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DEFENSIVE SYMBIOSIS

Bioactive alkaloids in vertically transmitted fungalendophytesDaniel G. Panaccione*,1, Wesley T. Beaulieu2 and Daniel Cook3

1Division of Plant & Soil Sciences, West Virginia University, 1090 Agricultural Sciences Building, Morgantown, WV26506-6108 USA; 2Department of Biology, Indiana University, Bloomington, IN, USA; and 3USDA ARS PoisonousPlant Research Laboratory, Logan, UT, USA

Summary

1. Plants form mutualistic symbioses with a variety of microorganisms including endophyticfungi that live inside the plant and cause no overt symptoms of infection. Some endophyticfungi 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 Clavicipit-

aceae. These symbioses have been studied extensively because of their significant impacts oninsect 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 di!erent combinations among endophyte taxa.

3. Several legumes, including locoweeds, are associated with a toxic syndrome called locoismas a result of their accumulation of swainsonine. Species in two genera were recently found tocontain previously undescribed endophytic fungi (Undifilim spp., family Pleosporaceae) that

are the source of that toxin. The fungi are strictly vertically transmitted and have narrow hostranges.

4. Some plant species in the morning glory family (Convolvulaceae) also form symbioses withendophytic 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 thatproduce swainsonine. The swainsonine-producing endophytes associated with the Convolvula-

ceae are distinct from the Undifilum spp. associated with locoweeds and the Clavicipitaceousfungi associated with Convolvulaceae.5. In the establishment of vertically transmitted symbioses, fungi must have entered the symbi-

osis with traits that were immediately useful to the plant. Bioactive metabolites are likely can-didates 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 rangesand that produce bioactive compounds are likely to be found as important mutualists in

additional plants.

Key-words: defensive mutualism, ergot alkaloids, indole-diterpenes, lolines, peramine,

plant-herbivore interactions, swainsonine, symbiosis

Introduction

Several classes of mutualisms have been recognized based

upon the benefits exchanged between the partners

including energetic (e.g. photosynthetic Chlorella in Anth-

ozoans), nutritional (e.g. mycorrhizae), transport (e.g. pol-

lination) 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*Correspondence author. E-mail: [email protected]

Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Journal of Applied Ecology 2013 © British Ecological Society

Functional Ecology 2013 doi: 10.1111/1365-2435.12076

that of the Ant-Acacia system in which voracious ants pro-

tect 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 e!ects on host success as well as commu-

nity 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 o!

yeasts that would otherwise degrade the ants’ fungal gar-

den (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 a!orded 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 caus-

ing overt detrimental symptoms and typically has no obvi-

ous 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 a!or-

ded 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 Clavici-

pitaceae.

Hereditary symbionts that are strictly vertically trans-

mitted 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 e!ect 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 com-

pletely 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 associ-

ated with defensive symbioses. Clavicipitaceous endo-

phytes 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 con-

trast, 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 e!ects 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 condi-

tions. 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). W!ali et al. (2006) showed that red fescue (Festuca

rubra) infected with Epichlo!e festucae in subarctic regions

su!ered 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 e!ects have been seen, it does not

preclude the existence of some positive benefit that has not

been measured that results in maintenance of the mutual-

ism, 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-endo-

phytic fungus associations – particularly those that fit well

with the definition of defensive mutualism – is the produc-

tion of bioactive secondary metabolites by the fungal sym-

bionts. Whereas fungi in general produce a wide array of

secondary metabolites, we propose that bioactive metabo-

lites are particularly important to vertically transmitted

fungi, which in the establishment of their fungus–plantsymbioses 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 transmit-

ted symbiotic fungi that produce noteworthy bioactive

chemicals in their symbiotic state but for which roles of


Journal of Applied Ecology 2013 © British Ecological Society, Functional Ecology

2 D. G. Panaccione et al.

the chemicals in the symbioses have not been experimen-

tally 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 Epichlo!e and Neotyphodium grow sym-

biotically with many cool season grasses. Neotyphodium

species are exclusively asexual and grow within the inter-

cellular spaces of their grass hosts (Fig. 1a). Epichlo!e spe-

cies represent the teleomorphic states of several

Neotyphodium species. In addition to the symbiotic phase

typical of Neotyphodium species, Epichlo!e species are capa-

ble of exiting their plant hosts via the formation of sexual

reproductive stroma on plant inflorescences. Those sexu-

ally reproducing Epichlo!e species are thus capable of verti-

cal 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.

In addition to direct e!ects on herbivores, epichloid en-

dophytes of grasses can significantly a!ect ecological

communities. Neotyphodium coenophialum-infected tall

fescue (Lolium arundinaceum) suppresses both plant (Clay

& Holah 1999) and arthropod diversity (Finkes et al.

2006), a!ects 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 relation-

ships between diversity and ecosystem properties such as

productivity (Rudgers, Koslow & Clay 2004).

The epichloae collectively produce four classes of bioac-

tive metabolites in their symbiotic associations with plants:

ergot alkaloids, indole-diterpenes, loline alkaloids and per-

amine. 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 representa-

tives of all four classes; most epichloae produce metabo-

lites belonging to one to three of the chemical classes

(Schardl et al. 2011).

ERGOT ALKALO IDS

Ergot alkaloids are a diverse family of secondary metabo-

lites produced by certain epichloae, ergot fungi (Claviceps

spp.) and related species of Balansia and Periglandula (dis-

cussed 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 vasoconstric-

tion, 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 e!ects 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 alka-

loids also a!ect insects and nematodes, which contain

homologous neurotransmitters. Activities in insects include

feeding deterrence, delayed development and increased

(a)

(c) (e)(d)

(b)

Fig. 1. Class 1 endophytic fungi. (a) Ani-line blue-stained hypha of Neotyphodiumcoenophialum growing intercellularly in apeeled leaf sheath of tall fescue (Loliumarundinaceum); (b) Hyphae of GFP-expressing Undifilum oxytropis in locoweedvascular tissue; (c) Numerous, smallcolonies of Periglandula ipomoeae growingepiphytically on the adaxial leaf surface ofIpomoea asarifolia; (d) Fungicide-treatedI. asarifolia leaf lacking fungal colonies;(e) Aniline blue-stained colonies ofP. ipomoeae from the adaxial leaf surfaceof I. asarifolia. (Photos: the authors)



Bioactive alkaloids of fungal endophytes 3

mortality (Clay & Cheplick 1989; Ball, Miles & Prestidge

1997; Potter et al. 2008).

The ergot alkaloid pathway is notable for its accumula-

tion 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 ine"-

ciency 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 di!ers from the bene-

fit(s) provided by the pathway end product. Di!erences in

activities of clavine intermediates and spur products com-

pared 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 Epichlo!e typhin-

a 9 Neotyphodium lolii isolate Lp1 (hereafter simply Lp1)

provide more support for this hypothesis. A knockout

mutant that accumulated certain clavines but not ergopep-

tines or simple amides of lysergic acid deterred rabbit feed-

ing on infected grasses as well as or better than the wild

type of the fungus (Panaccione et al. 2003, 2006b). In con-

trast, 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 alka-

loids were restricted to earlier pathway intermediates and

spur products. Thus, the accumulation of both intermedi-

ates 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 di!erent

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.

INDOLE -D ITERPENES

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 Tricho-

comaceae (e.g. Aspergillus and Penicillium spp.) (Saikia

et al. 2008). Indole-diterpenes have been studied inten-

sively because certain members of this class of metabolites

have strong tremorgenic activity in mammals. For exam-

ple, the lolitrems produced by Neotyphodium lolii in peren-

nial ryegrass cause ryegrass staggers (Gallagher, White &

Fig. 2. Diversification of ergot alkaloids associated with endophyte–plant symbioses. Double arrows indicate one or more omitted inter-mediates. Dashed arrows indicate uncharacterized steps. Relevant enzymes associated with catalysis at branch points are indicated. At thefirst branch point, alternative forms of EasA form festuclavine (not pictured) and agroclavine (Coyle et al. 2010); additional alternativeforms are hypothesized to produce cycloclavine and lysergol in Periglandula-infected Ipomoea spp. At the second branch point, combina-tions 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.




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 diver-

sification of indole-diterpenes can be based on the oxida-

tion and prenylation of intermediate terpendole I and its

subsequent metabolites independently, resulting in di!er-

ent end products including terpendoles, lolitrems and

janthitrems (Fig. 3).

Much of the early analysis of grass endophytes for

indole-diterpenes focused intensively on lolitrem B. Evi-

dence 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 natu-

rally occurring isolates that vary in indole-diterpene pro-

files (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 (Gate-

nby 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 for-

age 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 e!ects 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 negligi-

ble 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 biogeni-

cally related yet structurally distinct compound nodulisp-

oric acid. Nodulosporic acid is produced in culture by

Nodulisporium sp. (an anamorphic fungus in the Xylaria-

ceae 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 accu-

mulate 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.

Fig. 3. Diversification of indole-diterpenes associated with endophyte-plant symbioses. Double arrows indicate one or more omitted inter-mediates. Dashed arrows indicate uncharacterized steps. LtmE/LtmJ and LtmF/LtmK represent separate prenyl transferase/monooxygen-ase (respectively) combinations that work on opposite ends of members of the indole-diterpene family (Young et al. 2006, 2009). Eachcombination can act on multiple substrates.




LOL INES

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 (Faba-

ceae) (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 deter-

mined. The biosynthesis and activities of lolines has been

reviewed in detail by Schardl et al. (2007). Lolines occur in

exceptionally high concentration in Neotyphodium coeno-

phialum-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 pres-

ence or absence of methyl, formyl or acetyl groups on the

homoserine-derived amine group (Fig. 4a).

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). Rie-

dell 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 di!erent aphid species and loline pro-

duction in a genetic cross among Epichlo!e 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 e!ect on insects,

lolines also are nematicidal (Bacetty et al. 2009a). The

e!ects of lolines appear to be restricted to invertebrates.

Schardl et al. (2007) carefully reviewed studies on verte-

brate toxicity of lolines and concluded that anti-vertebrate

e!ects of lolines were likely to be negligible, because studies

indicating such e!ects were either confounded by the

presence of ergot alkaloids in the same plant tissues or con-

ducted with exceptionally high concentrations of lolines.

PERAMINE

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 epi-

chloae (Clay & Schardl 2002; Tanaka et al. 2005). Its ori-

gin as a fungal metabolite was shown by its production by

isolated fungi in vitro (Rowan 1993), and more convinc-

ingly 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 sin-

gle 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 e!ects of peramine are not univer-

sal, 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 inacti-

vated 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 endo-

phyte (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 endo-

phyte-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 cuti-

cle. 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.

(a)

(b)

Fig. 4. Structures of (a) lolines and (b) peramine. Variationamong lolines derives from substituents on the indicated nitrogen(Schardl et al. 2007).




DISTR IBUT ION OF BIOACT IVE ALKALO ID CLASSES

AMONG EP ICHLOAE

The capacity to produce the four classes of bioactive alka-

loids varies among epichloae taxa. Two classes of epi-

chloae-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 uncer-

tain). 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. coe-

nophialum 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 symbi-

ota 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.

The distribution of lolines, ergopeptines (but not other

ergot alkaloids) and peramine among sexual stroma-pro-

ducing Epichlo!e spp. (those capable of horizontal transmis-

sion) 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 en-

dophytes. The reduced level of anti-insect alkaloids in

grasses hosting sexually reproducing epichloae is consistent

with the dependence of the sexual Epichlo!e 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 epibiot-

ic fungal symbionts of the Convolvulaceae (morning

glories) that produce ergot alkaloids in the seeds and, in

Table 1. Distribution of anti-vertebrate and anti-insect alkaloids among epichloae–grass symbiota in which all four classes of bioactivealkaloids have been assayed*

Fungus Host plant Anti-vertebrate alkaloids† Anti-insect alkaloids‡

Epichlo!e elymi Elymus canadensis ERG ERG, PEREpichlo!e festucae Festuca longifolia ERG, IDT ERG, PERE. festucae Festuca ovina ERG ERG, PERE. festucae Festuca rubra subsp. commutata ERG ERG, PERE. festucae F. rubra subsp. commutata – PERE. festucae F. rubra subsp. rubra IDT –E. festucae F. rubra subs. rubra ERG ERGE. festucae Lolium giganteum ERG ERG, LOLEpichlo!e typhina Lolium perenne – PERNeotyphodium aotearoae Echinopogon ovatus – LOLNeotyphodium coenophialum Lolium arundinaceum ERG ERG, LOL, PERN. coenophialum L. arundinaceum – LOL, PERNeotyphodium huerfanum Festuca arizonica – PERNeotyphodium gansuense Achnatherum inebrians ERG ERGNeotyphodium lolii Lolium perenne ERG, IDT ERG, PERN. lolii L. perenne IDT PERN. lolii L. perenne IDT –N. lolii 9 E. typhina isolate Lp1 L. perenne ERG, IDT ERG, PERNeotyphodium siegelii Lolium pratense – LOL, PERNeotyphodium starrii Bromus anomalus ERG ERG, PERNeotyphodium sp. E55 Poa autumnalis – LOL, PERNeotyphodium sp. E4074 Lolium sp. – LOL, PERNeotyphodium sp. E4078 Lolium sp. ERG, IDT ERG, PERNeotyphodium sp. Festuca paradoxa – PERNeotyphodium sp. Festuca subverticillata – –Neotyphodium sp. Hordelymus europaeus – –Neotyphodium tembladerae F. arizonica – PERNeotyphodium typhinum Poa ampla – PERNeotyphodium uncinatum L. pratense – LOL

*Refer to Schardl et al. (2011) for details on symbiota.†ERG, ergot alkaloids; IDT, indole-diterpenes.‡ERG, ergot alkaloids; LOL, loines; PER, permine.




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 vis-

ible 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 demon-

strated 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 alka-

loids in the foliage (Kucht et al. 2004). Unlike other occur-

rences of plant-associated Clavicipitaceae, Periglandula

species are the only clavicipitaceous fungi known to associ-

ate 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 dissimi-

lar and occur on di!erent host plants (Steiner et al. 2011).

A third, undescribed, Clavicipitaceae from Ipomoea tri-

color, which does not form epiphytic mycelia, was detect-

able 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 occur-

rence of ergot alkaloids in the Convolvulaceae was thought

to be a case of convergent evolution or horizontal gene

transfer (Steiner, Hellwig & Leistner 2008). Although pub-

lished 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 dis-

covery 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 ritualisti-

cally for divination. Two decades later, Albert Hofmann

isolated the lysergic acid amides ergine and lysergic acid

a-hydroxyethylamide from T. corymbosa (Hofmann &

Tscherter 1960; Hofmann 1961), which were likely respon-

sible for the hallucinogenic e!ects 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 alka-

loid-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), origi-

nally discovered in the clavicipitaceous fungi Balansia ob-

tecta which form epiphytic infections of Cenchrus echinatus

(Sandbur Grass) (Cyperaceae) (Powell et al. 1990). In

addition to ergot alkaloids known from other Clavicipita-

ceae, unique ergot alkaloids have been discovered in the

Convolvulaceae, notably cycloclavine from Ipomoea hilde-

brandtii, an African shrub (Stau!acher 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); how-

ever, the phylogeny of this large family is still not clearly

resolved. Both major clades within the Ipomoeeae (Stefa-

novic, Krueger & Olmstead 2002) have ergot alkaloid-posi-

tive representatives, and there is variation within sections

with respect to the presence of ergot alkaloids (Eich 2008).

Expanded sampling of the Convolvulaceae for ergot alka-

loids will surely reveal more species infected by Periglandu-

la. 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, how-

ever, reports of tremorgenic symptoms in livestock caused

by grazing of foliage from ergot alkaloid-positive Convol-

vulaceae, 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 contain-

ing indole-diterpenes also su!er from tremorgenic symp-

toms (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 e!ects of Periglandula infection or ergot

alkaloid presence in the Convolvulaceae has yet to be stud-

ied. As Periglandula species are closely related to Neoty-

phodium 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 sea-

son 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 &

Hu"aman 1996). Many ergot alkaloid-positive Convolvula-

ceae 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 demon-

strate increased resistance to foliar herbivory, resistance to

seed predation may be an important dimension of the Peri-

glandula-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 parasit-

ized 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 bru-

chine 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 di!erences 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 Periglan-

dula 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 live-

stock in the Americas, Asia and Australia (Marsh 1909;

Marsh & Clawson 1936; Gardiner, Linto & Aplin 1969;

Huang, Zhang & Pan 2003). Locoism, a neurologic dis-

ease, 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 loco-

ism are similar in animals intoxicated by locoweed species

and Swainsona species (James, Van-Kampen & Hartley

1970; Panter et al. 1999). Swainsonine (Fig. 5), a trihydr-

oxyindolizidine alkaloid, was first identified as the active

principle in Swainsona canescens, a legume native to Aus-

tralia (Colegate, Dorling & Huxtable 1979), and subse-

quently identified as the active principle in locoweeds

(Molyneux & James 1982). Swainsonine inhibits the

enzymes a-mannosidase and mannosidase II resulting in

lysosomal storage disease and altered glycoprotein synthe-

sis (Hartley 1971; Dorling, Huxtable & Vogel 1978).

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 Undifi-

lum 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. Undifi-

lum species have been found to be associated with swainso-

nine-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 di!erent plant species are associated with unique Undifi-

lum species (Pryor et al. 2009; Baucom et al. 2012).

In addition to the legumes, swainsonine occurs sporadi-

cally 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 carpino-

folia 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 (Clavici-

pitaceae) (Schneider et al. 1983; Patrick, Adlard & Kesha-

varz 1993). Rhizoctonia leguminicola is a fungal pathogen

of red clover (Trifolium pratense) that causes black patch

disease in the plant. Metarhizium anispoliae is an entomo-

pathogen 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

Fig. 5. Proposed pathway for swainsonine and slaframine biosyn-thesis. Pathway is based on studies conducted in Rhizoctonia legu-minicola (Harris et al. 1988b).




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). Swainso-

nine is derived from lysine which is converted into pip-

ecolic 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 biosynthet-

ic 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 follow-

ing observations: (i) locoweed plants infected with Undifi-

lum 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 concentra-

tions 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 swains-

onine 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 di!erent 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 concen-

trations >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

di!er significantly in the amount of endophyte they

contain which may explain the di!erence in swainsonine

concentrations (Cook et al. 2009, 2011).

Swainsonine and endophyte amounts have been investi-

gated in di!erent plant parts at di!erent 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 concen-

trations 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 intoxi-

cated (Ralphs et al. 2002; Pfister et al. 2003). In fact ani-

mals 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 stud-

ies, although preliminary results show that swainsonine

has no e!ect on some insect species (Parker 2008).

Legumes are known for forming symbioses with N-fixing

bacteria and investigators found that swainsonine concen-

trations 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 symbi-

onts. An alternative interpretation is that the improved

nitrogen status of the host may have increased substrate

availability for swainsonine production; however, no con-

sistent di!erences 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 canes-

cens, in the Convolvulaceous genera of Ipomoea and Turbi-

na, 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 car-

nea (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

di!erences (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 endo-

phyte 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 chemo-

types in native plant populations that di!er greatly in the

amount of swainsonine and extent of endophyte coloniza-

tion. Second, a small amount of Undifilum species myce-

lium 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 clavicipita-

ceous 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 verti-

cally transmitted endophytic fungi that produce ergot alka-

loids, indole-diterpenes, lolines or peramine are derived

from Clavicipitaceae, regardless of the plant family with

which they are associated, whereas strictly vertically trans-

mitted endophytes that produce swainsonine are derived

from di!erent fungal families that form relationships with

specific plant families. In regard to this observation, the

evolutionary history of the swainsonine biosynthetic path-

way in these diverse fungi is particularly intriguing.

Concluding remarks

In this study, we have focused on the bioactive metabolites

of vertically transmitted endophytes and the e!ects 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 verti-

cally transmissible endophytes, they need to be advanta-

geous for their host plants, otherwise they would be

outcompeted by non-infected conspecifics, which typically

exist among endophyte-infected populations because endo-

phyte transmission is rarely 100% (Afkhami & Rudgers

2008). The advantage provided to the host by the endo-

phyte 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 clavicipita-

ceous endophytes of grasses and morning glories may pro-

vide an illustration of this point. Spatafora et al. (2007)

demonstrated through phylogenetic analyses that the

plant-infecting Clavicipitaceae (which includes the epi-

chloae and the Periglandula spp.) likely evolved from

insect-infecting Clavicipitaceae. Insect-pathogenic Meta-

rhizium spp. share a recent common ancestor with the

clade that contains the epichloae and related plant-infect-

ing Clavicipitaceae; these fungi are part of a monophyletic

group (Clavicipitaceae clade A) whose most recent com-

mon ancestor was likely an insect pathogen (Spatafora

et al. 2007). The genomes of M. anisopliae and Metarhizi-

um 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 produc-

tion has not been demonstrated experimentally in these

species. Considering the distribution of ergot alkaloid

Table 2. Important characteristics of plant–fungal symbiota considered in this review

Grasses Morning Glories Locoweeds

Associated FungalFamily

Clavicipitaceae Clavicipitaceae Unknown Pleosporaceae

Bioactive ChemicalsProduced

Ergots, lolines, peramine,indole-diterpenes

Ergots, lolines2, indole-diterpenes3 Swainsonine Swainsonine

Major Clade Monocots Eudicots EudicotsGrowth Form Herbaceous Mostly woody vines (also shrubs,

trees, herbaceous vines)Herbaceous

Distribution Mostly temperate Mostly tropical & subtropical Semiarid to temperateEconomicImportance

Forage, crops Some crops1, agricultural pests Agricultural pests

Pollination System Wind Pollinated Insect pollinated Insect pollinatedMode(s) ofTransmission

Strictly vertical, mixed(vertical and horizontal),and strictly horizontal

Strictly vertical (?) Strictlyvertical (?)

Strictly vertical (?)

1Ipomoea batatas (sweet potato) and Ipomoea aquatica (water spinach) are crop species but do not contain ergot alkaloids (Eich 2008) andhave never been reported to produce swainsonine.2Lolines were reported from A. mollis (Tofern et al. 1999), but it has not been demonstrated that they are produced by Periglandula.3Indole diterpenes have not been reported from morning glories, but there have been reports of tremorgenic symptoms, characterisitc ofindole diterpene poisoning, in livestock feeding on species infected by Periglanudla (Araújo et al. 2008).




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 Clavicipita-

ceae. The biosynthetic capacity may have then diversified

to include other forms that are more e!ective against ver-

tebrate 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 M. anisopliae produces swainso-

nine 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 e!ort has gone into understanding ecologi-

cal impacts of endophytes (reviewed in Clay & Schardl

2002; Schardl et al. 2009). Similarly, molecular and bio-

chemical approaches have improved our understanding of

endophyte-produced bioactive metabolites and their bio-

synthetic pathways (reviewed in Lorenz et al. 2009; Panac-

cione 2010; Schardl et al. 2011). Future research should

connect these two lines of investigation to molecularly dis-

sect the roles of various chemicals in fungus–grass–herbi-vore 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 associa-

tions 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 e!ects 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 trans-

mission) may facilitate their discovery. Vertically transmit-

ted symbionts are observed at much higher infection

frequencies than horizontally transmitted symbionts, pos-

sibly 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 verti-

cally transmitted Neotyphodium spp. reach infection fre-

quencies 40–130% greater than Epichlo!e species that have

mixed horizontal and vertical transmission. In aphid–bac-terial interactions, the vertically transmitted Buchnera spe-

cies is fixed in most populations and considered obligate,

whereas there are many horizontally transmitted symbio-

nts 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 charac-

teristics 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 clas-

ses 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 Rodri-

guez 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.

Acknowledgements

Funding from the U.S. Department of Agriculture National Institute ofFood and Agriculture (2012-67013-19384) to D.G.P. is gratefully acknowl-edged. We thank Keith Clay for helpful guidance on the content of thisreview, Christopher Schardl and an anonymous reviewer for constructivecomments on a previous version of this article, and Sarah Robinson forassistance with the bibliography. This article is published with permissionof the West Virginia Agriculture and Forestry Experiment Station asscientific article number 3155.

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Received 20 October 2012; accepted 21 January 2013Handling Editor: Edith Allen




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