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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
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.
Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
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.
Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
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*
*Refer to Schardl et al. (2011) for details on symbiota.†ERG, ergot alkaloids; IDT, indole-diterpenes.‡ERG, ergot alkaloids; LOL, loines; PER, permine.
Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
(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).
Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
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).
Published 2013. This article is a U.S. Government work and is in the public domain in the USA.
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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