence of ergot alkaloids (Eich 2008). Expanded sampling of the Convolvulaceae for ergot alkaloids will surely reveal more species infected by Periglandula. Extrapolation from available data would suggest upwards of 250 species of ergot alkaloid-positive Ipomoeeae.

Interestingly, loline and ergot alkaloids have been detected in the seeds and foliage of one species, Argyreia mollis (Tofern et al. 1999), suggesting Periglandula may also produce other classes of secondary metabolites found in other Clavicipitaceae. There have been no published reports from the Convolvulaceae of the other two major classes of clavicipitaceous secondary metabolites – indole-diterpenes and peramine – although we do not know of any studies that explicitly tested for them. There are, however, reports of tremorgenic symptoms in livestock caused by grazing of foliage from ergot alkaloid-positive Convolvulaceae, specifically caused by sheep feeding on Ipomoea muelleri in Australia (Gardiner, Royce & Oldroyd 1965) and sheep and cattle feeding on I. asarifolia in Brazil (Araújo et al. 2008). Livestock grazing on grasses containing indole-diterpenes also suffer from tremorgenic symptoms (Belesky & Bacon 2009). Because the tremoring symptoms were caused by grazing on these two ergot alkaloid-positive species (one of which is host to P. ipomoeae), the possibility that some Periglandula species produce indole-diterpenes should be investigated.

The ecological effects of Periglandula infection or ergot alkaloid presence in the Convolvulaceae has yet to be studied. As Periglandula species are closely related to Neotyphodium species endophytes and share some important characteristics (e.g. vertical transmission and production of ergot alkaloids), their presence also may confer similar benefits to their convolvulaceous hosts such as resistance to herbivory (Clay & Schardl 2002). Unlike the cool season pooid grasses which host epichloae, there is striking variation in life history and habitat even among the limited number of known ergot alkaloid-positive Convolvulaceae. They include herbaceous twining vines (e.g. I. tricolor), woody lianas (e.g. Argyreia nervosa), sprawling vines (e.g. I. pes-caprae) and shrubs (e.g. Ipomoea leptophylla and I. hildebrandtii) which can be found worldwide in deserts, sand dunes, forests and grasslands in North and South America, Africa, Asia and Australia (Verdcourt 1978; Devall 1992; Austin, Jarret & Johnson 1993; Austin & Huáman 1996). Many ergot alkaloid-positive Convolvulaceae are restricted to a single continent, but one species, Ipomoea pes-caprae, is found on tropical and subtropical beaches worldwide where it grows as a pioneer species just above the high tide line (Devall 1992).

While many studies in epichloae-grass systems demonstrate increased resistance to foliar herbivory, resistance to seed predation may be an important dimension of the Periglandula-Convolvulaceae symbioses. Some studies have indicated that alkaloids can be present in the seeds but not the foliage (Chao & Der Marderosian 1973; Jirawongse, Pharadai & Tantivatana 1977). Convolvulaceae seeds can be large, often >5 mm in diameter (Verdcourt 1978), and presumably represent a large investment of plant resources. Additionally, many species are highly parasitized by bruchine beetles (Coleoptera: Bruchinae) (Reyes, Canto & Rodriguez 2009). The larvae of these beetles can bore into the seed, consume the cotyledons or embryo and leave a characteristic circular exit hole. Reports of bruchine parasitism rates range from 4 to 85% in I. pes-caprae (Wilson 1977; Devall & Thien 1989), and 34 to 100% in I. leptophylla, a shrub found in the short grass prairies of the Central United States (Keeler 1980, 1991). Whereas several factors may contribute to this variation, especially visitation by ants to extra floral nectaries (Keeler 1980), population differences in ergot alkaloid content may play a role. Other possibilities are that the specialized bruchines associated with ergot alkaloid-positive Convolvulaceae have overcome the plant's acquired defence from Periglandula or that there is ongoing co-evolution between the plant–fungal symbiota and the beetles.

Endophytes of locoweeds and related taxa

Several species in the legume genera Astragalus, Oxytropis and Swainsona have been found to be toxic to grazing livestock in the Americas, Asia and Australia (Marsh 1909; Marsh & Clawson 1936; Gardiner, Linto & Aplin 1969; Huang, Zhang & Pan 2003). Locoism, a neurologic disease, was first noted by the Spanish conquistadors, and again during the settlement of Western North America by pioneers (Marsh 1909; Marsh & Clawson 1936; Jones, Hunt & King 1997). Clinical signs and pathology of locoism are similar in animals intoxicated by locoweed species and Swainsona species (James, Van-Kampen & Hartley 1970; Panter et al. 1999). Swainsonine (Fig. 5), a trihydroxyindolizidine alkaloid, was first identified as the active principle in Swainsona canescens, a legume native to Australia (Colegate, Dorling & Huxtable 1979), and subsequently identified as the active principle in locoweeds (Molyneux & James 1982). Swainsonine inhibits the enzymes α-mannosidase and mannosidase II resulting in lysosomal storage disease and altered glycoprotein synthesis (Hartley 1971; Dorling, Huxtable & Vogel 1978).

Figure 5.

Proposed pathway for swainsonine and slaframine biosynthesis. Pathway is based on studies conducted in Rhizoctonia leguminicola (Harris et al. 1988b).

Recently, a fungal endophyte, Undifilum oxytropis (Pryor et al. 2009), previously described as an Embellesia species (Wang et al. 2006), was reported to produce swainsonine in locoweeds (Braun et al. 2003). The Undifilum genus (Pleosporaceae) is closely related to the genera Alternaria, Embellesia and Ulocladium (Pryor et al. 2009). Undifilum species are only associated with swainsonine-containing Astragalus and Oxytropis species with one exception, Undifilum bormuelleri, a pathogen of the legume Securigera varia that does not contain swainsonine. Undifilum species have been found to be associated with swainsonine-containing Astragalus and Oxytropis species in North America and China (Pryor et al. 2009; Yu et al. 2010; Baucom et al. 2012). Like many epichloae and the known Periglandula species, Undifilum species associated with locoweeds are vertically transmitted and have no apparent sexual stage (Oldrup et al. 2010; Ralphs et al. 2011). Undifilum species also appear to have a narrow host range as different plant species are associated with unique Undifilum species (Pryor et al. 2009; Baucom et al. 2012).

In addition to the legumes, swainsonine occurs sporadically in two other plant families, the Convolvulaceae and the Malvaceae. In the Convolvulaceae, some Ipomoea and Turbina species are reported to contain swainsonine, for example, I. carnea and T. cordata (de Balogh et al. 1999; Dantas et al. 2007), while in the Malvaceae, Sida carpinofolia is reported to contain swainsonine (Colodel et al. 2002). Like the legumes, swainsonine was identified in the plant species associated with these families due to livestock poisoning and subsequent economic impact.

Swainsonine is also reported to be produced by two phylogenetically disjunct fungi, Rhizoctonia leguminicola (Ceratobasidiaceae) and Metarhizium anisopliae (Clavicipitaceae) (Schneider et al. 1983; Patrick, Adlard & Keshavarz 1993). Rhizoctonia leguminicola is a fungal pathogen of red clover (Trifolium pratense) that causes black patch disease in the plant. Metarhizium anispoliae is an entomopathogen that attaches to the outside of an insect, grows internally and causes death. The roles swainsonine plays in either of these biological systems have not been elucidated. Like the ergot alkaloids, swainsonine appears to be more widely distributed in fungi other than seed-transmitted endophytes.

The biosynthesis of swainsonine has been investigated in the fungus R. leguminicola (Harris et al. 1988b). Swainsonine is derived from lysine which is converted into pipecolic acid. Two precursors of swainsonine in the fungal biosynthetic pathway were detected in the shoots of Diablo locoweed (Astragalus oxyphysus) (Harris et al. 1988a,b); as a result, Harris et al. (1988a) proposed that the biosynthetic pathway of swainsonine in R. leguminicola is similar to the pathway in locoweeds, where swainsonine was later found to be produced by Undifilum species.

Support for the fact that swainsonine is a fungal-derived secondary metabolite in locoweeds is based on the following observations: (i) locoweed plants infected with Undifilum species contain swainsonine; (ii) plants derived from Astragalus and Oxytropis embryos in which the seed coat, the primary location of Undifilum, was removed have no detectable swainsonine, or have concentrations less than 0·001% (Oldrup et al. 2010; Grum et al. 2012); (iii) plants derived from fungicide-treated Astragalus and Oxytropis seeds have no detectable swainsonine or have concentrations less than 0·001% (Grum et al. 2012); (iv) Undifilum species isolated from locoweeds produce swainsonine in pure culture (Braun et al. 2003); (v) plants derived from embryos that were inoculated with Undifilum have swainsonine concentrations greater than 0·01% (Grum et al. 2012); and (vi) rats fed U. oxytropis developed lesions and clinical signs similar to those fed swainsonine-containing Oxytropis lambertii (McLain-Romero et al. 2004).

Swainsonine concentrations vary greatly among species, varieties and populations. For example, Astragalus species generally have greater swainsonine concentrations than do Oxytropis species in North America (Ralphs et al. 2008) while different varieties of O. lambertii vary greatly in their swainsonine concentrations (Gardner, Molyneux & Ralphs 2001). Additionally, in toxic populations of locoweeds, two chemotypes of plants have been identified, namely chemotype one plants, which contain swainsonine concentrations >0·01%, and chemotype two plants, which have concentrations <0·01% (generally near 0·001% or not detected) (Cook et al. 2009, 2011). These two chemotypes differ significantly in the amount of endophyte they contain which may explain the difference in swainsonine concentrations (Cook et al. 2009, 2011).

Swainsonine and endophyte amounts have been investigated in different plant parts at different phenological stages (Cook et al. 2012). Swainsonine is found in all plant parts although concentrations are greater in above-ground parts than in below-ground parts (Cook et al. 2009, 2011). Endophytic Undifilum species also are found in all plant parts with only small quantities found in the root (Cook et al. 2009, 2011). The root crown appears to be a major reservoir for the endophyte during the following year's growth as many locoweeds are perennial plants (Cook et al. 2009, 2011), and swainsonine and endophyte amounts increase in above-ground parts as the plant matures (Cook et al. 2012). Finally, swainsonine concentrations are greatest in floral parts and seeds (Grum et al. 2012), consistent with the optimal defence theory.

There are few studies regarding the ecological role of swainsonine and how it responds to environmental changes. Swainsonine concentrations do not change in respond to clipping used to simulate herbivory, nor does it deter grazing as animals become progressively more intoxicated (Ralphs et al. 2002; Pfister et al. 2003). In fact animals take 2–3 weeks to show clinical signs and continue grazing locoweeds after becoming intoxicated (Pfister et al. 2003). Activity of swainsonine against insects, fungi or bacteria has not been definitively tested in published studies, although preliminary results show that swainsonine has no effect on some insect species (Parker 2008). Legumes are known for forming symbioses with N-fixing bacteria and investigators found that swainsonine concentrations were greater in plants inoculated with one strain of Rhizobium but not others (Valdez Barillas et al. 2007), suggesting an interaction between the two classes of symbionts. An alternative interpretation is that the improved nitrogen status of the host may have increased substrate availability for swainsonine production; however, no consistent differences in swainsonine concentrations were observed in locoweed plants, whether nitrogen deficient or adequate, when nitrogen was supplied through fertilizer (Delaney et al. 2011). Lastly, swainsonine concentrations were shown to increase slightly in response to water stress in some locoweed species but not others (Vallotton et al. 2012).

It has not been determined whether swainsonine is plant- or fungal-derived in the legume Swainsona canescens, in the Convolvulaceous genera of Ipomoea and Turbina, or in Sida carpinfolia, a species of the Malvaceae family. The presence of swainsonine in these species may be a case of convergent evolution or horizontal gene transfer; however, due to the sporadic occurrence of swainsonine in these genera, it seems probable that a swainsonine-producing fungal endophyte is associated with these taxa that contain swainsonine. In fact, recent research suggests that a fungal endophyte is present in the swainsonine-positive taxa, S. canescens and Ipomoea carnea (Cook and Beaulieu, unpublished data). The fungal endophyte associated with S. canescens appears to be a novel Undifilum species, while the endophyte associated with I. carnea appears to belong to an Ascomycete family not related to the Pleosporaceae family that contains Undifilum. Preliminary data suggest that these endophytes produce swainsonine in vitro, are vertically transmitted, and have a narrow host range.

A comparison of the locoweed/swainsonine-producing endophyte system(s) to those involving clavicipitaceous endophytes reveals some similarities but also significant differences (Table 2). (Note that the Convolvulaceae are interesting in that individual species have symbioses with Periglandula species, presently undescribed swainsonine producers, or in some cases no endophytes.) First, in the grass-epichloae and Convolvulaceae-Periglandula species endophyte symbioses, plants completely free of the endophyte are occasionally encountered (Schardl et al. 2009; Schardl 2010; Beaulieu, Clay & Panaccione, unpublished), whereas in the locoweed system, an endophyte-free plant has not yet been detected in a natural population (Cook et al. 2009, 2011). Instead, locoweeds occur in two chemotypes in native plant populations that differ greatly in the amount of swainsonine and extent of endophyte colonization. Second, a small amount of Undifilum species mycelium is detected in below-ground plant parts, whereas epichloae typically are not reported to be in below-ground parts (Schardl, Leuchtmann & Spiering 2004). Third, swainsonine is found in all tissues in symbiotic plants (Cook et al. 209; Cook et al. 2011) as is the clavicipitaceous water-soluble alkaloid peramine, while the ergot and indole-diterpene alkaloids produced by the clavicipitaceous endophytes are found in above-ground parts only (Ball et al. 1997a,b; Spiering et al. 2002, 2005). Lastly, all vertically transmitted endophytic fungi that produce ergot alkaloids, indole-diterpenes, lolines or peramine are derived from Clavicipitaceae, regardless of the plant family with which they are associated, whereas strictly vertically transmitted endophytes that produce swainsonine are derived from different fungal families that form relationships with specific plant families. In regard to this observation, the evolutionary history of the swainsonine biosynthetic pathway in these diverse fungi is particularly intriguing.

Table 2. Important characteristics of plant–fungal symbiota considered in this review
 GrassesMorning GloriesLocoweeds
  1. a

    Ipomoea batatas (sweet potato) and Ipomoea aquatica (water spinach) are crop species but do not contain ergot alkaloids (Eich 2008) and have never been reported to produce swainsonine.

  2. b

    Lolines were reported from A. mollis (Tofern et al. 1999), but it has not been demonstrated that they are produced by Periglandula.

  3. c

    Indole diterpenes have not been reported from morning glories, but there have been reports of tremorgenic symptoms, characterisitc of indole diterpene poisoning, in livestock feeding on species infected by Periglanudla (Araújo et al. 2008).

Associated Fungal FamilyClavicipitaceaeClavicipitaceaeUnknownPleosporaceae
Bioactive Chemicals ProducedErgots, lolines, peramine, indole-diterpenesErgots, lolinesb, indole-diterpenescSwainsonineSwainsonine
Major CladeMonocotsEudicotsEudicots
Growth FormHerbaceousMostly woody vines (also shrubs, trees, herbaceous vines)Herbaceous
DistributionMostly temperateMostly tropical & subtropicalSemiarid to temperate
Economic ImportanceForage, cropsSome cropsa, agricultural pestsAgricultural pests
Pollination SystemWind PollinatedInsect pollinatedInsect pollinated
Mode(s) of TransmissionStrictly vertical, mixed (vertical and horizontal), and strictly horizontalStrictly vertical (?)Strictly vertical (?)Strictly vertical (?)

Concluding remarks

In this study, we have focused on the bioactive metabolites of vertically transmitted endophytes and the effects of these metabolites on herbivores. Certainly, there are other ways that endophytes can contribute to the symbioses in which they engage; however, toxins are noteworthy because they have a clear impact on humans, grazing animals and/or insect pests. For fungi to persist as vertically transmissible endophytes, they need to be advantageous for their host plants, otherwise they would be outcompeted by non-infected conspecifics, which typically exist among endophyte-infected populations because endophyte transmission is rarely 100% (Afkhami & Rudgers 2008). The advantage provided to the host by the endophyte may be sporadic in nature, resulting in the infected and non-infected hosts within a population. Bioactive metabolites are a pre-adaption that fungi can bring with them in the establishment of a symbiosis.

Pathways for many of the metabolites discussed here may have evolved in fungi prior to their lineage becoming associated with plants as endophytes and have been retained because the toxins provided the free-living fungus protection from insects or other animals. The clavicipitaceous endophytes of grasses and morning glories may provide an illustration of this point. Spatafora et al. (2007) demonstrated through phylogenetic analyses that the plant-infecting Clavicipitaceae (which includes the epichloae and the Periglandula spp.) likely evolved from insect-infecting Clavicipitaceae. Insect-pathogenic Metarhizium spp. share a recent common ancestor with the clade that contains the epichloae and related plant-infecting Clavicipitaceae; these fungi are part of a monophyletic group (Clavicipitaceae clade A) whose most recent common ancestor was likely an insect pathogen (Spatafora et al. 2007). The genomes of Manisopliae and Metarhizium acridum (Gao et al. 2011) contain gene clusters very similar to the ergot alkaloid gene clusters of other ergot alkaloid-producing fungi, although ergot alkaloid production has not been demonstrated experimentally in these species. Considering the distribution of ergot alkaloid biosynthetic capacity among the epichloae, Periglandula spp., and Metarhizium spp., and the toxicity of certain ergot alkaloids to insects, ergot alkaloid biosynthetic capacity may have been present in the insect-pathogenic ancestor that evolved into the plant-infecting Clavicipitaceae. The biosynthetic capacity may have then diversified to include other forms that are more effective against vertebrate herbivores. So long as there is some initial benefit to harbouring the fungal endophyte, the fungus may be selected for and then evolve other bioactive metabolites or beneficial mechanisms apart from secondary compounds. The ability of fungi in this lineage to colonize both plants and insects is demonstrated by Metarhizium robertsii, which can grow endophytically in switch grass (Panicum virgatum) and bean (Phaseolus vulgaris) roots, in addition to parasitizing insects (Sasan & Bidochka 2012). It also is interesting to note that Manisopliae produces swainsonine despite being phylogenetically disjunct from the other swainsonine-producing fungi. Thus, it is possible that swainsonine may have first arisen in an insect pathogen as well.

Considerable effort has gone into understanding ecological impacts of endophytes (reviewed in Clay & Schardl 2002; Schardl et al. 2009). Similarly, molecular and biochemical approaches have improved our understanding of endophyte-produced bioactive metabolites and their biosynthetic pathways (reviewed in Lorenz et al. 2009; Panaccione 2010; Schardl et al. 2011). Future research should connect these two lines of investigation to molecularly dissect the roles of various chemicals in fungus–grass–herbivore interactions and assess the impact of eliminating or adding specific chemicals via genetic modification of the endophyte. Moreover, these approaches need to be applied to a broader range of vertically transmitted endophytes, including the clavicipitaceous endophytes of the Ipomoeeae and the swainsonine-producing endophytes of locoweeds and related legumes.

Finally, the possibility that similar endophyte associations are more widely present among plant taxa should be considered. The examples described herein were evident to us because of their impact on animal agriculture or because of the effects of the bioactive metabolites on humans. Other such symbioses may occur in plants that are not food for humans or livestock and, thus, we have not been confronted with them. While searching for other plant–fungal symbiota with these characteristics may be a daunting task, a high level of endophyte prevalence among populations of a plant species and low genetic diversity of the endophyte (due to asexual, vertical transmission) may facilitate their discovery. Vertically transmitted symbionts are observed at much higher infection frequencies than horizontally transmitted symbionts, possibly as a result of their generally more beneficial nature (Ewald 1987). Support for this generalization comes from Clay & Leuchtmann (1989) who surveyed several grass–endophyte combinations and found much higher infection frequencies among vertically transmitted species. Rudgers et al. (2009) expanded upon this work and showed vertically transmitted Neotyphodium spp. reach infection frequencies 40–130% greater than Epichloë species that have mixed horizontal and vertical transmission. In aphid–bacterial interactions, the vertically transmitted Buchnera species is fixed in most populations and considered obligate, whereas there are many horizontally transmitted symbionts that exhibit variability in their presence (Oliver et al. 2010). Finally, the rapidly expanding cache of genomic data available to researchers may further facilitate the search for endophytes involved in defensive mutualisms. Genomic studies of plants may provide an opportunity to search in silico for evidence of these symbionts by looking for gene clusters of fungal metabolites or conserved fungal genes.

In summary, plant–fungal associations with the characteristics of vertical transmission, narrow host range, and the production of bioactive secondary metabolites resulting in a generally mutualistic association extend beyond the well-studied grass–epichloae systems. As other major classes of plant–microbial symbioses involve diverse taxa – for example, mycorrhizae are formed by Glomeromycota and Basidiomycota while nitrogen-fixing associations are formed by Rhizobium (Proteobacteria) and Frankia (Actinobacteria) – class 1 endophytes as defined by Rodriguez et al. (2009) are not limited to the Clavicipitaceae. Considering advances in knowledge from work in the grass–epichloae symbiota, studying additional endophyte symbioses may provide key insights into the organization of ecological communities and provide excellent case studies on the evolution and diversification of mutualisms.


Funding from the U.S. Department of Agriculture National Institute of Food and Agriculture (2012-67013-19384) to D.G.P. is gratefully acknowledged. We thank Keith Clay for helpful guidance on the content of this review, Christopher Schardl and an anonymous reviewer for constructive comments on a previous version of this article, and Sarah Robinson for assistance with the bibliography. This article is published with permission of the West Virginia Agriculture and Forestry Experiment Station as scientific article number 3155.